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Table of Contents
Introductions
Section One
High-Altitude
Medicine
3.... High-Altitude Medical Research in China:
Importance and Relevance
Wu Tianyi, M.D.
4.... Research Atop the Roof of the World
Alan Leshner, Ph.D.
6.... A Unique Challenge in High-Altitude Medicine:
The Qinghai-Tibet Railroad
7.... Human Performance Engineering at High Altitude
9.... Intrinsic Characteristics in Tibetans of Tolerance to Hypoxia
Following Long Periods at Sea Level
10.... Exploration and Evidence of High-Altitude Adaptation
in Tibetan Highlanders
12.... Peopling of the Tibetan Plateau and Genetic Adaptation
to High-Altitude Hypoxia in Tibetans
14.... New Approaches for Facilitating High-Altitude Acclimatization
15.... Evidence for Genetic Contribution to High-Altitude Pulmonary
Edema in Chinese Railway Construction Workers
17.... Studies on the Prevention of Acute Mountain Sickness
in People Entering High Altitudes by Airplane
18.... Adaptive Responses of the Brain to High-Altitude
20.... Mechanism of Chronic Intermittent Hypoxia-Induced Impairment
in Synaptic Plasticity and Neurocognitive Dysfunction
22.... Chinese Herbs and Altitude Sickness: Lessons from
Hypoxic Pulmonary Hypertension Research
24.... Fast Acclimatization to High Altitude Using
an Oxygen-Enriched Room
25.... A Comparison of Perimenopausal Sex Hormone Levels Between
Tibetan Women at Various Altitudes and Han Women at Sea Level
26.... Diagnosis and Treatment of HAPE and HACE in
the Tibet High-Altitude Region in the Last Decade
27.... Cardiac Surgery on the Tibetan Plateau: From Impossible
to Successful
28.... Acute Mountain Sickness on the Tibetan Plateau: Epidemiological
Study and Systematic Prevention
29.... Study on Erythrocyte Immune Function and Gastrointestinal
Mucosa Barrier Function After Rapid Ascent to High Altitude
30.... Basic Methods and Application of Altitude Training
on the Chinese Plateau
31.... Hypoxic Preconditioning at High Altitude Improves
Cerebral Reserve Capacity
33.... The Dynamic Balance Between Adaptation and Lesions of the
Cardiovascular System in Tibetans Living at High-Altitude
34.... Establishment of an Improved Bundle Therapy Procedure for
Acute High-Altitude Disease
36.... Differences in Physiological Adaptive Strategies to Hypoxic
Environments in Plateau Zokor and Plateau Pika
1
Table of Contents
Section Two
Hypoxic
Physiology
38.... Cardioprotective Effect of Chronic Intermittent
Hypobaric Hypoxia
40.... Corticotropin-Releasing Factor Type-1 Receptors Play a Crucial Role in the Brain-Endocrine Network Disorder Induced by High-Altitude Hypoxia
42.... The Key Role of Vascular Endothelial Dysfunction in Injuries Induced by Extreme Environmental Factors at High Altitude
45.... Targeting Endothelial Dysfunction in High-Altitude Illness with a Novel Adenosine Triphosphate-Sensitive Potassium Channel Opener
47.... Adaptation to Intermittent Hypoxia Protects the Heart from Ischemia/
Reperfusion Injury and Myocardial Infarction
49.... Mild Hypoxia Regulates the Properties and Functions
of Neural Stem Cells In Vitro
51.... Hypobaric Hypoxia or Hyperbaric Oxygen Preconditioning Reduces
High-Altitude Lung and Brain Injury in Rats
53.... Mitochondria: A Potential Target in High-Altitude Acclimatization/
Adaptation and Mountain Sickness
55.... Mimicking Hypoxic Preconditioning Using Chinese
Medicinal Herb Extracts
57.... Molecular Path Finding: Insight into Cerebral Ischemic/Hypoxic Injury and Preconditioning by Studying PKC-isoform Specific Signaling Pathways
59.... Hypoxic Preconditioning Enhances the Potentially Therapeutic Secretome from Cultured Human Mesenchymal Stem Cells in Experimental Traumatic Brain Injury
61.... Mitochondrial Adaptation and Cell Volume Regulation in Hypoxic Preconditioning Contribute to Anoxic Tolerance
62.... The Effects of Ratanasampil, a Traditional Tibetan Medicine, on β-amyloid Pathology in a Transgenic Mouse Model and Clinical Trial
of Alzheimer’s Disease
63.... Duoxuekang, a Traditional Tibetan Medicine, Reduces Hypoxia-Induced High Altitude Polycythemia in Rats
64.... k-opioid Receptor and Hypoxic Pulmonary Hypertension
66.... Paracrine-Autocrine Mechanisms in the Carotid Body Function
at High Altitude and in Disease
ABOUT THE COVER: Mount Qomo Lhari, which stands 7,314 m high and is known
in Tibetan as the “Goddess Peak,” has yet to be conquered by humans. The sharp and
forbidding peak, with its encircling white clouds, carries the message of good luck to those
setting out to explore the unknown.
Photo credit: Gesang Luobu and Shilie Jiangca
This booklet was produced by the Science/AAAS Custom Publishing Office and sponsored
by the National Key Basic Research Program of China (“973” Program). Materials that
appear in this booklet were commissioned, edited, and published by the Science/AAAS
Custom Publishing Office and were not reviewed or assessed by the Science Editorial staff.
This booklet was produced in association with the Beijing Institute of Basic Medical Sciences.
Editors: Sean Sanders, Ph.D.; Fan Ming, Ph.D.
Assistant Editor: Lingling Zhu, Ph.D.
Proofing: Yuse Lajiminmuhip; Design: Amy Hardcastle
2
© 2012 by The American Association for the Advancement of Science.
All rights reserved. 14 December 2012
High-Altitude Medical Research in China:
Importance and Relevance
Tibetans are considered
to be well-adapted to
hypoxic conditions and
form a unique group for
the study of the chronic
effects of hypoxia on
human physiology
and disease.
In the last 30 years, great strides have been made in high-altitude medical research in China
due in large part to the unique set of circumstances in the country. China encompasses a vast
and mountainous region with four high plateaus (the Qinghai-Tibet, Inner Mongolia, Yun-Gui,
and the Yellow Land Plateaus). The Qinghai-Tibet Plateau, the Earth’s largest and highest, is
sometimes called the “the roof of the world.” Altitude-related health problems are particularly
important in China since nearly 80 million people live above 2,500 m with more than 12 million
residing on the Qinghai-Tibet Plateau alone. Additionally, large, rich deposits of valuable ores,
precious metals, and oil have been discovered recently in Tibet, where the most important
mines are located above 4,000 m and the miners living there experience chronic hypoxia
(reduced oxygen supply). Finally, to support industrial development in western China, the new
1,142 km long Qinghai-Tibet Railway has been recently completed. Over 85% of the rail line
is above 4,000 m, even reaching 5,072 m. During construction of the railway from 2001 to
2005, approximately 140,000 workers were required to labor in a severely hypoxic environment,
emphasizing the need to understand and treat altitude-specific illnesses.
In Tibet, native populations of differing origin have been living at high altitude for varying
lengths of time, making the Tibetan plateau a natural location for comparing the effect of high
altitude on biologically distinct populations. Han Chinese inhabitants are newcomers to these
higher elevations, having come from low altitudes within the past one to three generations. They
therefore typically tolerate hypoxia poorly and are only weakly acclimatized to high altitude.
By contrast, the Tibetans are an indigenous Himalayan population who are reproductively
isolated and genetically stable due to limited intermarriage. Archaeological evidence indicates
that primitive societies have existed in northern Tibet for 25,000 to 50,000 years. Tibetans are
therefore considered to be well-adapted to hypoxic conditions and form a unique group for the
study of the chronic effects of hypoxia on human physiology and disease.
Recently studies have shown that Tibetans, compared with Han lowlanders, maintain higher
arterial oxygen saturation at rest and during exercise with increasing altitude, and show reduced
loss of aerobic performance. Tibetans have greater hypoxic and hypercapnic ventilatory
responsiveness, large lungs, better lung function, and greater lung diffusing capacity than Han
lowlanders. Additionally, Tibetans develop only minimal hypoxic pulmonary vasoconstriction
and have higher levels of exhaled nitric oxide. The sleep quality of Tibetans at altitude is better
than Han lowlanders and their blood oxygen levels drop less at night. These findings are all
indicative of remarkable high-altitude adaptation.
The Tibetan and Han Chinese populations also provide an ideal opportunity to study genetic
predisposition to high-altitude disease. Chronic mountain sickness (CMS) in particular is a
public health problem in Qinghai-Tibet. Epidemiological data indicates that CMS is found in
Han immigrants at a rate of 5% to 10%. In contrast, CMS is rare in Tibetans (0.5% prevalence).
Physiological data from multiple studies supports the possibility that the Tibetans carry
protective genetic factors. Of particular interest is the lower average hemoglobin concentration
in Tibetans compared with Han Chinese living at the same altitude. Excessive hemoglobin,
known as polycythemia, is a hallmark of CMS and is caused by the body’s overreaction to
altitude hypoxia, resulting in characteristically viscous blood. Tibetans maintain relatively low
hemoglobin at high altitude, a trait that makes them less susceptible to CMS than immigrants. To
pinpoint the genetic origin underlying Tibetans’ relatively low hemoglobin levels, recent research
in China, England, Ireland, and the United States comparing DNA from Tibetans with their Han
lowland counterparts, found variations in a gene called EPAS1 (endothelial PAS domain protein
1, also known as HIF2A, hypoxia inducible factor 2A). These genetic differences are thought
to be responsible for the low blood hemoglobin and resulting CMS protective effects. Although
much work remains to determine if other physiological factors may be at work, these findings
have opened a new era in our understanding of genetic adaptation among Tibetans.
With an increasing number of people moving to the higher altitudes, the study of physiological
adaptation to hypoxia and related diseases is growing in importance, making life on the Tibetan
plateau one of the most relevant research fields in our region.
Wu Tianyi, M.D.
Member of the Chinese Academy of Engineering
Professor, High Altitude Medical Research Center, University of Tibet, Lhasa, China
Director, High Altitude Medical Research Institute, Qinghai, China
3
Research Atop the Roof of the World
So whether by genetic
adaptation or through
the application of
our knowledge,
experience, and
intellect, humans are
continuing to adjust
to harsh conditions
on the Tibetan plateau
and expand our
understanding of
the effect of extreme
environments on
our bodies.
It would make sense that a primary reason for the success of the human race at populating
almost every corner of the planet’s surface is our ability to adapt to the majority of climates
and environmental conditions. From the hottest deserts to the coldest and most barren arctic
landscapes, humans have made their homes. It hasn’t always been easy, though.
One of the more extreme climes that humans have settled must surely be the QinghaiTibet region of western China, the world’s largest and highest plateau. This often inhospitable
landscape offers its courageous inhabitants frigid temperatures, thin atmosphere, and hypoxic
(low oxygen) conditions.
This booklet, an editorial collaboration between the Custom Publishing Office at the journal
Science and top researchers in the field of high-altitude medicine and hypoxic physiology in
China, provides scientists around the globe with a window into some of the fascinating research
being carried out in this field, using the Qinghai-Tibet Plateau as a test bed. Brief reviews in an
array of different areas of study are presented, together highlighting the many advances that
have been made in the understanding, treatment, and prevention of high-altitude sickness.
Each year, chronic and acute mountain sickness claims the lives of many unsuspecting or
even well-prepared travelers to these high-altitude regions, and a greater number are sickened
or permanently disabled. Extensive efforts are under way by doctors and researchers in China
to develop improved treatments and preventative measures that will allow for safer travel and
long-term habitation in the region. An array of studies are under way attempting to elucidate the
underlying mechanism for high-altitude illnesses and thereby development suitable treatments.
Interventions range from the use of conventional Western medicine to specialized physical
exercise regimens that speed acclimation and minimize potential health issues. Traditional
Chinese medicines that, in some cases, have been used for many hundreds of years are
also being more closely and systematically studied for their efficacy in preventing or reducing
altitude-related ailments.
Also intensively studied is the role that genetics and evolution might play in the adaptation of
long-term plateau dwellers. Although hard evidence of sustained occupation is scarce, native
Tibetans are believed to have lived on the Qinghai-Tibet Plateau for upward of 25,000 years,
potentially enough time for them to gain a genetic advantage over their lowland ancestors.
Elucidation of the particular DNA changes they might have acquired may provide researchers
with some clues about where to look for possible drug targets.
So whether by genetic adaptation or through the application of our knowledge, experience, and
intellect, humans are continuing to adjust to harsh conditions on the Tibetan plateau and expand
our understanding of the effect of extreme environments on our bodies. What researchers learn
will have implications for the health and well-being of all high-altitude populations.
Alan Leshner, Ph.D.
CEO, AAAS
Executive Publisher, Science
4
CREDIT: © ISTOCKPHOTO.COM/DEIMAGINE
Section One: High-Altitude Medicine
High-Altitude Medicine
A Unique Challenge in High-Altitude Medicine:
The Qinghai-Tibet Railroad
Wu Tianyi1,*, Ding Shou Quan2, Liu Jin Liang3, Bengt Kayser4
A
s a result of industrial development in western China, the
Chinese government decided to build the Qinghai-Tibet
Railway (QTR) in 2001. This railroad, between Golmud (2,808 m) and Lhasa (3,658 m), is 1,142 km long
and over 85% of the rail line is above 4,000 m. The highest pass is
5,072 m, through the Mt. Kun Lun and Tanggula ranges, making the
QTR the highest railroad in the world (1, 2). From 2001 to 2005, the
new railroad was built by more than 140,000 workers, of whom 80%
traveled from their lowland habitat to an altitude of approximately
5,000 m. Construction of the railroad represented a unique challenge
in high altitude medicine. Initially, the overall incidence of acute
mountain sickness (AMS), high-altitude pulmonary edema (HAPE),
and high-altitude cerebral edema (HACE) in workers was approximately 45% to 95%, 0.49%, and 0.26%, respectively (2). The challenge in terms of treatment and prevention of high altitude sickness
was significant.
Our research team worked continuously for five years in three of the
highest local hospitals along the rail line in the Fenghoushan (altitude:
4,779 m, barometric pressure (PB) ~417 Torr), Kekexili (4,505 m, PB
~440 Torr) and Dangxiang areas (4,292 m, PB ~447 Torr). The study
was approved by the Qinghai High Altitude Research Institute Committee on Human Research.
Preexisting Conditions
The construction of the QTR resulted in several challenging problems
in high-altitude medicine (1–8). First, identifying which individuals are
not suited for high altitudes is not easy for patients with preexisting disorders, thus making it difficult for physicians to give clear advice. We
studied the medical conditions of 14,050 high-altitude workers, paying
particular attention to preexisting illnesses. All subjects were observed
at low and high altitude. Based on our findings, we believe that neither
taking a rather permissive stance nor setting rigid rules of contraindication is correct. The former may put some persons at risk whereas the
latter may exclude too many subjects from traveling to high altitudes,
even when this may be safe. Obviously, conditions that are related to
hypoxia at low altitude will be exacerbated at high altitude. Such conditions include chronic obstructive pulmonary disease with arterial desaturation, recent cardiac infarction or heart failure, obesity with sleep
apnea, or severe hypertension. Subjects with such conditions should be
advised against travel to high altitude. Conversely, patients with mild
anemia or allergic asthma do not appear to have increased risk of developing ailments at high altitudes and their conditions may even improve.
We have suggested that careful evaluation of preexisting chronic ill-
National Key Laboratory of High Altitude Medicine,
High Altitude Medical Research Institute, Xining, China;
2
Qinghai-Tibet Railroad Hospital at Fenghuoshan, Qinghai, China;
3
Qinghai-Tibet Railway Hospital at Kekexili, Qinghai, China;
4
Institut des Sciences du Movement et de la Macute Medécine du Sport,
Faculté de Médecine, Université de Genève, Geneva, Switzerland.
*
Corresponding author: [email protected]
1
6
nesses allows the prevention of high-altitude–induced deterioration of
a preexisting health condition (3).
AMS Risk Assessment
A more significant question is which individuals are at greater risk of
AMS, as an understanding of the risk factors may affect clinical management by providing measures for intervention and prevention. A total of 11,182 workers were surveyed and a risk model was developed
using multiple logistic regression. Our findings suggest that multiple
risk factors usually affect individuals who are at risk. Combinations
of rapid ascent, a higher altitude reached, and greater physical exertion increase the likelihood that illness will develop. Newcomers
from sea-level areas, obese persons, and younger people are advised
to take care when traveling to high altitude (4). Additionally, altitude
exposure was a risk factor for upper gastrointestinal tract bleeding,
especially in combination with alcohol, aspirin, and dexamethasone
intake (5). Risk factors that can be modified should be attended to,
and physicians should perform check-ups and tests to identify subjects who are at greater risk, to effectively control the risk factors of
AMS (4).
AMS and Smoking
It has been suggested that smokers have a lower risk of AMS at high
altitudes (6). However, the relationship between cigarette smoking and
AMS is not clear. To assess AMS risk and altitude acclimatization in
relation to smoking, 200 healthy nonsmokers and 182 cigarette smokers were recruited from a population of male Han Chinese lowlanders.
These subjects were without prior altitude exposure, were matched for
age, health status and occupation, and were transported to an altitude of
4,525 m. AMS scores, smoking habits, arterial saturation, hemoglobin,
lung function, and mean pulmonary artery pressure were assessed upon
arrival, and after three and six months at high altitude. Interestingly,
smokers may initially be at less risk at altitude, but not in the long term
(6). This study allowed us to advise smokers on altitude exposure using
the epidemiological data and suggested new avenues for research on
AMS pathophysiology.
HACE Studies
HACE is a serious type of acute altitude sickness with a high mortality
rate. An early diagnosis is therefore critical. We observed 66 lowland
railway workers suffering from HACE who had ascended to altitudes
of greater than 4,000 m. Ataxia was present in 48 workers (73%) and
was observed to have occurred earlier than the most common signs
of HACE such as disturbance of consciousness (79%) in the majority of patients. There was a high concordance (96%) between ataxia
and computed tomography scans or magnetic resonance imaging in the
diagnosis of HACE. Ataxia can be measured in mountainous regions
by simple coordination tests including a modified Romberg test. These
tests can serve as an early diagnostic predictor of HACE, indicating that
death due to HACE can be avoided if the early symptoms and signs are
recognized (2).
Section One
Intermittent Altitude Exposure
The construction of the QTR also provided a unique opportunity to
study the relationship between intermittent altitude exposure and AMS
(7). For five years, workers spent seven-month periods at high altitude
interspersed with five-month periods at sea level. The incidence, severity, and risk factors of AMS were prospectively investigated. A group
of 600 lowlanders who commuted between sea level and 4,500 m for
five years was compared with 600 lowland workers recruited each year
upon their first ascent to high altitude. AMS was assessed using the Lake
Louise Scoring System. We noted that a long-term, 7/5 month commuting pattern led to a gradual reduction in the incidence and severity of
AMS, and thus reduced susceptibility. This suggested that exposure to
high altitude may help minimize the development of high altitude sickness during each subsequent exposure (7). These data support clinical
guidelines for lowlanders periodically ascending to high altitude for
work and may help prevent illness and improve performance.
Occasional Altitude Exposure
After completion of the railroad in June 2006, about two million passengers each year are rapidly exposed to high-altitude travel on this
train. How would people tolerate traveling at high-altitudes by the
QTR? An initial study observed that the AMS incidence varied from
16% to 31% in passengers even when an oxygen concentrator was present in the train. To curb the health risk of rapid travel at high altitudes
by train, prospective travelers should be better informed, medical personnel aboard the train should be well trained, and a staggered travel
schedule with one to two days at intermediate altitudes should be suggested to non-acclimatized subjects (8).
Future Research
After the completion of the QTR, the Chinese government launched
several other important engineering projects in Tibet including
the construction of a new railroad from Lhasa to Xigatse (altitude:
3,890 m) (7). These projects put many subjects at risk for altitude
sickness, and it remains to be investigated if the incidence of altitude sickness can be reduced further using the results obtained from
our studies.
REFERENCES
1. T. Y. Wu, High Alt. Med. Biol. 5, 1 (2004).
2. T. Y. Wu et al., High Alt. Med. Biol. 7, 275 (2006).
4. T. Y. Wu et al., Chin. Med. J. (Engl.) 125, 1393 (2012).
5. T. Y. Wu et al., World J. Gastroenterol. 13, 774 (2007).
6. T. Y. Wu et al., Thorax 67, 914 (2012).
ACKNOWLEDGMENTS
This work was supported by the National “973” Program of China (Grant
No. 2006 CB708514 and 2012CB518202) and the National Natural Science
Foundation of China (Grant No. NNSF-30393130).
Human-Performance Engineering at High Altitude
Yu Mengsun
H
uman-performance engineering can be regarded as humancentered system engineering focused on maintaining and improving the homeostatic level in humans to improve quality
of life and develop natural potential (1). High-altitude health
care is an example of human-performance engineering, the goal of
which is to solve human-performance problems in high-altitude environments.
Our research group carried out human-performance engineering at
high altitudes in accordance with the principles of system engineering,
which regards human beings as large, open, and complex systems, as
first proposed by Qian Xuesen (2, 3). Based on the initial idea of human-performance engineering, the focus of research has shifted from
the “disease” (altitude sickness) to the process of altitude acclimatization in a hypoxic environment. In other words, there has been a shift in
focus from a “cure” to the “dynamic regulation” of homeostasis during
altitude acclimatization, which can improve the synergy of the physical system with the hypoxic environment to achieve normal function at
high altitudes.
Institute of Aviation Medicine Beijing
No. 28, Fucheng Road, Haidian, Beijing, China.
Studies have shown that sleep is crucial for maintaining optimal metabolic performance and homeostasis (4). Aviation medicine has demonstrated that a change in sleep quality is a common feature in response
to various psychological, physical, and environmental stressors. Thus,
managing stress reactions may improve acclimatizing ability. Altituderelated hypoxic stress can result in sleep disorders, as well as physical
and psychological reactions, when the environmental change (increasing hypoxia) is too rapid for the body’s self-organizing process, which
attempts to compensate and maintain homeostasis. Additionally, it may
prolong the time needed to adapt and could result in an inability to fully
acclimate. This scenario can be represented as follows:
Environment Variation Rate (EVR) >> Physical
Self-organizing and Self-Adapting Rate (PSSR) (1)
As shown by equation 1, altitude stress can be prevented in two ways.
First, a lower EVR could be artificially induced. Second, the PSSR
could be increased. Thus, the relationship between EVR and PSSR
could be changed from 1 to 2, as follows:
EVR>>PSSR
(1)
EVR≤PSSR
(2)
A technical approach is therefore proposed to prevent altitude-related
stress; (i) Administering progressive, intermittent hypoxic exposure
7
Figure 1. System model
of altitude acclimatization.
SSE, error sum of squares;
HR, heart rate. G1, G 2, G 3 are
transfer functions: G1, instant
response to hypoxic environment; G 2, feedback parameter related to the ability for
regaining homeostasis; G 3,
characterstic parameter related to the personal acclimatization process.
Figure 2. Two patterns of IHE training.
(IHE) training in which the environmental conditions are adjusted to
be more in line with the time constant―a term that describes how fast
the system can react following a trigger―required for self-organizing
adaption by the body; (ii) Evaluating the reaction of the body during
training using sleep monitoring techniques in order to maintain the EVR
as close to the PSSR as possible. As a result, the efficiency of training
will be improved.
In this study, we attempted to clarify the mechanisms at work during human altitude acclimatization. The changes seen in physiological
parameters (e.g., arterial blood oxygen saturation, heart rate, and deep
sleep time) due to altitude stress can be approximately fitted to first-order function curves (5). Figure 1 shows the arterial blood oxygen saturation (SaO2) curves for a team of four men flying to 3,800 m and the corresponding dynamic model. Here, τ is the time constant or time scale.
In principle, the acclimatizing process during IHE training should
approximate a first-order time function. Because of the intermittent nature of the training, the model expression includes four other variables,
besides τ, related to intermittent training, such as the training intensity
each time point, the interval of training, frequency of training, and the
rate at which adaptation changes (“fading factor”). Additionally, it involves an individual optimal training intensity, S0(H), which is a function of the degree of adaptation achieved (5). The model expression of
IHE training is explained in full in reference 3. It has been suggested
that the training intensity should be as close as possible to S0(H) for each
exercise course, and the efficiency of training will reach a maximum
value when EVR equals PSSR. We designed two similar protocols for
incremental IHE training. One is IHE training before going to a high
altitude area, while the other is IHE training at high altitude after acute
hypoxia exposure. The two training patterns are illustrated in Figure 2.
Results showed that the two types of IHE training were equivalent
both in principle and in their practical effects (6). In our study, the time
constant, τ, of the untrained group during the process of acclimatization was 3.2 days at 3,800 m above sea level. The τ value of the group
8
trained at altitude was significantly decreased, to 0.633 days, while for
the group trained before hypoxia, it was still less than 0.633 days at
0.434 days. This demonstrated that subjects could completely avoid
reactions to altitude and ensure minimal health impact on exposure to
hypoxia if a suitable amount of training is done.
To maintain homeostasis in a hypoxic environment, the management
of diet and physical exercise is important. Permanent residents at high
altitudes risk suffering from oxygen toxicity when moving to lower altitudes, caused by the rate of environmental change being greater than
that of acclimatization to the hyperoxic environment of the plains. In
these cases, we recommend introducing intermittent hyperbaric oxygen
training to prevent the risk of severe disease resulting from acute oxygen exposure.
This research describes the first practice of human performance engineering at high altitude. It has shown that maintaining homeostasis in
a human system can be achieved when we fully understand the limits
and self-organizing ability of human beings, such as acclimatizing to
environmental changes and recovering from illness.
REFERENCES
1. Z. L. Tao, “Comprehensive Report” (Report on Advances in Biomedical
Engineering (2011-2012), China Science and Technology Press, Beijing,
2012).
2. X. S. Qian, Systemic Engineering (Shanghai Jiao Tong Univ. Press,
Shanghai, 2007), pp. 288-299.
3. X. S. Qian, Establish Systematology (Shanghai Jiao Tong Univ. Press,
Shanghai, 2007), pp. 125-129.
4. M. S. Yu, H. J. Zhang. China Medical Device Information 3, 4 (2003).
5. M. S. Yu, “Human performance engineering at high altitude” (Report on
Advances in Biomedical Engineering (2011–2012), China Science and
Technology Press, Beijing, 2012).
6. J. Yang, M. S. Yu, Z. T. Cao. Chinese Journal of Aerospace Medicine.
doi:10.3760/cma.j.issn.1007-62392012.03.004.
Section One
Intrinsic Characteristics in Tibetans of Tolerance to
Hypoxia Following Long Periods at Sea Level
Zhou Zhaonian*, Zhuang Jianguo, Zhang Yi
F
or thousands of years, the overwhelming majority of Tibetans in the Tibetan group. The results suggested that physiological adaptahave lived at very high elevations, characterized by an aver- tion to hypoxia in Tibetans at high altitude did not depend on systemic
age altitude in excess of 4,000 m. It is necessary that mech- functions (2, 3, 8).
anisms develop in both humans and animals to compensate
Maximal oxygen uptake (VO2 max) and oxygen transfer to tissues
for low oxygen levels at high altitude and to facilitate metabolism and were compared between Tibetan and Han subjects. There was no signifother physiological functions in this hypoxic environment. It is unclear icant difference in VO2 max between the groups at sea level (p>0.05).
whether hypoxic tolerance in Tibetans is a result of physiological ac- During acute hypoxia, the VO2 max was decreased in both groups, but
climatization or due to an intrinsic tolerance (genetic adaptation), or significantly higher (p<0.05) in the Tibetan group (1.41 ± 0.04 L/min/
both. A comparison of the differences in the physiological responses M2) than in the Han group (1.25 ± 0.04 L/min/M2), indicating a better
to hypoxic stress under certain circumstances between Tibetans and physical work capacity in the Tibetans (4, 8).
lowlanders may reveal essential facts about
the possible mechanisms underlying the hyTable 1. Basic physiological parameters of Tibetan and Han subjects (mean ± SE).
poxic tolerance of native Tibetans.
A study was carried out to investigate
VE
RR
HR
SaO2
whether superior hereditary tolerance was
(L/min/M2)
(breaths/min)
(beats/min)
(%)
responsible for acute hypoxia tolerance (1).
Tibetan
Physiological changes observed in highland0m
4.88 ± 0.24
16.1 ± 1.2
74.6 ± 4.6
99.4 ± 0.3
ers after long-term sea-level residence may
3,700 m
6.46 ± 0.43
21.0 ± 2.0
83.9 ± 4.6
91.7 ± 0.6
provide a clearer understanding of the process of adaptation to high altitudes. It was
Han
hypothesized that if Tibetans have acquired
0m
4.71 ± 0.23
15.8 ± 1.1
87.3 ± 2.7
99.2 ± 0.4
a hereditary adaptation, then this would be
3,700 m
6.78 ± 0.94
17.2 ± 2.0
102.9 ± 3.0*
86.9 ± 0.6*
retained during habitation in the lowlands.
If, however, the benefit were a physiologiVE, minute ventilations; RR, respiration frequency; HR, heart rate; SaO2, arterial blood oxygen
cal acclimatization, then the hypoxic resissaturation. *p<0.05 compared with the Tibetan group.
tant capacity of Tibetans after living at sea
level for many years would be similar to that
of lowland residents. A comparison of physiological responses to hyOxygen transfer to tissues was similar in the Han and Tibetan groups
poxia between native highlanders and native lowlanders may highlight at sea level (p>0.05). During acute hypoxia, the oxygen extraction in
mechanisms of hypoxia tolerance that can be used to prevent hypoxia- tissues (O2 EXT) was significantly higher in Tibetans (55.0% ± 4.2%)
induced damage caused by disease and the environment.
than in Han subjects (47.3 ± 9.1%) (p<0.01). Arterial oxygen pressure
The study subjects consisted of the following cohorts: lowlander and saturation were also higher in Tibetans (7.2 ± 0.6 kPa and 87.9
(Han Chinese) males born at sea level without high altitude exposure ± 3.3%, respectively) than in Han subjects (5.5 ± 0.2 kPa and 78.2 ±
and Tibetan males raised in an environment above 3,600 m who had 1.6%, respectively, p<0.05). Thus, Tibetans could adapt better to acute
migrated to Shanghai (sea level) and not returned in the previous four hypoxia than Han subjects, even after living at sea level for four years.
years. The age, body weight, and height were similar between the This process mainly depended on changes in oxygen uptake, transport,
groups. Acute hypoxia was induced by placing subjects into a hypo- and release at the tissue and cellular level. The genetic adaptation of
baric chamber for two hours at a simulated height of 3,700 m. At rest, Tibetans, through long term existence at the high altitude of the plarespiratory and blood indices measured before depressurization were teau, may therefore play a role in their capacity for survival in hypoxic
not significantly different between Tibetan and Han subjects. The basic environments (3, 5, 6).
physiological parameters at sea level were not significantly different
It is known that at high altitudes, adaptive and acclimatized indiat the resting baseline, but showed a higher response in the Han group viduals have better oxygen reserve capacity than individuals from sea
during rest at 3,700 m (Table 1). There were no statistically significant level environments. Therefore, comparison of reserve capacity should
changes in respiration and cardiac pump function due to acute hypoxia highlight differences between adaptive and acclimatized individuals
during acute hypoxia. Individuals with a higher reserve capacity should
have higher resistance to acute hypoxia. Hypoxic resistance was evaluated using oxygen reserve capacity (reserve VO2) during acute hypoxia,
which was found to be significantly higher in Tibetans than in Han subLaboratory of Hypoxic Cardiovascular Physiology, Shanghai Institutes
jects (Figure 1) (8).
for Biological Science (Shanghai Institute of Physiology),
Heart rate variability (HRV) of Tibetan and Han groups was
Chinese Academy Sciences, Shanghai, China.
*
measured in a resting supine position at sea level and again one hour
9
characteristics of autonomic control are inherited traits (7).
In conclusion, our studies demonstrated that superior tolerance
to acute hypoxia and better physical performance were still present
in Tibetans after living at sea level for four years, implying that the
intrinsic characteristics of hypoxic adaptation exist in native high
altitude-dwelling Tibetans.
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1. X. H. Ning, Z. N. Zhou, X. Z. Lu, X. C. Hu, In Proceedings of Symposium On Qinghai-Xizang (Tibet) Plateau (Beijing, China). Geological and Ecological Studies of Qinghai-Xizang plateau. (Gordon and Branch Science, Press, New York, 1981), p. 1407.
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no significant differences in the reserve of oxygen consumption (reserve
VO2) among the groups at sea level (0 m), but it was significantly higher in
Tibetans than in Han subjects during acute hypoxia (3,700 m). T, Tibetan
group; H, Han group. Results represent mean ± SE. *p<0.05 compared
to the Tibetan group.
after simulated ascent to 3,700 m in a hypobaric chamber. HRV may
better clarify levels of sympathetic and parasympathetic activity.
The results indicated that Tibetans exhibited greater parasympathetic
tone at rest at sea level, and ascent to an altitude of 3,700 m did not
significantly alter their heart beat. However, Han subjects at 3,700 m
had a significantly reduced vagal tonic activity of the heart. Therefore,
it is likely that Tibetans’ greater adaptation to hypoxia and their specific
202 (1995).
5. Z. N. Zhou, X. F. Wu, H. Y. Jiang, L. Q. He, Hypoxia Med. J. 3, 13 (1996).
6. Z. N. Zhou, J. G. Zheng, X. F. Wu, L. Q. He, In Progress in Mountain Medicine and High Altitude Physiology (Dogura & Co. Ltd. Kyoto. Press, 1998), p. 52.
7. J. G. Zhuang, H. F. Zhu, Z. N. Zhou, Jpn. J. Physiol. 52, 51 (2002).
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Physiol. Sci. 58, 167 (2008).
ACKNOWLEDGMENTS
This work was funded by a grant from the National Basic Research Program
of China, (Grant No. 2006CB504100 and 2012CB518200) and the National
Natural Science Foundation of China (Grant No.3927089 and 30393130).
Exploration and Evidence of High-Altitude Adaptation
in Tibetan Highlanders
Wuren Tana and Ge Ri-Li*
M
odern humans migrated from the African continent approximately 200,000 years ago and, during migration, humans
adapted to different extreme environments, including those
of high altitude (1). The Qinghai-Tibet Plateau is the largest
and highest plateau in the world and although there are controversial
theories about the origins of settlers on this plateau based on archeological findings (2), Tibetans may have resided in this harsh environment
for up to 3,000 years despite the physiological challenges associated
with chronic hypoxia and increased ultraviolet light exposure (3).
Research Center for High Altitude Medicine, Qinghai University,
Xining, Qinghai China.
*
10
Physiological Evidence for Tibetan Adaptation
Populations at high altitudes have evolved physiological adaptations to
counter the environmental hypobaric hypoxia at high altitudes. However, studies of hypoxia-related physiological traits in different high
altitude populations indicate independent patterns of adaptive phenotypes amongst them. For example, Tibetan women are relatively protected from hypoxia-influenced maternal physiological responses that
can cause low child survival rates and low birth weight (4). Studies
have demonstrated that placental growth and development are remarkably well protected among certain high altitude female populations (5).
In 1890, Francois-Gilbert Viault identified polycythemia in his blood
at 4,500 m in Peru, and in 1924, T. Howard Somervell observed that the
hemoglobin concentration in Tibetans was significantly lower than in
the expedition team during a climb of Mt. Everest (6). It has since been
shown that chronic exposure to hypoxia in lowland populations leads
Section One
to an elevation of hematocrit due to increased numbers of erythrocytes
(polycythemia) whereas the majority of Tibetan highlanders maintain
comparable hematocrit levels to populations living at sea level (7).
While increased hemoglobin concentration may be considered a
beneficial adaptation to hypoxia, at certain threshold levels the increased
number of erythrocytes results in higher blood viscosity, which could
impair capillary blood flow and oxygen delivery (8). Therefore, the
genetic basis of low hemoglobin levels in Tibetans warrants further
investigation.
Human energy demands and metabolic adaptation have been studied
extensively with respect to diet, but metabolic adaptation in response to
unique environments has only recently been closely examined. Previous
studies of native high altitude populations suggested that decreased fatty
acid oxidation could be a favorable adaptation to hypoxia (9), while a
study from our group demonstrated that Tibetans have comparatively
higher free fatty acid concentrations compared to individuals living at
sea level. This suggests that anaerobic glucose metabolism is increased
and fatty acid oxidation may be decreased in Tibetans (10). To better
understand the physiological significance of these patterns, larger
sample sizes, better controls, and broader studies at different altitudes
are needed. Meanwhile the genetic basis and metabolic implications of
high altitude adaptation requires further investigatation.
Genetic Evidence for Tibetan Adaptation
to High Altitude
To detect natural selection for particular genetic variants in high altitude populations during the evolution of high altitude adaptation, several population genetics methods have been employed.
In a previous study, we used two statistical tests, the High Integrated
Haplotype Score (iHS) and Cross Population Extended Haplotype
Homozygosity (XP-EHH) to determine whether Tibetans evolved
adaptively under positive selection. We found that among 240 genes
related to the hypoxia pathway in gene ontology categories, 10
genes were involved in high altitude adaptation in Tibetans, and
were present in regions of strong positive selection (11). The 10
candidate genes included endothelial PAS domain-containing protein
1 (EPAS1), prolyl hydroxylase domain-containing protein/Egl nine
homolog 1 (PHD2/EGLN1), and peroxisome proliferator-activated
receptor alpha (PPARA). These may be important because individuals
carrying additional copies of a putatively advantageous haplotype
of PHD2 [build 36 (Hg18), chromosome 1 positions 229793717,
229667980, and 229665156] and PPARA (Hg18, chromosome 22
positions 44827140, 44832376, and 44842095) have significantly
lower hemoglobin concentration, suggesting that these haplotypes
are associated with protection against polycythemia in Tibetan
highlanders (11).
A study of Andean and Tibetan populations also revealed that both
populations had experienced positive selection for hypoxia-inducible
factor (HIF) pathway genes, including PHD2/EGLN1 (11, 12). In normoxia conditions, PHD enzymes are involved in HIF-1α and HIF-2α
ubiquitinization and their rapid destruction in proteasomes (13). Thus,
PHD and Von Hippel–Lindau tumor suppressor proteins (VHL) are major negative regulators of HIFs (13). We identified a novel missense
mutation in the PHD2 gene, which together with another previously
reported but unvalidated PHD2 single nucleotide polymorphism (SNP)
that results in missense mutation, correlated with lower hemoglobin
levels in Tibetan highlanders (unpublished data).
Other studies have shown that Tibetans experienced positive selec-
tion for variants of EPAS1, which regulates expression of the erythropoietin gene. Based on phenotype/genotype association analysis, highly differentiated SNPs in the EPAS1 region were related to decreased
hemoglobin levels in two independent studies of high-altitude adaptation in Tibetans (14, 15).
A more recent study that analyzed another groups of Tibetans from
the Tuo Tuo River area suggested that EPAS1 and PPARA putative
adaptive haplotypes were associated with elevated serum lactate and
free fatty acid levels, which suggests that adaptation to decreased
oxygen availability may be enhanced by a shift in fuel preference
to glucose oxidation and glycolysis, at the expense of fatty acid
catabolism (10).
Considering the lack of genetic differences detected by analysis of
the protein-coding regions of Han Chinese and Tibetans (14), it is possible that many genetic targets of selection are in noncoding, regulatory
regions of the genome. Our analyses of individuals living in Maduo
County (elevation ~4,300 m), the highest county in China, have identified an miRNA near the PPARA gene and a noncoding, highly conserved region in a Tibetan population that may be involved in highaltitude adaptation (unpublished data).
Perspective
Studies from our group and others regarding indigenous Tibetans
have identified genes that may be involved in adaptation to hypoxia.
It is clear that during this adaptation process, Tibetans developed
unique genetic changes compared with neighboring lowland populations. Genetic and statistical analysis from these studies have provided interesting data, but to understand this complex process it will
be necessary to integrate these results with functional analyses to obtain a more complete picture of the mechanisms involved in hypoxia
adaptation. Ultimately, we hope that genetic and functional analyses may be used in the prevention and treatment of hypoxia-related
diseases.
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1. A. Lawler. Science 331, 387 (2011).
2. M. Aldenderfer, High Alt. Med. Biol. 12, 141 (2011).
3. M. Aldenderfer, World Archaeol. 38, 357 (2006b).
4. L. Postigo et al., J. Physiol. 587, 15 (2009).
5. L. G. Moore et al., Resp. Physiol. Neurobiol. 178, 181 (2011).
6. M. C. T. van Patot, M. Gassmann, High Alt. Med. Biol. 12, 157 (2011).
7. C. M. Beall, Resp. Physiol. Neurobiol. 158, 161 (2007).
8. J. T. Prchal, in Williams Hematology. K. Kaushansky, M.A. Lichtman, T.
J. Kipps, E. Beutler, U. Seligsohn, J. T. Prchal, Eds. (McGraw Hill, New
York, ed. 8, 2010), pp. 435-449.
9. J. E. Holden et al., J. Appl. Physiol. 79, 222 (1995).
10. R.-L. Ge et al., Mol. Genet. Metabol. 106, 244 (2012).
11. T. Simonson et al., Science 329, 72 (2010).
12. A. Bigham et al., PLoS Genetics 6, 1 (2010).
13. G. L. Semenza, Physiology 24, 97 (2009).
14. X. Yi, Y. Liang et al., Science 329, 75 (2010).
15. C. M. Beall et al., Proc. Natl. Acad. Sci. U.S.A. 107, 11459 (2010).
ACKNOWLEDGMENTS
This project was supported by the National Basic Research “973” Program
of China (Grant No. 2012CB518200), the Program of International S&T
Cooperation of China (Grant No. 0S2012GR0195), and the National Natural
Science Foundation of China (Grant No. 30393133).
11
Peopling of the Tibetan Plateau and Genetic Adaptation
to High-Altitude Hypoxia in Tibetans
Qi Xuebin1, Shi Hong1, Cui Chaoying2, Bianba2, Ouzhuluobu2, Wu Tianyi3, Su Bing1,*
T
he Tibetan Plateau, with a mean elevation of more than
4,000 m, is characterized by extremely harsh environmental
conditions such as cold temperatures during winter, strong
ultraviolet radiation, and low oxygen concentrations. For
people living in these inhospitable terrains, high-altitude hypoxia is a
condition that cannot be overcome by traditional treatments. Currently,
there are nearly five million indigenous Tibetans living on the plateau,
and two thirds of them live at an altitude exceeding 3,500 m (1). Modern
Tibetans have physiologically adapted to the high-altitude hypoxic
environment (2). For example, compared with lowlanders, Tibetans
have greater hypoxic and hypercapnic ventilatory responsiveness,
larger lungs, better lung function, greater lung diffusion capacity,
minimal hypoxic pulmonary hypertension, and higher levels of exhaled
nitric oxide (2). This environmental adaptation in Tibetans may result
from long-term natural selection that has been taking place since the
ancestors of modern Tibetans permanently settled on the plateau. To
elucidate the molecular mechanism underlying this genetic adaptation
to hypoxia, it is necessary to answer two key questions: (i) when did
the ancestors of modern Tibetans first permanently settle on the Tibetan
Plateau and (ii) how did genetic modifications in Tibetans improve their
physiological functions and endow them with the ability to thrive in
hypoxic conditions?
Regarding the question of when the Tibetan Plateau was populated,
no human fossils have been found on the plateau, and consequently no
direct biological evidence is available to infer that humans previously
inhabited the region. However, archaeological findings based on
limited cultural artifacts suggest that the earliest human occupation
likely occurred at relatively low altitude areas (<3,000 m) around 40–
30 thousand years ago (kya) during the early Upper Paleolithic period,
while the permanent occupation at high altitude (>3,000 m) did not
begin until the advent of farming and pastoral economy about 8.2–6
kya (3). The ice sheet hypothesis describing human occupation of the
Tibetan Plateau during the Last Glacial Maximum (LGM, 22–18 kya)
may support this notion (4, 5). It is generally believed that even though
modern humans might have successfully settled on the plateau during
the Upper Paleolithic period, the early settlers would not have survived
the LGM, and thus present-day Tibetans are likely descendants of postglacial immigrants.
Recent genetic studies of present-day Tibetan populations have
provided a different picture of the prehistoric peopling of the plateau.
Interestingly, by examining the genetic composition of the paternal (Y
chromosome) and maternal (mitochondrial DNA) lineages of Tibetans,
both ancient and recent genetic components were identified (6, 7).
This suggests that modern Tibetan populations may have been formed
genetically from two distinct ancestral populations that ventured into
the plateau region during both the Paleolithic and the Neolithic periods
(6, 7). We recently screened more than 6,000 Tibetan individuals from
41 geographic populations across the Tibetan Plateau. We found that
the majority of lineages in Tibetans (87.80% of Y-chromosomal and
90.99% of mitochondrial) were of East Asian lineages dating back to
51–18 kya, a coalescence age falling into the Upper Paleolithic period
(Figure 1). We also identified a molecular signature indicating a recent
population expansion within Tibetans around 10–7 kya during the early
Neolithic period, likely caused by a second migratory wave of modern
humans onto the plateau (Figure 1). Both the Paleolithic migration and
Neolithic expansion had a significant impact on the genetic makeup of
present-day Tibetan populations. The ancient peopling of the Tibetan
Plateau suggests that the ancestors of modern Tibetans had undergone a
lengthy natural selection process against hypoxic stress and may explain
why Tibetans have the most effective genetic adaptation to high-altitude
hypoxia in the world (2). Hence, Tibetans are an ideal population for
delineating the molecular mechanism of genetic adaptation to highaltitude hypoxia.
Regarding the question of how Tibetans improved their physiological
functions, we and other research groups have recently conducted genome-wide analyses aimed at identifying genes involved in the genetic
adaptation to hypoxia (8–10). These genome-wide studies revealed a
set of candidate genes that likely play important roles in physiological adaptation to hypoxia in Tibetans (Table 1). Of these, EPAS1 (also
called hypoxia-inducible factor 2α, HIF2α) and its negative regulator,
PHD2/EGLN1, appear to play major roles (8–10). However, functional
studies (both in vitro and in vivo) have yet to be conducted to delineate
the molecular pathways and physiological mechanisms at work.
REFERENCES
1. T. Wu, High Alt. Med. Biol. 2, 489 (2001).
2. T. Wu, B. Kayser, High Alt. Med. Biol. 7, 193 (2006).
3. M. Aldenderfer, High Alt. Med. Biol. 12, 141 (2011).
4. M. Kuhle, Universitas 27, 281 (1985).
5. Y.-F. Shi, B.-X. Zheng, S.-J. Li, Chinese Geographical Science 2, 293
(1992).
6. H. Shi et al., BMC Biol. 6, 45 (2008).
7. B. Su et al., Hum. Genet. 107, 582 (2000).
8. Y. Peng et al., Mol. Biol. Evol. 28, 1075 (2011).
9. C. M. Beall, High Alt. Med. Biol. 12, 101 (2011).
10. T. S. Simonson, D. A. McClain, L. B. Jorde, J. T. Prchal, Hum.
State Key Laboratory of Genetic Resources and Evolution, Kunming Institute
of Zoology, Chinese Academy of Sciences, Kunming, China;
2
High Altitude Medical Research Center, School of Medicine, Tibetan
University, Lhasa, China;
3
National Key Laboratory of High Altitude Medicine, High Altitude Medical
Research Institute, Xining, China.
*
1
12
Genet. 131, 527 (2012).
ACKNOWLEDGMENTS
This work was supported by the National Basic Research “973” Program
of China (Grant No. 2012CB518202 and 2011CB512107) and the National
Natural Science Foundation of China (Grant No. 91231203, 30870295, and
91131001).
Section One
Figure 1. The migratory route of the two proposed prehistoric migrations of modern humans onto the Tibetan Plateau. The shaded area represents
the entire region of the Tibetan Plateau, and the small red area indicates the earliest Neolithic site in China dated to 8,500 years ago.
Table 1. Candidate genes thought to be involved in high-altitude hypoxia adaptation in Tibetans.
Candidate genes
Gene Functions Predicted in UniProtKB/Swiss-Prot Database
EPAS1
Endothelial PAS domain protein 1, also known as hypoxia-inducible factor 2-alpha (HIF2α)
EGLN1
Egl nine homolog 1, also known as prolyl hydroxylase domain-containing protein 2 (PHD2) or hypoxia-inducible
factor prolyl hydroxylase 2 (HIF-PH2)
EP300
E1A binding protein p300
ARNT
Aryl hydrocarbon receptor nuclear translocator, also known as hypoxia-inducible factor 1-beta (HIF1β)
HBB
Hemoglobin subunit beta
HBG2
Hemoglobin subunit gamma-2
EPO
Erythropoietin, involved in erythrocyte differentiation and erythrocyte circulation.
EDN1
Endothelin 1, endothelium-derived vasoconstrictor peptides
EDNRA
Endothelin receptor type A
HMOX2
Heme oxygenase 2
ANGPTL4
Protein with hypoxia-induced expression in endothelial cells
ANGPT1
Angiopoietin 1, involved in angiogenesis, endothelial cell survival, proliferation, migration, adhesion, and cell
spreading
PPARA
Peroxisome proliferator-activated receptor alpha
TGFBR3
Transforming growth factor beta receptor III
RYR1
Ryanodine receptor 1 (skeletal)
ECE1
Endothelin converting enzyme 1
13
New Approaches for Facilitating High-Altitude
Acclimatization
Huang Qingyuan1,2,†, Zhang Gang1,2,†, Cui Jianhua3, Gao Wenxiang1,2, Fan Youming1,2, Huang Jian1,2, Cai Mingchun1,2,
Li Peng1,2, Liu Fuyu1,2, Zhou Simin1,2, Gao Yixing1,2, Li Xiaoli1,2, Gao Yuqi1,2,*
T
he study of practical measures to facilitate acclimatization to
high altitudes is of major importance, as poor acclimatization
can lead to severe deficits in physical and cognitive performance, and to high-altitude diseases (1). A gradual ascent to
high altitude can reduce the incidence and severity of acute mountain
sickness (AMS) and improve working performance, and thus has been
widely accepted as a preventative measure (2). However, this measure
takes time and is unsuitable for rapid ascents. We studied ways in which
to facilitate high altitude acclimatization, particularly in large groups.
These approaches were implemented either before ascent, within a
short time after arrival, or after a certain duration at high altitude.
Hypoxia preconditioning can protect organisms against subsequent
severe hypoxia-induced injury (3). Hypobaric hypoxia-induced (12,000
m, four hours) brain injury in mice was significantly ameliorated by
hypoxia pretreatment (7,000 m, 2.5 hours/day for three days) (4). We
recruited young males to validate the effects of hypoxia preconditioning on the human body. We found that hypoxic gas (15% O2) inhalation combined with an up-and-down stepping exercise (10 minutes, six
times/day for three days) could decrease AMS incidence and improve
physical performance at a simulated altitude of 4,300 m. Based on these
findings, we applied this procedure in the field at high altitude. We first
designed a portable hypoxic respirator according to the principle of a
rebreathing circuit (Chinese Patent ZL 02 222805.5), in which carbon
dioxide is absorbed by soda lime. Forty young men wore the hypoxic
respirators and walked rapidly (10 minutes walking followed by five
minutes resting, four times in the morning and repeated in the afternoon)
for five days at sea level. Oxygen saturation was reduced from 97.3 ±
1.2% to 88 ± 5.4% and the heart rate increased from 70 ± 9 beats/minute
to 126 ± 16 beats/minute during walking. One day or five days after cessation of training, the training groups (20 subjects per group) and the
control group (without training; 20 subjects) then traveled to an altitude
of 4,300 m by bus. We observed that AMS incidence and severity were
significantly reduced in the hypoxia preconditioned groups compared
with the control group. Physical working performance, determined by
maximal oxygen uptake (VO2 max) and physical working capacity at a
heart rate of 170 beats/minute (PWC170), was decreased significantly in
the control group, but not in the hypoxia preconditioned groups. This
suggested that five-day hypoxia preconditioning can reduce the risk of
AMS and improve physical working performance at 4,300 m, and that
the positive effects endure for at least five days after cessation of training (5). This simple method is suitable for large groups and can be ad-
College of High Altitude Military Medicine, Third Military Medical
University, Chongqing, China;
2
Key Laboratory of High Altitude Medicine, Ministry of Education, Third
Military Medical University, Chongqing, China;
3
Institute of High Altitude Medicine, 18th Hospital of Chinese People’s Liberation Army, Yecheng, Xinjiang, China.
*
†
Contributed equally to this work.
1
14
ministered over a short time before ascending to high altitude.
Reactive oxygen species (ROS) may be involved in stimulating the
protective pathways of hypoxia preconditioning. Since hyperoxia pretreatment can generate ROS, it may also induce similar protective outcomes. To test our hypothesis, PC12 cells were treated with 35% O2 for
three hours, followed by a 12-hour recovery period. We observed that
cell death induced by a subsequent 72-hour hypoxic exposure (1% O2)
was significantly reduced. Hyperoxia pretreatment increased the intracellular ROS level, ROS inhibitors diminished, and ROS supplements
can mimic the protective effects of hyperoxia pretreatment, indicating
that ROS contributes to the protective effects (6). In a separate experiment, young human males were subjected to hyperbaric oxygen (2.5
absolute atmospheres) two hours daily for three days before ascent to
an altitude of 5,000 m. This pretreatment lowered AMS incidence and
improved physical working performance, indicating hyperbaric oxygen
preconditioning is an effective measure for high altitude acclimatization (7).
Previous research has suggested that acetazolamide can be used as an
AMS prophylactic medication, but it has some side effects. We tested
methazolamide, an analogue of acetazolamide, and found that it prolonged the swimming time of mice in a hypobaric chamber (simulating an altitude of 5,000 m) and prolonged the survival time in sealed
150 mL containers containing 5 g soda lime, which gradually induces
hypoxia (8). In a field experiment, young human males were orally administrated 25 mg of methazolamide for seven consecutive days, twice
daily (starting two days prior to ascent). After arrival of an altitude of
4,300 m, they had higher resting oxygen saturation and lower incidence
of AMS compared with the placebo group (unpublished data). These results indicated that methazolamide could be used during the early phase
of acclimatization to high altitude.
Reduced exercise is favorable for oxygen supply/consumption balance in a hypoxic environment. We observed skeletal muscle atrophy in
rats living in a hypobaric chamber (simulating 5,000 m) for five weeks,
while those that undertook swimming training (one hour/day) under
a hypoxic environment showed no skeletal muscle atrophy. However,
these rats showed increased capillary density in the myocardium and
gastrocnemius muscle, increased metabolic enzyme activity and percentage of α-myosin heavy chain in the myocardium, and enhanced
cardiac function (9). Taken together, this indicates that appropriate
exercise could be beneficial for maintaining physical performance at
high altitude, but exact exercise prescriptions for optimal performance
requires further study.
Octacosanol is a nutritional supplement that has been reported to be
effective in improving athletic performance, suggesting it may be useful
at high altitudes. Chronic hypoxic rats treated with Octacosanol (5 mg/
kg daily for four weeks) showed a significant improvement in exercise
capacity and lower pulmonary arterial pressure at a simulated altitude
of 5,000 m (unpublished data). In a subsequent field test, human volunteers who had lived at an altitude of 3,700 m for one to two years were
administered 10 mg Octacosanol or placebo daily for 30 days. Blood
Section One
hemoglobin concentration and resting or exercising heart rates were
significantly decreased, and exercising oxygen saturation significantly
elevated, in those taking Octacosanol relative to the control group (10).
Traditional Chinese herbals, such as Panax notoginseng, Astragalus
membranaceus, and Phyllanthus emblica may also facilitate altitude
acclimatization, as suggested by animal and field experiments (11), indicating that Octacosanol and Chinese herbs could benefit people living
at high altitude over a prolonged period.
In conclusion, to facilitate acclimatization to high altitude, hypoxia/
hyperbaric oxygen preconditioning should be considered before ascending to high altitudes. In addition, methazolamide, Octacosanol, and
other traditional Chinese herbals could be taken in the early phase of
acclimatization. For people living at a high altitude for extended periods of time, appropriate exercise should be encouraged.
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ACKNOWLEDGMENTS
This work was supported by grants from the National Basic Research
“973” Program in China (Grant No. 2012CB518201), the Key Project of
the National Research Program of China (Grant No. 2009BAI85B06),
and National Natural Science Foundation of China (Grant No. 31071036,
30771043, and 39730190).
Evidence for Genetic Contribution to High-Altitude
Pulmonary Edema in Chinese Railway Construction Workers
Qiu Changchun1,3,*, Fan Ming2, Qi Yue3, Zhou Wenyu3, Liu Jicheng1,*
H
igh-altitude pulmonary edema (HAPE) is a rare and potentially fatal noncardiogenic pulmonary edema (1). The exact
mechanism underlying the development of HAPE remains
unclear. Although hypoxia is the main trigger, some individuals are more susceptible to HAPE than others when exposed to identical hypoxia conditions, suggesting a possible genetic predisposition (2,
3). Currently, it is not clear which genes are involved in the pathogenesis of HAPE. We therefore sought to identify susceptibility genes and
determine the synergistic effect of these genes (if any) on HAPE in a
large cohort of subjects.
Research Design and Subjects
The Chinese Government began work on the Qinghai-Tibet railway in
2001. The railroad stretches for 1,142 km with more than 960 km of the
track above 4,000 m. Over a period of five years, more than 140,000
people worked in high-altitude conditions, which included a cold
and unpredictable climate, dry weather, and low barometric pressure
Institute of Polygenic Diseases, Qiqihar Medical University, Qiqihar, China;
2
Institute of Basic Medical Sciences, Academy of Military Medical Sciences,
Beijing, China;
3
National Laboratory of Medical Molecular Biology, Institute of Basic Medical
Sciences, Chinese Academy of Medical Sciences/Peking Union Medical
College, Beijing, China.
*
Corresponding authors: Qiu Changchun ([email protected]) and
Liu Jicheng ([email protected])
1
resulting in a low ambient partial pressure of oxygen. This provided
us with an opportunity to collect data relating to the epidemiological
aspects of HAPE, samples from HAPE patients and controls, as well as
gain insight into genetic etiologic mechanisms.
To study the underlying mechanisms of HAPE in the absence of confounding factors, we planned a prospective cohort study. The entire cohort of approximately 140,000 individuals involved in the construction
of the railway were examined using a screening procedure that involved
two physical examinations. Subjects with cardiovascular, pulmonary
disease, asthma, diabetes, hepatitis, and/or other infectious diseases
were excluded.
We performed a candidate gene association study to identify HAPE
susceptibility genes. In this study, 23 genes were investigated for a potential role in HAPE. Of these 23, six genes and/or their haplotypes
presented some association with HAPE susceptibility.
Genes in The Renin-Angiotensin-Aldosterone
Pathway
The renin-angiotensin-aldosterone system (RAAS) plays a key role in
maintaining fluid balance and regulating blood pressure. Therefore, we
hypothesized that the pathogenesis of HAPE may be partially attributable to proteins in the RAAS cascade. To address this, we genotyped 12
gene polymorphisms evenly interspersed in six RAAS candidate genes.
Single locus analysis showed that polymorphisms C-344T and K173R
in the cytochrome P450 family protein CYP11B2, and the A-240T
polymorphism in the angiotensin I converting enzyme (ACE) protein
15
A
Figure 1. Interaction dendrogram for the five polymorphisms modeled
by the multifactor-dimensionality reduction (MDR) method. (A) There
were strong synergistic (nonadditive) effects of ACE A-240T, A2350G,
and CYP11B2 C-344T polymorphisms. These three polymorphisms
comprised the best overall MDR model. The relationship of each pair with
the other was independent and additive. The distribution of HAPE (left bars)
and controls (right bars) are shown for each genotype combination in each pair
of interacting polymorphisms. (B) The ratio of the total number of HAPE cases
to the total number of controls in the database did not exceed the threshold
of 0.97; the boxes were labeled as low-risk or high-risk. Nonlinear patterns of
high-risk (dark grey) and low-risk (light grey) genotype combinations indicative
of interaction were observed.
were significantly associated with HAPE
after applying the Bonferroni correction
(p<0.005). Gene-gene interaction analysis found that the ACE A-240T, A2350G,
and CYP11B2 C-344T polymorphisms
had a strong synergistic effect on HAPE.
In particular, the homozygous genotype
combination of -240AA, 2350GG, and
-344TT conferred high genetic susceptibility to HAPE. Our results provided
further evidence for the synergistic effect
of RAAS gene polymorphisms on HAPE
susceptibility (4–6) (Figure 1).
B
Genes in the Heat Shock Protein 70 Family
Heat shock proteins (HSPs) are a group of intracellular proteins upregulated during hypoxic stress. We focused on the common gene polymorphisms in HSPA1A, HSPA1B, and HSPA1L in the HSP70 family to explore their potential interactions with HAPE. Significant differences in
alleles from the A-110C polymorphism of HSPA1A and alleles from the
A1267G polymorphism of HSPA1B were observed between Han Chinese railway workers with and without HAPE. Furthermore, using haplotype analysis to compare the relative risk of HAPE, we observed that
individuals with Hap 4 (G-C-A) (A1267G, G190C for HSPA1A, and A110C for HSPA1B), and Hap 5 (G-C-A) had a significantly reduced risk
(p=0.0009), whereas Hap 7 (A-C-C) resulted in a 2.43-fold increased
risk for HAPE. When considered as diplotypes, individuals with Dip5
(Hap1-Hap7) had a significantly higher risk for HAPE (OR=3.39; 95%,
CI=1.28-9.17; p=0.014). Functional assessment supported a role for the
A-110C polymorphism of HSPA1A in the development of HAPE via a
change in HSPA1A promoter activity (7).
Endothelial Nitric Oxide Synthase Gene
The endothelial nitric oxide synthase (eNOS) gene encodes the enzyme
responsible for the production of NO, a signaling molecule involved in
vasodilation. Some variants of the eNOS gene associated with HAPE
have been reported (7). We conducted the largest, nested, case-controlled study to explore the genetic contribution to HAPE in railway
construction workers living in Qinghai-Tibet at an altitude of 4,000 m
above sea level. We found that the allele 894T and heterozygous G/T
of the 894G/T variant of the eNOS gene was positively associated with
susceptibility to HAPE. Furthermore, haplotype analysis comparing the
relative risk of HAPE among co-inherited alleles, demonstrated that
individuals with Hap 3 (T-T-b) and Hap 6 (C-G-a) were more susceptible to HAPE compared to those with other haplotypes, suggesting the
interaction of multiple genetic loci within eNOS might be a major determinant for susceptibility to HAPE (8).
16
Other Candidate Genes
Other specific candidate genes involving in the complex traits of HAPE
have been investigated, including HLA-DR, HLA-DQ, GNB3, ADD1,
ADRB2, CAT, GSTP1, CuZnSOD, MnSOD, HiF1, EPAS1, and mtDNA. However, no significant association of these genes with susceptibility or resistance to HAPE was identified (9, 10).
HAPE is thought to be a multifactorial disorder resulting from the
interaction of genetic and environmental factors. The combined study
design of genome-wide association and epigenetic analysis should be
undertaken in the future to elucidate the pathogenesis of HAPE and the
complex interactions between the genome and hypoxic environment.
This project was approved by the Ethics Committee of the Institute
of Basic Medical Sciences, CAMS/PUMC, and informed consent was
obtained from all patients and healthy volunteers.
REFERENCES
1. P. H. Hackett, R. C. Roach, N. Engl. J. Med. 345, 107 (2001).
2. H. Mortimer, S. Patel, A. J. Peacock, Pharmacol. Ther. 101, 183 (2004).
3. M. J. MacInnis, M. S. Koehle, J. L. Rupert, High Alt. Med. Biol. 11, 349
(2010).
4. Y. Qi, W. Niu, T. Zhu, W. Zhou, C. Qiu, Eur. J. Epidemiol. 23, 143 (2008).
5. Y. Qi et al., J. Renin Angiotensin Aldosterone Syst. 12, 617 (2011).
6. T. Stobdan et al., J. Renin Angiotensin Aldosterone Syst. 12, 93 (2011).
7. Y. Qi et al., Clin. Chim. Acta 405, 17 (2009).
8. S. Yu-jing et al., Chin. Med. Sci. J. 25, 215 (2010).
9. Q. Shen et al., Bull. Med. Res. 38, 29 (2009).
10. Y. Qi et al., Basic Clin. Med. 29, 811 (2009).
ACKNOWLEDGMENTS
The authors thank the volunteers for participating in this study. This work was
supported by grants from the National Basic Research “973” Program (Grant
No. 2006CB504103), the Key Projects in the National Science and Technology
Pillar Program (Grant No. 2006CB1190B), the National Laboratory Special
Fund (Grant No. 2060204) and the National Natural Science Foundation of
China (Grant No. 30393130, 30470615, and 31171146).
Section One
Studies on the Prevention of Acute Mountain Sickness
in People Entering High Altitudes by Airplane
Niu Wenzhong*, Fan Quanshui, Wu Qian, Yin Xudong, Pu Yonggao, Tang Bin
T
housands of people enter high altitude (HA) areas by airplane every year. In the past, acute mountain sickness (AMS)
was the most common disease in those lacking the time for
gradual acclimatization (1). To prevent AMS, a series of measures were studied and adopted including HA health education, physical examinations, standardization of AMS preventive measures (2), and
screening of medications for AMS (3, 4).
We studied the effects of a modified physical examination, popularization of health education, and disease prevention on the reduction
of the causative factors for AMS. These measures have been used to
draw up the five national standards for AMS prevention, which have
played an important role in the prevention and control of AMS, and
demonstrated that an obligatory medical management system is more
4,300 m above sea level (three men and two women) by airplane. Participants were 28 to 55 years old. After entering the HA area, they immediately performed low-intensity labor for more than eight hours a
day under medical surveillance. Symptoms of reactions to HA were
observed and scored daily for the first three days at HA and when they
had finished the work. Based on Yin’s AMS Scoring System (9), mild
symptoms and signs of reaction to HA with scores of two to four were
observed in five subjects, but none suffered from AMS. No obvious
symptoms and signs of HA reaction occurred in the other six subjects.
All subjects satisfactorily finished their scheduled work with no abnormal changes observed. Thus, it is not necessary to stop work completely in order to prevent of AMS; low-intensity labor could also be
performed under proper medical supervision.
Table 1. Incidence of AMS in people rapidly entering HA areas by airplane since 1987.
Year
1987
1993
1994
1998
2001
2003
2005
2007
2009
2011
Altitude (m)
3,500
3,680
3,680
3,200
3,900
3,680
3,680
3,680
3,680
3,900
Incidence (%)
48.5
38.0
31.4
20.0
22.8
10.8
5.6
3.0
2.6
1.7
Table 2. AMS Hospitalization rate in people rapidly entering HA areas by airplane since 1993.
Year
1993
1994
1998
2001
2004
2005
2007
2009
2011
Altitude (m)
3,680
3,680
3,200
3,900
3,680
3,680
3,680
3,680
3,900
Hospitalization (%)
2.18
1.58
0
0
0.20
0.14
0.13
0.10
0.12
effective than prophylactic medication. There was a significant reduction in both the incidence of AMS from 48.5% to 1.7% (Table 1) and
the hospitalization rate from 2.18% to 0.12% (Table 2) (5–7) in those
rapidly entering HA areas over the past 18 years.
As shown in Tables 1 and 2, AMS is no longer a severe threat to
people who rapidly enter HA areas under normal circumstances. However, if physical work is undertaken immediately and without enough
rest after entering HA areas, AMS is still the most common risk factor
(8). Therefore, several field trials at HA were performed to observe the
incidence of AMS in people who worked in the plain region without
a rest period after they were rapidly exposed to HA. This allowed for
the study of preventive strategies for AMS in people from the plain regions who have to work at HA. Two groups of human volunteers were
sent to either 3,680 m above sea level (five men and one woman) or
REFERENCES
1. Y. Q. Gao, High Altitude Military Medicine (Chongqing Publisher,
Chongqing, China, 2005), p. 251.
2. W. Z. Niu, the XXXVI World Congress on Military Medicine, St.
Petersburg, Russia, June 2005.
3. Y. Wang, W. Z. Niu, J. J. Zhang, H. J. Wang, N. R. Chen, J. Preventive
Medicine of Chinese People’s Liberation Army 22, 110 (2004).
4. W. Z. Niu, Y. Wang, Z. W. Cao, S. X. Yu, L. Zhang, J. High Alt. Med. 16,
6 (2006).
5. W. Z. Niu et al., Medical Journal of National Defending Forces in
Southwest China 17, 822 (2007).
6. W. Z. Niu, L. Fang, X. D. Yin, Q. Y. Zhai, Chin. J. Public Health Manage.
27, 416 (2011).
7. W. Z. Niu, Y. Wang, J. J. Zhang, N. R. Chen, J. High Alt. Med. 12, 12
(2002).
8. W. Z. Niu, Q. S. Fan, L. Fang, X. F. Nie, W. J. Wei, J. High Alt. Med. 21,
Laboratory of Prevention of High Altitude Disease, Center for Disease
Prevention and Control, Chengdu Military Command, Chengdu, Sichuan, China.
*
62 (2010).
9. Z. Y. Yin et al., J. Preventive Medicine of Chinese People’s Liberation
Army 15, 395 (1997).
17
Adaptive Responses of the Brain to High Altitude
Zhang Jiaxing1,*, Yan Xiaodan2, Zhang Haiyan1, Weng Xuchu2, Gong Qiyong3, Fan Ming4
T
he brain is one of the heaviest consumers of oxygen in the
body. Hypoxia challenge at high altitude (HA) usually puts
unacclimatized individuals at risk of acute mountain sickness
that can cause neurological impairments including cerebral
edema, cortical atrophy, and cortical and subcortical lesions, accompanied by behavioral compromises such as decline of cognitive performance and hallucinations (1). In contrast, under prolonged HA exposure, peripheral physiological systems typically employ adaptative
mechanisms such as changes in respiratory and circulatory function,
hemoglobin concentration, and arterial oxygen saturation. Such alterations change oxygen transport in the cerebral blood flow, leading to cumulative changes in brain structure and function. Our primary research
focus is the use of a multimodal magnetic resonance imaging (MRI)
approach to investigate cerebral adaptation to HA. Quantitative analysis methods such as voxel-based morphometry and Tract-Based Spatial
Statistics were employed to measure gray matter (GM) and white matter (WM) microstructural changes, while task-based and resting-state
functional MRI (fMRI) were used to study functional changes.
Every year, the number of travelers to HA regions increases. We conducted a pre-post MRI study on 14 young, amateur mountain climbDepartment of Physiology and Neurobiology, Medical College of Xiamen
University, Xiamen, China;
2
Laboratory for Higher Brain Function, Institute of Psychology, Chinese
Academy of Sciences, Beijing, China;
3
Huaxi Magnetic Resonance Research Center, West China Hospital, Sichuan
University, Chendu, China;
4
Department of Brain Protection and Plasticity, Institute of Basic Medical
Sciences, Beijing, China.
*
1
ers (19–23 years of age) who were without neurological complications
before and after their travel to an altitude of approximately 6,206 m
(1). No significant changes were observed in the total volumes of GM,
WM, and cerebrospinal fluid after mountain climbing, although structural alterations were seen in WM, but not GM. Significantly decreased
WM fractional anisotropy (FA) values were observed at multiple sites
of WM tracts (Table 1). Furthermore, compromises were found in the
microstructural integrity of WM tracts (but not in WM volumes), suggesting that cytotoxic edema had occurred. We did not observe any cognitive changes in this population.
Every year people move from the lowlands to HA regions for work,
study, or training, staying for several months to several years. An example of such a population is college students. In a previous study, we recruited 52 college students originally from areas at sea level, who were
studying at a moderate altitude of 2,260 m over a seven-month period,
with a return to sea level for 30 days in the middle of this period (2).
We administered a battery of neuropsychological tests for comprehensive memory functions, which included short- and long-term memory,
examining explicit and implicit visual and auditory memory. Results
showed comparable performance with the matched sea-level control
group except for a short-term visual, construction task. In another study,
we obtained MRI data from 16 young healthy men (20–22 years of age)
who had immigrated to the Qinghai-Tibet Plateau (2,300 to 4,400 m)
for 2 years (unpublished data). Compared with matched sea level residents, they showed changes in GM volumes, accompanied by changes
in anisotropy and diffusivity at multiple sites of the WM tracts (Table
1). Increased GM volume in some regions had a significant positive
correlation with altitude. Moreover, HA subjects developed ventilatory
depression and deficits in mental rotation performance and reaction time
Table 1. Changes in grey and white matter, and behavior in mountain climbers, adult immigrants, and immigrant descendants.
Mountain climbers
HA adult immigrants
HA immigrant descendants
Gray matter
volume
No significant changes (2)
Increases in: right middle frontal gyrus, right
parahippocampal gyrus, right inferior and
middle temporal gyri, bilateral inferior ventral
pons, and right cerebellum Crus. (unpublished
data)
Decreases in: right postcentral gyrus and right
superior frontal gyrus
Decreases in: bilateral anterior insula, right
anterior cingulate cortex, bilateral prefrontal
cortex, left precentral cortex, and right
lingual cortex (4)
White matter
FA values
Decreases in: CC
(anterior and posterior
body, splenium), bilateral
CRT, right PCB, left SLF,
and left middle cerebellar
peduncle (2)
Increases in: CC (anterior body), left
corticonuclear tract, bilateral SLF, and right ILF.
Decreases in: CC (posterior body), bilateral
hippocampus, right SCR, and right SLF
(unpublished data)
Increases in: bilateral ALIC, bilateral
anterior external capsule, CC, right CRT,
right PCB, bilateral SLF and ILF, and
bilateral SCR (4)
Decreases in: the left optic radiation and
left SLF (4)
No significant changes (2)
Deficits in: reaction time in memory search,
mental rotation, number search tasks and SRTT
and mental rotation task (unpublished data),
and in ROCF and visual reproduction (3)
Deficits in: digit span, ROCF, reaction time
in SWM and VWM (5)
Behavioral
tests
ALIC, anterior limb of internal capsule; CC, corpus callosum; CRT, corticospinal tract; FA, fractional anisotropy; HA, high altitude; ILF, inferior
longitudinal fasciculus; PCB, posterior cingulum bundles; ROCF, Rey-Osterrieth Complex Figure; SCR, superior corona radiata; SLF, superior
longitudinal fasciculus; SRTT, serial reaction time task; SWM, spatial working memory; VWM, verbal working memory.
18
Section One
tasks. GM volume in the parahippocampal gyrus and middle frontal gyrus
in HA subjects was negatively correlated with vital capacity. GM in the
superior frontal gyrus had a significant
positive correlation with mental rotation and GM in the postcentral gyrus
was negatively correlated with number search reaction time and memory
reaction time.
Immigrant descendants are a suitable population for studying developmental adaptation. Thus, 28 college
students (17–23 years of age) born and
raised in the Qinghai-Tibet Plateau region (2,616–4,200 m) for at least 17
years were recruited. Their families
had migrated from sea level areas to
HA regions two to three generations
ago. The control group consisted of Figure 1. Statistical parametric map for grey matter volume correlates with altitude (p<0.05). Red indicates
positive correlation; green indicates negative correlation. Upper left numbers indicate the normalized
matched subjects living at sea level. sagittal slice number (y-value).
All subjects were from Han Chinese
populations. HA subjects showed
decreased GM volume in a number of brain regions accompanied by
In summary, GM changes occur in a number of regions of the brain
changes in FA values in multiple fiber pathways (Table 1) (3). HA sub- responsible for HA respiratory and cardiovascular control. The WM
jects also showed significant differences in resting-state brain activity in microstructural alterations in the corpus callosum, cerebellar WM, and
multiple brain regions (4). A separate study demonstrated a decrease in corticospinal tract might be related to changes in motor skills following
cerebrovascular reactivity and a delay in hemodynamic response dur- acclimatization to HA. HA adaptation occurred at the cost of increased
ing a visual-cue guided maximum inspiration task (5). Since decreased reaction time and deficits in working memory, as well as visual spatial
appetite and weight loss were reported among travelers ascending to construction. Observed changes in GM may help elucidate the mechaHA regions, we used pictures of food to elicit gustatory processing nisms involved. The brains of youth and adults exhibit a different adapduring an fMRI experiment. HA subjects showed decreased activation tive response to HA, which is in agreement with previous studies showwithin the neural circuit for food craving such as the insula, accompa- ing that spatial memory was changed in pups, but not in adults, exposed
nied by increased activation in regions for emotional processing such to intermittent hypobaric hypoxia (10). In addition to developmental
as the cingulate gyrus (6). A number of cognitive deficits were found influences, the effects of genetic inheritance on the brain and whether
in HA subjects, including working memory and mental construction brain changes recover to normal after a return to sea level should be
(Table 1). An fMRI experiment with a verbal working memory task investigated in future studies.
revealed decreased activation in many brain regions and decreased
connectivity strength to and from the precentral cortex (7). The activaREFERENCES
tion and connectivity strength significantly correlated with behavioral
1. H. Zhang et al., High Alt. Med. Biol. 13, 118 (2012).
performance. In contrast, an fMRI experiment with spatial working 2. J. Zhang, H. Liu, X. Yan, X. Weng, High Alt. Med. Biol. 12, 37 (2011).
memory revealed an adaptive compensatory neural mechanism, show3. J. Zhang et al., PLoS One 5, e11449 (2010).
ing no significant difference in activation in HA subjects compared 4. X. Yan, J. Zhang, J. J. Shi, Q. Gong, X. Weng, Brain Res. 1348, 21
with controls (8).
(2010).
Based on the above studies, we observed that GM in the anterior insu- 5. X. Yan, J. Zhang, Q. Gong, X. Weng, BMC Neurosci. 12, 94 (2011).
lar cortex, anterior cingulate cortex, superior prefrontal gyrus, premotor 6. X. Yan, J. Zhang, Q. Gong, X. Weng, Exp. Brain Res. 209, 495 (2011).
cortex, inferior and middle temporal cortex, ventral pons, parahippo- 7. X. Yan, J. Zhang, Q. Gong, X. Weng, Exp. Brain Res. 208, 437 (2011).
campus, and posterodorsal portion of the cerebellum and WM in the 8. X. Yan, J. Zhang, Q. Gong, X. Weng, Brain Cogn. 77, 53 (2011).
corpus callosum, corticospinal tract, and frontal cortex were most sus9. J. Zhang, H. Zhang, S. Liu, Q. Gong, M. Fan, Abstract presented at
ceptible to chronic hypoxia. To confirm this, 77 Tibetan natives (14–18
18th Annual Meeting of the Organization for Human Brain Mapping.
years of age) from the Qinghai-Tibet Plateau (2,300–5,300 m) were
Beijing, 10 June 2012.
recruited for a study (9). Whole-brain analysis was conducted based on 10. J. Zhang et al., J. Neurosci. Res. 84, 228 (2006).
the mean GM volumes and WM FA values to identify significant associations of structural responses with increasing altitude. The results ACKNOWLEDGMENTS
revealed that altitude significantly correlated with GM volume in a large This work was supported by the National Natural Science Foundation of
number of areas as observed in the previous study (Figure 1). Addition- China (Grant No. 30425008, 60628101, and 31071041), China Postdoctoral
ally, altitude had a significant positive correlation with GM volume in Science Foundation (Grant No. 20060390129) and the National Key Project
the occipital visual cortex, superior temporal gyrus, and temporal pole.
(Grant No. 2012CB518200).
19
Mechanism of Chronic Intermittent Hypoxia-Induced
Impairment in Synaptic Plasticity and Neurocognitive
Dysfunction
Ke Ya1, Chan Ying-Shing2, Yung Wing-Ho1,*
O
bstructive sleep apnea (OSA) is a common but often
overlooked sleep and breathing disorder that has a similar
prevalence across different geographic regions and ethnic
groups, found in approximately 4% of men and 2% of
women (1). In OSA, the periodic obstruction of the upper airway
leads to intermittent hypoxia and subsequent hypoxemia. There is
substantial evidence to suggest that intermittent hypoxia, either alone
or in combination with sleep deprivation and fragmentation caused
by microarousals during sleep, can lead to an array of problems in
OSA patients such as cardiovascular morbidity, insulin resistance,
hypertension, and dyslipidemia (1, 2). Apart from being a breathing
disorder with metabolic consequences, the repeated hypoxia/
reoxygenation cycles imposed on the body during OSA can impair
brain performance. Thus, OSA is a major cause of sleepiness and sleeprelated traffic accidents. OSA can also result in decreased attention and
vigilance, increased irritability, and impaired executive functions and
long-term memory (1, 3). Understanding the mechanism of intermittent
hypoxia-induced neurocognitive deficit is a question of scientific and
clinical importance.
Over the last decade, numerous studies have investigated the relationship between OSA-associated intermittent hypoxia and cognitive dysfunction using experimental animal models. Exposure to an intermittent
hypoxia paradigm during the sleep cycle of adult rats is associated with
spatial learning deficits, accompanied by neuronal loss within susceptible brain regions such as the hippocampus and cortex (4). Subsequent
studies confirmed that chronic intermittent hypoxia treatment could
impair the spatial memory functions of rodents to different degrees (5).
Studies from our group and other investigators suggest that chronic
intermittent hypoxia-induced apoptosis, oxidative stress, endoplasmic
reticulum (ER) stress, and reduced neuronal excitability, particularly in
the hippocampus, contribute to these observations (4, 6–8).
Using a mouse model of OSA, we examined the effect of intermittent
hypoxia on the magnitude of early phase long-term potentiation (ELTP) in the hippocampal CA3-CA1 pathway, the prototypical pathway
for the study of memory-related synaptic plasticity. Adult mice were
exposed to either normoxia or intermittent hypoxia treatment that lasted
three to 14 days. To mimic the intermittent hypoxia experienced by
human subjects, the regimen of hypoxia consisted of cycles of oxygen
levels between 10% and 21% every 90 seconds during the daytime for
eight hours. We observed a significant decrease in E-LTP in both the
seven-day and 14-day intermittent hypoxia groups compared with the
control group (Figure 1A). Of particular significance, we demonstrated
School of Biomedical Sciences, Faculty of Medicine, The Chinese University
of Hong Kong, Shatin, Hong Kong, China;
2
Department of Physiology & Research Centre of Heart, Brain, Hormone and
Healthy Aging, Li Ka Shing Faculty of Medicine, The University of Hong
Kong, Sassoon Road, Hong Kong, China.
*
1
20
Figure 1. Elucidation of the key role and underlying cause of reduced
brain-derived neurotrophic factor (BDNF) expression in intermittent
hypoxia-induced impairment in hippocampal plasticity. (A) Field
excitatory postsynaptic potentials (fEPSPs) were recorded in the CA1
area of hippocampal slices and E-LTP was induced by conventional
tetanic stimulation. Both seven-day and 14-day intermittent hypoxia
treatment impaired E-LTP. The insets show raw traces from representative
experiments. The right panel is a summary of data from 16–20 slices. (B)
Chronic intermittent hypoxia resulted in reduced expression of mature
BDNF (mBDNF) but not pro-BDNF revealed by western blot analysis.
Pooled data from 9–11 samples are summarized in the right panel. (C)
Intermittent hypoxia-induced impairment was prevented by surgical
replenishment of BDNF via multiple intracerebroventricular injections of
BDNF during intermittent hypoxia exposure. (D) Reduction in plasmin
expression strongly suggested that reduced conversion of pro-BDNF
to mBDNF is a factor underlying reduced mBDNF expression. *p<0.05;
**p<0.01; ***p<0.001 compared with the corresponding control. A–C
from reference 6.
Section One
that intermittent hypoxia significantly impaired
conventional E-LTP and to a greater extent
late-phase LTP, which better correlates with the
formation of long-term memory (6). These data
indicate that neurocognitive deficits observed in
OSA may be caused by intermittent hypoxiainduced impairment of hippocampal LTP.
We previously showed that brain-derived neurotrophic factor (BDNF) is crucial for the consolidation of long-term synaptic plasticity (9). We
speculated that BDNF levels in the brain might
be altered by intermittent hypoxia. Indeed, in the
mouse model, we found that BDNF expression
was ��
significantly ��
reduced after chronic intermittent hypoxia treatmen��
t��
(Figure 1B)��
. Addition��
ally, exogenous application of BDNF restored
the magnitude of LTP in hippocampal slices
from hypoxia-treated mice, and microinjection
of BDNF into the brain of hypoxic mice prevented the LTP impairment (Figure 1C). Thus, a
decrease of BDNF could be a crucial factor contributing to the absence of normal hippocampal
plasticity and therefore memory function in the
Figure 2. Proposed interactions between BDNF reduction and other pathological processes
that lead to neuronal injury and decreased neuroplasticity in obstructive sleep apnea (OSA).
intermittent hypoxia model. In line with this hyChronic intermittent hypoxia causes decreased neuronal excitability, decreased BDNF
pothesis, we recently showed that administration
expression and generation of oxidative stress and ER stress. These factors act synergistically
of ampakines, a group of AMPA receptor moduto increase apoptosis and impair long-term synaptic plasticity, resulting in impaired memory
lators, restored BDNF levels in the hippocampus
and other neurocognitive deficits. In this model, decreased expression of BDNF plays a pivotal
and prevented the decrease in LTP magnitude
role in ROS generation, ER stress, apoptosis, and impairment of synaptic plasticity.
caused by intermittent hypoxia (unpublished
data). Thus, by targeting BDNF expression, ampakine administra- in generating the cascade of events leading to neurocognitive deficits in
tion could be a potential therapeutic treatment for the neurocognitive OSA, as depicted in Figure 2. In this model, OSA-associated chronic
symptoms of OSA subjects.
intermittent hypoxia results in reduced neuronal excitability, decreased
BDNF levels are upregulated during some pathological conditions BDNF levels, increased oxidative stress, and ER stress. These factors
of the central nervous system, a phenomenon usually regarded as interact and operate in a synergistic manner to increase apoptosis and
a compensatory mechanism beneficial to the survival of neurons. cell injury and weaken long-term synaptic plasticity that together
However, during prolonged and repeated hypoxia/reoxygenation under cause memory impairment and other neurocognitive dysfunctions.
the chronic intermittent hypoxia paradigm, the ability of brain cells to Careful dissection of the interrelationship among these factors may
express BDNF may be compromised. The duration-dependent decrease enable us to have a greater understanding of the pathogenesis of OSA
in BDNF levels observed in the intermittent hypoxia model supports this neurobehavioral symptoms.
notion. We believe that the decrease in BDNF is not simply the result
of neuronal loss because other neurotrophic factors, such as NT4/5,
REFERENCES
were not affected. Additionally, we observed the reduced expression
1. W. Lee, S. Nagubadi, M. H. Kryger, B. Mokhlesi, Expert Rev. Respir.
of plasmin, an extracellular enzyme that cleaves pro-BDNF to form
Med. 2, 349 (2008).
mature BDNF, in the chronic intermittent hypoxia model (Figure 1D).
2. P. Levy, M. R. Bonsignore, J. Eckel, Eur. Respir. J. 34, 243 (2009).
This is consistent with our observation that pro-BDNF levels were
3. M. L. Jackson, M. E, Howard, M. Barnes, Prog. Brain Res. 190, 53
not affected by chronic intermittent hypoxia treatment, and suggests
(2011).
that proteolytic cleavage of pro-BDNF to mature BDNF was affected,
4. D. Gozal, J. M. Daniel, G. P. Dohanich, J. Neurosci. 21, 2442 (2001).
rather than transcription of the BDNF gene.
5. B. W. Row, Adv. Exp. Med. Biol. 618, 51 (2007).
Our studies shed new light on the causes underlying intermittent
6. H. Xie et al., Neurobiol. Dis. 40, 155 (2010).
hypoxia-induced impairment in long-term synaptic plasticity and
7. B. W. Row, R. Liu, W. Xu, L. Kheirandish, D. Gozal, Am. J. Respir. Crit.
neurocognitive deficits. Reduced neuronal excitability and other
Care Med. 167, 1548 (2003).
pathological processes such as the generation of reactive oxygen species 8. Y. Zhu et al., J. Neurosci. 28, 2168 (2008).
(ROS) and ER stress may contribute to neuronal damage under chronic
9. P. Pang et al., Science 305, 487 (2004).
intermittent hypoxia condition. Since BDNF can prevent or suppress
these processes, it is possible that lack of BDNF in chronic intermittent ACKNOWLEDGMENTS
hypoxia not only underlies impaired long-term synaptic plasticity but This work was supported by the Research Grants Council of Hong Kong and
also fails to prevent apoptosis and other neuronal injuries induced by the National Natural Science Foundation of China (Grant No. CUHK478308,
ROS and ER stress. Thus, the lack of BDNF could play a pivotal role 2900336, and 30931160433).
21
Chinese Herbs and Altitude Sickness: Lessons from
Hypoxic Pulmonary Hypertension Research
Ke Tao1,3, Li Zhichao2, Zhang Wenbin1,3, Wang Jiye1,3, Luo Wenjing1,3,*, Chen Jingyuan1,3,*
H
igh-altitude pulmonary edema (HAPE) and high-altitude pulmonary hypertension (HAPH) are two forms of altitude sickness that endanger the lives of people climbing or migrating
to high altitudes. Hypoxic pulmonary hypertension (HPH) is
a hallmark of HAPE and HAPH. Although several drugs that are used
for the prevention and treatment of pulmonary hypertension (PH) are
potent in preventing or treating HPH, they are not ideal. Since large
areas of China are at a high altitude, there is a perfect opportunity for
scholars to observe the effects of Chinese herbs on the treatment of altitude sickness. The development of traditional Chinese medicine (TCM)
for altitude sickness in parallel with standard drug therapies opens an
avenue to study TCM in mountain medicine. Here, we summarize the
history of TCM used in mountain medicine in China with a brief review
of our work on HPH.
TCM and Altitude Sickness
In TCM theory, altitude sickness is a disequilibrium state of the human
body that continually interacts and exchanges resources and energies
with the surrounding high-altitude environment of hypoxia, cold, irradiation, and dryness. The development and prognosis of the illness
is dependent on two opposite and complementary factors in the human
body, namely “yin” and “yang,” which are important in the philosophy
of TCM and ancient China. Many Chinese herbs believed to be capable
of adjusting “yin” or “yang” have been used to treat altitude sickness
for nearly half a century. New information regarding these medicines
has been obtained from local medical journals as well as anecdotal evidence. The selection of candidate drugs for these trials was often experience-based and most of the studies performed did not follow protocols
or methodology favored by modern medicine. The herbs were usually
prescribed as a mixture, with the appropriate ratio of individual herbs
decided by the different states of “yin” and “yang.” The treatments used
were from high-altitude plants (such as Dracocephalum heterophyllum
Benth and Gymnadenia conopsea), a class of herbs to treat cardiovascular ailments (Salvia miltiorrhiza, Radix Codonopsis, and Radix Angelicae Sinensis), adaptogens (Rhodiola rosea and Panax ginseng) or
antioxidants (Ginkgo biloba and Lycium chinense), with some adjuvant
herbs. It is thought that the different compounds play complementary
roles to produce the overall effect, although evidence for beneficial effects of the herbs is usually doubted or neglected by researchers who
favor a reductionist approach. However, it should be noted that more
robust clinical trials and experimental studies of Chinese herbs and single molecule entities have been conducted in recent years. These trials
Department of Occupational and Environmental Health,
Fourth Military Medical University, Xi’an, Shaanxi, China;
2
Department of Pathophysiology, Fourth Military Medical University,
Xi’an, Shaanxi, China;
3
The Ministry of Education Key Lab of Hazard Assessment and Control in
Special Operational Environment, School of Public Health,
Fourth Military Medical University, Xi’an, Shaanxi, China.
*
Corresponding authors: Chen Jingyuan ([email protected])
and Luo Wenjing ([email protected])
1
22
report a beneficial effect of Chinese herbs on altitude sickness, such as
acute mountain sickness (AMS), HAPH, or chronic mountain sickness
(CMS) (Table 1) (1). Among these, S. miltiorrhiza and R. rosea were
the two herbs most commonly prescribed and evaluated, and a series of
studies using these herbs supported the potential for TCM in the treatment of altitude sickness.
Salvia Miltiorrhiza and Rhodiola Rosea:
Candidate Drugs for HPH
The pathogenesis of HPH describes a process ranging from acute hypoxic pulmonary vasoconstriction to medial hypertrophy and remodeling of muscular pulmonary arteries, in which oxidative stress, altered
expression of ion channels in pulmonary arterial smooth muscle cells
(PASMCs), dysfunction of pulmonary vascular endothelium and calcium mobilization, and influx in PASMCs are believed to be underlying
mechanisms. Using isolated pulmonary arteries and cultured PASMCs
from chronically hypoxic rats, we found that Tanshinone IIA, one of
the active components found in S. miltiorrhiza, had an inhibitory effect
on HPH and pulmonary vascular remodeling. Chronic hypoxia-induced
increases in the mean pulmonary arterial pressure (mPAP), right ventricular hypertrophy (RVH), and thickening of distal pulmonary arteries, were all attenuated by pretreatment with Tanshinone IIA. This
was further supported by the Tanshinone IIA-induced suppression of
hypoxia-induced PASMC proliferation and prevention of hypoxia-induced downregulation of voltage-activated potassium channel mRNA
and protein expression in pulmonary arteries and PASMCs (2). An
antioxidant effect of Tanshinone IIA was observed as an increase in
superoxide dismutase and reduction in malondialdehyde levels in the
homogenates of lungs exposed to hypoxia (3). Moreover, in pulmonary
artery rings from normal rats, Tanshinone IIA eliminated acute hypoxiainduced vasoconstriction and potentiated vasorelaxation. In pulmonary
artery rings from hypoxic pulmonary hypertension rats, Tanshinone IIA
reversed sustained constriction induced by phenylephrine and led to
sustained vasodilation (4). Tanshinone IIA, which mimics the action
of acetazolamide (5), achieved its effect by inhibiting hypoxia-induced
Ca2+ responses, although it was endothelium independent and partially
mediated by opening Ca2+-activated K+ channels.
Another potent drug for treating HPH is Rhodioloside, one of the active components of R. rosea. Rhodioloside research has benefited from
multidisciplinary studies. Similar to treatment with Verapamil, a calcium channel blocker, Rhodioloside inhibited acute hypoxia-induced
proliferation of PASMCs in rabbit lung (6), and increased the expression of nitric oxide synthase and inhibited KCl-induced cell contraction
in vascular smooth muscle cells (7). Pretreatment with Rhodioloside
increased arterial oxygen saturation by 3% in a group of young men
acutely exposed to an altitude of 3,658 m (unpublished data). Together,
this provides evidence supporting a possible anti-HPH effect of Rhodioloside. Our ongoing research with R. rosea is focused on mechanistic studies and active-site chemistry using chemically engineered
analogues. Preliminary results demonstrate that one analogue, benzyl
galactosidase, has an effect similar to Rhodioloside (8).
Section One
Table 1. Clinical studies on Chinese herb compounds used for treating altitude sickness in China.
Publication year
Compound name
(Latin name)
Study type
Numbers of
participants
Disease
82
CMS
Randomized
controlled trial
Baojianwan (Senna tora /Polygonatum sibiricum /Cortex
cinnamomi/Salvia miltiorrhiza /Radix Angelicae Sinensis)
Rensheng (Panax ginseng)
150
AMS
1996
Controlled trial
Fufangtianji (Rhodiola rosea /Lycium chinense /Fructus
hippophae)
439
CMS
1997
Self-controlled trial
Fufangdangsheng pian (Radix Codonopsis)
26
Cognitive
impairment
1999
Randomized
controlled trial
Yiyeqinglan jiaonang (Dracocephalum heterophyllum Benth)
52
AMS
2003
Randomized doubleblind controlled trial
Hongjingtian jiaonang (Rhodiola rosea)
600
AMS
2005
Clinical observation
Ciwujiazhushe ye (Eleutherococcus setulosus)
102
CMS
2005
Randomized
controlled trial
Fufangdangsheng jiaonang (Radix Codonopsis /Adenophora
stricta/Salvia miltiorrhiza)
45
AMS
2005
Randomized
controlled trial
Sanpuhongjingtian jiaonang (Rhodiola rosea /Fructus
hippophae / Lycium chinense)
122
AMS
2006
Self-controlled trial
Lishukang jiaonang (Rhodiola rosea /Gymnadenia conopsea /
Dracocephalum tanguticum Maxim /Cortex phellodendri
chinensis /Rhododendron anthopogonoides Maxim)
104
CMS
2006
Randomized
controlled trial
Shulikang jiaonang (Rhodiola rosea /Senna tora /Radix
Angelicae Sinensis /Flos rosae rugosae)
150
AMS
2008
Xingnaojing zhusheye (Moschus moschiferus /Borneolum
synthcticum/Radix curcumae /Fructus gardeniae)
78
AMS
2011
Ershiweichenxiang wan (Aquilaria agallocha Roxb /Syzygium
aromaticum /Chaenomeles sinensis)
27
HAPH
2011
Randomized
controlled trial
Danhong zhusheye (Salvia miltiorrhiza /Flos carthami)
76
AMS
1996
1996
Controlled trial
Randomized
Yingxingye pian (Ginkgo biloba); Hongjingtian jiaonang
236
controlled trial
(Rhodiola rosea)
CMS, chronic mountain sickness; AMS, acute mountain sickness; HAPH, high-altitude pulmonary hypertension.
2011
Future Development of Chinese Herbs
for Treatment of HPH
HAPE and HAPH are two potentially fatal high-altitude pulmonary
diseases with high mortality in high-altitude immigrants and travelers.
For socio-economic reasons, there will be increasing numbers of
people exposed to high altitudes worldwide; thus developing new
effective drugs will be a priority to overcome resulting diseases.
Chinese herbs form an incomparable collection of substances for
developing anti-HPH drugs, and because the natural plant components
of Chinese herbs seldom cause side effects, they can be continuously
administered to patients. In addition, many compounds have a broad
spectrum of pharmacological activities for the treatment of altitude
sickness. Although the herbs investigated in our laboratories and others
show promise, the mechanisms of the individual components and
the interactions between them are still unclear. The challenge for the
development of Chinese herbs is to provide convincing translational
evidence of the effects of individual components. Furthermore, more
evidence in support of TCM is needed and may be obtained through
new strategies using cooperative research and multidisciplinary studies.
As there are many active components in an extract of S. miltiorrhiza,
it is a major challenge to research them individually; thus, we have
focused on several main active components using high throughput
screening technology. One disadvantage of this system is that some
active components of S. miltiorrhiza are present in the plant at very low
quantities. Thus, it may be simpler to develop analogues and study the
bioactive sites of the active components, which can be designed and
AMS
synthesized as research tools using chemical engineering technology.
Because the effect of S. miltiorrhiza on altitude sickness is not only
due to its direct actions on damaged organs, but also indirectly by
improving the functions of other organs and/or systems, it can be
difficult to interpret results from studies using modern medical theory.
Therefore, developing and advancing the theory of TCM based on
systems biology may help to interpret the phenomenon and lay a
solid foundation for future research of Chinese herbs for treating
altitude sickness.
REFERENCES
1. Y. K. Zhang et al., J. High Alt. Med. 21, 57 (2011).
2. Y. F. Huang et al., J. Ethnopharmacol. 125, 436 (2009).
3. M. L. Liu et al., Chin. Pharm. Bull. 24, 723 (2008).
4. J. Wang et al., Eur. J. Pharmacol. 640, 129 (2010).
5. L. A. Shimoda et al., Am. J. Physiol.-Lung C. 292, L1002 (2007).
6. S. X. Lin et al., Chin. J. Pathophysiol. 17, 968 (2001).
7. H. L. Zhi et al., Chin. Heart J. 15, 86 (2003).
8. J. Zhang et al., J. Fourth Mil. Med. Univ. 30, 1916 (2009)
ACKNOWLEDGMENTS
This work was supported in part by the National Key Technology R&D
Program (Grant No. 2009BAI85B04), the National Natural Science
Foundation of China (Grant No. 30770925, 30700265, and 81172621), and
the Program for Changjiang Scholars and Innovative Research Team in
University (PCSIRT and IRT1112).
23
Fast Acclimatization to High Altitude Using
an Oxygen-Enriched Room
Xiao Huajun*, Deng Changlei, Zhang Zhaorui, Wang Guiyou, Zang Bin, Wen Dongqing, Liu Xiaopeng, Zhang Bo
A
n artificial oxygen-enriched environment at high altitudes
can protect people from hypoxia and is especially suitable
for flight crews (1) and tourists (2) who have had little time
to acclimatize to low oxygen conditions. Here, we report a
new way to prevent high-altitude pulmonary edema using an oxygenenriched room with a diffusion oxygen-supply system (3, 4).
To determine a suitable standard procedure for the application of
the oxygen-enriched room, animal and human experiments were performed. Wistar rats were divided into five groups and placed at simulated altitudes as follows: 0 m, 6,000 m, 6,000 m with 35% or 30%
continuous oxygen (physiologically equivalent to an altitude of approximately 2,500 m or 3,500 m, respectively), and 6,000 m supplied
with 35% oxygen for four hours at a time, with breaks of four hours.
The water ratio, endothelin-1, and nitric oxide synthase (NOS) content
of the lungs were remarkably different between the groups (4, 5). The
group receiving 35% oxygen alternately every four hours had the best
results of all hypoxic groups.
At an altitude of 3,500 m, an oxygen-enriched room with an oxygen
concentration of 25.49% ± 0.26% (physiologically equivalent to an altitude of approximately 2,200 m) was developed by using a high altitude
diffusion oxygen-supply system based on a molecular sieve oxygen
generating system with pressure swing adsorption (1). Young human
volunteers were divided into three groups: the oxygen rich group (O)
was in the oxygen-enriched room at night, without oxygen enrichment
during the day; the hypoxia group (H) was without oxygen enrichment;
and a group representing low altitude plain conditions (control, P) was
given no treatment. After group O and H reached a high altitude by
airplane, heart rate (HR) and oxygen saturation (SaO2) levels were recorded before oxygen was supplied, while group P remained on the
plain. From 22:00 to 9:00 hours the next day, group O and H slept in the
oxygen-enriched room or a normoxic room, respectively.
Results showed that the SaO2 of group O was 92.3% ± 1.0% after
oxygen was supplied, which was higher than that before oxygen was
supplied (82.9% ± 4.2%). The SaO2 of group O was also higher than
that for group H (79.3% ± 5.9%; p<0.01), but was lower than that for
group P (97.3% ± 0.8%; p<0.05). There was no significant difference
in HR between group O and H before or after administration of oxygen. However, the HR of group H and O was higher than that of group
P (p<0.01). Measurement of heart rate variability (8) showed that the
normalized low frequency and low frequency/high frequency ratio of
group O and H were 89.3 ± 2.9 ms2, 9.4 ± 2.8 and 90.2 ± 1.8 ms2, 9.9 ±
1.9 ms2, respectively, which was not significantly different. However,
these values were significantly higher than those of group P (85.8 ± 2.9
ms2 and 6.4 ± 1.4 ms2; p<0.05).
There was no significant difference in sleep structure between group
O and H, although both groups had significantly more light sleep and
less deep sleep (p<0.01), as measured by a portable sleep monitor, compared with group P. The results of a questionnaire showed that group
P had the best sleep quality and group H had the worst, while group O
was in between (6).
In summary, we have developed a new high altitude diffusion oxygen-supply system, which is especially suitable for flight crews (1) and
tourists (2). The oxygen concentration of the oxygen-enriched room
should be 25% ± 0.5%, which results from an oxygen partial pressure
of inspired air (75±2 mmHg) and a physiologically equivalent altitude
(2,200±150 m) (7). Additionally, an intermittent oxygen supply is better than a continuous one (6). These results represent the foundation for
the application of an oxygen-enriched room for use at night, which is
an efficient solution for people entering Tibet who lack the time to acclimatize gradually (9).
REFERENCES
1. H. J. Xiao, Physiology of Aviation Oxygen Protective Equipment (Military
Medicine Science Publisher, 2005) pp. 291-293.
2. H. J. Xiao, High Alt. Med. Biol. 5, 226 (2004).
3. H. J. Xiao et al., China-Japan Medical Conference, Beijing.Nov,36(2002).
4. H. J. Xiao et al., J. Chin. Aerospace Med. 22, 259 (2011).
5. Z. R. Zhang et al., J. Chin. Aerospace Med. 22, 34 (2011).
6. C. L. Deng et al., J. Chin. Aerospace Med. 21, 51 (2010).
7. C. L. Deng, H. J. Xiao, J. Alt. Med. S1, 42 (2009).
Institute of Aviation Medicine, No.28, Fucheng Lu, Beijing, China.
*
24
8. H. J. Xiao et al., High Alt. Med. Biol. 5, 233 (2004).
9. H. J. Xiao et al., J. Chin. Aerospace Med. 19, 266 (2008).
Section One
A Comparison of Perimenopausal Sex Hormone
Levels Between Tibetan Women at Various Altitudes
and Han Women at Sea Level
Zhang Jianqing*, Wei Chunmei, Xiao Hong, Zhang Shuna
P
eople from Tibet are well adapted to high altitudes. One example of adaptation can be found in the maternal-placentalfetal system in Tibetan women, where placental mechanisms
ensure an adequate oxygen supply to the fetus even in hypoxic
conditions (1–5). However, few studies have looked at sex hormone
levels during perimenopausal stages in women living at moderate or
high altitudes. We therefore compared sex hormone levels in groups of
perimenopausal Tibetan women living at various altitudes with perimenopausal Chinese Han women living at sea level to investigate the
correlation between altitude and sex hormone levels.
2009. The age and BMI values are shown in Table 1 and venous sex
hormone testing results in Table 2.
It is interesting that no significant difference in sex hormone levels
was observed among the two groups of perimenopausal Tibetan women
and the group of Han women living at sea level, verifying the study
results of previous researchers (6, 7). These results suggested that a
different oxygen partial pressure (PaO2) at moderate and high altitude,
or at sea level, has no effect on the Tibetan female hormonal reproductive system.
Studies on the maternal-placental-fetal system in Tibetan women
Table 1. Age and BMI comparison among Tibetan women at moderate and high altitudes and Han women at sea level.
Group
Number of Cases
Age (years)
BMI (kg/m2)
Sea level
2,260 m
198
180
48.9±2.2
48.0±2.1
23.2±2.3
23.4±2.6
4,200 m
180
47.0±2.7
23.1±2.5
No statistically significant difference was found amongst the three groups.
Table 2. Venous E2, FSH, T, P, PRL, and LH levels in Tibetan women at moderate and high altitudes and Han
women at sea level.
Target
E2 (pg/ml)
FSH (IU/L)
T (nmol/L)
P (pg/ml)
PRL (IU/L)
LH (IU/L)
Sea level group
(database)
74.29±67.23
5.80±3.41
2.11±1.56
10.9±10.6
390.8±230.0
25.8±24.1
2,260 m group (n=180)
4,200 m group (n=180)
72.86±68.74
74.96±66.72
1.84±1.83
1.82±1.74
5.61±3.81
9.80±10.4
378.8±228.0
28.5±24.7
6.00±3.78
9.0±10.8
400.0±228.1
26.6±26.1
(n=180 for each Tibetan group). E2, estradiol; FSH, follicle-stimulating hormone; T, testosterone; P, progesterone;
PRL, prolactin; LH, luteinizing hormone.
Tibetan women were divided into two groups (180 women per
group), one living at a moderate altitude (2,260 m), and the other at
high altitude (4,200 m). The age of onset of menarche was 14.01 ±
1.60 years old. The data for Han women at sea level were obtained
from a study entitled “Clinical significance of measuring six female sex
hormones” published in the journal Chinese Clinical Professionals (8).
The data were collected from residents in Beijing who were the same
age and had the same body mass index (BMI) as the Tibetan women
we studied.
Venous blood samples were obtained early in the follicular phase and
two months after the cessation of menses from April 2008 to October
have suggested that people living at high altitudes for generations might
develop genetic adaptations against hypoxia. Our study has identified
a similar adaptive phenomenon, providing further evidence to support
the belief that Tibetans are highly adapted to high-altitude living.
REFERENCES
1. C. M. Beau et al., Proc. Natl. Acad. Sci. U.S.A. 99, 17215 (2002).
2. C. M. Beau et al., Am. J. Phys. Anthropol.106, 385 (1998).
3. F. C. Villafuerte et al., J. Appl. Physiol. 96, 1581 (2004).
4. Y. Zhang, Ed. Human and Plateau (Qinghai People’s Publication,
Qinghai, 1996), pp. 1995-2911.
5. H. Liu, Medicine Innovation Research 04, 139 (2007).
6. J. Li, Chinese J. Birth Health Heredity 22, 113 (2006).
7. Z. Nong, Guide of China Medicine 08, 926 (2005).
Qinghai Red Cross Hospital, Xining, Qinghai, China.
*
8. Y. Dai et al., Journal of Beijing University of Traditional Chinese
Medicine 27, 80 (2004).
25
Diagnosis and Treatment of HAPE and HACE in
the Tibet High-Altitude Region in the Last Decade
Li Suzhi‡, Zheng Bihai‡, Huang Yue, Yan Chuncheng, Xie Xiaomian*, Zhou Xiaobo, Tao Chengfang
H
undreds of thousands of people visit the high-altitude regions
of Tibet for business or recreational travel each year, despite
the threat that severe acute high altitude illnesses—such as
high-altitude pulmonary edema (HAPE) and high-altitude
cerebral edema (HACE)—represent to their health (1, 2). Although
diagnostic criteria for severe acute high-altitude illnesses have been
established, the specific diagnosis of illness at a very early stage is
still difficult and, therefore, the best opportunity for treatment may be
missed (3). Many reports have discussed the treatment for severe acute
high altitude illnesses, but there is still a lack of standardization regarding medication (4). Our center is located at an altitude of 3,658 m and
receives many patients every year. In the last decade, the center has
emphasized early-stage diagnosis and standardized treatment of HAPE
and HACE, and much progress has been made.
To analyze the efficacy of early-stage diagnosis of HAPE and HACE,
screening of 24,200 subjects who rapidly ascended to a high altitude
was completed using a symptom scoring system for acute mountain
sickness (AMS). Subsequent observation and follow-up visitation was
completed for subjects strongly suspected of being sick, looking at early symptoms, physical manifestations of AMS, and auxiliary examination results. Through comparative analysis of suspected individuals and
diagnosed subjects, we identified a series of clinical features of early
stage HAPE, including dyspnea, bilateral or unilateral/local respiratory
crackles, and X-ray characteristics of decreased radiolucent lungs, increased or hazy lung markings, and ground-glass opacity (5). We also
identified a series of clinical features of early stage HACE, including
headache and vomiting with progressive severity not relieved by oxygen therapy, and magnetic resonance imaging features of decreased
T1WI signals and increased T2WI signals of cerebral parenchyma with
patchy appearance (6). Finally, we established the early-stage diagnostic criteria for HAPE and HACE.
For the treatment of HAPE and HACE, we set up several treatment
regimens based on the major medications used in clinical practice
worldwide. The results showed that a treatment regimen using oxygen,
dexamethasone, aminophylline, and furosemide was the most effective and safe for the treatment of HAPE (7), while a treatment regimen using oxygen, dexamethasone, mannitol, and furosemide was
the most effective and safe for the treatment of HACE (8). We established standardized treatment regimens for both HAPE and HACE
that took into account general treatment principles, oxygen therapy,
drug indications, medication doses and delivery method, prevention of
complications, control of liquid intake, and discharge instructions. Additionally, we applied ultrashort wave therapy and digitally controlled
hypothermic blanket/cap in the treatment of HAPE and HACE. We also
balanced exogenous nitric oxide for treatment of HAPE, by using a
normal composition of air instead of a mixture of N2 and pure oxygen
or 80% oxygen. By looking at the hemodynamics, oxygen metabolism
dynamics, clinical presentation of symptoms, improvements in patient
wellness, and mean duration of treatment, we found that treatment with
medication was more effective when combined with the therapies outlined above.
Over the last decade, we have successfully performed research on
early-stage diagnosis and standardized treatment of HAPE and HACE,
and have provided a scientific basis for the early recognition, diagnosis,
and therapy in order to generate a best practices regimen for clinical
treatment. Through the dissemination of our research results, we have
improved the medical services provided to soldiers working at high
altitude and also made important contributions to providing medical
services to the Qinghai-Tibet railway construction workers and to the
YuShu mountain earthquake relief teams. None of the latter two groups
died of AMS and no soldier has succumbed to AMS in the last decade,
both considerable achievements in high-altitude medicine.
REFERENCES
1. C. Sartori et al., N. Engl. J. Med. 346, 1631 (2002).
2. M. Maggiorini et al., Prog. Cardiovasc. Dis. 52, 500 (2010).
3. F. Z. Wang et al., Clinical Focus (Chinese) 15, 426 (2000).
4. D. S. Geng et al., Medical Recapitulate (Chinese) 13, 1623 (2007).
5. S. Z. Li et al., Military Medical Journal of South China 24, 161 (2010).
6. S. Z. Li et al., Military Medical Journal of South China 24, 167 (2010).
Center for Prevention and Treatment of High Altitude Illness, Tibet General
Hospital of PLA, Lhasa, Tibet, China.
*
‡
26
7. S. Z. Li et al., Medical Journal of National Defending Forces in
Section One
Cardiac Surgery on the Tibetan Plateau:
From Impossible to Successful
Li Suzhi‡, Xie Xiaomian‡,*, Huang Yue, Liu Houdong, Yan Chuncheng, Zheng Bihai, Wu Qianjin, Huang Wenchao
A
n important question in cardiology is, “Can cardiac surgery be carried out at high altitude in a hypoxic environment?” The answer is yes, as we have carried out a large
number of cardiac surgeries on the Tibetan plateau since
late 2000. Mild-hypothermia beating open heart surgery at high altitude, with the support of cardiopulmonary bypass, was shown to be
possible when we performed the first cardiac surgery in the Tibet
high altitude region (at 3,658 m) on November 10, 2000. Since then,
we have performed a variety of open heart surgeries on the Tibetan
plateau, including surgery for atrial septal defects, ventricular septal
defects, tetralogy and trilogy of Fallot defects, transposition of great
arteries, and valvular heart disease. The total success rate is approximately 98.9%. Cardiac surgery at a high altitude involves a number
of high altitude medical and hypoxia-related complications, which
may undermine the surgical results and greatly increase the risks due
to the severe hypoxic environment (1, 2). By carefully identifying
the hypoxia problems and treating hypoxia-related symptoms correctly, we have succeeded in almost all cardiac surgeries carried out
at high altitude, which provides a basis for future advancement in high
altitude surgery.
The average altitude of the Tibet plateau is above 4,000 m (3). In
such a high altitude region, the severe hypoxic environment is characterized by a sharp fall in atmospheric pressure and a significant drop in
partial pressure of atmospheric oxygen (4). Congenital heart disease
is frequently encountered in this region and the incidence is approximately two to three times that of the lowlands (5). Before 2000, doctors
and patients were concerned about the incidence of congenital heart
disease, mostly because cardiac surgery could not be performed in
high-altitude regions at that time. Some children with severe congenital
cardiac disease in remote areas died shortly after birth because of a
lack of surgical treatment options. Since 2000, we have visited schools
and villages annually throughout the high altitude region to screen
for congenital heart disease among local children, and have provided
free surgical treatment for affected children. We have also performed
cardiac surgeries for a variety of non-congenital cardiovascular disorders, such as rheumatic valvular heart disease, with satisfying surgical
treatment results.
Environmental hypoxia poses challenging problems for cardiac surgery at high altitude including severe hypoxemia, metabolic acidosis,
and pulmonary artery hypertension. Even if the cardiac defects can be
repaired, the environmental hypoxia can still lead to severe hypoxemia
and a series of pathophysiological changes in the body (6). Chronic
hypoxia can also lead to polycythemia, higher blood viscosity, and
pulmonary hypertension secondary to pulmonary vasoconstriction and
Figure 1. (A) Doctors screening for
congenital heart
disease among local children at a
primary school on
the Tibetan plateau.
(B) Doctors performing a mild-hypothermia beating
open heart surgery
at an altitude of
3,658 m, with the
support of cardiopulmonary bypass
remodeling of arterioles (1, 6). Therefore, patients were given highflow oxygen via a face mask daily for seven days during the preoperative stage to correct severe hypoxemia in the body. A 5% sodium
bicarbonate solution was used preoperatively, intraoperatively, and
postoperatively, by continuous intravenous drip, to correct metabolic
acidosis of tissue cells. Sodium nitroprusside was used preoperatively
and postoperatively to correct pulmonary hypertension and to decrease
pulmonary pressure to a normal level. Hyperbaric oxygen was used if a
patient had very severe pulmonary hypertension. Since myocardial protection is very important in cardiac surgery performed at high altitude,
mild-hypothermia beating open heart surgery was used in most cases,
without aortic cross-clamping. Oxygen consumption of the slowly
beating, empty heart decreased sharply (7–9). The operation schedule
was simplified without perfusion of cold crystalloid cardioplegic solutions, myocardial damage was reduced, and cardiopulmonary bypass
time and total operation time were shortened. All of these were important for myocardial protection and the success of cardiac surgery in a
hypoxic environment.
REFERENCES
1. C. Sartori et al., N. Engl. J. Med. 346, 1631 (2002).
2. M. Maggiorini et al., Prog. Cardiovasc. Dis. 52, 500 (2010).
3. P. Tapponnier et al., Science 294, 1671 (2001).
4. A. J. Peacock, BMJ 317, 1063 (1998).
5. Q. H. Chen et al., Chin. Med. J. (Engl) 121, 2469 (2008).
Center for Prevention and Treatment of High Altitude Illness, Tibet General
Hospital of PLA, Lhasa, Tibet, China.
‡
*
6. C. Imraya et al., Prog. Cardiovasc. Dis. 52, 467 (2010).
7. A. Mo et al., Heart Lung Circ. 20, 295 (2011).
8. F. I. Macedo et al., Semin. Thorac. Cardiovasc. Surg. 23, 314 (2011).
9. D. F. Loulmet et al., Ann. Thorac. Surg. 85, 1551 (2008).
27
Acute Mountain Sickness on the Tibetan Plateau:
Epidemiological Study and Systematic Prevention
Li Suzhi, Huang Xuewen, Huang Yue, Liu Houdong, Yan Chuncheng, Zheng Bihai, Zheng Jianbao, Xie Xiaomian*
T
he Himalayas stretch over 2,400 km from east to west with
a mean altitude over 6,000 m above sea level, and contain
a large population that works and lives at a high altitude.
Since July 2006, with the successful opening of the QinghaiTibet railway, the highest railway in the world, increasing numbers of
travelers have visited Lhasa and the Himalayan high-altitude regions.
Environmental hypoxia has always presented medical challenges for
the prevention, on-site rescue, and clinical treatment of acute mountain sickness (AMS) including acute mild altitude illness, high-altitude
pulmonary edema (HAPE), and high-altitude cerebral edema (HACE).
AMS is induced or exacerbated by various causes including overwork, extreme cold, rapid ascent to a high altitude, alcohol consumption, psychological stress, acute respiratory infection, chronic mountain
sickness (CMS), and cardiopulmonary diseases (1, 2). During the last
15 years, we have collected information on 19,118 cases of high-altitude illnesses, and analyzed the relationship between the morbidity
rates of all the types of AMS and CMS. The results suggested a high
correction between these types of high-altitude illnesses (1). The morbidity rate of the AMS group (45.15%) and CMS group (17.40%) was
significantly higher than that of the control group (4.02%), and the risk
of AMS for people who had previously suffered from AMS or CMS
was 11-fold or four-fold higher, respectively, than people who had not
previously suffered from these disorders (3).
Through decades of epidemiological study on AMS on the Tibetan
plateau, we have proposed a concept of systematic prevention of AMS
in the Tibetan high-altitude region. This concept is intended to improve
AMS prevention outcomes, and takes into account all elements and fac-
tors related to prevention, including the regions where AMS occurs, the
current effective approaches for treatment, the various levels of preventative care, and the occasion when AMS is most likely to occur. The
five aspects of the concept of systematic prevention are: (i) physicians
specialized in high-altitude illnesses; (ii) grass-roots health workers;
(iii) medical examinations before entering high altitude; (iv) dissemination of knowledge to the populace; and (v) prophylactic medications for
the prevention of AMS (4). Since inception of this systematic prevention strategy, the total incidence of AMS decreased significantly from
50%–60% to 2%–3%, and the total rate of successful rescue increased
from 85.5% to 99.7% (5, 6). In our practice of treatment of AMS, we
have greatly improved treatment outcomes by clinical application of
the most effective medications and methods, and have also developed
early-stage diagnostic criteria and standardized treatment regimens to
decrease mortality and increase the rate of successful rescue of severe
acute high-altitude illnesses (7).
This comprehensive, multilevel prevention system for AMS, the result of over 15 years of epidemiological research, has resulted in significant medical progress and dramatically improved quality of life for
many people arriving in the Tibetan plateau region.
REFERENCES
1. Y. Huang et al., J. Epidemiol. (Chinese) 24, 74 (2003).
3. X. W. Huang et al., Medical Journal of National Defending Forces in
4. L. Pan et al., High Alt. Med. (Chinese) 17, 32 (2007).
5. X. W. Huang et al., Medical Journal of National Defending Forces in
Center for Prevention and Treatment of High Altitude Illness,
Tibet General Hospital of PLA, Lhasa, Tibet, China.
*
28
6. T. S. Xie et al., Journal of Military Surgeon in Southwest China 10, 9
(2008).
7. S. Z. Li et al., People’s Military Surgeon (Chinese) 52, 97 (2009).
Section One
Study on Erythrocyte Immune Function and
Gastrointestinal Mucosa Barrier Function After
Rapid Ascent to High Altitude
Shi Quangui, Li Suzhi*, Zheng Bihai
S
ince an American immunologist developed the theory of
erythrocyte immunology in the early 1980s, great progress
has been made in this field (1, 2). Erythrocytes are able to
recognize, adhere to, and destroy antigens, and can eliminate
immune complexes (2). Moreover, erythrocytes play a role in immune
regulation in the body, constituting a subsystem of the immune system
(3). To elucidate the role of erythrocyte immunology in the mechanisms
of human acclimatization to high altitude and the its effect on acute
mountain sickness (AMS), we have researched the characteristics of
erythrocyte immunologic function in people who rapidly ascended to
high altitudes, as well as the gastrointestinal mucosa barrier function in
people and rabbits afflicted with acute anoxia at high altitudes.
Changes in Erythrocyte Immune Function
by Rapid Ascent to High Altitude
The erythrocyte C3b receptor rosette (E-C3bRR) and erythrocyte immune complex rosette (E-ICR) levels were monitored and analyzed in
40 low-altitude natives who rapidly ascended to high altitude (3,600
m) by airplane (test group), as well as in 36 low-altitude natives living
at high altitude and 30 high-altitude natives living at the same high
altitude who had not experienced a recent ascent (controls). In the test
group, E-C3bRR levels decreased sharply while E-ICR levels increased
dramatically after they arrived at the high altitude. E-C3bRR and EICR levels recovered gradually to the levels of the controls in 30 days,
indicating that AMS correlates with impaired erythrocyte immune
function and improving erythrocyte immune function before rapid ascent to high altitudes may reduce the incidence of AMS (3). In another
study, E-C3bRR and E-ICR levels in 36 high-altitude natives who rapidly ascended to high altitude after a long stay at low altitude were also
monitored and analyzed. The E-C3bRR levels decreased, while E-ICR
levels increased, after they arrived at high altitude, but the magnitude
of changes in E-C3bRR levels and E-ICR levels was significantly less
than that of the low-altitude natives test group mentioned above. The
results indicated that high-altitude natives have an advantage in their
adaptation to high-altitude hypoxia, which is possibly related to erythrocyte structure, erythrocyte immune function, and hereditary factors
as well (4, 5).
Gastrointestinal Mucosa Barrier Function Is
Impaired by Rapid Ascent to High Altitude
By noting the digestive symptoms of 1,753 individuals who rapidly
ascended to high altitude, we observed that 1,097 individuals (62.58%)
suffered different degrees of gastrointestinal disorders. Gastroscopic
examinations in 20 individuals afflicted with acute anoxia showed
slow peristalsis (60%), bile reflux (50%), and mucosal damage (85%)
(6). Significant changes in gastrointestinal hormones, inflammatory
mediators, and oxygen free radicals after the rapid ascent to high altitude
were found (7). In another study, electron microscope analysis of small
intestinal mucosa from rabbits exposed to anoxia identified damage to
the small intestinal villi with leakage of fibrin and erythrocytes. The
activity and concentration of the serum diamine oxidase (DAO) and
malondialdehyde (MDA) in rabbits at high altitude were higher than
those in the control group remaining at low altitude. However, the
activity and concentration of small intestinal mucosal DAO, glutamine,
serum superoxide dismutase, nitric oxide, and glutamine decreased
under anoxic conditions when compared with the control group (7, 8).
Taken together, these results indicate that rapid exposure to a hypoxic
environment causes secondary reduction of erythrocyte immune function and damage of gastrointestinal mucosal barrier function. Improving erythrocyte immune function and protecting gastrointestinal mucosal function may be of clinical importance in reducing the incidence of
high-altitude diseases and improving treatment regimens.
REFERENCES
1. L. Siegel et al., Lancet 2, 566 (1981).
2. F. Guo, Immunological Journal (Chinese) 6, 60 (1990).
3. Q. G. Shi et al., Immunological Journal (Chinese) 11, 178 (1995).
4. Q. G. Shi et al., Southwest Defense Journal of Medicine (Chinese) 10,
303 (2000).
5. Q. G. Shi et al., High Alt. Med. (Chinese) 10, 11 (2000).
Center for Prevention and Treatment of High Altitude Illness,
Tibet General Hospital of PLA, Lhasa, Tibet, China.
*
6. S.Z. Li et al., Occupational Health (Chinese) 27, 427 (2011).
7. S.Z. Li et al., South Military Medical Journal (Chinese) 25, 273 (2011).
8. B.H. Zheng et al., South Military Medical Journal (Chinese) 25, 4 (2011).
29
Basic Methods and Application of Altitude
Training on the Chinese Plateau
Ma Fuhai*, Fan Rongyun, Yu Xiaoyan, He Yingying
T
he physiological function, tissue structure, and biochemical metabolism of native high-altitude plateau athletes show
adaptive changes to hypoxia environments when compared
with low-altitude plain athletes undergoing an identical training load and intensity (1). In competitive sports, especially endurance
events, athletes living in plateau and mountain areas of China (at 2,000
to 3,000 m) have achieved great success. Thus, studies on altitude training of plateau athletes may have a positive effect on athletic training in
general.
The “high-high” training method, besides training in a high-altitude
region and living in a low-altitude region, also makes use of other beneficial effects of the plateau, while avoiding the disadvantages. Han et
al. (1) determined that athletes who had lived and trained at an altitude
of 1,900–2,000 m for many years could readily cope with training at
2,500 to 3,400 m three to four times a week. Liu et al. (2) described
the advantages of alternative altitude training: 35 days at an elevation
of 2,260 m, 28 days at 396 m, then altitude training for 22 days at
2,260 m. Both the high-high training and the alternative altitude training methods had positive effects on the physiological function and athletic capacity of subjects. Ma et al. (3) did research on seven native
female middle-distance runners who undertook six weeks of intermittent altitude training between 2,260 m and 3,150 m. They found that
the cardiopulmonary function, VO2 max (peak oxygen uptake), anaerobic threshold speed, and hemogram of the runners showed significant
improvement when they training at the higher altitude (3,150 m) after
training at 2,260 m, which indicated that their aerobic capacity had improved. However, when the native athletes arrived at low altitude after
intermittent attitude training, their movement ability and some exercise
physiology indexes were negatively impacted compared with the native
plain athletes and non-altitude training athletes. As a result of these differences, defining the time that athletes might reach their peak competition fitness was challenging. Li et al. (4) compared the effect of training
at high altitude on the Chinese plateau with the same on the Japanese
plains. The results showed that for better physiological function and
performance it is necessary to train at an altitude of 3,200 m for a short
period. Additionally, race walkers in Mexico, at an altitude of 2,300 m,
developed an alternative altitude training method that has allowed them
to become world leaders in the sport (5).
In 1991, Levine (6) first proposed a particular “live high, train low”
(Hi-Lo) method where athletes lived at a higher elevation to encourage
the body to adapt to high-altitude hypoxia environment. This allowed
athletes to achieve a larger training load and intensity in a low elevation
zone. It was observed that native plateau athletes undergoing intermittent hypoxic training at 4,000 m failed to stimulate the kidney to release
erythropoietin, while the responses of vascular endothelial growth factor (VEGF) were more sensitive. This response suggests that improved
muscle capillarization rather than increased red blood cell production
would be a major adaptation to intermittent hypoxic training. Running
at an altitude of 3,000 m resulted in increased erythropoietin release in
the hypoxia group, but not in the control group.
Through the theory and practice of altitude training both at home
and abroad, most research has shown some benefit in improving the
performance of athletes worldwide and, in particular, of native plateau
athletes. Studies are rare in the areas of optimal elevation for altitude
training, the training time needed to achieve the most benefit, the timing of performance peaks following altitude training, and physiological
changes during altitude training. Although consensus on some of the
major issues of altitude training has been reached, there are many unanswered questions, especially regarding the best altitude training regimens, the ideal training load and intensity, pre-altitude training preparation, optimal altitude training time pre-competition, and the specific
focus on strength, speed, aerobic, and anaerobic qualities.
Further studies will be valuable for improving athlete performance
and will also have positive effects on developing individualized altitude
training protocols for native plateau athletes. Additionally, such studies
can provide a scientific basis for coaches to determine the ideal duration
and intensity of training to capitalize upon the beneficial altitude training effects discussed above.
REFERENCES
1. Z. Han et al., Track and Field 6, 25 (1995).
2. Z. Liu et al., Sports Science 6, 34 (1999).
3. F. Ma et al., Sports Science 6, 34 (2000).
4. H. Li et al., Sports Science 5, 30 (1995).
Qinghai Institute of Sports Science, Xining, Qinghai, China.
*
30
5. Y. Shang et al., Sports Science 3, 11 (1996).
6. B. D. Levine. High Alt. Med. Biol. 3, 177 (2002).
Section One
Hypoxic Preconditioning at High Altitude Improves
Cerebral Reserve Capacity
Wu Shizheng1,*, Zhang Shukun1, Chang Rong1, Li Na1, Wu Yao-An2, Feng Yuliang3, Wang Yigang3
T
he Qinghai-Tibet Plateau is a natural laboratory for mountain
sickness research, especially cerebral hypoxic/ischemic tolerance, because its unique geographic environment (alpine,
hypobaric, and hypoxic climate) results in a complex succession of pathophysiologic alterations. When exposed to hypoxia at high
altitudes, the brain initiates autoregulation of blood flow, formation
of collateral circulation, and preservation of metabolism, to maintain
Figure 1. (A), Phase contrast image of HUVECs at 24 hours of hypoxic
treatment showing rounding up and shrinkage of cells with nonhypoxic preconditioning (non-HPC), which was prevented by hypoxic
preconditioning (HPC). (B), Quantitative analysis of TUNEL positivity
showing decreased numbers of TUNEL+ nuclei in HPCHUVECs (*p<0.01
versus normoxiaHUVECs, # p<0.05 versus non-HPCHUVECs). (C, D) Quantitative
analysis of RT-PCR showing that expression of Ang-2 (C) and VEGF (D)
was significantly elevated in HPCHUVECs (*p<0.01 versus normoxiaHUVECs,
#
p<0.05 versus non-HPCHUVECs). All values expressed as mean ± SEM,
n = 8 for each group. (E) Heat map of miRNA microarray showing the
enrichment of angio-miRs in HPCHUVECs. (F, G) Graph showing that the
percentage of cells positive for Bcl-2 (F) and NGB (G) was significantly
higher in HPCHUVECs (*p<0.01 versus normoxiaHUVECs, # p<0.05 versus
non-HPC
HUVECs). All values expressed as mean ± SEM, n = 10 for each
group. (H, I) Quantitative analysis of RT-PCR showing Bcl-2 (H) and NGB
(I) expression was significantly elevated in HPCHUVECs (*p<0.01 versus
normoxia
HUVECs, # p<0.05 versus non-HPCHUVECs). All values expressed
as mean ± SEM, n = 6 for each group. NOR, normoxia; non-HPC,
non-hypoxic preconditioning; HPC, hypoxic preconditioning; GAPDH,
glyceraldehyde 3-phosphate dehydrogenase.
normal blood flow and avoid impairment of cerebral reserve capacity
induced by hypoxia/ischemia (1). Cerebral reserve capacity describes
the ability to maintain adequate blood flow in the face of decreased
perfusion pressure��
and oxygen��
suppl��
��
y. It has been identified as the major predictive indicator for the risk of subsequent cerebral infarction. It
has been previously reported that repeated, short episodes of hypoxic
preconditioning protect the brain against subsequent hypoxic insult (2).
Over the past few years, we have investigated the effect of hypoxic
preconditioning at high altitudes. Using an in vitro hypoxic model consisting of Human Umbilical Vein Endothelial Cells (HUVECs), phase
contrast microscopy revealed a hypercontracted morphology of nonhy-
poxic preconditioned HUVECs (non-HPCHUVEC, at 37ºC, 1% O2 + 5%
CO2 + 94% N2) whereas restored morphology was observed in hypoxic
preconditioned HUVECs (HPCHUVEC, at 37ºC, 1% O2 + 5% CO2 + 94%
N2 for 30 min). Following hypoxic treatment, the viability of non-HPCHUVECs was reduced by 40%, 52%, and 59% at 8, 12, and 24 hours,
respectively, compared to HPCHUVECs (25% at 24 hours). Furthermore,
terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positivity, which measures the incidence of apoptosis, was markedly decreased in HPCHUVECs (15.2%) compared with non-HPCHUVECs
Qinghai Provincial People’s Hospital, Xining, China;
The University of York, York, UK;
3
Department of Pathology and Laboratory Medicine,
University of Cincinnati, Cincinnati, OH, USA.
*
1
2
31
Figure 2. Immunohistochemical quantification in cerebral tissues on
days 1, 3, 7, and 14 after cerebral infarction showed downregulation of
ASPP1 expression in the Qaidam group (3,500 m) compared with the
Xining group (2,260 m) and Tuotuo River group in chronological order
(4,500 m) (* p<0.01 versus Xining or Tuotuo River Group). All values
expressed as mean ± SEM, n = 60 for each group.
(25.3%), but was higher than the NormoxiaHUVECs (4.3%, at 37ºC, 5%
CO2), implying that hypoxic preconditioning can elicit cytoprotection
against hypoxic stress. Reverse transcription polymerase chain reaction
(RT-PCR) analysis demonstrated significant upregulation of vascular
endothelial growth factor (VEGF) and angiopoietin (Ang)-2 in non-HPC
HUVECs compared with NormoxiaHUVECs, and even greater increases
in HPCHUVECs. VEGF and Ang-2 are mediators in angiogenesis and
formation of collateral circulation (3). Our microRNA microarray data
identified a cluster of angiogenesis-related microRNAs (angio-miRs)
that are significantly enriched in HPCHUVECs ��
(unpublished data)��
. Similarly, hypoxic preconditioning could protect cultured neonatal neurons
(NN) against lethal hypoxic insult as determined by TUNEL positivity:
1.1% in NormoxiaNN, 44.1% in non-HPCNN, and 9.8% in HPCNN (4). mRNA
and protein levels of Bcl-2 and neuroglobin (NGB) were also significantly upregulated in HPCNN. This implied that coordination between
the critical anti-apoptotic protein Bcl-2 (5) and oxygen carrying protein
NGB (6) was involved in the significant improvement of neuronal survival and cerebral reserve capacity (Figure 1).
To verify our hypothesis in vivo, 180 healthy Wistar rats were
divided into three groups at different altitudes: Xining group, 2,260 m
above sea level; Qaidam group, 3,500 m; Tuotuo River group, 4,500 m.
All groups were placed at their specific altitude for 30 days to induce
acclimatization, after which middle cerebral artery occlusion (MCAO)
was induced. Immunohistochemistry of cerebral tissues on days 1, 3, 7,
and 14 after cerebral infarction showed that the presence of apoptosisstimulating protein 1 of p53 (ASPP1), a key proapoptotic protein, was
decreased in the Qaidam group, relative to the others (Figure 2) (7). This
32
indicated that hypoxia is a proverbial “double-edged sword,” for which
and an appropriate level of hypoxia can reduce apoptosis and increase
cerebral reserve capacity, but severe hypoxia had no such effect.
Cerebrovascular reactivity (CVR) is a physiological characteristic of
brain arteries to alter their size in response to a vasoactive stimulus.
Impaired CVR is a predictive factor of imminent stroke. In a clinical
study, CVR was examined using transcranial doppler technology in
patients with symptomatic stenosis of the anterior intracranial artery, and
compared with that in normal healthy individuals living permanently in
the Xining area (2,260 m). CVR was significantly reduced in patients
with anterior artery stenosis compared with the normal control.
Moreover, multivariate stratified analysis revealed that patients with
symptomatic anterior circulation artery stenosis had a greater reduction
in CVR compared with the asymptomatic patients at the same altitude.
In symptomatic patients, CVR of the stenosis side was reduced as
compared with the non-stenosis side. Furthermore, we found CVR
positively correlated with serum levels of nitric oxide (NO), Na+/Ca2+
exchanger type-1 (NCX1), and granulocyte colony-stimulating factor,
all of which correlate with the risk of stroke, indicating its potential
value for the early detection of cerebral infarction.
Interestingly, we observed that administration of butylphthalide and
Mailuoning (a Chinese herbal medicine) could increase cell viability
and reduce apoptosis by activating the same signaling molecules
(VEGF and Ang-2) as those observed in hypoxic preconditioning.
Thus, these drugs might be potential preconditioning agents that can
stimulate the biochemical pathways of hypoxic preconditioning and
therefore protect vascular endothelial functions (8, 9).
Taken together, our in vitro and in vivo experiments, combined with
the clinical study, demonstrated that hypoxic preconditioning at high
altitudes by intermittent exposure to hypobaric condition improved
cerebral reserve capacity, and structural and functional restoration of
the brain upon return to normoxia or low altitude. Investigating the
characteristics of CVR and cerebral autoregulation in populations living at different altitudes to identify the underlying mechanisms of the
formation of collateral circulation and hypoxic/ischemic tolerance may
provide novel insights for stroke prevention. Thus, intermittent and optimal hypoxic preconditioning by intermittent exposure to high altitude
might be a new paradigm for neuroprotection and restoration of cerebral reserve capacity in patients with ischemic stroke.
REFERENCES
1. H. Zhou, G. M. Saidel, J. C. LaManna. J. Adv. Exp. Med. Biol. 614, 371
(2008).
2. S. Z. Wu, Chin. J. Stroke 2, 965 (2007).
3. Z. G. Zhang et al., J. Cereb. Blood Flow Metab. 22, 379 (2002).
4. G. L. Rong, S. Z. Wu. Chin. J. New Drug 21, 06 (2012).
5. Z. M. Ding et al., Int. J. Mol. Sci. 13, 6089 (2012).
6. E. Fordel et al., IUBMB Life 56, 681 (2004).
7. J. Cheng, S. Z. Wu, S. K. Zhang. Chin. J. Pract. Prev. Med. 18, 04
(2011).
8. G. L. Rong, S. Z. Wu, S. K. Zhang. Chin. J. New Drug 20, 1015 (2011).
9. L. Na, S. Z. Wu, S. K. Zhang. Chin. J. New Drug 21, 06 (2012).
ACKNOWLEDGMENTS
This work was supported by grants from the Qinghai Provincial High Altitude
Medicine Science Research Programs (Grant No. 200908-01 and 20111101) and National Institutes of Health (Grant No.HL089824 and HL110740;
Yigang Wang).
Section One
The Dynamic Balance Between Adaptation
and Lesions of the Cardiovascular System in
Tibetans Living at High Altitude
Gesang Luobu
T
ibetans have inhabited the Tibetan plateau for approximately
25,000 years and appear to be well adapted to high altitudes
with higher arterial oxygen saturation, low incidence of
chronic mountain sickness, and minimal hypoxia pulmonary
hypertension (1). We feel that it is important to better understand the
damage caused by chronic hypoxia and the mechanisms of human adaptation to high-altitude environments through studying the impact of
high-altitude environment on native Tibetans.
At high altitudes, changes in the cardiovascular system are necessary
in order to transport sufficient oxygen to tissues to help the body adapt
to low oxygen environments. We have previously assumed that there
was a certain dynamic balance between adaptation of, and damage to,
the cardiovascular system in high-altitude environments. When this
balance is altered, the incidence of various cardiovascular problems—
including arrhythmia, high blood pressure, pulmonary hypertension,
and coronary artery disease—increases. Our early research (2) showed
that the mean heart rate of Tibetans in the Lhasa area (3,658 m) over a
24 hour period was 71 beats per minute, significantly lower than the 87
beats per minute observed in Han Chinese individuals at the same altitude. Additionally, the prevalence of bradycardia (slowing of the heart,
which reduces myocardial oxygen consumption, increases stroke volume, and improves the mechanism of adaptability to an anaerobic environment) was increased in healthy Tibetans in the Lhasa area. However,
the low heart rate could also increase the incidence of cardiac events
such as angina and, in the most serious cases, cardiac arrest. Further
studies indicated that the sinus nodal recovery time, sinus atrial conduction time, and corrective sinus nodal recovery time were much longer
in healthy Tibetans at high altitude than in Han individuals at sea level.
This suggests that the direct influence of high-altitude–related hypoxia
on the sinus node and adjustment of the sympathetic nervous system
has reached a certain balance, which increases the adaptability of Tibetans to high-altitude environments.
Systemic Blood Pressure
High-altitude environments cause a significant change in the cardiovascular system including the increased occurrence of pulmonary hypertension (3). We performed right heart catheterization in healthy Tibetans living in Lhasa and found that the mean pulmonary arterial pressure
was similar to that of individuals at sea level. Pulmonary hypertension
was uncommon among these individuals, because native Tibetans have
shown great adaptability to high-altitude environments. However, studies on systemic blood pressure in hypoxic environments are relatively
rare. A study from Peru showed the prevalence of hypertension was
greater at sea level than at high altitude (4). This does not appear to hold
true in Tibet. The results of a hypertension survey that we conducted in
1991 showed that native Tibetans have the highest prevalence of hypertension (5). The incidence of stroke in Tibet was the highest in China
according to a national survey and a recent epidemiological survey of
stroke in Lhasa performed by our group demonstrated that hypertension
was the primary risk factor among Tibetan patients (6). Therefore, hypertension is a significant health hazard for Tibetan plateau natives. Besides genetic factors, high-altitude–related hypoxia may also be a risk
factor. Although the adaptability of native Tibetans influences pulmonary circulation, further effort is required to find a relationship between
the high morbidity rate of hypertension and adaptability to high-altitude
environments.
Coronary Artery Disease (CAD)
Acute hypoxia increases coronary blood flow in direct proportion to the
reduction in arterial oxygen concentration. Conversely, coronary blood
flow is reduced in permanent residents of high altitudes in Peru compared with people at sea level. Few data exist that quantify adverse cardiac events at high altitudes although some studies showed that casts of
the coronary vessels had a greater density of peripheral branching than
those of sea level controls, perhaps explaining the relatively lower incidence of angina and myocardial ischemia in the Peruvian population
(4). However, it remains unclear to what degree hypoxia impacts the
process of coronary artery disease (CAD) at high altitudes, and whether
it is safe for patients with CAD to travel to high altitudes.
As the regional infrastructure has developed rapidly, it is more convenient for people to enter high-altitude regions. Official statistics show
that more than 8.6 million people entered the Tibet region in 2011 (7).
Thus, it is crucial to evaluate the risk of cardiac events for travelers to
Tibet, in order to provide more precise prevention plans and to determine whether the high-altitude environment might have a positive or
negative effect on those with CAD.
We have studied the status of CAD in native Tibetans to determine
the relationship between hypoxia and CAD. In 1977, our study reported
a 66.9% incidence rate of coronary atherosclerosis in Lhasa residents,
and a 12% incidence of ischemia-like electrocardiogram changes or
coronary insufficiency, as determined by a positive exercise test (8).
With the rapid development of the Chinese economy, the population’s
dietary habits have changed greatly and risk factors for CAD have increased in concert with the increase in patients with dyslipidemia and
diabetes. From 1986 to 2003, the portion of the in-hospital patients with
CAD increased from 4.33% to 9.1% and acute myocardial infarction
was found to be the main reason for hospitalization (9). The fatality
rate in this group was 28.5%. In Tibetan patients with CAD, the left
anterior descending artery was most commonly involved, followed by
the right coronary artery and then the left circumflex; however, the left
main coronary artery was involved to a lesser degree (10). The distribution of atherosclerotic lesions did not differ from sea level patients.
Department of High Altitude Sickness, People’s Hospital of Tibet Autonomous
Region, and the Tibet Institute of High Altitude Medicine, Lhasa, Tibet, China.
33
We speculated that the adaptation to high altitude, including decreased
heart rate and reducing oxygen consumption, might induce the onset of
angina pectoris and acute myocardial infarction in Tibetans. As the incidence rate of dyslipidemia (11) and diabetes increases (12), the balance
between the advantages of adaptation and resulting increase in atherosclerotic lesions may be altered. This hypothesis is supported by the
observation that large numbers of Tibetan in-patients have acute myocardial infarction (9). It is not uncommon for native Tibetans to suffer
from CAD. Studying CAD in the Tibetan population not only helps to
identify possible risk factors, but it also helps to reveal the mechanism
of CAD, and to aid in the development of novel prevention methods.
Genetic Research
Research initiated by our group and the University of Colorado School
of Medicine in the United States found that native Tibetan newborns
had higher arterial oxygen saturation at birth and during the first four
months of life than Han Chinese newborns. Additionally, healthy Tibetan’s resting pulmonary arterial pressure was normal by sea-level standards and they exhibited minimal hypoxic pulmonary vasoconstriction.
This implied that the Tibetan adaptation to high altitudes had a genetic
basis. In 2010, in cooperation with the company BGI-Shenzhen, we
found that the frequency of one single nucleotide polymorphism in the
endothelial Per-Arnt-Sim (PAS) domain-containing protein 1 (EPAS1)
gene differed in frequency between Tibetans and Han Chinese by 78%,
providing strong genetic evidence for high altitude adaptation in Tibetans (13). Besides traditional risk factors such as dietary habits and
smoking, interactions between hypoxia and genetic factors may play a
significant role in the etiology of hypertension in Tibetans. Our research
showed a significant association between the D allele of the angiotensin-converting enzyme gene and hypertension in Tibetans, and the frequency of the G allele was significantly higher in hypertensive than in
normotensive Tibetans, but not Han Chinese (14). Thus, we speculate
that the increased incidence of hypertension among native Tibetans
may be due to interactions between hypoxia and genetic factors, which
helps us to further understand the mechanism involved in the adaptation
of native Tibetans to high-altitude environments, as well as how these
mechanisms manifest clinically.
Summary
It is well documented that Tibetans are adapted to high altitudes. However, they also suffer from cardiovascular system damage caused by
hypoxia. We speculate that the increased economic activity and improvement in living standards in China will greatly affect the balance
between adaptation and cardiovascular problems in Tibetans at high
altitude. Insight into this balance might be helpful to better treat the
Tibetan population as well as understand the basis of human adaptation
to hypoxia.
REFERENCES
1.T. Y. Wu. High Alt. Med. Biol. 5, 1 (2004).
2. L. H. Yang et al., Tibet J. Med. 22, 1 (2001).
3. A. L. Baggish et al., High Alt. Med. Biol. 11,139 (2010).
4. J. B. West, R. B Schoene, J. S Milldge, in High Altitude Medicine and
Physiology, (Hodder Arnold, London, ed. 3, 2007), pp. 93-94.
5.Y. C. Hu et al., J. Hygiene Res. 35, 5 (2006).
6. Y. H. Zhao et al., Stroke 41, 2739 (2010).
7. http://www.chinatibetnews.com/xizang/node-9151.htm.
8. W. J. Cen et al., Tibet Hygiene 5,4 (1977).
9.L. B. Gesang et al., Tibet J. Med. 25, 80 (2004).
10 C. R. Dawa et al., Chinese Circ. J. 27, 3 (2012).
11 K. Li et al., Med. J. West. China 24, 3 (2012).
12 L. H. Yang et al., Chin. J. Endocrinol. Metab. 19, 5 (2003).
13 X. Yi et al., Science 329, 75 (2010).
14 L. B. Gesang et al., Hypertens. Res. 25, 3 (2002).
Establishment of an Improved Bundle Therapy
Procedure for Acute High-Altitude Disease
Zhang Xuefeng1,*, Ma Si-Qing2, Jin Guoen3, Pei Zhiwei1, Guo Zhijian4
F
or many years, the acute high-altitude diseases, pulmonary
edema (HAPE) and cerebral edema (HACE), did not have
standard treatment procedures. Over the last 30 years, we have
continually improved the normal treatment programs and established an improved bundle therapy strategy for acute high-altitude
diseases (Figures 1 and 2).
Department of High Altitude Diseases, Golmud People’s Hospital,
Golmud, Qinghai, China;
2
Department of Critical Care Medicine, Qinghai Province Hospital,
Xining, Qinghai, China;
3
Research Center for High Altitude Medicine, Qinghai University
Medical College, Xining, Qinghai, China;
4
Qinghai Traffic Hospital, Xining, Qinghai, China.
*
1
34
Patients suffering from high-altitude disease (n=203), including 181
males and 22 females with a mean age of 33.64 ± 11.40 years, from
different regions in the Tibetan Plateau (3,000 to 5,130 m above sea
level) were selected in a prospective study. All patients were treated
in Golmud (2,800 m). Diagnoses were in line with HAPE and HACE
diagnostic criteria of the Chinese Medical Association Third National
High Altitude Medicine Symposium (1). Cases were selected by a random number table and divided into a bundle scheme therapy group (125
cases) and a control group receiving ordinary therapy (78 cases).
HAPE diagnostic criteria were as follows. First phase (light edema):
changes in interstitial lung edema determined by X-ray; secondary
phase (medium edema): X-ray changes in the occurrence of unilateral lung edema; third phase (heavy edema): bilateral interstitial lung
edema determined by X-ray; fourth phase (extremely heavy edema):
Section One
Table 1. Comparison of therapeutic effects on acute severe high-altitude sickness.
Number
of Cases
Hospitalization time
in Days
(mean ± SD)
Percentage Cured (No.)
Percentage Mortality (No.)
Bundle therapy group
125
5.28 ± 3.17*
96.80 (121)*
3.20 (4)*
Control group
78
6.94 ± 4.05
89.74 (70)
10.26 (8)
Group
*p<0.05 compared with the control group.
Table 2. Comparison of therapeutic effects on acute high-altitude sickness.
Group
Bundle therapy group
Control group
Degree of Edema
Number of
Cases
Number of
deaths
Percentage Mortality
light/medium
50
0
0
heavy
55
1
1.28
extremely heavy
20
3
15
light/medium
38
0
0
heavy
27
2
7.41
extremely heavy
13
6
46.15
The above results suggest that we have developed an improved bundle therapy strategy for acute high-altitude diseases. However, further
work is required.
congestive heart failure (CHF), respiratory failure, acute respiratory distress syndrome (ARDS), and multiple organ dysfunction syndrome (MODS) (2). HACE
staging and classification were based on
all cases according to the diagnostic criteria as follows. First phase (light edema):
no significant change in X-ray and computed tomography (CT) exam; secondary
phase (medium edema): CT showed mild
cerebral edema and shallow sulci; third
phase (heavy edema): CT showed large
areas of low density and disappearance
of the gyrus and ambient cistern; fourth
phase (very heavy edema): CHF, respiratory exhaustion, ARDS, and MODS. There
was no statistically significant difference
(p>0.05) in age, height, weight, altitude,
blood pressure, heart rate, respiratory rate,
other physiological parameters by balancing tests, or incidence of light, medium,
heavy, or extremely heavy cerebral edema
and pulmonary cerebral edema between
the two groups before treatment.
The two groups were administered rest,
oxygen, conventional hormone treatment,
theophylline, vasoactive drugs, rehydration, and hyperbaric oxygen. The types of
general therapy used for the control group
were not determined based on patient staging and classification whereas the bundle
therapeutic group was treated according
to staging and typing. Results showed that
mortality and number of days in hospital
were decreased while the total cure rate increased (Tables 1 and 2) when the bundle
therapy approach was used.
Figure 1. Bundle therapy strategy for high-altitude pulmonary edema (HAPE).
a: rest; b: high pressure oxygen; c: 654-2; d: respirator; e: intensive care unit.
ARDS, acute respiratory distress syndrome; MODS, multiple organ dysfunction syndrome.
Figure 2. Bundle therapy strategy for high-altitude cerebral edema (HACE).
a: rest; b: high pressure oxygen; c: 654-2; d: respirator; e: intensive care unit; f: reduce intracranial pressure.
ARDS, acute respiratory distress syndrome; MODS, multiple organ dysfunction syndrome; CT, computed
tomograpy.
REFERENCES
1. T. Y. Wu, J. High Alt. Med. 17, 3 (1995).
2. W. Wei et al., J. Fourth Military Medical University 26, 363 (2005).
ACKNOWLEDGMENTS
This work was supported by the Project of Qinghai
Province Science and Technology Program (Grant No.
2011-N-150).
35
Differences in Physiological Adaptive Strategies
to Hypoxic Environments in Plateau Zokor and
Plateau Pika
Wei Deng-Bang*, Wang Duo-Wei, Wei Lian, Ma Ben-Yuan
T
he plateau zokor (Myospalax baileyi) and plateau pika
(Ochotona curzoniae) are specialized rat species found on the
Qinghai-Tibet plateau. Plateau zokor is a blind subterranean
mole rat that spends its life underground in sealed burrows
(1). During the spring, summer, and autumn, the oxygen content in
their burrow at a depth of 18 cm was found to be 18.02%, 17.04%, and
18.43%, respectively, and the carbon dioxide level was 0.22%, 1.46%
and 0.81%, respectively (2). Plateau pika, which is a member of the
genus Ochotona of the Ochotonidae family, is a small, nonhibernating
rodent that lives in remote mountain areas at an elevation of 3,000 to
4,800 m (3). Both the zokor and pika have evolved a series of physiological adaptations that allow them to thrive in a hypoxic environment.
Both rodents have bigger lungs, a higher density and smaller area of
pulmonary alveoli, a thinner air-blood barrier and microvessel muscle,
higher red blood corpuscle counts, and lower hematocrit and mean corpuscular volume than normal rats (4). The oxygen pressure in zokor is
about 1.5-fold higher than pika in arterial blood, but only 0.36-fold that
of pika in venous blood. Partial pressure for carbon dioxide in arterial
and venous blood of zokor is 1.5-fold and 2.0-fold higher, respectively,
than in pika, while oxygen saturation of zokor is 5.7-fold lower in venous blood than that of pika. As a result, oxygen saturation in arterial
blood to venous blood is twofold higher in zokor than in pika (5). Zokor
have a strong oxygen uptake capacity and higher oxygen utilization
compared with pika (5).
Microvessel densities, the numerical density of mitochondria, and
the surface density of mitochondria in skeletal muscle of zokor are significantly higher than those of pika (6). The myoglobin content in skeletal muscle of zokor is also notably higher than that of pika (6). In the
skeletal muscle of pika, the expression of the gene encoding one subunit of heteromeric lactose dehydrogenase, Ldh-a, is markedly upregulated, and the main LDH isoenzymes found are LDH-A4, LDH-A3B,
and LDH-A2B2. However, in skeletal muscle of zokor, the expression of
Ldh-b, encoding a different LDH subunit, is upregulated, and the main
LDH isoenzymes are LDH-A4, LDH-AB3 and LDH-B4. The activity
of LDH in skeletal muscle of pika is significantly higher than that of
zokor. Taken together, this suggests that even though zokor inhabits a
hypoxic environment, its skeletal muscle can produce high energy lev-
els by aerobic oxidation, whereas pika skeletal muscle obtains energy
by anaerobic glycolysis. Furthermore, we found that Ldh-c, originally
thought to be expressed only in testis and spermatozoa in mammals
(7), was expressed in skeletal muscle of pika, but not in that of zokor.
LDH-C4 is a lactate dehydrogenase that catalyzes the interconversion
of pyruvate to lactate. It has a low Km for pyruvate (~0.030 mM) and a
high Km for lactate (~2.0 mM) compared with LDH-A4 (8). This finding implies that LDH-C4 has an affinity for pyruvate that is 60-fold
higher than for lactate, suggesting that pyruvate turnover may be higher
even at high concentrations of endogenous or extracellular lactate.
This notion is supported by studies using a human spermatozoa incubation system in which the addition of excess lactate (50-fold excess
in relation to pyruvate) did not influence ATP production in capacitating spermatozoa (9). Therefore, skeletal muscle produces ATP mainly
by aerobic glycolysis in zokor, and mainly by anaerobic glycolysis
in pika.
In conclusion, plateau zokor and plateau pika adopt different strategies to adapt to hypoxic environments. Plateau zokor
has an efficient system for energy production by aerobic oxidation even though they inhabit a hypoxic environment, whereas plateau pika has an efficient system for energy production by anaerobic
glycolysis.
REFERENCES
1. N. C. Fan, Y. Z. Shi, Acta Theriol. Sinica 2, 180 (1982).
2. J. X. Zeng, Z. W. Wang, Z. X. Shi, Acta Biol. Plateau Sinica 3, 163
(1984).
3. Z. J. Feng, C. L. Zheng, Acta Theriol. Sinica 5, 269 (1985).
4. X. J. Wang et al., Acta Zool. Sinica 54, 531 (2008).
5. D. B. Wei, L. Wei, J. M. Zhang, H. Y. Yu, Comp. Biochem. Phys. A 145,
372 (2006).
6. S. Zhu et al., Sheng li xue bao:[Acta Physiol. Sinica] 61, 373 (2009).
7. A. Blanco, W. H. Zinkham, Science 139, 601 (1963).
8. C. E. Coronel, C. Burgos, N. M. Gerez de Burgos, L. E. Rovai, A.
Blanco, J. Exp. Zool. 225, 379 (1983).
9. T. H. Hereng et al., Hum. Reprod. 26, 3249 (2011).
ACKNOWLEDGMENTS
This work was supported by the National Natural Science Foundation of
Department of Biology, Qinghai University, Xining, Qinghai, China.
*
36
China (Grant No. C030302 and 30960054), and the Key Project of the
Chinese Ministry of Education (Grant No. 209132).
CREDIT: © ISTOCKPHOTO.COM/A330PILOT
Section Two: Hypoxic Physiology
Hypoxic Physiology
Cardioprotective Effect of Chronic Intermittent
Hypobaric Hypoxia
Zhang Yi1,* and Zhou Zhaonian2,*
I
schemic heart disease is a leading cause of cardiovascular mortality
in developed countries (1). Ischemia/reperfusion (I/R) injury is a
common cardiovascular problem and has no satisfactory treatment.
Thus, identifying new approaches that will reduce I/R injury is clinically important. We have studied the protective use of chronic intermittent hypobaric hypoxia (CIHH) systemically since 1996 and have
demonstrated that CIHH treatment has a significant cardioprotective
effect against I/R injury.
In a previous study, adult male Sprague-Dawley rats were exposed
to hypobaric hypoxia in a hypobaric chamber, simulating an altitude
of 5,000 m (PO2= 84 mmHg) over 28 or 42 days, for six hours each
day. CIHH-treated adult or neonate rats displayed cardiac protection
against global and regional I/R, which lasted for two weeks after the
end of CIHH treatment. CIHH treatment alleviated the inhibition of
ventricular function, limited the infarct area, promoted the recovery
of cardiac function, and prevented arrhythmia during I/R (2). In addition, CIHH decreased the activity of lactate dehydrogenase, prevented
mitochondrial structural damage and mtDNA deletion, and inhibited
calcium overload during I/R (3). Furthermore, I/R-induced apoptosis
in cardiomyocytes was significantly attenuated in CIHH-treated rats (4)
(Figure 1).
Several mechanisms in the heart may be involved in CIHH-mediated cardiac protection. Our electrophysiological studies suggested that
CIHH treatment causes prolongation of the action potential (AP) duration and effective refractory period in a time-dependent manner under
normoxic conditions. Additionally, CIHH efficiently prevents the inhibition of AP in the ventricular papillary muscles during simulated ischemia. Whole-cell patch clamp analysis demonstrated that CIHH did not
alter the activity of the Ica-L channel in normal recording conditions, but
resisted the decrease of Ica-L current and a positive shift of the steadystate inactivation curve under simulated ischemic conditions (5). Thus,
the aforementioned effects of CIHH might determine the electrophysiological basis for CIHH antiarrhythmia.
Oxygen free radicals are a major cause of I/R-induced injury in cardiomyocytes (6). Endogeneous antioxygenation systems, such as superoxide dismutase (SOD) and catalase, can block the cascade reactions
of oxygen free radicals and activation of lipid peroxidation, resulting
in cardiac protection. Our study showed that myocardial SOD activity
was increased and malonaldehyde levels were decreased during I/R in
CIHH-treated rats, suggesting that CIHH can improve myocardial antioxidation capacity (2).
Stress proteins or heat shock proteins (HSPs) can be induced by
Department of Physiology, Hebei Medical University and Hebei Key Laboratory of Medical Biotechnology, Shijiazhuang, China;
2
Shanghai Institute for Biological Sciences, Chinese Academy of Sciences,
Shanghai, China.
*
Corresponding authors: Zhang Yi ([email protected]) and Zhu Zhaonian
([email protected])
1
38
various stress stimuli such as hypoxia or ischemia, and have an important protective role in the body. A previous study demonstrated
that CIHH induced the augmentation of cardiac HSP70 mRNA expression, which was maintained for approximately two weeks after CIHH treatment (7). The augmentation of cardiac HSP70
mRNA expression was inversely correlated with the incidence rate
of arrhythmia, suggesting that HSP is important in CIHH cardiac
protection (7).
Opening of the ATP-sensitive potassium channel (KATP) both in
cell membranes and mitochondrial membranes is involved in the cardiac protection of ischemia preconditioning (IP). Additionally, glibenclamide, an inhibitor of KATP in the cell membrane, and 5-HD, an
inhibitor of KATP in the mitochondrial membrane, eliminated the improvement of cardiac function caused by I/R and shortened the timeto-peak contracture of ischemic hearts in CIHH-treated rats compared
with control animals. This suggests that KATP, especially KATP in mitochondrial membranes, is involved in the cardiac protection rendered
by CIHH (8).
CIHH was shown to promote the expression of particulate fraction PKC-a, -b and -d isozymes after I/R. In isolated rat heart, chelerythrine, a PKC inhibitor, significantly inhibited the improvement
of cardiac functional recovery from I/R in CIHH-treated rats, but had
no effect on control heart function. Further research on the mechanism of PKC effects showed that chelerythrine treatment increased
intracellular sodium ([Na+]i) and calium ([Ca2+]i) in cardiomyocytes,
and aggravated the overloading of Na+ and Ca2+ caused by I/R in
CIHH-treated rats. Additionally, CIHH inhibited ischemia-induced
acidosis in a PKC-dependent manner (9). Together, these data suggest that PKC contributes to the cardiac protection afforded by CIHH
against I/R.
Treatment with aminoguanidine, a specific inhibitor of inducible
nitric oxide synthase, reversed the cardioprotective effect against I/R
injury in CIHH-treated rats, but cardiac function in control rats was
unchanged. This result suggested that nitric oxide might be involved in
the cardiac protection of CIHH (10).
To study the role of mitochondria in cardiac protection with CIHH
treatment, atractyloside (Atr), a specific agent that opens the mitochondrial permeability transition pore (MPTP), was used in Langendorff
isolated rat heart preparations. The results showed that CIHH reduced
myocardial [Ca2+]i, delayed the time for myocardial MPTP to open and
contract, and decreased mitochondrial cytochrome C leakage. The Atr
pretreatment abolished the cardioprotective effect in CIHH-treated rats.
In contrast, Atr aggravated myocardial [Ca2+]i overloading and contracture in control rats. These results suggested that mitochondria play a
pivotal role in cardiac protection of CIHH by inhibiting MPTP (11).
Proteomic studies of mitochondrial proteins in CIHH-treated and normoxic control rats showed that more than 14 protein spots were altered
at least three-fold. Among the 11 proteins identified by mass spectrometry, nine were involved in energy metabolism, of which seven were
increased and two were decreased after CIHH treatment. Biochemical
tests of energy metabolism in mitochondria supported the proteomic
Two
S e cSection
t i o n Two
A
B
D
E
C
F
G
Figure 1. The protective effect of chronic intermittent hypobaric hypoxia (CIHH) on the heart
against ischemia and reperfusion (I/R) injury in rats. Data were expressed as mean±SD. (A)
Representative recording of left ventricular pressure. (B) Representative photos of infarct size. (C)
Representative photomicrographs showing apoptotic cardiomyocytes. Brown staining (TUNELpositive) indicates apoptotic myocytes (arrows). (D) Recovery of left ventricular developing
pressure (LVDP), maximal differentials pressure (±LVdp/dtmax) after 30 minute ischemia and 60
minute reperfusion. N=8 for each group, *p<0.05, **p<0.01 vs Control. (E) Arrhythmia score after
30 minute ischemia and 60 minute reperfusion. N=8 for each group, **p<0.01 vs Control. (F) Infarct
size of heart after 30 minute ischemia and 120 minute reperfusion. N=5 for each group, **p<0.01
vs Control. (G) Mean number of cardiomyocytes undergoing apoptosis per slide. N=6 slides for
each group, I/R represents 30 minute ischemia and 60 minute reperfusion. **p<0.01 vs Control,
##
p<0.01 vs corresponding I/R.
results. CIHH also increases the expression of a molecular chaperone,
HSP60 and an antioxidant protein, peroxiredoxin 5. This suggests that
adaptive alterations of the expression of enzymes involved in material
and energy metabolism in mitochondria play a role in the cardioprotective effect of CIHH (12).
It is generally recognized that CIHH treatment offers cardiac protection against I/R injury. Multiple mechanisms or pathways have been
suggested to contribute to the cardioprotection of CIHH. In conclusion,
CIHH treatment is useful because it is safe, simple and easy to apply,
economical, and can be applied to a broad range of disorders. Thus, it
has important clinical value.
REFERENCES
1. C. J. Murray, A. D. Lopez, Lancet 349, 1436 (1997).
5. Y. Zhang, N. Zhong, Z. N. Zhou, High Alt. Med. Biol. 11, 61 (2010).
6. M. K. Ozer et al., Mol Cell Biochem. 273, 169 (2005).
7. N. Zhong, Y. Zhang, Q. Z. Fang, Z. N. Zhou, Acta. Pharmacol. Sin. 21,
467 (2000).
8. H. F. Zhu, J. W. Dong, W. Z. Zhu, H. L. Ding, Z. N. Zhou, Life Sci. 73,
1275 (2003).
9. H. L. Ding, H. F. Zhu, J. W. Dong, W. Z. Zhu, Z. N. Zhou, Life Sci. 75,
2587 (2004).
10. H. L. Ding et al., Acta. Pharmacol. Sin. 26, 315 (2005).
11. W. Z. Zhu, Y. Xie, L. Chen, H. T. Yang, Z. N. Zhou, J. Mol. Cell. Cardiol.
40, 96 (2006).
12. W. Z. Zhu, X. F. Wu, Y. Zhang, Z. N. Zhou, Eur. J. Appl. Physiol.
112,1037 (2012).
2. Y. Zhang, N. Zhong, H. F. Zhu, Z. N. Zhou, Acta. Physiol. Sin. 52, 89
ACKNOWLEDGMENTS
3. W. Z. Zhu, J. W. Dong, H. L. Ding, H.T. Yang, Z. N. Zhou, Eur. J. Appl.
Program (Grant No. 2006CB504106 and 2012CB518200), the National
(2000).
Physiol. 91, 716 (2004).
4. J. W. Dong, H. F. Zhu, W. Z. Zhu, H. L. Ding, Z. N. Zhou, Cell Res. 13,
385 (2003).
This work was supported by grants from the National Basic Research “973”
Natural Science Foundation of China (Grant No. 30572086, 31071002, and
31271223), and the Natural Science Foundation of Hebei Province (Grant
No. C2012206001).
39
Hypoxic Physiology
Corticotropin-Releasing Factor Type-1 Receptors Play
a Crucial Role in the Brain-Endocrine Network Disorder
Induced by High-Altitude Hypoxia
Du Jizeng and Chen Xuequn
A
pproximately 140 million people around the world live at
altitudes above 2,500 m, including on the Qinghai-Tibet
Plateau in China. The increasing ability to travel rapidly to
high altitudes results in millions of people being exposed
to the risk of mountain sickness every year, but the underlying mechanism of this disorder is still not fully understood. The hypothalamopituitary-adrenal (HPA) axis is critical in maintaining homeostasis and
in physiological, endocrine, and behavioral responses to stress (1–4).
Our findings have shown that corticotropin-releasing factor (CRF) and
CRF type-1 receptor (CRFR1) play a crucial role in hypoxia-induced
brain-endocrine-immune network disorder (2, 3, 5). The mechanisms
include activation of the HPA axis by CRF and CRFR1 signaling in
the paraventricular nucleus (PVN); suppression of the growth hormone
axis (6); inhibition of the reproductive and metabolic axis (7, 8); disturbance of immune function; alteration of cognition (9, 10); and induction of anxiety-like behavior (4), as well as reduced sensitivity of the
HPA axis and a multimodal pattern of responses to severe hypoxic challenge in mammals of the Tibetan Plateau (11). This minireview extends
these findings.
Hypoxia Activates the HPA Axis
Hypoxia can acutely activate the HPA axis by CRF and endothelin-1
(ET-1) release. CRFR1 mRNA expression is upregulated in the PVN
where CRF and ET-1 neurons are stimulated through an ultrashort (autocrine and/or paracrine) positive feedback loop to activate CRFR1 signaling. Morphologically, CRFR1 is co-localized with CRF and ET-1
(12). Moreover, CRF release is positively regulated by norepinephrine
(NE) (13) and angiotensin II (AII) (14), and negatively regulated by
arginine vasopressin (AVP) and β-endorphin (β-EP). In the pituitary,
hypoxia, cold, and restraint stress, alone or in combination, trigger differential expression of CRFR1 and CRFR2 mRNA, suggesting that this
may lead to distinct endocrine responses not only in the HPA axis but
also in other endocrine systems. Interestingly, restraint stress boosts hypoxia-induced responsiveness, but not in combination with cold stress
(2). Hypoxia chronically induces a phase alteration of the HPA axis
cascade amplification, including CRF expression in the PVN, as well as
ACTH and corticosterone levels in plasma (Figure 1) (1, 3, 12).
Hypoxia Suppresses the Neuroendocrine Network
In addition to activation of the HPA axis by hypoxia, a series of shifts
Division of Neurobiology and Physiology, Department of Basic Medical Sciences, School of Medicine, Zhejiang University, Hangzhou, China;
Key Laboratory of Medical Neurobiology of The Ministry of Health, China;
Key Laboratory of Medical Neurobiology of Zhejiang Province,
Hangzhou, China.
Corresponding authors: Du Jizeng ([email protected]) and Chen Xuequn
([email protected])
40
in physiological activity occurs. Hypoxia, particularly at very high altitude, can acutely induce neuroendocrine network disorder and significantly suppress growth and development (6), reproduction (7), metabolism (8), and immunity, along with marked stimulation of prolactin
(PRL) (3), oxytocin (OXT), dynorphin (DNY), and enkephalin (ENK)
secretion (Figure 1). All of these effects are modulated by the central activation of CRF and CRFR1 in the PVN and by increased corticosteroid
feedback due to stress. In support of this, morphological observations
show that CRF and CRFR1 are present throughout the brain, endocrine
organs, and immune tissues, and our findings show that their expression is upregulated following hypoxia. Mimicking ascent to an altitude
of 5,000 m in a hypobaric chamber not only activates the HPA axis but
also causes a concomitant decline in body weight, reduced food intake,
and decreased growth hormone release in adult male and female rats
(6). These effects correlate with increased somatostatin gene expression
and secretion in the periventricular nucleus and upregulated CRFR1
expression in the pituitary. Although CRFR1 can stimulate insulin-like
growth factor 1 (IGF-1) mRNA expression in the liver, it did not alter
the downregulated growth hormone (GH) response (6).
Hypobaric hypoxia chronically inhibits the normal development
of the testes in neonatal rats, especially at the critical age for gonadal
development. Notably, hypoxia reduces testosterone secretion and induces swelling of the testicular interstitium and enlargement of the mitochondria in Leydig cells at postnatal day 21 (7). CRF inhibits gonadotropin-releasing hormone (GnRH) and testosterone levels. However,
AVP coordinates the inhibition of GnRH by CRF. Moreover, β-EP can
also suppress GnRH release, but NE and acetylcholine (Ach) stimulate
GnRH release.
Interestingly, acute hypoxia stimulates thyrotropin-releasing hormone (TRH) release from the PVN and median eminence, enhancing
plasma thyrotropin-stimulating hormone (TSH) levels. NE is also involved in TRH release via the α2 receptor (8), whereas chronic hypoxia
suppresses TRH mRNA expression in the rat PVN (1), and β-EP modulates TRH release in rats.
Hypoxia inhibits T-lymphocyte proliferation, suppresses humoral
immune function and alters initial antigen processing via CRF. Chronic
hypoxia elicits a dose- and time course-dependent change in CRF and
CRFR1 mRNA expression in the PVN, and in CRFR1 mRNA expression in the pituitary (2, 12). During sustained hypoxia, CRF mRNA
expression reaches a peak at day five and returns to normal levels at
day 10. The peak of CRF and CRFR1 secretion inhibits the release of
insulin, which can be blocked by a specific CRFR1 antagonist, suggesting CRFR1 is important in glucose metabolism in rats chronically
exposed to hypoxia (Figure 2) (15). Hypoxia decreases insulin release,
suggesting its potential use in organ transplantation and preservation.
Hypoxia Alters Cognitive Ability
and Induces Anxiety Behavior
Hypoxia can reduce cognitive ability. Hypoxic injury correlates to a
Two
S e cSection
t i o n Two
large degree with the severity of hypoxia.
Interestingly, intermittent hypoxia improves spatial learning and memory in
postnatal mice, particularly at two to three
weeks after birth. This is related to spine
growth, its increased density, enhanced
spine-associated Rap-specific GTPaseactivating protein and postsynaptic density-95 protein expression, which enhances
long term potentiation and activation of
cAMP response element-binding protein
(CREB) expression in the hippocampus
(9,10). CRFR1 is also involved in these
improvements.
Importantly, hypoxia induces an anxiety-like behavior in adult male offspring
subjected to gestational intermittent hypoxia. This is gender-specific and associated with hypoxia-induced sensitization
of the HPA axis and activation of CRF
and CRFR1 in the PVN, but not in the
central nucleus of the amygdala in adult
rat male offspring. Furthermore, the dopamine level in the locus coeruleus is
upregulated (4). These findings suggest
that gestational hypoxia at high altitude
requires serious consideration.
Figure 1. Neuro-endocrine-immune network under hypoxia. Ach, Acetylcholine; ACTH,
adrenocorticotropic hormone; AVP, arginine vasopressin; AII, angiotensin II, CRF, corticotropinreleasing factor; Corts, corticosterone; DNY, dynorphin; ENK, enkephalin; β-EP, β-endorphin;
E2, estradiol; FSH, follicle stimulating hormone; GnRH, gonadotropin-releasing hormone; GH,
growth hormone; LH, luteinizing hormone; NE,norepinephrine; OXT, oxytocin; PRL, prolactin; SS,
somatostatin; T, testosterone; T3, thiiodothronine; T4, thyroxine; TRH, thyrotropin-releasing hormone;
TSH, thyrotropin-stimulating hormone.
Multimodal Strategy Against
Hypoxia in Tibetan Mammals
How animals on the Tibetan plateau acclimatize to hypoxia is an interesting and
important topic. Our findings show that
the HPA axis in the mammals Ochotona
curzoniae and Microtus oeconomus,
which live in the Qinghai-Tibet Plateau
alpine meadows (O. curzoniae had been
proposed as a well-acclimated animal
model for research) (1), has a low sensitivity to extreme altitude hypoxic challenge because of differences in CRFR1
molecular structure. Additionally, these
animals have a multimodal protective
mechanism against hypoxia damage
through the IGF-1 signaling pathway.
Moreover, they have extremely high oxygen utilization rates and low consumption of glycogen, thus maintaining stable
Figure 2. Crucial role of CRFR1 under hypoxia. Corts, Corticosterone; CRF,
corticotropin-releasing factor; CRFR1, CRF type-1 receptor; GH, growth hormone;
expression of hepatic lactate dehydroHPA, hypothalamo-pituitary-adrenal; PVN, paraventricular nucleus; PRL, prolactin.
genase-A and isocitrate dehydrogenase
mRNA, indicating that the normal balance between anaerobic glycolysis and Krebs cycle is preserved (11). under hypoxic stress in lowland animals. However, this is not the case
In contrast, in the lowland rat (Rattus norvegicus) hypoxia increases in plateau mammals that are acclimatized to hypoxia. HIF-1α is not
IGF-1 expression and enhances permeability of lysosomal membranes generally expressed, except under extreme hypoxia (11). The scale-less
in hepatic cells, which leads to the leakage of acid phosphatase and carp (Gymnocypris przewalskii) lives in the alkaline and saline waters
aryl sulfatase from lysosomes into the cytosol, resulting in a cytolysis of Qinghai Lake, at an altitude of 3,200 meters. The metabolic cost of
and necrosis. Evidence shows that HIF-1α, a hypoxia-inducible fac- living for these fish is 40% lower in lake water than it is in their freshtor, is commonly upregulated and involved in target gene transcription water spawning grounds—demonstrated by a reduced metabolic rate
41
Hypoxic Physiology
and O2 consumption—indicating that they are well adapted to hypoxic
conditions and experience a “metabolic holiday” when in Qianghai
Lake (16). Gill remodeling during hypoxia is a general characteristic of
cold-water carp species. The reduced magnitude of the response to hypoxia in G. przewalskii relative to goldfish and crucian carp may reflect
a more active lifestyle and the fact that it lives in a hypoxic environment at altitude. Low habitat temperatures have reduced the evolutionary pressure for selection of HIF-1α in G. przewalskii, whereas the cold
and hypoxic lake water has contributed to the evolution of the HIF-1α
gene (17).
In summary, activation of CRF and CRFR1 by high-altitude hypoxia
can result in activation of the HPA axis and a series of disorders of
growth, reproduction, metabolism, immunity, and behavioral responses. CRFR1 antagonists may be used to treat and prevent disorders of the
neuroendocrine network under hypoxic conditions.
REFERENCES
1. J. Z. Du, in Progress in Mountain Medicine and High Altitude
Physiology. H.Ohna et al., Eds. (Press committee of the 3rd world
congress on mountain medicine and high altitude physiology, Japan,
1999), pp. 416-417.
2. T. Y. Wang et al., Neuroscience 128, 111 (2004).
3. J. F. Xu, X. Q. Chen, J. Z. Du, Horm. Behav. 49, 181 (2006).
4. J. M. Fan, X. Q. Chen, H. Jin, J. Z. Du, Neuroscience 159, 1363 (2009).
5. J. F. Xu, X. Q. Chen, J. Z. Du, T. Y. Wang, Peptides 26, 639 (2005).
6. X. Q. Chen, N. Y. Xu, J. Z. Du, Y. Wang, C.M. Duan, Mol. Cell.
Endocrinol. 242, 50 (2005).
7. J. X. Liu, J. Z. Du, Neuro. Endocrinol. Lett. 23, 231 (2002).
8. T. D. Hou, J. Z. Du, Neuro. Endocrinol. Lett. 26, 43 (2005).
9. X. J. Lu et al., Neuroscience 162, 404 (2009).
10. J. X. Zhang, X. Q. Chen, J. Z. Du, Q. M. Chen, C. Y. Zhu, J. Neurobiol.
65, 72 (2005).
11. X. Q. Chen, S. J. Wang, J. Z. Du, X. C. Chen, Am. J. Physiol. Regul.
Integr. Comp. Physiol. 292, R516 (2007).
12. J. J. He, X. Q. Chen, L. Wang, J.F. Xu, J. Z. Du, Neuroscience 152,
1006 (2008).
13. X. Q. Chen, J. Z. Du, Y. S. Wang, Regul. Pept. 119, 221 (2004).
14. Y. Wu, J. Z. Du, Acta Pharmalogica Sin. 21,1035 (2000).
15. X. Q. Chen, J. Dong, C. Y. Niu, J. M. Fan, J. Z. Du, Endocrinology 148,
3271 (2007).
16. C. M. Wood, Physiol. Biochem. Zool. 80, 59 (2007).
17. Y. B. Cao, X. Q. Chen, S. Wang, Y. X. Wang, J. Z. Du, J. Mol. Evol. 67,
570 (2008).
ACKNOWLEDGMENTS
This work was supported by grants from the National Natural Science
Foundation of China (Grant No. 30393130) and the National Basic Research
“973” Program (Grant No. 2006CB504100 and 2012CB518200).
The Key Role of Vascular Endothelial Dysfunction
in Injuries Induced by Extreme Environmental Factors
at High Altitude
Long Chaoliang1, Liu Jiaying1, Yin Zhaoyun1, Liu Wei1, Wang Hai1,2,*
F
ar from being a passive lining of blood vessels as was initially
thought, endothelium is a dynamic tissue with numerous functions, which include regulation of blood fluidity, formation
of an active barrier between the vascular lumen and tissue,
modulation of local vascular tone, development of new vessels during
angiogenesis, and propagation and amplification of the inflammatory
response (1). Extreme environmental factors at high altitude such as
hypoxia and cold can result in endothelial dysfunction, which plays
key roles in the initiation and progression of high-altitude sickness and
frostbite. Vascular endothelial cells (ECs) are the first to respond to low
oxygen partial pressure (PO2) experienced at high altitudes. The endothelial response to hypoxic challenge induces a highly reproducible
cascade of events, which includes modification of cellular phenotype
Environmental Medicine Research Center, Institute of Health and
Environmental Medicine, Academy of Military Medical Sciences,
Tianjin, China;
2
Thadweik Academy of Medicine, Beijing, China.
*
1
42
leading to capillary leakage, activation of prothrombosis, and initiation
of inflammation (1, 2).
Hypoxia and Endothelial Dysfunction
We previously demonstrated that hypoxia could induce structural
and functional injury of ECs. When cultured pulmonary artery ECs
(PAECs) were exposed to hypoxia, the rates of von Willebrand factor
(vWF) and prostaglandin I2 (PGI2) release were significantly decreased,
while endothelin-1 (ET-1) release was increased, suggesting that PAEC
functionality as a permeability barrier was impaired (3). Vascular endothelial growth factor (VEGF), a potent and specific mitogen for ECs,
can initiate angiogenesis and increase vascular permeability (2). Under hypoxic conditions, PAEC monolayer permeability to bovine serum albumin and human albumin was enhanced and VEGF levels were
significantly increased, indicating that PAEC permeability may play
an important role in the occurrence of high-altitude pulmonary edema
(HAPE) (3). In vivo studies showed decreased levels of nitric oxide and
nitric oxide synthase activity in the brain and lungs from rats exposed to
hypoxia. High-altitude cerebral edema (HACE) in rats was induced by
Two
S e cSection
t i o n Two
Figure 1. The endothelial dysfunction hypothesis of frostbite. Cold exposure induces endothelial cell (EC) freezing and
ischemia/reperfusion (I/R) injuries, resulting in EC- polymorphonuclear leukocyte (PMN) adhesion and EC dysfunction.
The imbalance of vasoactive factors and inflammatory mediators released from damaged ECs induce coagulation disorders including blood hemorheological disorders, vasomotor dysfunction, edema, and hypercoagulability. These events
result in microcirculation disorder and thrombosis, which lead to tissue injuries such as cell necrosis at sites of frostbite.
NO, nitric oxide; PGI2, prostaglandin I2; vWF, von Willebrand factor; ET-1, endothelin-1; ACE, angiotensin converting enzyme; TXA 2, thromboxane A 2; RBC, red blood cell.
exposure to a simulated altitude of 8,000 m, and electron micrographs
showed capillary endothelial cell swelling, irregular thickening of capillaries, and widening of the perivascular space in the blood–brain barrier (BBB). Gene expression and protein levels of VEGF, aquaporin-1,
and aquaporin-4 in ECs were significantly increased (4, 5). Changes in
ECs during cerebral edema caused by hypoxic exposure might induce
BBB injury, and be involved in the pathogenesis of HACE. Based on
these findings, we proposed that management of endothelial dysfunction could be an important strategy for the prevention and treatment of
HAPE and HACE.
Cold and Endothelial Damage
In addition to hypoxia, cold is another extreme environmental factor; frostbite is sometimes observed among individuals exposed to
43
Hypoxic Physiology
low temperatures at high altitude, caused by a combination of tissue
freezing and hypoxia. Tissue freezing depends on environmental factors including wind, duration of exposure, and air moisture, resulting
in tissue necrosis (3, 6). Research over the past 20 years has led to a
new understanding of the pathophysiology of cold injury. Elucidation
of the roles of inflammatory mediators such as prostaglandin F2 (PGF2),
thromboxane A2 (TXA2), platelet aggregation, and thrombosis has led
to active medical regimens for the treatment for frostbite, such as the
use of ibuprofen and aloe vera, heparin, low molecular weight dextran,
and vasodilating agents, as well as the widespread acceptance of the
importance of rapid rewarming (6).
Our previous studies demonstrated that exposure to cold could induce structural and functional damage, as the number of ECs in circulatory blood was significantly increased, suggesting they had been
damaged and separated from the arterial wall lamina (7). Concurrently,
the levels of TXA2, fibronectin, the TXA2:PGI2 ratio, and angiotensin
converting enzyme activity in serum were increased, while PGI2 levels
and antithrombin-III activity were significantly decreased. Histological examination showed that cold induced microvascular EC degeneration, necrosis, and detachment. Platelet aggregation, bleeding, and
thrombosis were also observed in microvessels. These data suggested
that cold induced structural and functional damage to ECs, resulting
in vasoconstriction, promotion of blood coagulation, circulation disorders, and tissue anoxia or necrosis (7, 8). Consistent with the in vivo
results, in vitro experiments showed that ECs were sensitive to cold
injury, which caused a decrease in the number of ECs and the PGI2
levels, while lactate dehydrogenase (LDH) activity and TXA2 levels in
the culture media increased (3).
The characteristics of cold acclimation include enhanced tolerance
to cold exposure and lower susceptibility to cold injury. To confirm
the role of ECs in the pathogenesis of frostbite, the effects of cold
acclimation on EC functions in rats affected by frostbite were investigated. Frostbite caused damage to ECs, resulting in their detachment, as well as vasoconstriction, blood coagulation, and microcirculation disorders, which could exacerbate the effects of frostbite.
When cold acclimation was induced, these parameters were temporarily changed, and the area of tissue survival significantly increased. This suggested that adaptive changes after cold acclimation,
such as increased metabolic turnover rate and improved function of
ECs, were beneficial to enhancing repair responses and resistance to
frostbite (9, 10).
We further investigated the molecular mechanisms of EC damage
induced by cold and found that freezing/thawing of ECs and polymorphonuclear leukocytes (PMNs) could elicit increased expression of intercellular adhesion molecule-1 (ICAM-1) on the surface of ECs and
lymphocyte function-associated antigen-1 (LFA-l) on the surface of
PMNs. This may trigger EC-PMN adherence and subsequently lead to
EC damage. After freezing/thawing, tumor necrosis factor α (TNF-α)
release from ECs was increased, which was promoted by EC-PMN interactions. TNF-α can promote the expression of LFA-l (on the surface
of frozen/thawed PMNs) and ICAM-1 (on normal ECs), thus encouraging EC-PMN interactions and further induction of EC damage. TNF-α
can also promote apoptosis or necrosis in normal ECs and frozen/
thawed PMNs, causing further tissue damage. Monoclonal antibodies
against LFA-1 and ICAM-1 could partly block EC-PMN adhesion and
thereby attenuate EC damage (11–13).
During the frostbite process, hemagglutination is enhanced as the
blood becomes hypercoagulable due to increased platelets and rising
44
plasma levels of TXA2, which could promote platelet aggregation and
enhance vasoconstriction. Altered hemorheological behavior, such as
increased levels of hematocrit and whole blood viscosity, and decreased
red blood cell deformability, resulted in slower blood flow and thrombosis, and led to microcirculation disorder. Ultrastructural analysis of
frozen tissue showed that the most obvious change was characterized
by vascular EC injury that was consistent with the degree of cold (9,
10). Thus, we proposed that dysfunctional ECs have a key role in the
pathogenesis of frostbite (Figure 1). Indeed, our new medical regimen
for treating frostbite, based on this hypothesis, was highly effective.
Summary
Hypoxia and cold coexist at high altitudes. Tissue injuries induced by
hypoxia and cold together were more serious than damage caused by either factor alone. Blood circulation disorders reduced tissue blood and
oxygen supply, and the rate of tissue metabolism, which exacerbated
tissue damage and delayed the repair of damaged tissue, causing extensive tissue necrosis. EC damage induced the synthesis and secretion of
PGI2 and TXA2, which induced vasoconstriction, platelet aggregation,
and exacerbated hemagglutination and thrombosis (10). However, we
found that EC damage was more serious and the tissue survival area
was reduced in frostbitten rats acclimated to hypoxia when compared
to frostbite at normoxia or frostbite during acute hypoxia.
In summary, extreme environmental factors, such as hypoxia and
cold, induced structural damage and dysfunction of ECs. Inflammatory responses from ECs further aggravated dysfunction, forming a
feedback loop resulting in the imbalance of biologically active factors
synthesized and released by ECs, causing high altitude pulmonary hypertension, HAPE, HACE, and frostbite. Endothelial dysfunction plays
a key role in injuries induced by extreme environmental factors at high
altitudes. Thus, ECs may be important targets for the prevention and
treatment of high-altitude sickness.
REFERENCES
1. V. S. Ten, D. J. Pinsky, Curr. Opin. Crit. Care 8, 242 (2002).
2. D. Shweiki, A. Itin, D. Soffer, E. Keshet, Nature 359, 843 (1992).
3. H. Wang et al., Sci. Sin. (Vitae) 41, 822 (2011).
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5. Y. M. Tian et al., Chin. J. Appl. Physiol. 27, 7 (2011).
6. C. Imray, A. Grieve, S. Dhillon, the Caudwell Xtreme Everest Research
Group, Postgrad. Med. J. 85, 481 (2009).
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10, 280 (1994).
8. F. Z. Li et al., Chin. J. Appl. Physiol. 16, 204 (1996).
9. Z. R. Yang, J. Y. Liu, P. H. Yan, Clin. Hemorheol. Microcirc. 29, 103
(2003).
10. Z. R. Yang et al., Clin. Hemorheol. Microcirc. 20, 189 (1999).
11. J. Y. Liu, Q. L. Shan, Z. R. Yang, P. H. Yan, F. R. Sun, Chin. J. Appl.
Physiol. 22, 153 (2006).
12. M. Wang, J. Y. Liu, Z. R. Yang, P. H. Yan, W. Cao, Chin. J. Appl.
Physiol. 19, 52 (2003).
13. L. Y. Jin, J. Y. Liu, Z. R. Yang, P. H. Yan, Chin. J. Appl. Physiol. 21, 393
(2005).
ACKNOWLEDGMENTS
Program (Grant No. 2012CB518200) and the State Key Research Project of
China (Grant No. AWS11J003).
Two
S e cSection
t i o n Two
Targeting Endothelial Dysfunction in High-Altitude
Illness with a Novel Adenosine Triphosphate-Sensitive
Potassium Channel Opener
Pan Zhiyuan1, Cui Wenyu1, Zhang Yanfang1, Long Chaoliang1, Wang Hai1,2,*
T
here are at least 140
million individuals
living or working in
high-altitude areas
worldwide who suffer from
high-altitude illnesses (1).
The limitations of available
therapies against these diseases have been recognized and
effective medications are urgently needed (2). A growing
body of evidence suggests that
endothelial cells play a critical
role in the pathophysiological
events related to the initiation
and progression of high-altitude sickness (3). Hypoxia exposure rapidly induces energy
metabolism
abnormalities,
activation of vascular endothelial cells, and subsequently
results in endothelial dysfunction. Dysfunctional endothelial cells secrete lower levels
of nitric oxide (NO) and prosFigure 1. Iptakalim activates SUR2B/Kir6.1-type K ATP channels to protect against endothelial dysfunction induced
taglandin I2 (PGI2), and higher
by environmental factors. By opening endothelial SUR2B/Kir6.1-type K ATP channels, thereby permitting plasma
membrane K+ efflux, iptakalim induces hyperpolarization, increases Ca 2+ entry, and consequently produces a
levels of endothelin-1 (ET-1)
series of cellular and molecular events to improve endothelial dysfunction: (i) An increase in cytosolic free Ca 2+
compared with normal cells,
activates cellular phospholipase A 2 (cPLA 2) and promotes arachidonic acid conversion to prostaglandin I2 (PGI2);
and overexpress multiple ad(ii) Free Ca 2+ binds to calmodulin and the Ca 2+–calmodulin complex activates endothelial nitric oxide synthase
hesion molecules and vascular
(eNOS) to produce nitric oxide (NO), an effective vasodilator that can synergize with PGI2 to relax resistance
of vessels and pulmonary arteries; (iii) Increased NO induced by iptakalim can suppress the expression of
endothelial growth factor (3).
proinflammatory mediators by inhibiting the activation of the transcription factor nuclear factor-κB (NF-κB);
This eventually leads to loss
(iv) Iptakalim can lead to secretion of chemerin and enhancement of the chemerin/ChemR23 signaling system;
of vasomotor control, pro(v) Iptakalim inhibits the expression of endothelin-1 (ET-1) and vascular endothelial growth factor (VEGF) to
motion of inflammation, and
prevent cardiovascular remodeling and increased capillary permeability. COX, cyclooxygenase; cPLA 2, cytosolic
cardiovascular
remodeling
phospholipase A 2; HIF, hypoxia inducible factor; MCP-1, monocyte chemotactic protein-1; ICAM-1, intercellular
adhesion molecule-1; VCAM-1, vascular cell adhesion molecule-1.
(2–4). Therefore, developing
a therapeutic target that affords protection against endothelial dysfunction is of great value for the distinct subunits. KATP channels vary among different tissues, suggesttreatment of high-altitude sickness.
ing structurally and functionally distinct subtypes. In endothelial cells,
KATP channels consist of SUR2B and Kir6.1 subunits and contribute to
The Role of K ATP Channels in Endothelium
maintaining resting membrane potential (5, 6). Although activated KATP
Adenosine triphosphate-sensitive potassium (KATP) channels regulate channels regulate intracellular Ca2+ levels that affect the production of
cell energy metabolism by controlling membrane potentials and play
important roles in modulating cell functions (5). KATP channels are
formed from two dissimilar subunits. The pore-forming subunit is an
1
Cardiovascular Drug Research Center, Institute of Health and
inwardly rectifying potassium channel that has two subtypes: Kir6.1
Environmental Medicine, Academy of Military Medical Sciences,
and Kir6.2. The regulatory subunit is the sulfonylurea receptor (SUR) Beijing, China;
that has three subtypes: SUR1, SUR2A, and SUR2B. The KATP chan- 2Thadweik Academy of Medicine, Beijing, China.
nel is a hetero-octameric complex composed of four pairs of these two *Corresponding author: wh9588@ yahoo.com.cn
45
Hypoxic Physiology
endothelial autacoids, it is not clear whether the opening of endothelial
KATP channels can modulate the process of endothelial dysfunction (4).
We showed that administration of a KATP channel opener (KCO) to
cultured endothelial cells increased NO release and free intracellular
Ca2+ levels. Additionally, KATP channel activation inhibited ET-1 synthesis and secretion that correlated with a reduction in gene expression of ET-1 and endothelin-converting enzyme. Notably, our results
demonstrated for the first time that KCO could reverse the imbalance between decreased NO release and increased ET-1 production
in dysfunctional endothelial cells (7). ET-1, a potent vasoconstrictor
and proliferative factor, is implicated in hypoxic pulmonary hypertension and cardiovascular remodeling, while NO, a potent vasodilator, can reduce the resistance of blood vessels. Reversing a NO/
ET-1 imbalance by activation of endothelial KATP channels is the
main factor protecting against endothelial dysfunction. Previously, it
was shown that opening of KATP channels could modulate endothelial resting membrane potentials and increase intracellular Ca2+ levels to activate cellular phospholipase A2. This promoted arachidonic
acid conversion to PGI2, and synergized with increased bioavailability
of NO to reduce pulmonary hypertension and ameliorate endothelial
function (3).
Interestingly, in endothelial cells under metabolic disturbances, we
demonstrated that KCO inhibited the overexpression of monocyte chemotactic protein-1, intercellular adhesion molecule-1, and vascular cell
adhesion molecule-1, which are proinflammatory proteins commonly
controlled by the transcription factor nuclear factor-κB (NF-κB) (7).
Because NO can inhibit the activity of NF-κB, increased NO by KCO
may decrease the expression of proinflammatory proteins. Recently, we
also reported that KCO upregulated endothelial chemerin secretion and
receptor ChemR23 gene/protein expression to inhibit inflammation mediated by endothelial cell activation (Figure 1) (8).
Treating High-Altitude Sickness Using
K ATP Channel Openers
Based on these investigations, we hypothesized that improving endothelial dysfunction with a KCO may be a useful way to prevent the
development of high-altitude sickness. However, currently available
KCOs, such as minoxidil, pinacidil, and diazoxide, cannot selectively
activate the endothelial SUR2B/Kir6.1-type KATP channel, and induce
many undesirable side effects that severely restrict their clinical utility
(6). Therefore, we used a heterologous expression technique and patch
clamp recordings to screen KCOs with selectivity for SUR2B/Kir6.1
channels. We discovered that iptakalim, a novel KCO, exhibited high
selectivity for the SUR2B/Kir6.1 channel. Clinical trials of iptakalim in
China have shown that in addition to its potent antihypertensive efficacy, it has a favorable safety and tolerability profile (6). Interestingly, our
previous studies indicated that iptakalim could potently protect against
endothelial dysfunction by activating endothelial KATP channels (7). We
further investigated the efficacy of iptakalim in treating high-altitude
cerebral edema in rats in a simulated high-altitude (8,000 meters) environment (9). As predicted, it prevented hypobaric hypoxia-induced
brain injury in a dose-dependent manner, attenuated permeability of
the blood brain barrier and resulting brain edema, reversed abnormalities in Na+ and Ca2+ levels, and normalized the activities of Na+/K+ATPases, Ca2+-ATPases, and Mg2+-ATPases in the rat cerebral cortex.
Furthermore, we found that iptakalim could increase cell survival and
decrease lactate dehydrogenase release in cultured endothelial cells
46
under oxygen-and-glucose-deprived conditions (9). In hypoxic pulmonary hypertensive rats (10), iptakalim decreased blood pressure in the
pulmonary circulation, and attenuated remodeling in the right ventricle
and pulmonary arteries. In monocrotaline-induced pulmonary arterial
hypertensive rats (11), iptakalim treatment reduced the high right ventricle systolic pressure and the increase in weight ratio of right ventricle to left ventricle plus septum, decreased the mean arterial pressure,
and inhibited right ventricle myocardial tissue cell apoptosis. It also
prevented pulmonary edema and inflammation, and reduced ET-1 and
tumor necrosis factor-α levels in lung tissue (11). We also investigated
the effects of iptakalim on the progression of cardiac hypertrophy failure in a rat model of pressure overloading caused by abdominal aortic banding (AAB) (12). In AAB-treated rats, iptakalim attenuated left
ventricular hypertrophy, lowered blood pressure, improved systolic and
diastolic cardiac dysfunction, and prohibited the progression of heart
failure. Because endothelial dysfunction is pivotal to cardiac hypertrophy and failure induced by pressure overload, we further explored
the effects of iptakalim on endothelial dysfunction in vivo. Following
iptakalim administration, the downregulation of the NO signaling system was reversed, whereas the upregulation of the endothelin signaling
system was inhibited, resulting in normalization of the balance between
these two systems (12). Importantly, a clinical trial of iptakalim for the
treatment of pulmonary hypertension is currently ongoing in China.
The preliminary statistical analysis implies that iptakalim significantly
ameliorates high-altitude heart disease by lowering pulmonary arterial
pressure (unpublished data). Iptakalim also appears to have a favorable
safety and tolerability in patients with high-altitude pulmonary hypertension and heart disease (unpublished data).
In summary, we have provided evidence to support the hypothesis
that improvement in endothelial dysfunction by activation of endothelial KATP channels could be an important strategy for the treatment of
high-altitude sickness. Iptakalim, a recently developed SUR2B/Kir6.1type KCO, has been shown to be efficacious in patients with high-altitude sickness.
REFERENCES
1. D. Penaloza, J. Arias-Stella, Circulation 115,1132 (2007).
2. B. Basnyat, D. R. Murdoch, Lancet 361,1967 (2003).
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(2000).
4. T. Minamino, M. Hori, Cardiovasc. Res. 73, 448 (2007).
5. C. G. Nichols, Nature 440, 470 (2006).
6. Z. Y. Pan et al., J. Cardiovasc. Pharmacol. 56, 215 (2010).
7. H. Wang et al., Cardiovasc. Res. 73, 497 (2007).
8. R. J. Zhao, H. Wang, Acta Pharmacol. Sin. 32, 573 (2011).
9. H. L. Zhu, W. Q. Luo, H. Wang, Neuroscience 157, 884 (2008).
10. H. Wang, Y. Tang, Y. L. Zhang, Cardiovasc. Drug. Rev. 23, 293 (2005).
11. J. S. Li, C. L. Long, W. Y. Cui, H. Wang, J. Cardiovasc. Pharmacol.
Ther. (2012), doi:10.1177/1074248412458154.
12. S. Gao, C. L. Long, R. H. Wang, H. Wang, Cardiovasc. Res. 83, 444
(2009).
ACKNOWLEDGMENTS
This work was supported by grants from the National New Drug Research
and Development Key Project of China (Grant No. 2008ZX09101-006,
2008ZXJ09004-018, 2009ZX09301-002, and 2010ZX09401-307-1-9) and
the National Basic Research “973” Program (Grant No. 2012CB518200).
Two
S e cSection
t i o n Two
Adaptation to Intermittent Hypoxia Protects the Heart from
Ischemia/Reperfusion Injury and Myocardial Infarction
Yang Huang-Tian
H
ypoxia is a life-threatening disorder that occurs as a natural consequence of exposure to high altitude or in clinical
diseases including ischemic heart disease. Multiple endogenous adaptive responses to minimize the injurious
effects of hypoxia have allowed many species to thrive in hypoxic
environments. Adaptation to intermittent hypobaric hypoxia (IHH)
has been shown to increase myocardial tolerance to subsequent severe ischemia/reperfusion (I/R) injury (1). This form of protection
is non-invasive and persists longer than ischemic preconditioning
with fewer side effects compared with chronic hypoxia, and thus, is
an attractive concept for treatment regimens. Therefore, understanding the mechanisms of IHH-induced cardioprotection and determining whether IHH has therapeutic benefits in myocardial infarction
(MI) are of basic and clinical importance. Here, we review our recent findings on the therapeutic effects of IHH on MI and the cellular mechanisms that underlie IHH-induced cardioprotection against
myocardial I/R injury.
Effect of IHH on Calcium Homeostasis
Intracellular Ca2+ overload due to abnormal Ca2+ homeostasis in cardiomyocytes is one of the main factors involved in I/R injury. Animals
subjected to 28 to 42 days of IHH (four to six hours per day, 24–45
days, 5,000 m) demonstrated an improved post-ischemic recovery of
contractile function (1, 2). We then investigated whether this was a direct effect of IHH on cardiomyocytes via maintenance of Ca2+ homeostasis by simultaneously examining the baseline intracellular free Ca2+
concentration ([Ca2+]i), Ca2+ transients, and cell contraction in isolated
ventricular myocytes. IHH (PO2 of 84 mmHg, corresponding to an altitude of 5,000 m, six hours per day, for 42 days) did not alter preischemic baseline [Ca2+]i and the dynamics of Ca2+ transients and cell
contraction, but it markedly suppressed I/R-induced intracellular Ca2+
overload, and improved I/R-suppressed Ca2+ transients and cell contraction (3). Consistently, IHH markedly protected the heart from lethal
myocardial injury caused by severe Ca2+ overload (4). This protection
involved the following aspects: (i) IHH completely inhibited ischemiasuppressed inward and outward sarcolemmal Na+/Ca2+ exchanger
(NCX) currents and protected the apparent reversal potential (3); (ii)
IHH attenuated I/R-induced depression of the sarcoplasmic reticulum
(SR) Ca2+ release channels/ryanodine receptors (RyRs) and Ca2+-pump
ATPase (SERCA2) protein contents and activity, and improved phosphorylation of phospholamban (PLB) during I/R. Therefore, it relieved
SERCA2 from inhibition and subsequently improved SR Ca2+ release
and uptake during I/R (2, 3); and (iii) IHH suppressed I/R-induced mitochondrial Ca2+ overload and enhanced the mitochondrial tolerance
to Ca2+ overload by prolonging the time taken to open permeability
transition pores (mPTPs) via opening mitochondrial ATP-sensitive potassium (mitoKATP) channels (4, 5). It also increased the expression of
ATP synthase subunit beta and mitochondrial aldehyde dehydrogenase
during I/R and significantly attenuated the reduction of myocardial ATP
content, mitochondrial ATP synthase activity, membrane potential, and
respiratory control ratios due to I/R (6).
Figure 1. Schematic representation of cellular and molecular
mechanisms underlying long-lasting IHH-induced cardioprotective
effects. Ischemia/reperfusion (I/R) causes abrupt increases in cytosolic
Ca 2+ ([Ca 2+]c) and mitochondrial Ca 2+ ([Ca 2+]m), and the subsequent
opening of mPTP, which are the main factors involved in reperfusion
injury. Long-lasting IHH induces adaptive responses in the Ca 2+-handling
proteins in cardiomyocytes, such as maintaining the activity of NCX,
RyR2, and SERCA2a and upregulating PLB phosphorylation during I/R.
It also inhibits the activation of mitoK ATP channels that subsequently
inhibit the opening of mPTP. These adaptive regulations inhibit
[Ca 2+]c and [Ca 2+]m overload and preserve ATP, resulting in improvement
of myocardial contractile function during I/R through the activation
of the ROS, Akt, PKC, PKA, and CaMKII pathways. Red lines: effects
of I/R injury; blue lines, effects of IHH. The figure was modified from
reference 1.
Key Laboratory of Stem Cell Biology & Laboratory of Molecular
Cardiology, Institute of Health Sciences, Shanghai Institutes for Biological
Sciences, Chinese Academy of Sciences & Shanghai Jiao Tong University
School of Medicine, Shanghai, China.
47
Hypoxic Physiology
Effect of IHH on Kinase Signaling
Protein kinases play important roles in mediating the transduction of
stress signals from the plasma to various cellular organelles. Activation
of protein kinase B (Akt/PKB), protein kinase Ce (PKCe), and protein
kinase II (CaMKII) during reperfusion, and activation of protein kinase
A (PKA) at the end of ischemia and early reperfusion, have been shown
to contribute to IHH-induced cardioprotection in SR and/or mitochondria and subsequently to the maintenance of intracellular Ca2+ homeostasis and improvement of post-ischemic myocardial performance (1,
2, 4, 7). Recently, we observed that IHH further elevated mitochondrial
reactive oxygen species (ROS) production during early reperfusion,
which contributed to IHH-induced cardioprotection by activating the
Akt and PKCe pathways and inactivating glycogen synthase kinase-3b
(8). Thus, a moderate increase in ROS during early reperfusion may be
required to efficiently activate pro-survival signaling pathways.
IHH as a Therapy
To date, most studies have focused on the preventative effects of IHH
on myocardial I/R injury, but little is known regarding the therapeutic
effect of IHH exposure after MI has occurred. We found that exposure
to 14 or 28 days of IHH (PO2 of 84 mmHg, six hours per day) 7 days
after the onset of MI significantly reduced the scar area and improved
myocardial viability and left ventricular function (9). These results
were associated in part with an anti-apoptotic effect, improved coronary flow by increased vascular endothelial growth factor expression
and capillary density, and reduction of collagen content in the peri-infarct region. These data raise the intriguing possibility that a relatively
simple intervention—intermittent exposure to a simulated altitude initiated days after coronary artery occlusion—may offer profound benefits
to patients with acute MI (10).
Published studies have indicated that long-lasting IHH might provide
a unique and promising preventive and therapeutic approach for treating ischemic heart disease (1, 2, 9, 10). IHH appears to protect the heart
by activating an intrinsic defensive system and integrating multiple targets to deal with injurious stimuli (Figure 1), but further studies are
48
required to dissect the mechanisms involved, especially those related to
transcriptional, post-transcriptional, and post-translational regulation.
More work is also required to confirm the therapeutic effect on MI and
to identify a suitable cycle length, number of hypoxic episodes per day,
degree and duration of IHH, and suitable MI candidates for IHH. The
knowledge derived from these studies should provide new insights into
understanding the intrinsic defensive mechanism and new therapeutic
approaches for protecting the heart against ischemic diseases and other
stresses.
REFERENCES
1. H.-T. Yang, Y. Zhang, Z. H. Wang, Z. N. Zhou, in Intermittent Hypoxia
and Human Diseases, L. Xi, T. V. Serebrovskaya, Eds. (Springer, New
York, 2012), pp. 47-58.
2. Y. Xie et al., Am. J. Physiol. Heart. Circ. Physiol. 288, H2594 (2005).
3. L. Chen et al., Am. J. Physiol. Cell. Physiol. 290, C1221 (2006).
4. Y. Xie et al., Life Sci. 76, 559 (2004).
5. W. Z. Zhu, Y. Xie, L. Chen, H. T. Yang, Z. N. Zhou, J. Mol. Cell. Cardiol.
40, 96 (2006).
6. Z. H. Wang et al., Exp. Physiol. 97, 1105 (2012).
7. Z. Yu, Z. H. Wang, H. T. Yang, Am. J. Physiol. Heart. Circ. Physiol. 297,
H735 (2009).
8. Z. H. Wang et al., Am. J. Physiol. Heart. Circ. Physiol. 301, H1695
(2011).
9. W. Q. Xu et al., Basic. Res. Cardiol. 106, 329 (2011).
10. K. Przyklenk, P. Whittaker, Basic. Res. Cardiol. 106, 325 (2011).
ACKNOWLEDGMENTS
These studies were supported by the Major State Basic Research
Development Program of China (Grant No. 2012CB518203 and
2006CB504106), the National Science and Technology Major Project
(Grant No. 2012ZX09501001), Major and General Programs of the
National Natural Sciences Foundation of China (Grant No. 81170119,
30393133, and 30370536), and the Knowledge Innovation Program of
the Chinese Academy of Sciences (Grant No. KSCX2-YW-R-75).
Two
S e cSection
t i o n Two
Mild Hypoxia Regulates the Properties and Functions
of Neural Stem Cells In Vitro
Zhu Ling-Ling, Wu Li-Ying, Wu Kui-Wu, Fan Ming*
S
elf-renewing
neural stem
cells (NSCs)
are present
throughout the developing and adult mammalian
brain and can differentiate in vitro into neurons,
astrocytes, or oligodendrocytes (1). The proliferation of NSCs in vitro
is regulated by various
factors. However, studies
using oxygen microelecFigure 1. Hypoxia promoted the proliferation of embryonic NSCs. (A) Phase contrast images of neurospheres formed
trodes have shown that
under normoxic (20% oxygen) conditions. (B) Phase contrast images of neurospheres formed under hypoxic (10%
oxygen) conditions. (C) The number of neurospheres produced under hypoxic conditions, especially 10% oxygen,
the mammalian embryo
increased significantly compared with controls. The data represent the mean ± SD (n = 4). *p<0.05, **p<0.01, compared
develops in the uterus in a
with controls (20% oxygen). Scale bar = 100 μm.
hypoxic environment and
NSCs in the brain are in a
hypoxic niche (2, 3). The mean oxygen concentration is approximately evaluated by immunohistochemistry, flow cytometry, and high-perfor1%–5% in tissues. Currently, standard in vitro culture studies using mance liquid chromatography. NSCs cultured in hypoxia (3% oxygen)
NSCs have primarily been performed under atmospheric conditions of displayed an increase in the percentage of neurons, especially the per20% oxygen (4). This indicates that oxygen concentrations used in tra- centage of tyrosine hydroxylase (TH)-positive neurons, compared with
ditional cell culture systems may form a hyperoxic environment, but NSCs grown in normal conditions. Dopamine (DA) levels in the sunot the conditions of “physiological hypoxia” that most cells are ex- pernatant of the hypoxia culture group were two-fold higher than in
posed to in situ. Herein, we describe a series of experiments that tested the normoxia group (Figure 2). Mild hypoxia may also promote the
whether exogenous hypoxia could impact the growth, regulation, and differentiation of MSCs and P19 cells into dopaminergic neurons, as
function of NSCs and to identify the molecular mechanisms involved.
demonstrated by TH staining and DA measurement (3, 7). Thus, this
First, we compared the effect of different exogenous oxygen concen- study identified a new approach to yield DA neurons by manipulating
trations on NSCs growth in vitro. Embryo-derived NSCs were cultured the physical environment.
under 3%, 10%, or 20% oxygen concentrations for one, two, or three
Taken together, we demonstrated that low oxygen conditions signifidays (Figure 1). There was an approximately two- to five-fold increase cantly promotes the proliferation of NSCs and supports self-renewal in
in the number of NSCs cultured after exposure to hypoxia conditions vitro. However, the molecular mechanisms underlying hypoxia-driven
(10% oxygen) compared with normal conditions (20%). Mild hypoxia proliferation are yet unknown. To address this question, a cDNA microdramatically promoted the proliferation of NSCs and decreased lev- array containing 5,704 rat genes was used to characterize gene expresels of apoptosis (5, 6). These results were confirmed using mouse and sion patterns during hypoxia-driven proliferation of NSCs (9). Of the
human-derived NSCs from embryos and adults, which revealed that 5,704 genes examined, 49 were downregulated less than 0.5-fold and
the growth of NSCs in vitro is optimal in a culture environment of 1% 22 were upregulated more than two-fold at 24 hours. At 72 hours, 60
to 10% oxygen. Additionally, mild hypoxia could also dramatically in- genes were upregulated and 11 were downregulated. The percentage
crease the in vitro proliferation of mesenchymal stem cells (MSCs) and of genes with altered expression at each time point was approximately
myoblasts while inhibiting the proliferation of embryonic stem cells 1.24%. A greater number of differentially expressed genes were down(ES) and P19 cell lines when grown in 3%, 5%, or 10% oxygen (3, 7). regulated at 24 hours, while at 72 hours, more where upregulated. Of
Thus, mild hypoxia (1%–10% oxygen) is a more potent trigger to pro- the 71 differentially expressed genes identified at 24 hours, the greatmote the proliferation of adult stem cells than normoxia (20% oxygen), est number were involved in glycolysis and metabolism (36%), folsuggesting that mild hypoxia could provide a novel methodology for lowed by transcriptional regulation (15%), and cell organization and
the expansion of various adult stem cells in vitro.
We further examined the differentiation ability of NSCs expanded
under hypoxia conditions in vitro (8). NSCs were cultured in a 3%
oxygen environment for three days, and differentiated with 1% fetal Beijing, China.
bovine serum (FBS) for another five–seven days. The cell lineage was *Corresponding author: [email protected]
49
Hypoxic Physiology
Figure 2. Lowered oxygen concentration
increased the yield of tyrosine hydroxylase (TH)positive neurons and dopamine (DA) content. (A)
Representative images of TH and Tuj1 doublelabeled cells differentiated from NSCs under
normal conditions. (B) Representative images of
TH and Tuj1 double-labeled cells differentiated
from NSCs under 3% oxygen. Scale bar = 25
μm. (C) The number of TH-positive neurons. (D)
Lowered oxygen concentration increased DA
yield in supernatants, as detected by HPLC.
Each bar represents the mean ± SD (n = 4).
**p≤0.01 compared with controls.
biogenesis (10%). NSCs under low oxygen consumed more glucose
and produced energy by glycolysis (9). This gene expression pattern
indicates that although the expression of most genes does not change
under conditions of hypoxia, NSCs are able to adapt quickly to low
oxygen environments. The information gained from gene expression
and metabolic changes of NSCs under low oxygen conditions will provide new approaches for the evaluation of NSCs as potential in vivo
cellular therapeutics.
Hypoxia-inducible factor (HIF)-1, a key transcription factor during
hypoxia, is important in mediating a variety of adaptive cellular and
systemic responses to hypoxia by regulating the expression of more
than 50 genes. We also demonstrated that the effects of mild hypoxia
on proliferation and differentiation of NSCs were mediated by the hypoxia-inducible transcription factor-1 alpha (HIF-1α) pathway in vitro
by overexpression and downregulation of HIF-1α in the cultured NSCs
following hypoxia (5, 6). The above differentially expressed genes
were regulated directly or indirectly by HIF-1. Additionally, hypoxiainduced small non-coding RNA (ncRNA) was shown to be involved
in the regulation of NSC proliferation (10). The results of significance
analysis of microarrays revealed that 15 small RNAs were upregulated
at least three-fold and 11 were downregulated in NSCs after being subjected to hypoxic conditions. MiR-210 was highly expressed in NSCs
in a time- and oxygen-dependent manner, and was directly regulated by
HIF-1α (10). Hypoxia-induced expression of miR-210 may be involved
in regulating apoptosis and proliferation of NSCs under hypoxia. However, further study is required to understand the hypoxia-induced expression of ncRNAs in NSCs.
In summary, our studies demonstrate that mild hypoxia not only
50
promotes the self-renewal ability of NSCs in vitro, but also increases
their differentiation ability into neurons. Therefore, mild hypoxia could
become a potential approach for allograft cell transplantation by inducing the expansion of NSCs in vitro or by modifying NSC properties in
situ. We hope these findings will aid in the transplantation of NSCs to
treat neurodegenerative diseases such as Parkinson’s disease and brain
trauma.
REFERENCES
1. G. Kempermann, L. Wiskott, F. H. Gage, Curr. Opin. Neurobiol. 14, 186
(2004).
2. K. Zhang, L. L. Zhu, M. Fan, Front. Mol. Neurosci. 4, 1 (2011).
3. L. L. Zhu, L. Y. Wu, D. T. Yew, M. Fan, Mol. Neurobiol. 31, 231 (2005).
4. A. Mohyeldin, T. Garzón-Muvdil, A. Quiñones-Hinojosa, Cell Stem Cell
7, 150 (2010).
5. T. Zhao et al., FEBS J. 275, 1824 (2008).
6. L. Xiong et al., Cell Stress and Chaperones 14, 183 (2009).
7. L.Y. Wu et al., Neurochemical Res. 33, 2118 (2008).
8. C. Zhang, T. Zhao, L. Wu, L. L. Zhu, M. Fan, Neurosignals 15, 259
(2007).
9. L. L. Zhu et al., Cell Reprogram. 13, 113 (2011).
10. Z. H. Liu et al., Cell. Mol. Neurobiol. 31, 1 (2011).
ACKNOWLEDGMENTS
Program (Grant No. 2011CB910800 and 2012CB518200) and the National
Natural and Sciences Foundation of China (Grant No. 90919025 and
31271205).
Two
S e cSection
t i o n Two
Hypobaric Hypoxia or Hyperbaric Oxygen Preconditioning
Reduces High-Altitude Lung and Brain Injury in Rats
Lin Hung-Jung, Chang Ching-Ping, Niu Ko-Chi, Lin Mao-Tsun*
H
igh-altitude exposure (HAE) brain and lung edema are common problems among people who ascend to altitudes greater
than 2,500 m (1). Currently, the most common method for
preventing brain and lung edema is a gradual ascent, but
there are serious drawbacks to this process that make the need for alternative methods quite urgent.
After hyperbaric oxygen therapy (HBO2T), the body experiences
relative hypoxia as the oxygen levels return to a normal level of 21%
(normoxia). Therefore, repeated HBO2T treatments may produce a cycle of hyperoxia/normoxia, contributing to the accumulation of hypoxia
inducing factor (HIF)-1a (2).
Heat shock protein 70 (HSP-70) is upregulated during hypoxia and
mediates cell protection and survival (3). Preinduction of HSP-70 promotes hypoxic tolerance and facilitates acclimatization to acute HAE in
the mouse brain. Hyperbaric oxygen preconditioning (HBO2P), similar
to hypobaric hypoxia, significantly reduces pulmonary edema in rats
caused by HAE (4). Thus, it is likely that HBO2P or hypobaric hypoxia
preconditioning (HHP) can reduce lung and brain edema, and cognitive
dysfunction in HAE by upregulating HSP-70 expression.
In our study, rats were randomly assigned to one of three treatment
groups as follows: (i) HHP (18.3% O2 at 0.66 atmosphere absolute
(ATA) for five hours per day, five consecutive days for two weeks); (ii)
non-HHP (21% O2 at 1.0 ATA for five hours per day, five consecutive
days for two weeks); (iii) NBA (normobaric air; 21% O2 at 1.0 ATA).
One week after HHP, the HHP group was subjected to simulated HAE.
A neutralizing polyclonal rabbit anti-mouse HSP-70 antibody (Ab; 0.2
mg/kg of body weight) dissolved in non-pyrogenic sterile saline was
intravenously administered to some HHP or non-HHP rats 24 hours before simulation of HAE (6,000 m; 9.8% O2 at 0.47 ATA) in a hypobaric
chamber for 24 hours (4, 5). Western blot analyses revealed that HSP70 protein expression in lung tissue was significantly higher in the HHP
group compared with the non-HHP group (6), and significantly lower
in the HHP+HSP-70 Ab group compared with the HHP group (6). The
injury scores, including edema, neutrophil infiltration and hemorrhage,
were significantly higher in the non-HHP group than in NBA controls
after 24 hours of HAE (Figure 1). However, the HHP group had significantly lower scores for acute lung injury than the non-HHP group (6).
Additionally, HHP reduced acute pleurisy and decreased lung myeloperoxidase activity and bronchoalveolar fluid levels of pro-inflammatory cytokines, glutamate, glycerol, 2,3-dihydroxybenzoic acid (DHBA),
and nitric oxide metabolites after HAE (6). These protective effects
of HHP were significantly reduced by administration of a neutralizing
anti-HSP-70 Ab (Figure 1). Thus, HHP-induced upregulation of lung
HSP-70 might attenuate HAE-induced acute lung injury or edema.
In a second experiment, groups of eight rats were randomly assigned
to the following groups: non-HBO2P+non-HAE; HBO2P+non-HAE;
non-HBO2P+HAE; HBO2P+HAE; and HBO2P+HSP-70Ab+HAE. The
HBO2P groups were administered 100% O2 at 2.0 ATA for 1 hour daily
for five consecutive days. The HAE groups were exposed to simulated
HAE in a hypobaric chamber for 24 hours. Immediately after returning
to NBA, the rats were given cognitive performance tests, overdosed
A
B
Figure 1. Histological examination of lung tissue from normobaric
air (NBA), non-hypobaric hypoxia preconditioning (HHP), HHP and
HHP+HSP-70 Ab rats. (A) Representative images of rat lung from NBA
(upper left panel), non-HHP (upper right panel), HHP (lower left panel)
and HHP+HSP-70 Ab groups (lower right panel). (B) Acute histological
score for NBA (white bar), non-HHP (striped bar), HHP (hatched bar), and
HHP+HSP-70 Ab groups (black bar). The non-HHP rats had interstitial
edema, neutrophil accumulation, and hemorrhage. The lung pathological
changes caused by HAE were significantly attenuated by HHP (p<0.05).
Results are mean ± S.D. (n = 8). *p<0.05, compared with the NBA group;
+
p<0.05, compared with the non-HHP group; §p<0.05 compared with the
HHP+HSP-70 Ab group.
Department of Medical Research, Chi Mei Medical Center and Department
of Biotechnology, Southern Taiwan University of Science and Technology,
Tainan, Taiwan, China.
*
51
Hypoxic Physiology
Figure 2. Effect of high-altitude exposure on cognition,
edema, and serum oxidative stress molecules. (A)
Latency period before entering the dark compartment in
a passive avoidance test; (B) percentage change of brain
weight, and oxidative stress markers; (C) 2,3-DHBA; (D)
lipid peroxidation; and (E) nitric oxide metabolites (NOx)
for all five groups were measured. *p<0.05, compared
with the non-HBO2P+NBA group; +p<0.05, compared with
the non-HBO2P+HAE group; §p<0.05, compared with the
HBO2P+HAE group. Each bar represents the mean ± SEM
of eight rats per group.
with a general anesthetic, and then their brains were excised for water content measurement and biochemical
evaluation and analysis. Western blot analysis demonstrated that the HBO2P group had significantly higher
hippocampal expression of HSP-70 than the non-HBO2P
group (p<0.01) (7), and the HBO2P+HSP-70 Ab group
had significantly lower hippocampal HSP-70 protein
expression levels than the HBO2P group without HSP70 Ab (p<0.05) (7). Behaviorally, the non-HBO2P+HAE
group had a significantly lower latency in cognitive
function tests than the non-HBO2P controls (Figure 2A).
HAE-induced cognitive dysfunction was significantly
attenuated in the HBO2P+HAE group, but HBO2P benefits were significantly attenuated in the HBO2P+HSP-70
Ab group. After three days of HAE, when the brain had
developed edema, the brain weight was higher in the
non-HBO2P+HAE group than in the non-HBO2P+NBA
control group (Figure 2B). Brain weight was significantly lower in the HBO2P+HAE group than in the nonHBO2P+HAE group (p<0.05). Additionally, the HBO2P
benefits were significantly attenuated (p<0.05) in the
HBO2P+HSP-70 Ab pretreatment group. Oxidative
stress markers including DHBA, mono-nitrogen oxides
(NOx), and lipid peroxidation levels in the hippocampus of the non-HBO2P+HAE group were significantly
increased (p<0.05) after 24 hours of HAE (Figure 2C, D,
E) compared with the NBA control group. Hippocampal
levels of DHBA, NOx, and lipid peroxidation were significantly lower
(p<0.05) in the HBO2P+HAE group than in the non-HBO2P+HAE
group. The HBO2P+HSP-70 Ab pretreatment group showed a reduction in the benefit of HHP treatment (Figure 2), strongly indicating that
the increased HSP-70 levels before HAE injury might be beneficial.
This suggested that the HSP-70 Ab could cross the blood-brain-barrier
and neutralize HSP-70 expressed in the brain before inhibiting antiapoptosis (5).
These results confirm the findings from previous studies. For example, prolonged and intermittent normobaric hyperoxia preconditioning
caused ischemic tolerance in rat brain tissue (8); preinduction of HSP70 by geranylgeranylacetone improved survival rate of mice exposed
to sublethal hypoxia for six hours, prevented acute hypoxic brain damage, and was involved in mediating these benefits (9); and hypobaric
hypoxia preconditioning in rats attenuated experimental heatstroke
syndromes by preinducing HSP-70 (10).
In conclusion, HHP or HBO2P might attenuate the occurrence of pulmonary edema, inflammation, ischemic and oxidative damage, brain
edema and oxidative damage, and cognitive deficits caused by HAE
partly by upregulating HSP-70 in the lungs and brain.
52
REFERENCES
1. P. Bärtsch, R. C. Roach, N. Engl. J. Med. 245, 107 (2001).
2. A. Quiñones-Hinojosa et al., J. Com. Neurol. 494, 415 (2006).
3. D. K. Das, N. Maulik, I. I. Moraru, J. Mol. Cell Cardiol. 27, 181 (1995).
4. Z. Li et al., J. Trauma 71, 673 (2011).
5. B. Liebelt et al., Neuroscience 166, 1091 (2010).
6. H. J. Lin et al., Clinical Sci. 121, 223 (2011).
7. H. Lin, C. P. Chang, H. J. Lin, M. T. Lin, C. C. Tsai, J. Trauma 72, 1220
(2012).
8. M. R. Bigdeli et al., Brain Res. 1152, 228 (2007).
9. K. Zhang et al., Cell Stress Chaperones 14, 407 (2009).
10. L. C. Wang et al., Am. J. Med. Sci. (2012).
Doi: 10.1097/MAJ.0b013e31824314fe (2012).
ACKNOWLEDGMENTS
This work was supported in part by the National Science Council of China
(Grant No. NSC 99-2314-B-384-006-MY2, NSC 99-2314-B-384-004-MY3,
and NSC 98-2314-B-218-MY2) and the Department of Health of China
(Grant No. DOH99-TD-B-111-003, the Center of Excellence for Clinical Trial
and Research in Neuroscience).
Two
S e cSection
t i o n Two
Mitochondria: A Potential Target in High-Altitude
Acclimatization/Adaptation and Mountain Sickness
Gao Wenxiang1,2,†, Luo Yongjun1,2,†, Cai Mingchun1,2, Liu Fuyu1,2, Jiang Chunhua1,2, Chen Jian1,2, Gao Yuqi1,2,*
H
ypobaric hypoxia is a primary cause of pathophysiological
changes at high altitude, and affects oxygen intake, transportation, and utility. Mitochondria are critical organelles that
consume high levels of oxygen and generate ATP via substrate dehydrogenation and oxidation, as well as oxidation phosphorylation (1). Therefore, we hypothesized that the mitochondrion may be at
the center of hypoxic responses in high altitude acclimatization/adaptation and mountain sickness.
Effect of Hypoxia on Mitochondria
First, we found that acute hypoxia could impair mitochondrial structure
and function. In acute hypoxic rats (housed at the equivalent of 4,000
m for three days), cerebral cortex mitochondria increased in size with
swelling, rupture, and lysis of cristae, but mitochondrial numbers were
not affected. Oxygen consumption state 3 (ST3, with ADP), respiratory
control rate (RCR), and the activities of inner membrane complexes
were decreased, while state 4 (ST4, ADP depleted) and uncoupling
protein (UCP) activity was elevated. ATP synthesis efficiency was
impaired as shown by decreased phosphate-oxygen ratios (P/O), F0F1ATPase activity, and ATP levels in cellular and mitochondrial adenine
nucleotide pools (2). Furthermore, acute hypoxic exposure can depress
mitochondrial ATP/ADP transportation, as shown by decreased ATP/
ADP carrier (AAC) activity and mitochondrial membrane potential
(MMP) (3). Mitochondrial RNA and protein synthesis was also
impaired in isolated rat brain mitochondria under conditions of acute
hypoxia (4).
During prolonged hypoxia, the organism acclimatizes to the hypoxic
environment and mitochondria exhibit gradual acclimatization changes.
Mitochondrial ultrastructure and numbers were not different compared
with normal controls in chronic hypoxic rat brain (4,000 m for 30 days).
In the heart (4,000 m for 30 days) and brain (5,000 m for 30 days) of
rats, ST3 and RCR levels were still low, but ST4 returned to normal
levels. Mitochondrial complex activity, P/O, ATP synthesis, the mitochondrial adenine nucleotide pool, AAC activity, and MMP were higher
in rats with chronic hypoxia than with acute hypoxia and lower than in
normal rats (2, 3). Mitochondrial RNA and protein synthesis in isolated
mitochondria from chronic hypoxic rats were also partially restored (4).
The Role of Genetics
Constant hypobaric hypoxia exposure over many generations has been
hypothesized to induce long-term genetic adaptation to high altitude.
Native Tibetans, who have resided at high altitude for thousand of years,
are considered the population best adapted to high altitudes. We found
placental mitochondria to be considerably swollen in unacclimatized
Han Chinese individuals who migrated to high altitudes, while mitochondria in native Tibetans who live at a similar high altitude were intact with only partial swelling and vacuolization (Figure 1). Measures
of placental mitochondrial oxidative phosphorylation function, such as
ST3, RCR, the oxidative phosphorylation ratio (OPR), and ATP synthesis were higher in native Tibetans than in the immigrant Han population, and only ST4 was not different between the two groups (Figure
1) (5). The activity of mitochondrial complexes I, II, and III; placental
energy charge; and mitochondrial ATP/ADP ratios were higher in the
native Tibetans than in the immigrant Han, probably because of higher
AAC activity and MMP in the placental mitochondria of native Tibetans
(Figure 1) (unpublished data). The differences between the two groups
may result from dissimilar gene and protein expression, as a set of differentially expressed genes related to pathways of energy metabolism,
signal transduction, cell proliferation, electron transport, cell adhesion,
and nucleotide-excision repair were identified by cDNA array (6). Furthermore, differences in the mitochondrial proteomes of skeletal muscle
were also observed between the plateau pika (5,000 m) and Wistar rats
(5,000 m for 30 days).
We further analyzed mitochondrial DNA (mtDNA) sequences from
native high altitude residents, including Tibetans, as well as the plateau
pika and the Tibetan wild donkey. We found that the nt3010G-nt3970C
haplotype was positively associated with high-altitude adaptation in
Tibetans, while the D4 haplogroup was negatively associated (7). In
the pika, there were 15 species of pika-specific amino acids in mtDNA
encoded proteins, including two α-helix amino acid replacements (positions 146 and 408), which caused a polarity switch from hydrophilic to
hydrophobic amino acids in cyclooxygenase-1 (COX1), and an amino
acid change at locus 47 in the mitochondrial matrix region. These amino
acid substitutions can affect the generation of NO via modification of
complex IV structure and alteration of cytochrome c oxidase activity
(8). Moreover, 16 amino acid substitutions were found in mtDNA-encoded proteins in the Tibetan wild donkey when compared with Equus
asinus and Equus caballus, including three in NADH dehydrogenase
subunit 4 (ND4) and ND5 (9).
To confirm the critical role of mitochondria in hypoxic responses, we
analyzed the correlation of mtDNA variations and maladaptation at high
altitude (i.e., mountain sicknesses). We found that the mtDNA 3397G
and 3552A genotypes correlated with susceptibility to high altitude pulmonary edema (HAPE) (10), and a recently identified mtDNA genotype
significantly decreased the risk of high altitude polycythemia (HAPC)
in Han Chinese migrating to the plateau (unpublished data). This suggests that the mtDNA variations are involved in the pathogenesis of both
acute (HAPE) and chronic (HAPC) mountain sickness.
In addition, there is also evidence for changes in mtDNA biogenesis
during high altitude acclimatization. For example, low-altitude Han
sperm mtDNA content reached a peak one month after ascent to a high
altitude (5,300 m) (11), while liver mtDNA content was decreased in
chronic hypoxia rats (5,000 m for 30 days) (12).
College of High Altitude Military Medicine, Third Military Medical University;
2
Key Laboratory of High Altitude Medicine, Ministry of Education, Third
Military Medical University, Chongqing, China.
*
†
1
53
Hypoxic Physiology
Figure 1. Differences in placental mitochondrial ultrastructure and function in immigrant Han Chinese and native Tibetans at high altitude (3,650 to
4,450 m). (A) Representative photographs of placental ultrastructure in immigrant Han and native Tibetans. (B–I) The placental mitochondrial ST3 (B),
RCR (D), OPR (E), energy charge of placental adenylate pool (F), ATP/ADP ratios in the placental mitochondrial adenylate pool (G), MMP (H), and AAC
activity (I) were significantly higher in native Tibetans than in immigrant Han Chinese, while ST4 (C) was not statistically different. *p<0.05 relative to
immigrant Han, **p<0.01 relative to immigrant Han.
Summary
In summary, mitochondrial structure, oxidative phosphorylation, and
ATP synthesis efficiency are involved in the processes of acute impairment and chronic acclimatization of migrating (Han Chinese) lowlanders, as well as genetic adaption of native (Tibetan) residents at high altitudes. However, individuals with certain mtDNA variations are prone
to show maladaptation to hypoxia and are susceptible to mountain
sicknesses. Further research focusing on the mitochondrial genome and
function is required, which may lead to new strategies for the prediction
and treatment of mountain sickness.
REFERENCES
(2007).
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1. V. Donald et al., Fundamentals of Biochemistry, 2nd Edition, (John Wiley
ACKNOWLEDGMENTS
2. G. Luo, Z. Xie, F. Liu, G. Zhang, Acta. Pharmacol. Sin. 19, 351 (1998).
of China (Grant No. 2012CB518201), the National Natural Science
and Sons, Inc. 2006), p. 547.
3. C. Li, J. Liu, L. Wu, B. Li, L. Chen, World J. Gastroenterol. 12, 2120
(2006).
4. J. Liu, et al., Sheng Li Xue Bao 54, 485 (2002).
54
5. X. Zhao, W. Gao, Y. Gao, L. Suo, J. Chen, Nat. Med. J. China 87, 894
This work was supported by the National Basic Research “973” Program
Foundation of China (Grant No. 81071610 and 30971426), and the Key
Project of National Science and Technology Ministry of China (Grant No.
2009BAI85B01).
Two
S e cSection
t i o n Two
Mimicking Hypoxic Preconditioning Using Chinese
Medicinal Herb Extracts
Qian Zhongming1 and Ke Ya2,*
P
reconditioning was first described in a dog model of myocardial injury in which sublethal ischemia enabled cells to better
tolerate subsequent induction of usually lethal ischemia (1).
A number of studies have demonstrated that preconditioning
induced by ischemia or hypoxia can produce a significant protective effect for neurons in experimental animals and humans (2). Ischemic tol-
nomenon of preconditioning has received much attention because of
its potential therapeutic importance (4). However, hypoxic or ischemic
preconditioning has not been used clinically because of safety concerns
(1). Therefore, it is desirable to find a safe preconditioning stimulus
that is both practical and effective, or a biological agent that can mimic
preconditioning pharmacologically (4, 5).
Figure 1. Expression
of
p-GSK,
p-ERK/t-ERK, HIF1α, and EPO. (AD) Cells were preincubated with or
without 100 mmol/L
of PD98059 (a specific MEK inhibitor) or 50 mmol/L of
LY294002 (a specific
PI3K inhibitor) for
one hour and then
administered
hypoxia (0 or 16 hours)
or
ginkgolides
(37.5 mg/mL for 24
hours). The expression of p-GSK (A),
p-ERK/t-ERK
(B),
HIF-1a (C), and EPO
(D) was analyzed
by western blot. 0h:
control group; HP:
16-hour
hypoxia
preconditioning;
Gin: ginkgolides for
24 hours; PD/HP:
PD98059 one hour +
HP; LY/HP: LY294002
one hour + HP; PD/
Gin: PD98059 one
hour + ginkgolides
for 24 hours; and LY/
Gin: LY294002 one
hour + ginkgolides
for 24 hours. The
data are presented as mean ± SEM (n = 3). *p<0.005, **p<0.001 versus controls; #p<0.005 versus HP; @p<0.005 versus Gin. (E–G) Ligustilide (LIG)
protected brain from injury induced by ischemia-reperfusion in rats. (E) Neurological deficit score. (F) Representative images of brain slices stained
with 2,3,5-triphenyltetrazolium chloride. (G) Infarct volume expressed as the percentage of brain volume. Animals were subjected to sham operation
(i), administration of vehicle only (ii), nimodipine at a dose of 12 mg/kg (iii), ligustilide at a dose of 20 (iv), 40 (v), or 80 mg/kg (vi) at three hours and 0.5
hours before undergoing middle cerebral artery occlusion for two hours followed by 24 hour reperfusion. Parametric data are presented as mean
± SEM (n = 6) and non-parametric data as box and whisker plots with the minimum and maximum values (n = 9). *p<0.05, **p<0.01 versus vehicle.
erance induced by hypoxic preconditioning in rodent brains is at least
in part due to the induction of hypoxia-inducible factor-1 (HIF-1) and
its target genes (3). Indeed, it is now recognized that this phenomenon
can be induced in the central nervous system not only by ischemia and
hypoxia (1), but also by a number of other stimuli including hyperthermia (2) and hypothermia (3). During the last few years, the phe-
Department of Pharmacology, Fudan University School of Pharmacy,
Shanghai, China;
2
School of Biomedical Sciences, Faculty of Medicine, The Chinese University
of Hong Kong, Shatin, Hong Kong, China.
*
1
55
Hypoxic Physiology
Figure 2. Proposed mechanisms for the neuroprotective role of
ginkgolides and ligustilide. The neuroprotective effect of ginkgolides and
ligustilide against ischemia-reperfusion injury, similar to that of hypoxic
preconditioning, is hypothesized to be mediated by the upregulation
of erythropoietin (EPO) and activation of other target genes of HIF-1.
Ginkgolides and ligustilide promote the phosphorylation of ERK (p-ERK).
The increased p-ERK then induces an increase in the phosphorylation
of HIF-1a. The latter leads to an increase in the transcriptional activity
of HIF-1 and the increased expression of EPO and other target genes.
Ginkgolides are the main constituents of the nonflavone standardized extract (EGb 761) from Ginkgo biloba (Ginkgoaceae), which has
been used as a Chinese herbal medicine for thousands of years and
shown to exert a wide range of biological activities. There is substantial
experimental evidence to support a role for EGb 761 in the neuroprotective properties of the Ginkgo biloba leaf (6). Thus, we investigated
whether ginkgolides can act as a safe preconditioning agent to protect
ischemic/hypoxic brain cells. We found that ginkgolides could protect
brain cells subject to lethal hypoxia or ischemia treatment (6–8). We
hypothesized that this protective effect of ginkgolides, similar to that of
hypoxic preconditioning, was due to changes in the content or transactivity of hypoxia-inducible factor-1 alpha (HIF-1a) and its downstream
gene targets. Indeed, our experiments demonstrated that the protective
effect of ginkgolides pre-treatment in hypoxic cells was accompanied
by elevated levels of HIF-1a, HIF-1 DNA-binding activity, and erythropoietin (EPO) (6–8).
Additional studies demonstrated that both pretreatment with
ginkgolides and hypoxic preconditioning could increase the expression
of phosphorylated glycogen synthase kinase (p-GSK) and phosphorylated extracellular signal-regulated kinase (p-ERK) in brain cells (Figure 1, A–D). These results suggested that ginkgolides upregulate HIF-1
transcriptional activity, leading to increased expression of EPO, via the
MEK/ERK and PI3/AKT/GSK-3β pathways. We also showed that increased expression of these proteins and improved cell viability induced
by ginkgolides and hypoxic preconditioning could be significantly inhibited by PD98059, a specific inhibitor of mitogen-activated protein
kinase (MAPK), or LY294002, a specific inhibitor of phosphatidylinositol 3’-kinase (PI3K) (Figure 1, A-D). Previous studies demonstrated
that EPO, induced by HIF-1, plays a dominant role in neuroprotection
after ischemic stroke. Thus, it is likely that EPO and other target genes
of HIF-1 also mediate the preconditioning-like effect of ginkgolides.
In our search for more compounds present in traditional Chinese
56
medicinal herbs that can mimic preconditioning pharmacologically,
we tested whether ligustilide has a protective effect against ischemiareperfusion (I/R) injury in the cerebral circulation in I/R rats in vivo
and I/R neurons in vitro (9, 10). Ligustilide is the main constituent of
the oil fraction of Radix Angelicae Sinensise, and is the root of Angelica Sinensis (Oliv.) Diels (Umbelliferae). Additionally, it is believed
to be one of the main pharmacologically active compounds of Danggui
(9), a popular traditional Chinese medicinal herb that has long been
used as a medicinal plant and is included in a number of traditional
Sino-Japanese herbal prescriptions. We found that pretreatment with
ligustilide reduced the neurological deficit score and infarct volume in
a dose-dependent manner in I/R rats in vivo (Figure 1, E-G). In neurons
exposed to oxygen-glucose deprivation (OGD) in vitro, ligustilide pretreatment also increased cell viability with a corresponding decrease
in lactate dehydrogenase (LDH) release (9). These observations were
accompanied by a significant increase in EPO in I/R rats in vivo, and
EPO as well as p-ERK in cultured neurons exposed to OGD in vitro
(9). These findings provide evidence for the preconditioning effect of
ligustilide and imply that the ligustilide-induced increase in EPO may
be mediated by the phosphorylation of ERK.
Together, these results suggest that ginkgolides and ligustilide can
serve as pharmacological agents that mimic hypoxic preconditioning
and protect brain cells from ischemic/hypoxic injury. These compounds
and hypoxic preconditioning may operate through similar mechanisms.
As illustrated in the model in Figure 2, these agents and hypoxic preconditioning promote the phosphorylation of ERK, leading to an increase in the phosphorylation of HIF-1a. The latter, in turn, increases
the transcriptional activity of HIF-1 and hence EPO expression. However, the mechanism by which ginkgolides and ligustilide increase the
phosphorylation of ERK is unknown. These compounds may also have
effects on the stability of HIF-1a. Further research is required to clarify
these questions.
Our findings have demonstrated that compounds extracted from traditional Chinese medicinal herbs can mimic hypoxic preconditioning
pharmacologically, and have the potential to be developed into safe
and effective preventive or therapeutic agents in high-risk conditions
including ischemic disorders of the cardiovascular and cerebrovascular systems. Clinical trials based on these findings are warranted. Our
findings shed new light on the potential beneficial effects of these compounds on the nervous system.
REFERENCES
1. L. Yang et al., Biochim. Biophys. Acta Mol. Basis Dis. 1822, 500 (2012).
2. F. Du et al., Biochim. Biophys. Acta Mol. Basis Dis. 1802, 1048 (2010).
3. F. Du et al., Neurochem. Int. 55, 181 (2009).
4. T. W. Stone, Brit. J. Pharmacol. 140, 229 (2003).
5. D. Ma et al., J. Cereb. Blood Flow Metab. 26, 199 (2006).
6. L. Zhu et al., J. Cell. Biochem. 103, 564 (2008).
7. W. He et al., Int. J. Biochem. Cell Biol. 40, 651 (2008).
8. X. M. Wu et al., J. Cell. Mol. Med. 13, 4474 (2009).
9. X. M. Wu et al., Brit. J. Pharmacol. 164, 332 (2011).
10. X. Kuang et al., Brain Res. 1102, 145 (2006).
ACKNOWLEDGMENTS
These studies were supported by the National Natural Science Foundation
of China (NSFC) (Grant No. 31271132), the National Basic Research
Program of China (Grant No. 2011CB510004) and the Joint Research Grant
of the NSFC and the Hong Kong RGC (Grant No. N-CUHK433/08).
Two
S e cSection
t i o n Two
Molecular Path Finding: Insight into Cerebral Ischemic/
Hypoxic Injury and Preconditioning by Studying PKCisoform Specific Signaling Pathways
Zhang Nan, Li Yun, Li Junfa*
H
ypoxia, which is the failure to effectively use
and regulate oxygen, is involved in the onset
of several ischemia/hypoxia-related diseases
such as high-altitude sickness, myocardial
infarction, and stroke. Of these disorders, stroke has
the highest morbidity and mortality rate, often causing
death and disability. Although progress has been made
in understanding the pathophysiology of stroke and the
time window of thrombolysis has been increased to 4.5
hours, results of clinical trials of pharmacological neuroprotective agents have been disappointing (1, 2). Thus,
novel directions such as those involving endogenous
strategies are being considered. Ischemic/hypoxic preconditioning (I/HPC) is a series of sublethal ischemic/
hypoxic exposures that can allow a specific tissue or organ to become resistant to subsequent severe ischemic/
hypoxic insults (3). The protective mechanism induced
by I/HPC is so profound that it has the potential to be a
future target of clinical therapeutic approaches, but its
molecular mechanism is still unclear (3, 4). Studies have
indicated that ischemic/hypoxic exposure activates various intracellular signaling pathways followed by altered
gene and protein expression, which may contribute to
early and delayed I/HPC. To elucidate the signal transduction pathways activated during cerebral ischemic/
hypoxic injury and I/HPC, we chose to investigate protein kinase C (PKC), one of many protein kinases implicated, but also an important factor in several pathways
that are likely to be involved during ischemic/hypoxic
injury.
Figure 1. Proposed PKC-isoform–specific signaling pathways in cerebral ischemic/
hypoxic injury and I/HPC development. A series of extracellular signals and intracellular
second messengers induce the activation of cPKCbII, cPKCg, and nPKCe, followed
by phosphorylation of several downstream molecules. These PKC-isoform specific
signaling pathways, especially cPKCbII-CRMP-2 and cPKCg-synapsin pathways,
are neuroprotective during the early stages of cerebral I/HPC. Nineteen miRNAs,
notably miR-615-3p, may target genes encoding cPKCbII, cPKCg, nPKCe and their
interacting proteins during delayed cerebral ischemic/hypoxic injury and I/HPC. PIP2,
phosphatidylinositol 4,5-bisphosphate; IP3, inositol trisphosphate; DAG, diacylglycerol;
PLC, phospholipase C; BDP, breakdown product (of CRMP2).
Determination of PKC-Isoforms and Downstream
Members in Cerebral Ischemic/ Hypoxic
Injury and I/HPC Development
PKC has been suggested to be involved during cerebral ischemic/hypoxic injury and I/HPC development. However, because of the biological complexity and dual characteristics of PKC isoforms, there is a lack
of detailed information on individual PKC isoforms in relation to neural
protection or damage. Using our established “auto-hypoxia”-induced I/
HPC mouse model, and hypoxic neuronal cells, we found that among
the 10 PKC isoforms, conventional PKC (cPKC) bII, g, and novel PKC
epsilon (nPKCe) showed increased membrane translocation, even
though protein expression levels were unchanged (4, 5).
Kinases downstream of PKC that may also be important in cerebral I/
HPC include the mitogen-activated protein kinase (MAPK) family that
consists of three groups: extracellular signal-regulated kinases (ERK),
c-Jun N-terminal kinases (JNK), and p38 MAPK. During the early
phase of I/HPC, ERK1/2 phosphorylation decreased in the hippocampus and frontal cortex of mice, while in the late phase, protein levels
decreased when compared with normoxic controls (6). While JNK protein expression remained unchanged, its level of phosphorylation increased at Thr183 and Tyr185 in the hippocampus and frontal cortex of
early and delayed I/HPC mice. Additionally, phospho-Thr183/Tyr185
JNK co-localized with a neuron-specific protein in I/HPC mouse brain
(7). Regarding p38 MAPK, there was a significant increase in phosphorylation at Thr180 and Tyr182 in the frontal cortex, hippocampus,
and hypothalamus of I/HPC mice. Furthermore, p38 MAPK was activated in specific cell types in I/HPC mice: microglia in the cortex and
hippocampus, and neurons in the hypothalamus (8). We also observed
the possible involvement of PKC and MAPK downstream molecules,
such as p90 KD ribosomal S6 kinase (RSK) (9), mitogen- and stress-
Department of Neurobiology, Beijing Key Laboratory for Neural Regeneration
and Repairing, and Beijing Institute for Brain Disorders, Capital Medical
University, Beijing, China.
*
57
Hypoxic Physiology
activated protein kinase 1 (MSK1), cyclic AMP (cAMP) response element binding protein (CREB), and Ets-like transcription factor-1 (Elk1) (10).
To determine whether I/HPC could protect the brain against ischemic
injury, we used a middle cerebral artery occlusion (MCAO)-induced
focal cerebral ischemia mouse model. Evaluation of neurological deficits, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick
end labeling (TUNEL), and 2,3,5-triphenyltetrazolium chloride (TTC)
staining were performed to determine the extent of neurological injury,
cortical neuron apoptosis, and cerebral infarction, respectively in I/HPC
preconditioned MCAO mice. Six hours after MCAO induction, mouse
neurological functions were significantly impaired and showed symptoms such as hypomobility, flattened posture, unidirectional circling,
passivity, forelimb flexion, and motor incoordination. I/HPC treatment
significantly attenuated MCAO-induced neurological deficits, reduced
the percentage of apoptotic cells, and decreased the infarct volume and
edema ratio of MCAO mice (11, 12). Taken together, we concluded that
I/HPC mice were resistant to subsequent cerebral ischemic/hypoxic injury.
Determination of PKC-Isoform–Specific Signaling
Molecules in Cerebral Ischemic/hypoxic Injury
and I/HPC Development
Using a functional proteomics approach, we investigated the role of
activated PKC isoforms and associated signaling molecules in cerebral
ischemic/hypoxic injury and I/HPC development. First, we observed a
reduction in ischemia-induced cPKCbII and g membrane translocation
in the peri-infarction region of MCAO mice. Additionally, inhibition of
cPKCbII or g activation could abolish I/HPC-induced neuroprotection
(11, 12). Second, we separated and identified PKC-isoform–specific interacting proteins in the brain of I/HPC mice using proteomic analysis.
A total of 49 cPKCbII-interacting proteins, of which 15 were cytosolic
and 34 were from particulate fractions, were identified. Among these
proteins, expression of four in the cytosol and eight in the particulate
fraction changed significantly during I/HPC development (11). We
identified 41 cPKCg-interacting proteins in I/HPC mouse brains that
showed significantly altered expression, of which eight were cytosolic
and 15 were in the particulate fraction (12). Third, we chose several
proteins with the greatest variation to validate their PKC-isoform–specific interactions and potential roles in cerebral ischemic/hypoxic injury and I/HPC development. We determined that cPKCbII-collapsin
response mediator protein-2 (CRMP-2) and cPKCg-synapsin pathways
were responsible for I/HPC-induced neuroprotection against initial
ischemic injuries (11, 12).
We also investigated whether changes in protein levels in PKC
isoform-specific signal pathways caused delayed cerebral ischemic/
hypoxic injury and I/HPC development. Large-scale miRNA
microarrays and bioinformatics analyses were used to determine
58
differentially expressed miRNAs and their PKC-isoform specific gene
network in I/HPC and MCAO mouse brains. Nineteen miRNAs were
differentially expressed in I/HPC and MCAO mouse brains, notably
miR-615-3p, which may target genes encoding cPKCbII, g, and
nPKCe-interacting proteins. All of these proteins could potentially
be involved in I/HPC-induced neuroprotection. It should be noted
that downregulation of miR-615-3p during I/HPC had a detrimental
effect on oxygen-glucose deprivation (OGD)-induced N2A
cell injury (13).
Proposed PKC-isoform Specific Signaling
Pathways in Cerebral Ischemic/Hypoxic Injury
and I/HPC Development
Following the development of an I/HPC treatment modality that could
protect mouse brains against subsequent ischemic/hypoxic injury, we
proposed several PKC-isoform specific signaling pathways that may
be involved in cerebral ischemic/hypoxic injury and I/HPC development. As shown in Figure 1, a series of signaling molecules, especially
cPKCbII-CRMP-2 and cPKCg-synapsin pathways, have a neuroprotective role during the initial stage of cerebral I/HPC, whereas the 19
miRNAs, notably miR-615-3p, might target genes encoding cPKCbII,
g, and nPKCe-interacting proteins during delayed cerebral ischemic/
hypoxic injury and I/HPC. These results have expanded our understanding of the molecular mechanisms underlying cerebral ischemic/
hypoxic injury and I/HPC, and may assist in identifying new molecular targets for future clinical therapy of cerebral ischemic/hypoxic injuries such as stroke.
REFERENCES
1. J. D. Marsh, S. G. Keyrouz, J. Am. Coll. Cardiol. 56, 683 (2010).
2. A. Stemer, P. Lyden, Curr. Neurol. Neurosci. Rep. 10, 29 (2010).
3. B. M. Tsai et al., Shock 21, 195 (2004).
4. J. Li et al., Brain Res. 1060, 62 (2005).
5. J. Li et al., Brain Res. 1093, 25 (2006).
6. C. Long et al., Neurosci. Lett. 397, 307 (2006).
7. N. Zhang et al., Neurosci. Lett. 423, 219 (2007).
8. X. Bu et al., Neurochem. Int. 51, 459 (2007).
9. Z. Qi et al., Neurochem. Res. 32, 1450 (2007).
10. J. Jiang et al., Neurochem. Res. 34, 1443 (2009).
11. X. Bu et al., J. Neurochem. 117, 346 (2011).
12. N. Zhang et al., Neurochem. Int. 58, 684 (2011).
13. C. Liu et al., J. Neurochem. 120, 830 (2012).
ACKNOWLEDGMENTS
This work was supported by grants from the National Natural Science
Foundation of China (Grant No. 31071048 and 31171147), the National
Basic Research “973” Pre-Program (Grant No. 2011CB512109), and the
National Basic Research “973” Program (Grant No. 2012CB518200).
Two
S e cSection
t i o n Two
Hypoxic Preconditioning Enhances the Potentially
Therapeutic Secretome from Cultured Human Mesenchymal
Stem Cells in Experimental Traumatic Brain Injury
Chio Chung-Ching1, Chang Ching-Ping2, Lin Mao-Tsun1,*
T
raumatic brain injury (TBI) is primarily caused by
A
mechanical disruption, although secondary or delayed
mechanisms may be involved (1–5). Mesenchymal stem
cells (MSCs) derived from donor rats or humans can
improve dysfunction in a rat model of TBI (6), while paracrine
mechanisms mediated by factors secreted from stem cells have
been shown to be important for the repair of brain damage after
stem cell mobilization (7, 8). Therefore, we examined whether the
secretome from precultured MSCs prior to transplantation could
improve their tissue regenerative potential in a TBI rat model.
Human MSCs were isolated from commercially available bone
marrow aspirates. Conditioned medium (CM) was prepared by
collecting serum-free medium after 24 hours of culturing of an
optimal number of cells (2×106). Hypoxic cells subcultured 1:2
and cultured for three days until confluent were treated in a wellcharacterized, finely controlled ProOX-C-chamber system for 24
hours. The oxygen concentration in the chamber was maintained
at 0.5% with a residual gas mixture constituting 5% carbon dioxide and balanced nitrogen.
Under hypoxia, the
expression levels of vasFigure 1. NeuN-TUNEL double
cular endothelial growth stained cells in ischemic brain re- B
gions. (A) Representative NeuN (red)
factor (VEGF) and heand TUNEL (green) double staining
patocyte growth factor in brain sections from a sham-TBI
(HGF) in the MSC-CM
rat (£), a TBI+Normoxia-CM rat
(▨), a TBI+Normoxia-MSC-CM rat
or secretome were significantly higher than under (▩), and a TBI+Hypoxia-MSC-CM
normoxia (1). Cultured rat (▤). Sham-TBI: rats were given a
sham traumatic brain injury (TBI) opMSCs grown under noreration; TBI+Normoxia-CM: TBI rats
mal or hypoxic conditions treated with conditioned medium
and transplanted into TBI (CM) obtained under normoxic condition without MSC for three days
animals significantly re(n=6); TBI+Normoxia-MSC-CM: TBI
duced (p<0.05) the brain
rats treated with conditioned mevolume and occurrence of dium from MSCs cultured for three
brain damage compared days under normoxic conditions
with animals receiving (n=6); and TBI+Hypoxia-MSC-CM:
culture medium alone TBI rats treated with conditioned medium from MSCs cultured for three days under hypoxic conditions (n=6). The data
were obtained four days after injection. (B) Mean ± standard deviation values of NeuN-TUNEL double stained cells in
(1). Normoxic or hypoxic
the ischemic brain regions. *p<0.05 compared with the sham+TBI group; +p<0.05 compared with the TBI+NormoxiaMSC secretome-treated CM group; §p<0.05 compared with the TBI+Normoxia-MSC-CM group.
TBI animals had a reduced incidence of brain damage compared with TBI animals receiv- MSC secretome-treated TBI rats (1).
ing control medium. Importantly, the protective effect was significantly
There were fewer NeuN-TUNEL double stained apoptotic neurons
greater in the hypoxic MSC secretome-treated animals compared with in the peri-lesioned cortex of normoxic or hypoxic MSCs secretomenormoxic MSC secretome-treated animals (1).
Behaviorally, the normoxic or hypoxic MSC-CM treated TBI rats
had significantly better motor and cognitive functions than control ani- 1
Department of Medical Research, Chi Mei Medical Center;
mals treated with CM alone when evaluated 4 days after TBI induction. 2Department of Biotechnology, Southern Taiwan University of Science
Additionally, the hypoxic MSC secretome-treated TBI rats performed and Technology, Tainan, Taiwan, China.
significantly better in motor and cognitive functions than normoxic *Corresponding author: [email protected]
59
Hypoxic Physiology
Figure 2. NeuN/BrdU/DAPI triple stained cells in peri-ischemic brain
regions. (A) Representative NeuN (red)/BrdU (green)/DAPI (blue) triple
staining in brain sections from a sham-TBI rat (£), a TBI+Normoxia-CM
rat (£), a TBI+Normoxia-MSC-CM rat (▨), and a TBI+Hypoxia-MSC-CM
rat (▤). (B) Mean ± standard deviation values of NeuN/BrdU/DAPI triple
stained cells in the peri-ischemic brain regions (see Figure 1 legends
for group abbreviations). *p<0.05 compared with the sham-TBI group;
+p<0.05 compared with the TBI+normoxia-MSC-CM group (n=6 for
each group).
treated TBI rats than that of control vehicle solution-treated TBI rats
(Figure 1). These neurons were also fewer in number in the perilesioned cortex of hypoxic MSCs secretome-treated TBI rats than that
of normoxic MSCs secretome-treated TBI rats (Figure 1).
BrdU-NeuN-DAPI triple staining of newly formed neurons was
greater in peri-lesions of the cortex in normoxic or hypoxic MSC secretome-treated TBI rats than in control vehicle solution-treated TBI rats
(Figure 2). Also, the peri-lesioned cortex of hypoxic MSCs secretometreated TBI rats had significantly greater BrdU-NeuN-DAPI triple
stained neurons than normoxic MSC secretome-treated rats.
Thus, our study strongly supports a paracrine mechanism for neuroprotective repair, as administration of MSC secretomes recapitulated
the beneficial effects observed after stem cell treatment of TBI. We
demonstrated that systemically delivering normoxia- or hypoxia-preconditioned human MSC secretomes effectively and potently inhibited
brain damage and functional impairment in TBI rats. Our results were
relatively consistent with previous studies. For example, Wei et al., (9)
demonstrated that the conditioned media of adipose stromal cells diminished hypoxia/ischemia-induced brain damage in neonatal rats.
Adult stem cells, particularly MSCs, produce and secrete a wide
60
variety of cytokines, chemokines, and growth factors
that may potentially repair damaged tissues. Furthermore, hypoxic stress increases the expression of several
of these factors (8). Our study demonstrated that hypoxic preconditioning enhanced the capacity of cultured
human MSC secretomes to release several of these factors, indicating the therapeutic potential of the cultured
MSC secretome.
Generally, the adult mammalian brain retains neural stem cells that continually generate new neurons
within the subventricular zone (SVZ) of the lateral
cerebral ventricle and the dentate gyrus subgranular
zone (SGZ) of the hippocampus. We observed that hypoxia-cultured MSCs increased their rate of migration in vitro using
the standard “scratch test” technique. Since HGF and HGF receptor/
c-Met in adult human MSCs mobilize and are involved in repair of
tissues, we propose that migration of neural stem cells from the SVZ
and SGZ to the ischemic brain during TBI might be augmented by
the HGF/c-Met signaling system, which might benefit tissue engineering and human MSC therapy. Moreover, the use of hypoxia enhanced the capacity of cultured MSC secretomes to generate newlyformed neurons during TBI, which might aid in the restoration of
damaged tissues.
After TBI, brain repair requires a continuous supply of blood provided by enlarging pre-existing anastomotic channels or sprouting new
capillaries from existing vascular cells (angiogenesis). Following the
administration of human MSCs in rats to catalyze brain plasticity, brain
VEGF levels significantly increased, indicating that new blood vessels
may have been created to nourish the damaged area. VEGF is a key
vasculogenic and angiogenic regulator. The hypoxia-cultured MSCs
used in our study also had increased VEGF levels in the secretome and
systemic delivery improved TBI. Thus, activating endogenous HGF
and VEGF or administration of human HGF and VEGF might reduce
behavioral deficits and cerebral damage in TBI.
In conclusion, we demonstrated that MSCs could secrete bioactive
factors including HGF and VEGF that stimulate neurogenesis and improve behavioral deficits in a rat model of TBI. Hypoxic preconditioning enhanced the secretion of MSCs-produced bioactive factors, indicating the therapeutic potential of hypoxic cultured MSC secretomes
for treatment of TBI and other neurodegenerative diseases.
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1. C. P. Chang et al., Clinical Sci. (2012) doi: 10.1042/CS20120226.
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9. X. Wei et al., Stem Cells 27, 478 (2009).
ACKNOWLEDGMENTS
This work was supported in part by the National Science Council of China
(Grant No. NSC 99-2314-B-384-006-MY2, NSC 99-2314-B-384-004-MY3,
and NSC 98-2314-B-218-MY2) and the Department of Health of China
(Grant No. DOH99-TD-B-111-003, the Center of Excellence for Clinical Trial
and Research in Neuroscience).
Two
S e cSection
t i o n Two
Mitochondrial Adaptation and Cell Volume Regulation in
Hypoxic Preconditioning Contribute to Anoxic Tolerance
Wu Li-Ying, Zhu Ling-Ling, Fan Ming*
H
ypoxic preconditioning (HP) can be induced by a brief, sublethal exposure to hypoxia. Adaptive hypoxia-protective responses are the main characteristics of HP. Mitochondria are
the cellular organelles most sensitive to hypoxia and their
dysfunction is the main cause of hypoxic injury. In contrast, adaptation
of mitochondria to hypoxia improves the ability of a cell to survive severe hypoxia or anoxia (1). Here, we show that mitochondrial adaptive
responses induced by HP are sufficient to stimulate protective mechanisms against severe hypoxic or anoxic injury. Over the past ten years,
we found that HP could reduce the occurrence of apoptosis by regulating the functions of mitochondria, and inhibit necrosis by regulating the
volume of cells.
We first reported 12 years ago that HP decreased apoptosis in cultured hippocampal neurons in vitro after anoxia-reoxygenation and induced anoxic tolerance (2). The protective effect of HP against anoxia
or severe hypoxia was further confirmed in vivo. Animals exposed to
varying levels of HP displayed a delayed appearance of hypoxic injury potential and the disappearance of presynaptic volley when exposed to acute, lethal hypoxia, indicating that synaptic function was
enhanced by HP (3). To reveal the potential mechanisms of protection
afforded by HP against severe hypoxia or anoxia, we analyzed various mitochondrial functions to determine their unique roles in hypoxia
(4, 5). Mitochondrial membrane potential (MMP) is the potential difference across mitochondrial membranes and changes in MMP levels
have been proposed as an index of mitochondrial function (6). MMP
in acute anoxia was monitored in real-time under a laser-scanning inverted confocal microscope after neurons were exposed to HP or normoxia. We found that HP enhanced the ability of hypoxia-sensitive
hippocampal neurons or the less-sensitive hypothalamic neurons to
maintain normal mitochondrial function under acute anoxia (7, 8).
These results suggest that mitochondria are organelles with a strong
self-regulating capacity.
Since HP decreased apoptosis induced by anoxia in cultured hippocampal neurons, and Bcl-2 proteins, which respond to stress, are critical
in the prevention of apoptosis, we examined the expression of Bcl-2
Figure 1. Necrosis induced by acute anoxia (AA) and the prevention
of necrosis by hypoxic preconditioning (HP) assessed by cell volume
regulation and lactate dehydrogenase (LDH) release from PC12 cells.
(A) Necrosis under AA exposure. a, 1% agarose electrophoresis gel at
indicated times after AA; b, Flow cytometric analysis of cells treated with
propidium iodide (PI); c, Morphology assessed by electron microscopy.
Upper image shows a cell in normoxia and lower image shows a cell
exposed to AA for 24 hours; d, PI (blue) and Hoechst 33258 (red) double
staining assessed by fluorescent microscopy. (B) The process of cell
volume regulation during 24 hours of AA exposure. AA: cells cultured
in normoxia were directly exposed to AA; HP+AA: cells treated with HP,
followed by exposure to AA. (C) LDH leakage. Control: cells were cultured
in normoxia. AA: cells were exposed to anoxia for 24 hours. HP+AA: cells
were treated with HP, followed by 24 hours of anoxia. HP+BB: cells were
treated with HP in the medium containing 20 μg/mL berberine chloride
(BB), followed by AA exposure for 24 hours. *p<0.01 compared with AA
group; #p<0.05 compared with HP+AA group.
A
B
C
Beijing, China.
*
61
Hypoxic Physiology
by immunocytochemical staining, assessing protein levels using an
imaging analyzer or by flow cytometric analysis. The results demonstrated that HP increased Bcl-2 expression which, taken together with
the previous results, suggest that higher levels of Bcl-2 induced by HP
may maintain the higher levels of MMP during anoxia. However, we
still lack direct evidence that these effects are caused by Bcl-2 overexpression induced by HP.
Necrosis is another mechanism of cell death and occurs more frequently in acute anoxia when energy metabolism is at a low level. Using a hypoxia-sensitive PC12 cell line, we found that HP prevented
the occurrence of necrosis induced by anoxia and delayed the regulatory volume decrease (RVD) after cells were exposed to acute anoxia
(9). Moreover, we demonstrated for the first time that this protection
was related to the HP-induced increase in aldose reductase (AR) and
sorbitol levels. AR catalyzes the conversion of glucose to sorbitol in
the presence of nicotinamide adenine dinucleotide phosphate (NADP).
Sorbitol synthesis is induced by increasing the amount and activity of
AR. Changes in osmolarity cause sorbitol to leak rapidly into the external medium through a sorbitol permease transport pathway, which
prevents excessive cell swelling. The efflux of sorbitol was the primary
mechanism for RVD, which protects the cells by minimizing cell swelling (10). In our study, we showed that acute anoxia caused cell necrosis
(Figure 1A) and HP prevented this by inhibiting the increase in cell
volume (Figure 1B) and lactate dehydrogenase (LDH) leakage, when
the cells were exposed to acute anoxia. Additionally, berberine chloride
(BB), an inhibitor of AR, completely reversed the protective effects of
HP against LDH release (Figure 1C). This suggested that cell volume
regulation could be a potential mechanism for the protection exerted
by HP against acute anoxia. Sorbitol, synthesized from glucose and
catalyzed by AR, is directly related to cell volume regulation (11). We
demonstrated for the first time that HP significantly increased sorbitol
levels, while treatment with BB, an inhibitor of AR, attenuated the increase in sorbitol content induced by HP (data not shown). Thus, we
speculated that HP increased the activity of AR. Quinidine, a stronger
inhibitor of sorbitol, reversed the protection afforded by HP (data not
shown), which indicated that sorbitol contributes to the protection by
HP. Taken together, HP can prevent necrosis induced by anoxia, and
the protective mechanism involves the regulation of cell volume mediated by the AR-sorbitol pathway. Thus, we propose that hypoxia causes
changes in metabolic products that in turn enable cells to adapt to the
hypoxic environment, a change in process from passive stress to active
adaptation.
In conclusion, our studies have provided potential mechanisms
for HP against severe hypoxia or anoxia. HP reduces the occurrence of apoptosis by regulating the functions of mitochondria
and inhibiting necrosis by regulating the volume of cells. In turn,
these adaptive changes facilitated the formation of hypoxic or
anoxic tolerance.
REFERENCES
1. L.C. Heather et al., Basic Res. Cardiol. 107, 3 (2012).
2. A. S. Ding et al., Chin. J. Neuroanat. 16, 1 (2000).
3. T. Zhao et al., Acta Phys. Sinica. 53, 1 (2001).
4. J. Wang et al., J. Neurochem. 77, 3 (2001).
5. E. Er et al., Biochim. Biophys. Acta.1757, 9 (2006).
6. O Cazzalini et al., Biochem. Pharmacol. 62, 7 (2001).
7. L. Y. Wu et al., Brain Res. 999, 2 (2004).
8. L. Y. Wu et al., Neurosignals 14, 3 (2005).
9. L. Y. Wu et al., Cell Stress Chaperon. 15, 4 (2010).
10. H. Garty et al., Am. J. Physiol. 260, 5 (1991).
11. A. W. Siebens, K.R. Spring, Am. J. Physiol. 257, 6 (1989).
ACKNOWLEDGMENTS
Program (Grant No. 2012CB518200 and 2011CB910800) and the National
Natural Science Foundation of China (Grant No. 31271211 and 81071066).
The Effects of Ratanasampil, a Traditional Tibetan Medicine,
on β-amyloid Pathology in a Transgenic Mouse Model and
Clinical Trial of Alzheimer’s Disease
Zhu Aiqin1,*, Li Guofeng1, Zhong Xin1, Li Yinglan1, Liao Baoxia1, Zhang Jun2, Chu Yide1
A
lzheimer’s disease (AD) is a progressive neurodegenerative
disease. Our early survey results indicated that a subpopulation of middle-aged residents living on the Tibetan plateau
often suffers from sleep disorders and light memory loss, and
that these symptoms worsen with age. Additionally, β-amyloid peptide
Department of Geriatrics, Qinghai Provincial Hospital, Xining, Qinghai, China;
National Institute of Neurological Disorders and Strokes/NIH, Bethesda,
MD, U.S.
*
1
2
62
(Aβ) production was seen to increase following cardiac arrest, indicating that a chronic hypoxia environment might be involved in the pathogenesis of AD (1, 2). We also studied hyperhomocysteinemia and cognition disorders at high altitude (3). Although drugs for the treatment of
AD exist, no current treatment is curative or permanently arrests the disease course (4). Ratanasampil (RNSP), a compound formulation from
traditional Tibetan medicine, has been widely used as an anti-aging and
anti-hypoxic drug to treat cerebrovascular diseases and high-altitude
sickness in China. Our research group conducted a series of investigations on the effects of RNSP on Aβ pathology in an AD transgenic
Two
S e cSection
t i o n Two
mouse model (Tg2576) and in patients living at high altitudes.
The Tg2576 mouse has been widely used to simulate features of human AD (5). Tg2576 mice were administered RNSP at 0.14 mg/day for
eight weeks. Immunohistochemistry in RNSP-treated mice showed a
marked reduction in amyloid plaques in the cortex and hippocampus.
Consistently, both Western blot and ELISA analysis indicated significant decreases of Aβ40 and Aβ42 peptides in the brains of RNSP-treated
Tg2576 mice. Furthermore, RNSP treatment dramatically reduced the
level of Aβ in serum. Densitometric analysis of C-terminal fragments
(CTFs) showed that α-CTF (also called p3, the product of amyloid
precursor protein cleavage by α-secretase containing the C-terminal
portion of Aβ but not releasing the damaging Aβ peptide) was significantly increased in the brains of the drug treatment group, resulting
in a higher ratio of α-CTF/β-CTF than in the vehicle group (6). Since
α-secretase activity does not release Aβ, the significant increase in
α-CTF seen in our study suggests that RNSP treatment most likely increases α-secretase activity. Thus, a higher ratio of α-CTF/β-CTF may
indicate reduced release of the Aβ peptide. Animal behavior experiments suggested an improvement in memory and decreased anxiety in
RNSP-treated Tg2576 mice (7, 8).
We then conducted a clinical trial to further investigate whether
RNSP improves cognitive function in AD patients living in Xining
(2,000 to 3,000 m). 140 patients with mild-to-moderate AD were divided into three groups: high-dose RNSP (1 g/day), low-dose RNSP (0.3 g/
day), and control (placebo). All patients were assessed using the Mini
Mental State Examination (MMSE), Alzheimer’s disease Assessment
Scale cognitive subscale (ADAS-cog), and activity of daily living scale
(ADL) at three time points: before treatment, after four weeks of treatment, and at the end of 16 weeks of treatment (9). MMSE, ADAS-cog,
and ADL scores were significantly improved in the high-dose RNSP
group compared to scores before treatment. Additionally, the serum
concentrations of tumor necrosis factor (TNF)-α, interleukin (IL)-1β,
and IL-6 were decreased at 16 weeks but only in the high-dose RNSP
group. Significantly, Aβ42 positively correlated with the concentrations
of TNF-α, IL-1β, and IL-6 in serum. Furthermore, we found the superoxide dismutase activity was increased whereas nitric oxide (NO)
synthase activity was inhibited in the high-dose RNSP group (10). Since
AD incidence is thought to correlate with increased levels of oxygen
radicals and NO (11), this result suggests that high-dose RNSP could be
considered as an option for AD treatment. Side effects of RNSP were
not evident. The protocol was approved by the Qinghai Provincial Medical Ethics Committee.
Our group initiated an investigation into the anti-aging and anti-hypoxia mechanisms of RNSP for the treatment of AD at high altitude.
Although the mechanism for RNSP therapeutic activity is not fully understood, we postulate three putative modes of action: (i) Intervention in
the amyloid precursor protein system―RNSP may selectively increase
α-secretase activity and/or inhibit β-secretase activity, resulting in the
reduction of Aβ; (ii) Anti-inflammatory effects―RNSP might reduce
inflammatory factors by inhibiting inflammatory responses and microglia activation; and (iii) Anti-oxidative stress activity.
In summary, these potential multifunctional effects of RNSP suggest
a novel mechanistic target for Alzheimer therapeutics. Currently, we
are working on a large group of subjects to determine the consistency
of RNSP effects on mild to moderate AD at high altitudes. We hope
to identify a form of RNSP with enhanced therapeutic efficacy and reduced side effects. Furthermore, to explore the prevalence and risk factors of AD for the elderly in areas of high altitude, we will investigate
populations over the age of 50 living on the Qinghai-Tibet plateau.
REFERENCES
1. R. Yaari, J. Corey-Bloom. Semin. Neurol. 27, 32 (2007).
2. H. Zetterberg et al., PLoS One 6, e28263 (2011).
3. Q. X. Ma, A. Q. Zhu. High Alt. Med. 7, 230 (2009).
4. S. A. Jacobson, M. N. Sabbagh. Alzheimers Res. Ther. 4, 20 (2011).
5. S. Lesne et al., Nature 440, 352 (2006).
6. A. Q. Zhu et al., Chin. Pharmacol. Bull. 6, 720 (2009).
7. A. Q. Zhu, Y. D. Chu, C. L. Masters, Q. X. Li, Chin. J. Psych. 3, 69
(2010).
8. A. Q. Zhu, C. L. Masters, Q. X. Li, Chin. J. Geriatr. 11, 950 (2009).
9. A. Q. Zhu et al., J. Behav. Brain Sci. 2, 82 (2012).
10. B. X. Liao, A. Q. Zhu, A. Q. Xi, Y. D. Chu, Chin. J. Geriatr. 19, 2437
(2009).
11. D. Y. Choi, Y. J. Lee, J. T. Hong, H. J. Lee, Brain Res. Bull. 87, 144 (2012).
Duoxuekang, a Traditional Tibetan Medicine, Reduces
Hypoxia-Induced High-Altitude Polycythemia in Rats
Zhang Yi1,*, Meng Xianli1, Wu Wenbin1,2, Lai Xianrong1, Wang Yujie1, Zhang Jing1, Wang Zhang1
C
hina has a wide area of plateaus, about 17% of which reach
an altitude of more than 3,000 m. In typical plateaus such
as Qinghai, Xinjiang, and Tibet, various physical ailments
and organ damage caused by hypoxia can pose considerable, sometimes life-threatening, risks to people visiting these areas
for the first time (1).
High-altitude polycythemia (HAPC), named “Plethora” in Tibetan
medicine, is a chronic altitude-induced disease characterized by
hyperplasia of red blood cells due to hypoxia and can seriously
jeopardize the health of high-altitude plateau residents. HAPC in
College of Ethnomedicine of Chengdu University of Traditional Chinese
Medicine, Sichuan, China;
2
Teaching Hospital of Chengdu University of Traditional Chinese Medicine,
Sichuan, China.
*
1
63
Hypoxic Physiology
China has an incidence rate of between 2.5% and 5% in the plateau
areas, with a total of approximately 250,000 patients, but currently
there is no effective cure (2). However, the symptoms are commonly
alleviated by increasing the oxygen-carrying capacity of the blood,
improving hypoxia, and reducing the number of red blood cells. Other
methods have been adopted, such as dredging blood vessels to improve
microcirculation, oxygen inhalation, administration of blood-thinning
agents, migration to lower altitude areas, and treatment with traditional
medicine. However, the lack of an effective treatment for HAPC has
hindered the economic and political development of China’s plateau
areas (3).
Traditional Tibetan medicinal plants are endowed with unique physiological activity owing to the specific environment in which they are
grown and, therefore, has unique effects in the prevention and treatment of a variety of chronic altitude illnesses. Nevertheless, a poor understanding of the bioactive chemical constituents and mechanisms of
traditional Tibetan remedies for the treatment of altitude sickness has
restricted the modern development of Tibetan medicine. In this study,
we selected one traditional Tibetan medicine, Duoxuekang, and conducted a proof of concept study of its mechanism to provide scientific
evidence for the treatment and prevention of chronic altitude sickness.
Duoxuekang is derived from a secret recipe owned by the famous
Tibetan medicine master, Cuoru, and has been found to alleviate exhaustion and increase tolerance to hypoxia, greatly alleviating blood
stasis and hyperplasia of red blood cells. In this study, we first established a HAPC rat model by using a low pressure chamber to simulate
the high altitude plateau environment. Treatment of HAPC rats with
Duoxuekang reduced the red blood cell, hemoglobin, and serum eryth-
ropoietin (EPO) concentrations. It also reduced the fibrinogen concentration, inhibited erythrocyte costimulation, and improved erythrocyte
deformation, which possibly explains its effect on promoting blood
circulation and dissipating blood stasis (4). Additionally, it downregulated hypoxia-inducible factor (HIF)-1α protein expression in brain
tissue, decreased EPO mRNA expression in kidney tissues and serum, and inhibited erythroid hyperplasia in the peripheral blood (5).
A high-performance liquid chromatography (HPLC) method to detect
active compounds in Duoxuekang was developed for quality control.
A serum pharmacochemical study of Duoxuekang revealed that the
plants Phyllanthus emblica and Hippophae rhamnoides might provide
the active compounds responsible for the efficacy in the treatment of
HAPC.
This study is helpful for understanding the mechanisms of action of
traditional Tibetan medicines used for the prevention and treatment of
HAPC caused by hypoxia, and related chronic diseases. Duoxuekang
and the identified compounds can be further developed as new therapeutics for the prevention and treatment of HAPC.
REFERENCES
1. C. F. Merino, Blood 5, 1 (1950).
2. P. Li et al., Exp. Hematol. 39, 37 (2011).
3. W. B. Wu, X. L. Meng, Y. Zhang, X. R. Lai, J. Guangzhou. Univ. TCM.
27, 492 (2010).
4. W. B. Wu, X. R. Lai, Q. M. Suolang, X. L. Meng, Tib. Sci. Technol. 33,
39 (2009).
5. W. B. Wu, X. R. Lai, Q. M. Suolang, X. L. Meng, F. Y. Liu, Pharmacol.
Clin. Chin. Mate. Clin. Med. 25, 93 (2009).
k-opioid Receptor and Hypoxic Pulmonary Hypertension
Li Juan‡, Zhang Lijun‡, Fan Rong‡, Guo Haitao, Zhang Shumiao, Wang Yuemin, Pei Jianming*
H
ypoxic pulmonary hypertension (HPH) is critical in the
pathogenesis and development of many cardiovascular and
pulmonary diseases such as chronic obstructive pulmonary
disease, chronic plateau disease, and neonatal pneumonia
(1). The κ-opioid receptor (κ-OR) is the predominant opioid receptor
isotype and is present in the heart and peripheral blood vessels (2).
However, the role of κ-OR in HPH has not been previously studied.
Therefore, we recently attempted to elucidate the role of κ-OR in HPH
along with its underlying mechanisms. Here, we present a review of
our studies on the role of κ-OR and its possible mechanisms of action.
Department of Physiology, National Key Discipline of Cell Biology,
Fourth Military Medical University, Xi’an, China.
*
‡
64
In 1986, Seelhorst and Starke demonstrated that κ-OR is found in
the pulmonary artery (PA) of rabbits (3). For the first time, we recently
demonstrated that κ-OR is expressed in PAs of rats and its expression
increases in hypoxic states (4). To explore the effect of κ-OR on PAs
in rats, isolated PA rings were perfused and the tension of the vessel
was measured. κ-OR stimulation with U50,488H [(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide], a
selective κ-OR agonist, induced relaxation of PAs in vitro in a dosedependent manner (5). This effect was abolished by administration
of nor-binaltorphimine (nor-BNI), a selective κ-OR antagonist. The
relaxation effect of U50,488H in PAs was partially endothelium-dependent and was significantly attenuated in the presence of NG-nitroL-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor.
The relaxation effect of U50,488H was also significantly attenuated by
KV channel blocker 4-aminopyridine (4-AP) but not by glibenclamide
(an ATP-sensitive K+ channel blocker) or tetraethylammonium (TEA,
a Ca2+-activated K+ channel blocker). Further study demonstrated that
Two
S e cSection
t i o n Two
Figure 1. Effects of U50,488H on pulmonary artery
(PA) remodeling induced by hypoxia (magnification
×400; from reference 3). (A, D) control; (B, E): hypoxia for two weeks; (C, F) hypoxia for two weeks
+ U50,488H. (A–C) Hematoxylin and eosin staining and (D–F) elastin stain detected by light and
transmission electron microscopy, respectively.
The proliferation and migration of pulmonary artery
smooth muscle cells and the thickness of the PA
wall were significantly reduced in the group with
hypoxia for two weeks that received intraperitoneal
administration of U50,488H (1.25 mg/kg).
endothelium denudation and 4-AP have an additive inhibitory effect on PA relaxation induced by
U50,488H. Additionally, dynorphin A 1-13 (an endogenous κ-OR agonist) at the same concentration
as U50,488H also induced a significant relaxation
effect in rat PA rings that was lower (47.5 ± 4.8
%) than for U50,488H (61.4 ± 4.2 %). The vasorelaxant effect of dynorphin A 1-13 was abolished
by nor-BNI. The above results suggest that κ-OR
stimulation with U50,488H relaxes PAs through
two separate pathways: (i) endothelium-derived nitric oxide, and (ii)
KV channels in pulmonary artery smooth muscle cells (PASMCs).
Based on the above results, we further explored the role of κ-OR
in HPH and its potential application for treatment of HPH. The HPH
model was developed by exposing rats to hypobaric and hypoxic environments with 10% oxygen and indices for hemodynamics and
right ventricular (RV) hypertrophy were measured. We found that
intravenous U50,488H significantly lowered mean pulmonary artery
pressure (mPAP) in normoxic control rats and this effect was abolished by administration of nor-BNI. Hypoxia for longer than two
weeks induced severe HPH in rats and intraperitoneal administration
of U50,488H during chronic hypoxia reduced mPAP and attenuated
RV hypertrophy compared with the control group (6). Moreover, acute
intravenous administration of U50,488H after rats were subjected to
Figure 2. Stimulation of κ-OR–initiated pulmonary circulation protection (upper panel) and initiation of PI3K-Akt-eNOS-NO survival signaling (lower
panel). Upper panel: In PAs, U50,488H binds to κ-OR, then relaxes PAs by two separate pathways: endothelium-derived nitric oxide and K V channels in PASMCs. U50,488H treatment inhibits remodeling of PAs during hypoxia. In PASMCs, U50,488H inhibits proliferation of these cells during
hypoxia. In vivo, U50,488H decreases
mPAP, RVP, and RV hypertrophy by activating κ-OR during hypoxia. Additionally, U50,488H balances the concentration of NO, ET, and AII in blood and
lung tissues in HPH rats. Lower panel:
U50,488H binds to κ-OR, leading to the
activation of the PI3K-Akt-eNOS-NO
pathway and may elicit pro-survival and
pulmonary vascular protective effects
including vasodilatation, anti-inflammation, anti-oxidative/nitrative stress, and
anti-apoptosis. Abbreviations: κ-OR,
κ-opioid receptor; PA, pulmonary artery;
PASMC, pulmonary artery smooth cell;
PAEC, pulmonary endothelial cell; MTT,
monotetrazolium; 3H-TdR, [3H]-thymidine;
mPAP, mean pulmonary artery pressure; RVP, right ventricular pressure; RV/
(LV+S), right ventricle (RV)/left ventricle
(LV) + septum (S); RV/BW, right ventricle
(RV)/body weight (BW); NO, nitric oxide;
ET, endothelin; AII, angiotensin II; PI3K,
phosphatidylinositol 3’-kinase; Akt, protein kinase B; eNOS, endothelial nitric
oxide synthase; PMN, polymorphonuclear neutrophil; NADPH, nicotinamide adenine dinucleotide 2’-phosphate; ROS,
reactive oxygen species.
65
Hypoxic Physiology
chronic hypoxia for four weeks significantly lowered mPAP. Thus,
U50,488H has a significant vasorelaxant effect in rat PAs and may have
preventive and therapeutic applications for HPH.
To further investigate the underlying mechanism of the HPH protective effect of κ-OR stimulation with U50,488H, we investigated the effect of U50,488H on PA remodeling and proliferation of PASMCs and
on vasomotor factors such as nitric oxide (NO), endothelin (ET), and
angiotensin II (AII). We found that U50,488H inhibited PA remodeling
compared with the hypoxic group (Figure 1). Moreover, U50,488H also
dose-dependently inhibited proliferation of hypoxia-induced PASMCs.
Compared with the hypoxic group, NO content was higher, whereas
production of ET and AngII were lower in both blood and pulmonary
tissue in the group treated with U50,488H. Our results suggest that inhibition of PA remodeling, PASMC proliferation and beneficial regulation of vasomotor factors may be responsible for the depressive effect
of U50,488H in HPH (7).
In conclusion, we provide for the first time evidence of the precise
location of κ-OR expression in PAs of rats and that κ-OR expression is
upregulated during hypoxia. Further studies demonstrated that κ-OR
stimulation plays an important role in inhibiting remodeling of PAs in
adaptation to chronic hypoxia and inhibits the proliferation of PASMCs.
Additionally, κ-OR stimulation with U50,488H balances the concentration of NO, ET, and AngII in blood and lung tissues, which indirectly
prevents the development of HPH. These findings suggest a potential
preventive and therapeutic effect for κ-OR in a HPH rat model.
Studies suggest that HPH is initiated by hypoxia-induced endothelial
injury of the pulmonary arteriole followed by an imbalance of various
vasomotor factors, PA contraction, and remodeling of pulmonary vessels. NO and NO synthase (NOS) are involved in the occurrence and
development of HPH. We previously found that U50,488H effectively
relaxes the PA ring and depresses mPAP in HPH rats. Moreover, activation of κ-OR reduces myocardial ischemia-induced cell apoptosis
and inhibits inflammation (8, 9). All of these effects are associated with
NO pathways. On the basis of previous studies, we intend to further
investigate the signaling mechanisms underlying the κ-OR-induced
improvement of endothelial function and to confirm the possible role
of NO signaling pathways in the anti-HPH effect mediated by κ-OR.
These experimental results will provide a novel prospective of HPH
and will present evidence for new clinical strategies for the prevention
and treatment of HPH. Based on our studies, we generated a schematic
diagram indicating the κ-OR signaling pathway (Figure 2, upper panel)
and areas of further study (Figure 2, lower panel).
REFERENCES
1. T. S. Güvenç et al., Heart, Lung and Circ. (2012) doi: 10.1016/j.
hlc.2012.08.004.
2. K. K. Tai et al., J. Mol. Cell. Cardiol. 23, 1297 (1991).
3. A. Seelhorst, K. Starke, Arch. Int. Pharmacodyn. Ther. 281, 298 (1986).
4. P. Peng et al., Anat. Rec. 292, 1062 (2009).
5. X. Sun et al., Life Sci. 78, 2516 (2006).
6. J. M. Pei et al., J. Cardiol. Pharmacol. 47, 594 (2006).
7. J. Li et al., Vasc. Pharmacol. 51, 72 (2009).
8. G. Tong et al., Life Sci. 88, 31 (2010).
9. X. D. Wu et al., Cytokine. 56, 503 (2011).
ACKNOWLEDGMENTS
This work was supported by the National Natural Science Foundation
of China (Grant No. 30900535, 30971060, 31100827, 81270402,
and 31200875) and Shaanxi Province (Grant Nos. 2010K01195 and
2011JM4001). We thank Professor Sharon Morey for editing input.
Paracrine-Autocrine Mechanisms in the Carotid Body
Function at High Altitude and in Disease
Fung Man Lung1,3,*, George L. Tipoe2,3, Leung Po Sing4
C
hanges in the chemical composition and pharmacological
activity of substances in the bloodstream evoke reflexive responses of the central autonomic nuclei via the glomus chemoreceptors in the carotid bodies bilaterally located at the
bifurcation of the common carotid artery. Physiological stimuli, notably
Department of Physiology, The University of Hong Kong, Pokfulam, Hong
Kong, China;
2
Department of Anatomy, The University of Hong Kong, Pokfulam, Hong
Kong, China;
3
Research Centre of Heart, Brain, Hormone & Healthy Aging, Li Ka Shing
Faculty of Medicine, The University of Hong Kong, Hong Kong, China;
4
School of Biomedical Sciences, The Chinese University of Hong Kong, New
Territories, Hong Kong, China.
*
1
66
hypoxia, hypercapnea, or acidosis, in addition to circulating chemicals,
stimulate reflexogenic chemoreceptors that trigger afferent impulses
transmitted by the carotid sinus nerve to brainstem nuclei to induce ventilatory and circulatory responses. In addition to these stimuli, recent
data suggest that a number of vasoactive peptides, such as cytokines
and their ligand-binding receptors locally expressed in the carotid body,
could function in a paracrine-autocrine manner to modulate the excitability and activity of carotid chemoreceptors. Additionally, hypoxia
regulates the expression of paracrine-autocrine signaling molecules via
the activation of hypoxia-inducible factors, suggesting that these local
responses play an important role in the functional modulation of the carotid body under chronically hypoxic conditions (1). Thus, these local
regulatory mechanisms may have functional implications with respect
to ventilatory acclimation at high altitudes and the cardiopulmonary
responses to chronic hypoxemia in disease.
Two
S e cSection
t i o n Two
Carotid Body Function at High Altitude
and in Disease
Minute ventilation increases at high altitude and the hypoxic ventilatory response gradually increases over hours
and days at high altitudes. This ventilatory acclimation is
an important physiological adaptive response to chronic
hypoxia. It has been shown to be mediated by the carotid
body, since the response is abolished by the resection or
denervation of this structure. A number of mechanisms can
modulate carotid chemoreceptor activity under hypoxic
conditions, including but not limited to: (i) hypoxia-inducible factor (HIF) pathways responsible for the structural
and functional changes of the carotid body (1); (ii) changes
in circulating or locally produced vasoactive peptides or
factors that alter carotid chemoreceptor activity; (iii) the
multiplicative effects of combined stimuli relevant to sleep
apnea, which occurs in subjects at high altitude or in disease conditions; and (iv) overproduction of reactive oxygen species (ROS) leading to oxidative stress induced by
intermittent hypoxia or sleep apnea in disease conditions
(1).
HIF-1 Pathway in the Carotid Body at High
Altitude
Figure 1. Expression of angiotensin II (AII) type 11 receptors (A, red) in the glomic
clusters expressing tyrosine hydroxylase (TH, B, blue) of the rat carotid body during
chronic hypoxia. (C) Merged image shows localization of the AII type 11 receptor in
the chemosensitive glomus cells (circles). (D) Negative control.
The carotid body enlarges and changes in sensitivity in
response to hypoxic conditions in humans and animals living at high
altitudes, and in subjects exposed to chronic hypoxemia associated with
cardiopulmonary diseases or hematological disorders. In the carotid
body, chronic hypoxemia can modulate the excitability and sensitivity of the chemosensitive glomus cells to chemical signals as well as
stimulate their proliferation and induce remodeling of the vasculature.
HIFs―heterodimeric transcriptional factors―are directly induced by
cellular or tissue hypoxia and regulate HIF-target gene expression in
response. Some of the targeted gene products, including endothelin-1
(ET-1), type II nitric oxide synthase (iNOS), and vascular endothelial
growth factor (VEGF) have important physiological roles in the control
of vascular tone and angiogenesis (1).
HIF-1 target genes expressed in the carotid body are modulated by
chronic hypoxemia, suggesting an active role for HIF-1 during moderate levels of hypoxic stress (2, 3). We have examined the role of HIF-1
and its target genes in the vascular and physiological changes in the
carotid body of rats exposed to chronic hypoxia (inspired normobaric
10% oxygen or an altitude of 5,000 m, for four weeks). In chronic hypoxia, the majority of cells in the carotid body increased protein expression of HIF-1α, a heterodimeric partner of HIF-1 induced by hypoxia
(2, 3). The increased level of HIF-1 activated the transcriptional expression of genes encoding VEGF and VEGF receptors in the carotid
body (2, 3). These changes may mediate angiogenesis for vascular remodeling in the carotid body, which is important for limiting the diffusion distance between the capillary and the chemosensitive glomic
tissue. Additionally, we showed that intracellular calcium responses to
ET-1 were augmented in the chemosensitive glomus cells of the carotid
body with increased ET-1 protein expression during chronic hypoxia
(4). This suggested an enhancement of the paracrine-autocrine effects
of ET-1 on the excitability and mitotic activities of the chemosensitive
glomus cells during chronic hypoxia (4). Moreover, iNOS protein was
localized in glomic clusters in the carotid body. Nitric oxide (NO) concentration in the carotid body was significantly higher in hypoxia than
that under normoxic conditions. Hypoxia-induced NO production was
attenuated by NOS inhibitor L-NAME and by S-methylisothiourea,
a specific inhibitor of iNOS. Increased NO levels potentiated the inhibitory effect of NO on carotid chemoreceptor activity, and therefore
negatively modulated the chemoreflex response to hypoxia (5). Thus,
our results suggest that chronic hypoxemia induces the transcriptional
activity of HIF-1 and regulates the expression of target genes for the
structural remodeling and physiological adaptation of the carotid body.
Local Renin-Angiotensin System Regulated
by Hypoxia in the Carotid Body
Angiotensin receptors are localized in chemosensitive glomus cells in
the carotid body and angiotensin II (AII) can stimulate chemoreceptor activity (Figure 1). The carotid chemoreceptor response to AII is
enhanced in chronically hypoxic rats, which can be explained by increased expression of the AII type 1 receptor in glomic clusters of the
carotid body during chronic hypoxia (6, 7). Interestingly, in addition
to its presence in the blood, AII is also locally produced by the reninangiotensin system (RAS) in the carotid body (7). The RAS component
genes encoding angiotensinogen (an angiotensin precursor) and Ang
receptors are upregulated by hypoxia (7), which might represent an
adaptive response of the carotid body to chronic hypoxia.
Moreover, AII and ROS expressed in the carotid body are involved in
the pathophysiological response to intermittent hypoxia under disease
conditions. We examined the expression of AII type 1 receptor and the
by-products of oxidative stress in the carotid body of rats exposed to
intermittent hypoxia mimicking a severe, obstructive sleep apnea condition (i.e., inspired oxygen levels alternating between 5%–21% per
minute, eight hours per day for up to four weeks). The expression of
AII type 1 receptors was markedly elevated in the glomic clusters of
the carotid body in intermittent hypoxia. Additionally, intracellular calcium responses to AII were significantly enhanced in chemosensitive
glomus cells, which could be abolished by administration of the AII
67
Hypoxic Physiology
Figure 2. Schematic summary of the paracrineautocrine mechanisms that modulate carotid chemoreceptor activity under hypoxic conditions. Arrows (black) show the cascade in the physiological
adaptive response to chronic hypoxic conditions at
high altitude. Red arrows highlight the pathophysiological cascade induced by intermittent hypoxia.
Bidirectional arrows (brown) are proposed interactions or crosstalk among paracrine-autocrine signaling pathways and the interrelationship with oxidative
stress induced by intermittent hypoxia under disease
conditions.
type 1 receptor antagonist losartan. Furthermore,
local expression of nitrotyrosine in the carotid body
and levels of malondialdehyde and 8-isoprostane
in the serum were significantly elevated in intermittent hypoxia, suggesting that the upregulation
of AII type 1 receptor and oxidative stress are significantly involved in the augmented chemoreceptor activity in intermittent hypoxia under disease
conditions (8).
Local Inflammation in the Carotid Body
Recently, we demonstrated increased expression of proinflammatory
cytokines (interleukin-1β, interleukin-6, and tumor necrosis factor-α)
and their receptors in the chemosensitive glomus cells of the carotid
body. These mediate inflammation in the organ, macrophage infiltration, expression of NADPH oxidase subunits, and increased hypoxic
responses of the carotid chemoreceptor during chronic hypoxia (9) or
intermittent hypoxia (10). Importantly, the levels of oxidative stress, local inflammatory responses, and augmented chemoreceptor activity can
be normalized by the daily administration of anti-inflammatory drugs
such as dexamethasone or ibuprofen (10). These results suggest that
inflammatory cytokines functioning in an autocrine-paracrine manner
could be important in the augmented activity of the carotid chemoreceptor and in local inflammation associated with oxidative stress induced by intermittent hypoxia under disease conditions.
hypoxia (1, 3). Additionally, the upregulation of local RAS could be an
important link between oxidative stress and inflammation in the carotid
body. Future studies in these areas may elucidate the causal relationship
and interactions or crosstalk among these paracrine-autocrine signaling
pathways in the modulation of carotid body function at high altitude
and in disease.
Summary and Perspectives
6. P. S. Leung, S. Y. Lam, M. L. Fung, J. Endocrinol. 167, 517 (2000).
In summary, the carotid chemoreceptor, via the chemoreflex, plays an
important role in the ventilatory and circulatory responses to hypoxia
at high altitude. The paracrine-autocrine mechanisms involving HIFregulatory pathways and hypoxia-induced upregulation of the local expression of RAS components and inflammatory cytokines play important roles in the altered carotid chemoreceptor activity under chronically
hypoxic conditions. Under disease conditions, the paracrine-autocrine
mechanisms significantly contribute to augmented chemoreceptor activity, which is also closely linked with oxidative stress induced by intermittent hypoxia. Thus, these local mechanisms are significant parts of
the hypoxia-mediated maladaptive changes of the carotid body function,
which is important in the pathophysiology of sleep apnea (Figure 2).
There is evidence to suggest that HIF pathways mediated by HIF-1
and HIF-2 have different roles in the adaptive or maladaptive responses
of the carotid body, respectively, during chronic hypoxia or intermittent
68
REFERENCES
1. N. R. Prabhakar, G. L. Semenza, Physiol. Rev. 92, 967 (2012).
2. G. L. Tipoe, M. L. Fung, Respir. Physiol. Neurobiol. 138, 143 (2003).
3. S. Y. Lam, G. L. Tipoe, E. C. Liong, M. L. Fung, Histol. Histopathol. 23,
271 (2008).
4. Y. Chen et al., Pflügers Archiv 443, 565 (2002).
5. J. S. Ye, G. L. Tipoe, P. C. W. Fung, M. L. Fung, Pflügers Archiv 444,
178 (2002).
7. M. L. Fung, P. S. Leung, in Frontiers in Research of the Renin-
Angiotensin System on Human Disease, P. S. Leung, Ed. (SpringerVerlag, Heidelberg, 2007), vol. 7, chap. 8.
8. S. Y. Lam, G. L. Tipoe, Y. W. Tjong, E. C. Liong, M. L. Fung, in Life on
the Qinghai-Tibetan Plateau, R. L. Ge, P. Hackett, Eds. (Peking Univ.
Med. Press, China, 2007), pp. 323-329.
9. S. Y. Lam, G. L. Tipoe, E. C. Liong, M. L. Fung, Histochem. Cell Biol.
130, 549 (2008).
10. S. Y. Lam et al., Histochem. Cell Biol. 137, 303 (2012).
ACKNOWLEDGMENTS
Studies were supported by grants from the Research Grants Council,
Hong Kong (Grant No. HKU 766110M, HKU 7510/06M to M.L.F., and
CUHK468912 to P.S.L.) and internal funding from the University Research
Committee, HKU (to M.L.F. and G.L.T).
Beijing Institute for Brain Disorders
The Beijing Institute for Brain Disorders (BIBD) at Capital Medical University (CMU), funded by the Beijing Municipal Government, is a research center aimed at understanding the mechanisms underlying brain disorders, and at
translating results from the laboratory bench to the patient’s bedside, in order to reduce the burdens that the disorders impose on patients, families, and society.
C
MU is one of the largest medical schools in the world, consisting of 10 schools, 18 affiliated hospitals, and 10 teaching hospitals with over 24,000 open hospital beds and
more than 10 million outpatient visits per year. The university and
its affiliated hospitals have a staff of ~20,000, among them over
1,000 professors and over 2,000 associate professors. CMU has
more than 10,000 enrolled graduate and postgraduate students
on campus and provides a wide range of educational programs
for Doctorate, Master’s, and Bachelor’s degrees, as well as certificates. Translational neuroscience research is a particular strength
of CMU, with experienced research teams that have access to
a large and diverse patient population. Research programs and
clinical services in neuroscience and related fields are at the forefront in China with highly specialized hospitals
in neurology (Xuanwu Hospital), neurosurgery (Tiantan and Sanbo Brain Hospitals), psychiatry (Anding Hospital), and rehabilitation (Boai Hospital). The Department of Neurology of Xuanwu Hospital and the Department of
Neurosurgery at Tiantan Hospital have both been ranked as number one in China in their respective disciplines.
In addition, research centers on stroke, Parkinson’s disease, Alzheimer’s disease, epilepsy, brain tumors, and
psychiatric diseases are the best in China, boasting large clinical databases and tissue banks. These centers also
host National Key Laboratories and have been coordination centers for awards from the National Key Initiatives and
Programs. CMU is number one in China with respect to publications in the field of neuroscience in both clinic and
basic research over the past two years.
The creation of BIBD represents a meaningful step forward in term of collaboration and productivity, drawing together
leading neuroscientists from various disciplines and institutions, inside and outside of CMU, to develop better
treatments for a range of neurological and psychiatric conditions. A number of research themes covering various
brain disorders―including Parkinson’s disease, Alzheimer’s disease, stroke, epilepsy, cancer, and psychiatric
disorders―are being pursued using techniques ranging from molecular biology to advanced in vivo studies. BIBD
has established beneficial interactions with many internationally recognized institutions in the field of neuroscience.
In fact, BIBD is a founding member of The International Alliance for Translational Neuroscience (IATN) together
with Department of Neuroscience at Karolinska Institutet, Massachusetts General Hospital, the McGovern Institute
for Brain Research at the Massachusetts Institute of Technology, the Brain Research Center at the University of
British Columbia, and the Florey Institute of Neuroscience at the University of Melbourne. IATN aims to achieve
decisive advancements in neuroscience and associated fields by developing interactions between BIBD and these
prestigious international institutions. BIBD is currently the host institution for IATN.
BIBD is recruiting outstanding faculty members at full professor level in the research areas outlined above. We invite
candidates who are committed to the highest standards of scholarship and professional activities to apply. BIBD
provides a dynamic research environment with faculty actively engaged in translational research in neuroscience.
Applicants should have an established research program that can be expected to continue with a high productivity,
and a strong record of extramural funding. BIBD offers outstanding office, laboratory, and research facilities.
If interested, please e-mail [email protected].
Qinghai Provincial People’s Hospital, China
A modern comprehensive public hospital ranked first among hospitals on the Qinghai-Tibet Plateau
Third grade A-level general hospital
• Named one of the 100 best hospitals in China
• International Research Base for Anoxia Medicine
• National Clinical Research Center for High Altitude Medicine
• Regional Center for Healthcare on the Qinghai-Tibet Plateau
O VERVIEW
Founded in 1927, Qinghai Provincial People’s Hospital is a hospital of Western medicine on the
Qinghai-Tibet Plateau. With a long history of offering the full spectrum of first-aid in addition to inpatient /outpatient healthcare and rehabilitation services, the staff is dedicated to teaching and scientific research. Of the 2,089 permanent staff, 1,892 are healthcare professionals, 313 are professors
and assistant professors, 12 are academic leaders in natural sciences and engineering, and 27
are outstanding specialists awarded a Governmental Special Allowance by the State Council. The
hospital has 56 clinical and medical technology departments, four specialized state-level clinical
sections, 10 provincial key multidisciplinary healthcare centers, and 32 state/provincial diagnostic
centers and medical research/training bases. The clinical laboratory center is the first standardized
laboratory in Qinghai province certified according to ISO 15189 international standards. Qinghai Provincial People’s Hospital has been committed to the advancement of medical techniques
to provide the best health care to the diverse ethnic communities on the Qinghai-Tibet Plateau.
The researchers at the hospital are dedicated to the exploration of the unique characteristics of
high-altitude medicine. With the application of teleconsultation systems, computerized medical
information systems, and refined management mechanisms, they have built the hospital into a
well-functioning comprehensive public service.
ADVANTAGES AND STRENGTHS
Taking full advantage of its unique geographic location, the primary focus of the hospital is clinical
research on the diagnosis and treatment of mountain sickness, endemic disease, and critical care
in a harsh environment (low pressure, oxygen deficiency, and cold temperatures). Impressive advances have been made in the study of the pathophysiological changes stemming from these environmental challenges. Many publications have come out of this work, including the introduction
of the “Qinghai Criteria” as the new standard of diagnostic criteria for chronic mountain diseases.
The hospital holds a leadership position in delivering the best quality health care services on the
Qinghai-Tibet Plateau and has long been a pioneer in the application of advanced medical technologies and techniques.
The hospital has always been active in providing medical assistance for local communities. Following the earthquake in Yushu prefecture, Qinghai province, on April 14, 2010, medical rescue
staff from the hospital delivered timely and efficient medical relief to earthquake-stricken areas.
Notably, there were no injuries from transportation and no deaths from infections after emergency
treatment.
Making full use of the professional knowledge and skills of its specialists and capacity of its multidisciplinary care centers, the hospital regularly offers professional guidance and medical training
for local hospitals in agricultural and pastoral areas, and provides professional health care for a variety of large and medium scale events both within and outside of the country, including two scientific expeditions to the South Pole and ten International Road Cycling Races around Qinghai Lake.
SCIENTIFIC RESEARCH ACHIEVEMENTS
Researchers at the hospital study a broad range of ailments, including high-altitude pulmonary
edema, cerebral edema and functional failure, polycythemia, and other high-altitude–related heart
and kidney diseases. Consistent and unswerving effort has led to significant research achievements, including the publication of over 3,300 journal articles. Thus far, 1,473 hospital-based research programs associated with new services, new technologies, and new surgical advances
have been conducted. These studies have been awarded one first prize and two second prize
National Science and Technology Progress Awards, as well as seven first prize, nine second prize,
and 26 third and fourth prize provincial Science and Technology Progress Awards.
ACADEMIC EXCHANGES
Qinghai Provincial People’s Hospital is committed to training and mentoring medical students from
Qinghai, Lanzhou, Suzhou, and Ningxia Medical Universities in order to further enhance academic cooperation and communication with other national and international medical institutions.
The hospital currently has a long-term cooperative agreement with the Medical Center of Harvard
University in United States, Klinikum Bamberg in Germany, and McMaster University Hospital in
Canada. Within China, it also cooperates with Peking Union Medical College Hospital, Peking University People’s Hospital, China-Japan Friendship Hospital, Beijing Tiantan Hospital, and Beijing
Jishuitan Hospital.
Qinghai Provincial People’s Hospital continues to strive to build itself into one of the best clinical,
research-oriented hospitals in the world.

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