Fernando Vazquez Alaniz CIIDIR IPN Unidad Durango Doctorado

Transcription

INSTITUTO POLITÉCNICO NACIONAL
CENTRO INTERDISCIPLINARIO DE INVESTIGACIÓN PARA EL DESARROLLO
INTEGRAL REGIONAL
Identificación de un elemento regulador común para la expresión de los
genes KiSS1 y REN en placentas de mujeres con preeclampsiaeclampsia comparado con placentas de mujeres
con embarazo normo-evolutivo.
TESIS
QUE PARA OBTENER EL GRADO DE DOCTOR EN CIENCIAS EN
BIOTECNOLOGÍA
PRESENTA
FERNANDO VAZQUEZ ALANIZ
DIRECTORES DE TESIS
DR. CARLOS GALAVIZ HERNÁNDEZ
DRA. LAURENCE ANNIE MARCHAT MARCHAU
Victoria de Durango, Dgo., Julio de 2012
THIS RESEARCH WAS DONE IN THE MOLECULAR BIOLOGY CIIDIR-IPN DURANGO
UNIT, MOLECULAR BIOLOGY LABORATORY AT SCIENTIFIC RESEARCH INSTITUTE
OF UNIVERSIDAD JUAREZ DEL ESTADO DE DURANGO, MÉX. UNDER DIRECTION
OF PhD. CARLOS GALAVIZ HERNÁNDEZ AND PhD JOSÉ MANUEL SALAS PACHECO
AND
IN
THE
BIOCHEMISTRY
LABORATORY
AT
MEDICINE
SCHOOL
OF
UNIVERSIDAD AUTÓNOMA DE SAN LUIS POTOSÍ, MÉX. UNDER DIRECTION OF
PhD. CLAUDIA CASTILLO MARTÍN DEL CAMPO
European Journal of Obstetrics & Gynecology and Reproductive Biology 159 (2011) 67–71
Contents lists available at ScienceDirect
European Journal of Obstetrics & Gynecology and
Reproductive Biology
journal homepage: www.elsevier.com/locate/ejogrb
Comparative expression profiles for KiSS-1 and REN genes in preeclamptic
and healthy placental tissues
Fernando Vazquez-Alaniz a,b, Carlos Galaviz-Hernandez a,*, Laurence A. Marchat c,
José M. Salas-Pacheco d, Isaı́as Chairez-Hernandez e, José J. Guijarro-Bustillos f,
Alberto Mireles-Ordaz f
a
Academia de Genómica, Centro Interdisciplinario de Investigación para el Desarrollo Integral Regional, IPN Unidad Dgo., Durango, Zip Code 34220, Mexico
Becario PIFI, Doctorado en Ciencias en Biotecnologıá, Nodo Durango, SIP-IPN, Mexico
c
Escuela Nacional de Medicina y Homeopatıá IPN, Mexico City, Zip Code 07320, Mexico
d
Instituto de Investigación Cientı´fica, Universidad Juárez del Estado de Durango, Durango, Dgo., Zip Code 34000, Mexico
e
Academia de Entomologıá, Centro Interdisciplinario de Investigación para el Desarrollo Integral Regional, IPN Unidad Dgo., Durango, Zip Code 34220, Mexico
f
Servicio de Ginecologıá y Obstetricia, Hospital General de Durango, Durango, Dgo., Zip Code 34000, Mexico
b
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 8 March 2011
Received in revised form 4 July 2011
Accepted 11 July 2011
Objective: The aim of the present work was to look at differences in the placental tissue expression of
KiSS-1 and REN genes from preeclamptic and healthy pregnant women, that could account for a possible
synergistic function for both genes in the pathogenesis of preeclampsia.
Study design: This case–control study involved 27 preeclamptic women and 27 normoevolutive
pregnant women. cDNA was obtained from placental tissue to carry out qPCR for both KiSS-1 and REN
genes in order to compare mRNA expression levels in the studied groups. Statistical analysis showed
expression differences that correlate with clinical and/or biochemical variables.
Results: Higher expression for KiSS-1 in PEE vs. control woman (p = 0.001) was observed, whereas no
difference was observed for REN expression (p = 0.300) when all the subjects were included. However,
REN expression was significant higher when the samples were stratified according to preeclampsia
severity. For 18 mild preeclamptic patients the p-value was p = 0.001 compared to their controls, while
for the remaining nine with severe preeclampsia the expression became significant (p = 0.001).
Conclusion: Our results suggest that the high KiSS-1 expression seen in preeclamptic patients is in
accordance with its role as an inhibitor of trophoblast invasiveness and maintained until the end of
gestation. On the other hand, aggressive therapeutic management and/or severity status of patients have
a direct effect on placental REN expression levels, masking the natural high expression of this gene on
preeclamptic placental tissue. Therefore it was not possible to establish a real concordant expression
profile for KiSS-1 and REN genes.
ß 2011 Elsevier Ireland Ltd. All rights reserved.
Keywords:
Placental co-expressed genes
Preeclampsia
KiSS-1
REN
1. Introduction
Preeclampsia (PEE) is one the leading causes of fetal and
maternal morbimortality in the world [1]. PEE is classified as mild
[blood pressure (BP) 140/90 mm Hg and proteinury 30 mg/dL]
and severe (BP 160/110 mm Hg and proteinury 2000 mg/dL).
Many different theories [2] have been advanced to understand its
basis. However, a clear deficiency of trophoblast invasiveness (TI)
to maternal spiral arteries in placentas from early and term
* Corresponding author at: Academia de Genómica, CIIDIR-IPN Unidad Durango,
Sigma #119 Fracc 20 de Noviembre II Durango, Dgo, Mexico c.p. 34220, Mexico.
Tel.: +52 618 814 2091; fax: +52 618 814 4540.
E-mail addresses: [email protected], [email protected]
(C. Galaviz-Hernandez).
0301-2115/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.ejogrb.2011.07.019
pregnancies [3–6] is central to all of them. A number of genes have
been involved in trophoblast invasiveness (TI): LEP [7], MMP-9 [8],
INSL4, KiSS1-R and KiSS-1 [9], being the last highly expressed in
syncytiotrophoblast cells [6].
Kisspeptin 10, the protein product of KiSS-1 gene, reduces the
normal invasiveness of trophoblast [4] through control of its
migratory properties [9]. Decreased plasma levels of this peptide
were reported during pregnancy [10]. Normal TI processes guide
the normal transformation of spiral arteries in the myometrial
segments and the impairment of this transformation [11] is
noticed in preeclamptic patients [12]. It is suggested that TI of the
spiral arteries is a continuous process [13,14] until the end of
pregnancy.
An important finding was the discovery of a functional renin
angiotensin system [RAS] in human [15,16] and rodent [17]
placental tissue. The major extra-renal RAS producer during
68
F. Vazquez-Alaniz et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 159 (2011) 67–71
pregnancy is the placenta [18]. Renin, the protein product of REN
gene, is a natural vasoconstrictor expressed during all pregnancy
[19]. Increases in the expression of some components of RAS were
noticed in serum of healthy pregnant women compared to those of
preeclamptic patients [20]. Thus, poor TI and generalized
endothelial dysfunction [21,22] are key players in the physiology
of the disease.
On the other hand, Nistala et al. [23] isolated a PAC that includes
the adjacent genes REN and KiSS1. Further studies confirmed the
expression of those genes in placental tissue [24,25].
Some neighbor genes are co-expressed in common metabolic
pathways [26] through shared promoters and transcription factors
binding sites [27]. So it is tempting to think about the coparticipation of KiSS1 and REN genes in PEE development. The aim
of the present study was to look for differences in expression of
KiSS-1 and REN genes in placental tissue between PEE and healthy
pregnant women, assuming a possible concordant expression of
both genes in the pathogenesis of PEE.
2. Materials and methods
equipment, using FAMTM dye-labeled TaqMan1 MGB probes
(Applied Biosystems). KiSS-1 probe was based on RefSeq
NM_002256.3; assay ID Hs00158486_m, which targets exons 2–
3. REN probe was based on RefSeq NM_000537.3, assay ID
Hs00166915_m1, targeting exons 1–2.
The relative expression (RQ) of KiSS-1 and REN genes was
normalized to the constitutive and endogenous control human
gene beta-2-microglobulin (B2M) (FAM/MGB Probe, Non-Primer
Limited RefSeq NM_ 004048.2), (Applied BiosystemsTM). This
probe targets exons 2–3. Amplification efficiencies with values
between 90 and 110% allowed establishing an optimal working
concentration of 30 ng/mL. An expression test was also randomly
run for B2M gene in five patients and five control samples in
triplicate, to estimate significant differences between groups and
determine the optimal PCR cycle at which fluorescence gave the
better exponential cycle. The qPCR conditions were: 10 min of
initial hold at 95 8C, followed by 48 cycles of 15 s at 95 8C for
denaturing and 1 min at 60 8C for both annealing and extension.
Cycle threshold values were obtained by triplicate and each sample
was normalized with B2M endogenous control gene to calculate
DDCt
RQ values or (2
) for KiSS-1 and REN.
2.1. Selection of patients
2.4. Statistical analysis
This case–control study was approved by an investigation
ethical committee in the Hospital General de Durango México, in
accordance with The Code of Ethics of the Declaration of Helsinki.
Twenty-seven preeclamptic women under anti-hypertensive
treatment and the same number of healthy pregnant women,
agree to donate their placentas for the study after signing an
informed consent letter.
Pairing was based on chronological age, gestational age and the
number of pregnancies, as they are known to affect placental gene
expression [28,29].
Inclusion criteria were those established by the Working Group
Report on High Blood Pressure in Pregnancy and patients classified
according to severity status in mild preeclampsia [blood pressure
[BP] 140/90 mm Hg, measured twice within a 2 h interval and
proteinury 300 mg/dL or 1+ inlab stick urine analysis)] or severe
preeclampsia [BP 160/110 mm Hg, on two or more occasions 6 h
apart and proteinury 2 g or more in 24 h or 2+ qualitatively) with
new onset hypertension after 20 weeks gestation [30].
All patients under treatment with glucocorticoids, affected by
gestational hypertension, gestational diabetes, diabetes mellitus
types 1 and 2 and with hepatic or kidney diseases were excluded. A
questionnaire with clinical relevant data was also obtained from all
participants.
Media, range, median quartile and coefficient of variation were
calculated for all clinical and biochemical features of the
population. The expression of KiSS-1 and REN genes in placentas
from both PEE and normoevolutive pregnancies (NEP) women was
compared using the Student’s t-test one way for independent data.
A cluster was created to stratify patients using the significant
variables reported [31] in Mexican preeclamptic populations. A
neural network analysis feed forward was done to find the most
important variables. A new Student’s t-test was performed for
subgroups to compare REN expression between PEE patient’s
subgroups. A final correlation test was run out between relative
expression gene and clinical and biochemical features of the
patients. All statistical analysis was performed with Statistic1
software V.7.0.
3. Results
Twenty-seven placental samples from PEE patients and the
same number from healthy controls were studied. The media and
coefficient range for chronological age was 20.0 7.00 years. The
media and coefficient variation value for gestational age was
38.8 2.8 weeks. Nulliparity was found in 22 cases and 5 were
2.2. RNA extraction and cDNA synthesis
Within 30 min after delivery, placentas were washed with cold
PBS at pH 7.4. Twelve different 5 5 mm biopsies were taken from
maternal side of the placenta. Fifty to one hundred milligrams of
the sample was kept in RNAlater1 (AmbionTM) at 4 8C until RNA
extraction with TRIpure isolation reagent1 kit (ROCHETM). RNA
was treated with DNA-free1 (AmbionTM) and its integrity was
verified by 1% agarose gel electrophoresis. Samples were stored at
72 8C until cDNA synthesis, which was carried out using the High
Capacity cDNA Reverse Transcription1 (Applied BiosystemsTM) kit,
according to the manufacturer protocol; quantity and purity were
evaluated through spectrophotometry. The synthesized cDNA was
kept at 20 8C until further analysis.
2.3. qPCR
Gene expression was analyzed by semi-quantitative polymerase chain reaction (qPCR) in a SteptOneTM Applied Biosystems
Table 1
Clinical characteristics for PEE and NEP patients.
Clinical characteristics
PEE group
(n = 27)
NEP group
(n = 27)
p-Value
Age (years)
Gestational age (weeks)
PEE antecedent (%)
Primiparity/multiparity cases
Delivery form (cesarean/vaginal)
cases
Mean arterial pressure in mm Hg
PEE classification (mild/severe)
cases
Newborn weight (pounds)
Serum uric acid (mmol/L)
Treatment rate until delivery (h)
22.2 28.3a
38.3 2.8a
22%
19/8
22/5
22.2 28.3a
38.3 2.8a
3%
17–10
3/24
NA
NA
0.000b
0.300b
0.001b
120.9 9.9a
8/19
86.4 4.6a
NEP
0.000b
NA
5.88 19.5a
354.5 24a
5.5 39.6a
6.77 9.9a
154.9 23.3a
4.8 24.5a
0.001b
0.000b
0.300b
Significant differences were appreciated for PEE antecedent, delivery form, MAP,
newborn weight and serum uric acid.
a
Media coefficient variation.
b
Student’s t-test.
multiparous. Preeclamptic (PEE) patients displayed higher values for
mean arterial pressure (MAP) (120.9 9.9 vs. 86.4 4.6 mm Hg,
p = 0.000), and higher values for uric acid blood (354.9 24 vs.
154.9 23.3 mmol/L (p = 0.000) than normoevolutive pregnant
(NEP) women (Table 1).
The comparison of KiSS-1 and REN placental expression
revealed higher KiSS-1 expression levels in preeclamptic
(1.99 0.80 and 0.99 0.47; p = 0.001), than healthy women. In
contrast, no significant differences (p = 0.300) between PEE
(1.25 0.51) and NEP (1.06 0.46) for REN relative expression
analysis were found (Fig. 1).
As renin regulates blood pressure, it is expected that a
treatment aimed to control it could modify REN expression.
Therefore a more detailed cluster analysis for this gene was done.
The considered variables for this analysis were PEE classification,
MAP, uric acid, proteinuria, PEE history, creatinine, urea, edema,
gestational age, drugs used for hypertension control and management time which revealed the presence of two clearly distinguishable subgroups n = 18 and n = 9 (circularized) (Fig. 2).
Resulting subgroups were analyzed again, revealing a high
expression for REN in 18 mild preeclamptic women (1.44 0.51)
vs. their corresponding controls (0.86 0.39) (p = 0.001) (Fig. 3). The
Fig. 1. Comparative expression analysis for KiSS1 and REN genes. Student’s t-test
showed differences for KiSS1 but not for REN gene placental expression between
groups.
69
Fig. 3. REN gene expression level analysis for PEE subgroups. Over-expression was
observed in 18 mild preeclampsia affected patients subgroup (left). Subexpression
was advertised in the remaining 9 severe patients treated with as least three control
blood pressure drugs in regard to their control (right). Significative Student’s t-test,
p 0.005.
high expression observed correlated with MAP, low proteins, urea and
uric acid high concentrations in serum, newborn low weight, PEE
history, increase of osteotendinous reflex (OTR) and the number of
pregnancies (p 0.05).
A decreased REN expression was found to be statistically
significant (p = 0.001) in the remaining 9 severe preeclamptic
patients (0.87 0.51 and 1.46 0.30,) compared to their controls
(Fig. 3). These 9 affected patients were analyzed for all clinical and
biochemical variables through neural network analysis feed forward,
obtaining a classification of 100% for training data and 80% for test
data. The analysis revealed that the 5 most important variables were:
vomit, use of at least 3 hypertension control drugs, hepatalgy, alkaline
phosphatase and treatment time before pregnancy resolution. All
these data agree with those obtained in dendrogram.
The high KiSS-1 expression in PEE patients correlates with
serum uric acid, newborn low weight, PEE history and number of
pregnancies, meanwhile the high REN expression correlated with
mean arterial pressure (MAP), used anti-hypertensive drugs and
newborn weight (Table 2).
Table 2
Correlation test for distinct analyzed variables with gene expression.
Fig. 2. Cluster diagram for PEE and NEP patients. Dendogram grouped the whole
sample in two major groups. PEE sub-group was further divided, circle denotes the
subgroup of PEE patients with MAP 126 mm Hg and the use of anti-hypertensive
drugs as common variables.
Variable
Gene
Correlation
coefficient, r2
p-Value
Mean arterial pressure
KiSS1
REN
0.137
0.454
0.490
0.017
Serum uric acid
KiSS1
REN
0.399
0.067
0.039
0.730
PEE history
KiSS1
REN
0.386
0.071
0.05
0.72
Pregnancies number
KiSS1
REN
0.387
0.230
0.042
0.574
Used anti-hypertensive drugs
KiSS1
REN
0.172
0.490
0.390
0.009
Newborn weight
KiSS1
REN
0.412
0.421
0.035
0.030
OTR
KiSS1
REN
0.217
0.402
0.25
0.03
Correlation test for clinical variables and gene expression in PEE patients. As can be
appreciated, REN gene expression correlates tightly with the use of antihypertensive medications (significance p-value 0.05 and CI 95%).
70
Differences for gene expression were evaluated in the studied
groups in regard to pregnancy way resolution (cesarean section vs.
vaginal delivery). Observed p-values for KiSS-1 (p = 0.062) and REN
(p = 0.387) in PEE patients and control subjects KiSS1 (p = 0.276)
and REN (p = 0.582) did not reveal such differences.
4. Comment
Some examples are known of genes clustered [26,27] in the
same chromosomal regions. Those genes used to be linked
throughout evolution sharing expression and related functions
in certain organs [24,25]. In this case–control study, gene
expression profiles for two contiguous genes KiSS-1 and REN
were compared in placentas of preeclamptic vs. healthy pregnant
women.
The main clinical features in our population are in agreement
with those observed in previous reports. However, in the present
study most preeclamptic women delivered by cesarean section
whereas controls did it through normal labor. No differences in
KiSS-1 or REN gene expression were observed between PEE and
NEP subjects in regard to delivering way, despite it is known to
affect placental gene expression [32,33].
KiSS-1 is a metastatic inhibitor for many different types of
cancer [34]. We observed a higher expression of KiSS-1 in 24
among 27 preeclamptic placental tissues vs. those of normoevolutive pregnant women. Matrigel analysis [35] demonstrated an
altered migration pattern related to KiSS-1 and PIGF high
expression [9] in normal trophoblast cells and choriocarcinoma
cells. An increase of Kisspeptins in serum of patients with PEE [36]
has also been reported. Thus, the observed KiSS-1 high expression
could be related to impaired early trophoblast migration and
maintained throughout pregnancy.
The presence of a placenta-specific RAS [37] showing differences of gene expression [20] between PEE women and NEP has
been reported. We did not find statistically significant differences
for REN expression between PEE and NEP groups as a whole.
However, 18 mild affected patients showed a higher REN
expression with regard to their respective controls. This high
expression pattern has already been reported in preeclamptic
uterine placental beds [38] and also in maternal decidua of PEE
patients [39,40]. Our sampling technique removed all decidual
tissue, implying that the higher REN expression can also be found
in chorionic tissue.
Interestingly, the observed high expression coincides with a
mild disease state and the use of one or two of the following drugs:
alpha methyl dopamine and hydralazine. This suggests that
neither alpha methyl dopamine nor hydralazine alone or in
combination, affects the expression of REN at least in placenta.
All preeclamptic patients were subjected to different antihypertensive drugs regimes depending on severity status: mild/
severe. KiSS-1 expression remains high in most preeclamptic
women, independently of degree of severity and corresponding
treatment. This could be expected as anti-hypertensive drugs are
not aimed to affect TI driven by KiSS-1.
On the other side, a differential behavior for REN expression
was advertised in preeclamptic patients depending on severity
status and concomitant treatment. A higher expression of REN in
18 mild preeclamptic patients compared to the remaining 9
severe affected patients was observed. The last subgroup were
treated with three (alpha methyl dopamine, hydralazine and
magnesium sulfate) or 4 (alpha methyl dopamine, hydralazine,
magnesium sulfate and nifedipine) antihypertensive drugs. These
9 severe preeclamptic patients presented a decreased REN
expression. This lower expression pattern could be explained
by two main possibilities: (1) the lowering of REN expression is
due to the combined actions of the 3 or 4 antihypertensive drugs
or (2) the severity of the disease explains such decrease in
expression. To date, there is no report that supports the first
assumption. However, nine differentially expressed genes were
reported [41] between severe early onset vs. mild late onset
preeclampsia.
The high KISS-1 and REN expression was also found associated
[42] to high MAP values and newborn low weight, events
commonly seen in preeclampsia. This is in direct relationship
with a deficient uterus/placental blood perfusion, avoiding the
proper transfer of nutrients from the mother to growing foetus
[39,43].
One concern is that the observed changes in gene expression
could be considered more a consequence than a cause of the
disease. However this is difficult to prove due to ethical
constraints imposed by the time of onset of illness and the taking
of samples.
KiSS-1 and REN genes are involved in two of the main
phenomena related to preeclampsia: trophoblast invasiveness
impairment and hypertension respectively. So, it is possible to
suggest the existence of a common regulatory mechanism for both
genes based on (1) the physical contiguity for both genes on
chromosome 1q32.1 [23] and (2) the presence of a couple of
functional enhancers directing the expression of REN gene in
kidney and placenta [44]. Then high expression levels for both
genes would be expected in this pathology.
Here, we did not provide definitive evidence of a parallel
expression for both KiSS-1 and REN genes in preeclamptic
patients. However we cannot discard this phenomenon because
of the presence of two variables which could mask the real
expression profile for REN: severity status and concomitant
treatment.
In this sense we found that the milder the presentation of the
disease, the higher the expression of REN. Therefore a study
including preeclamptic patients with the same severity status
could support our hypothesis.
We also found that a less aggressive hypertension management
is coincident with a higher expression of REN in placenta. Thus, it is
possible that a treatment not affecting REN expression in placenta
would leave them as high as those observed for KiSS-1.
Further studies are needed that focus on the molecular
mechanism of transcriptional regulation of these genes. Our data
also evidenced that REN could be an interesting therapeutic target
to control PEE [45].
Funding
This work was supported by CONACyT/México (Grant Number
2009-01-111870) and Secretaria de Posgrado e Investigación, del
Instituto Politécnico Nacional (SIP 20090313).
Contributors
Vazquez-Alaniz F., first author, field and experimental work,
analysis of results, manuscript writing. Galaviz-Hernandez C.,
corresponding author, design of the study, results analysis,
manuscript writing. Marchat L.A., manuscript writing, results
analysis. Salas-Pacheco J.M., guidance during experimental work
development, results analysis. Chairez-Hernandez I., statistical
analysis of data. Guijarro-Bustillos J.J., patient collection data.
Mireles-Ordaz A., collecting placentas.
Acknowledgement
Dr. Ramaiah Nagaraja from Lab of Genetics NIA/NIH for its
critical reading and suggestions on the manuscript.
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[31] Peralta Pedrero ML, Gúzman Ibarra F. MdlA., Bassavilvazo Rodrı́guez MA, et al.
Elaboración y validación de un Índice para el diagnóstico de preeclampsia.
Ginecol Obstet Mex 2006;74:205–14.
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in labouring and non-labouring human placenta near term. Mol Hum Reprod
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human placenta during labor. Placenta 2010;31:698–704.
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kisspeptin (KiSS-1)/GPR54 system in cancer biology. Cancer Treat Rev 2008.
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extravillous trophoblast proliferation, migration and invasiveness. Placenta
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peptide system with a role in reproduction, cancer and the cardiovascular
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in normal and pre-eclamptic pregnancies. Ther Adv Cardiovasc Dis 2008;
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[44] Galea P, Barigye O, Wee L, Jain V, Sullivan M, Fisk NM. The placenta contributes
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[45] Ziebell BT, Galan HL, Anthony RV, Regnault TR, Parker TA, Arroyo JA. Ontogeny
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placental growth restriction. Am J Obstet Gynecol 2007;197(420):e421–5.
I, Fernando, thank my infinitely wise and
beautiful wife for patience, support, and
love throughout this project, and my
daughters Ana Lizeth and Andrea for
putting my life into the right perspective.
GENERAL INDEX
Glossary...................................................................................................................... I
ABBREVIATIONS.................................................................................................... VIII
FIGURES INDEX ....................................................................................................... XI
TABLES INDEX .......................................................................................................XIV
ABSTRACT...............................................................................................................XV
RESUMEN ...............................................................................................................XVI
BACKGROUND ......................................................................................................XVII
CHAPTER I ................................................................................................................. 1
ANTECEDENTS ......................................................................................................... 1
1.1 Pregnancy ............................................................................................................ 1
1.2 Placental Anatomy .............................................................................................. 1
1.3 Cell types of the placenta ................................................................................... 5
1.4 Implantation and Placentation ........................................................................... 6
1.5 The trophoblast invasion and spiral artery remodeling................................... 8
1.6 Placental disorders ............................................................................................. 9
1.7 Preeclampsia ....................................................................................................... 9
1.8 Etiology and classification of preeclampsia. .................................................. 14
1.9 Classification of preeclampsia-eclampsia ...................................................... 17
1.10 Complications of preeclampsia ..................................................................... 22
1.11 Genetic of preeclampsia ................................................................................. 22
1.12 Trophoblastic invasion ................................................................................... 27
1.13 Renin-Angiotensin System............................................................................. 28
1.14 REN gene ......................................................................................................... 31
1.15 KiSS-1 gene ..................................................................................................... 32
1.16 Transcriptional control of gene expression.................................................. 36
1.17 Enhancer elements ......................................................................................... 40
1.18 Enhancers related with REN gene expression ............................................. 42
CHAPTER II .............................................................................................................. 44
II. JUSTIFICATION ................................................................................................... 44
CHAPTER III ............................................................................................................. 45
III. OBJECTIVES....................................................................................................... 45
3.1 General objective .............................................................................................. 45
3.2.1 Specific objectives ......................................................................................... 45
3.3 Hypothesis ......................................................................................................... 46
CHAPTER IV............................................................................................................. 47
IV. MATERIAL AND METHODS............................................................................... 47
4.1 KiSS1 and REN Expression gene at placental tissues from preeclamptic
women .................................................................................................................. 47
4.1.1 Selection of patients ...................................................................................... 47
4.1.2 RNA extraction and cDNA synthesis ............................................................ 48
4.1.3 qPCR................................................................................................................ 48
4.2 Searching of possible regulator elements ...................................................... 49
4.2.1 PCR amplification and cloning...................................................................... 49
4.2.2 Bacterial strain and plasmids culture media ............................................... 51
4.2.3 Escherichia coli and culture media............................................................... 51
4.2.4 Development of constructs to assess regulation on promoter KiSS-1 gene
............................................................................................................................... 55
4.2.5 Construction of expression plasmids .......................................................... 55
4.2.6 BeWo culture cells ......................................................................................... 60
4.2.7 Viability and cellular count ............................................................................ 61
4.2.8 Transfection and luciferase assays ............................................................. 62
4.2.9 Transfection efficiency ................................................................................. 63
CHAPTER V.............................................................................................................. 64
V. RESULTS ............................................................................................................. 64
5.1 Sample selection ............................................................................................... 64
5.2 Expression of KiSS-1 and REN genes ............................................................. 65
5.3 Construction of recombinant plasmids containing ChE and KE enhancers 74
5.4 Transfection and luciferase expression analysis........................................... 79
CHAPTER VI............................................................................................................. 84
VI DISCUSSION........................................................................................................ 84
CHAPTER VII............................................................................................................ 89
VII. CONCLUSIONS. ................................................................................................ 89
Cited Bibliography .................................................................................................. 90
Acknowledgements............................................................................................... 113
ANNEXES ............................................................................................................... 115
ACHIEVEMENTS AND AWARDS RECEIVED DURING THIS THESIS................. 132
Glossary
Terminology Associated with Molecular Biology and Molecular Genetics, Cell
Tissue and Organ Culture.
Amplification efficiency (EFF %): Calculation of efficiency of the PCR amplification.
The amplification efficiency is calculated using the slope of the regression line in the
standard curve. A slope close to -3.32 indicates optimal, 100% PCR amplification
efficiency.
Amplification plot: Display of data collected during the cycling stage of PCR
amplification
Asepsis: Without infection or contaminating microorganisms.
Aseptic technique: Procedures used to prevent the introduction of fungi, bacteria,
viruses, mycoplasma or other microorganisms into cell, tissue and organ culture.
Although these procedures are used to prevent microbial contamination of cultures,
they also prevent cross contamination of cell cultures as well. These procedures may
or may not exclude the introduction of infectious molecules.
Attachment efficiency: The percentage of cells plated (seeded, inoculated) which
attach to the surface of the culture vessel within a specified period of time. The
conditions under which such a determination is made should always be stated.
Cell culture: Term used to denote the maintenance or cultivation of cells in vitro
including the culture of single cells. In cell cultures, the cells are no longer organized
into tissues.
Cell generation time: The interval between consecutive divisions of a cell.
Cell line: A cell line arises from a primary culture at the time of the first successful
subculture. The term "cell line" implies that cultures from it consist of lineages of cells
originally present in the primary culture.
I
Cell strain: A cell strain is derived either from a primary culture or a cell line by the
selection or cloning of cells having specific properties or markers. In describing a cell
strain, its specific features must be defined.
Cesarean birth: Delivery of a baby and the placenta through an incision made in a
woman’s abdomen and uterus.
Chemically defined medium: A nutritive solution for culturing cells in which each
component is specifiable and ideally, is of known chemical structure.
Clone: In animal cell culture terminology a population of cells derived from a single
cell by mitoses.
Cloning efficiency: The percentage of cells plated (seeded, inoculated) that form a
clone. One must be certain that the colonies formed arose from single cells in order to
properly use this term. (See Colony forming efficiency)
Colony forming efficiency: The percentage of cells plated (seeded, inoculated) that
form a colony.
Comparative CT ǻǻCT) method: Method for determining relative target quantity in
samples. With the comparative Cɬ (ǻǻCɬ) method, the StepOne™ software
measures amplification of the target and of the endogenous control in samples and in
a reference sample. Measurements are normalized using the endogenous control
Continuous cell culture: A culture which is apparently capable of an unlimited
number of population doublings; often referred to an as immortal cell culture. Such
cells may or may not express the characteristics of in vitro neoplastic or malignant
transformation.
Cryopreservation: Ultra-low temperature storage of cells, tissues. This storage is
usually carried out using temperatures below -100°C.
II
Differentiated: Cells that maintain, in culture, all or much of the specialized structure
and function typical of the cell type in vivo.
Dilution serial factor: In the StepOne™ software, a numerical value that defines the
sequence of quantities in the standard curve. The serial factor and the starting
quantity are used to calculate the standard quantity for each point in the standard
curve.
Eclampsia: Seizures occurring in pregnancy and liked to high blood pressure.
Endocrine cell: In animals, a cell which produces hormones, growth factors or other
signaling substances for which target cells, expressing the corresponding receptors,
are located at a distance.
Endogenous control or “Housekeeping”: A target or gene that should be
expressed at similar levels in all samples you are testing. Endogenous controls are
used in relative standard curve and comparative Cɬ (ǻǻCɬ) experiments to normalize
fluorescence signals for the target you are quantifying
Epigenetic event: Any change in a phenotype which does not result from an
alteration in DNA sequence. This change may be stable and heritable and includes
alteration in DNA methylation, transcriptional activation, translational control and
posttranslational modifications
Finite cell culture: A culture which is capable of only a limited number of population
doubling after which the culture ceases proliferation.
Habituation: The acquired ability of a population of cells to grow and divide
independently of exogenously supplied growth regulators.
Housekeeping gene: A gene that is involved in basic cellular functions and is
constitutively expressed. Housekeeping genes can be used as endogenous controls
III
Immortalization: The attainment by a finite cell culture, whether by perturbation or
intrinsically, of the attributes of a continuous cell line. An immortalized cell is not
necessarily one which is neoplasically or malignantly transformed.
Induction: Initiation of a structure, organ or process in vitro.
In vitro propagation: Propagation of cells in a controlled, artificial environment, using
plastic or glass culture vessels, aseptic techniques and a defined growing medium.
In vitro transformation: A heritable change, occurring in cells in culture, either
intrinsically or from treatment with chemical carcinogens, oncogenic viruses,
irradiation, transfection with oncogenes, etc. and leading to the acquisition of altered
morphological, antigenic, neoplastic, proliferative or other properties. This expression
is distinguished form. The type of transformation should always be specified in any
description.
Liposome: A closed lipid vesicle surrounding an aqueous interior; may be used to
encapsulate exogenous materials for ultimate delivery of these into cells by fusion
with the cell.
Mutant: A phenotypic variant resulting from a changed or new gene.
Negative control: In the StepOne™ software, the task for targets or SNP assays in
wells that contain water or buffer instead of sample. No amplification of the target
should occur in negative control wells. Previously called no template control (NTC).
Paracrine: In animals, a cell which produces hormones, growth factors or other
signaling substances for which the target cells, expressing the corresponding
receptors, are located in its vicinity, or in a group adjacent to it.
Passage: The transfer or transplantation of cell, with or without dilution, from one
culture vessel to another. It is understood that nay time cells are transferred from one
vessel to another, a certain portion of the cells may be lost and, therefore, dilution of
cells, whether deliberate or not, may occur.
IV
Passage number: The number of times the cells in the culture have been
subcultured or passaged. In descriptions of this process, the ration or dilution of the
cells should be stated so that the relative cultural age can be ascertained.
Pathogen free: Free from specific organisms based on specific tests for the
designated organisms.
Placenta: Tissue that provides nourishment to and takes away waste from the fetus.
Population density: The number of cells per unit area or volume of a culture vessel.
Preeclampsia: A condition of pregnancy in which there is high blood pressure, and
protein present in the urine.
Primary culture: A culture started from cells, tissues or organs taken directly from
organisms. A primary culture may be regarded as such until it is successfully
subcultured for the first time. It then becomes a "cell line".
Reference sample: Used in relative standard curve and comparative Cɬ (ǻǻCɬ)
experiments. The sample used as the basis for relative quantitation results. Also
called the calibrator.
Relative standard curve method: This method requires running a standard curve for
both the gene of interest and the endogenous control. The template for the standard
curve can be any cDNA sample that expresses both the gene of interest and
endogenous control. It is important to note that the expression level of your sample
should fall within the limits of your standard curve. In other words, if the sample CT
value is outside of the standard curve, you should dilute your sample such that the CT
value falls between the highest dilution and the lowest dilution of the standard curve
Reporter: A fluorescent dye used to detect amplification. If you are using TaqMan®
reagents, the reporter dye is attached to the 5' end.
V
Saturation density or Confluence: The maximum cell number attainable, under
specified culture conditions, in a culture vessel. This term is usually expressed as the
number of cells per square centimeter in a monolayer culture or the number of cells
per cubic centimeter in a suspension culture.
Standard curve: In standard curve and relative standard curve experiments: The
best-ILW OLQH LQ D SORW RI WKH &ɬ YDOXHV IURP WKH VWDQGDUG UHDFWLRQV SORWWHG DJDLQVW
standard quantities. See also regression line. A set of standards containing a range of
known quantities. Results from the standard curve reactions are used to generate the
standard curve. The standard curve is defined by the number of points in the dilution
series, the number of standard replicates, the starting quantity, and the serial factor.
See also standard dilution series.
Standard dilution series: a set of standards containing a range of known quantities.
The standard dilution series is prepared by serially diluting standards.
Sterile: Without Life.
Subculture: With culture cells, this is the process by which the tissue is first
subdivided, then transferred into fresh culture medium.
TaqMan® reagents: PCR reaction components that consist of primers designed to
amplify the target and a TaqMan® probe designed to detect amplification of the target.
Target: The nucleic acid sequence that you want to amplify and detect by PCR
method.
Tissue culture: The maintenance or growth of tissues, in vitro, in a way that may
allow differentiation and preservation of their architecture and/or function.
Transfection: The transfer, for the purposed of genomic integration, of naked, foreign
DNA into cells in culture. The traditional microbiological usage of this term implied
that the DNA being transferred was derived from a virus.
VI
Transformation: In cell culture, the introduction and stable genomic integration of
foreign DNA into a cell by any means, resulting in a genetic modification.
Threshold: The level of fluorescence above the baseline and within the exponential
growth region
Threshold cycle: The PCR cycle number at which the fluorescence meets the
threshold in the PCR cycle number at which the fluorescence meets the threshold in
the amplification plot
(Schaefer 1990).
VII
ABBREVIATIONS
μg
Microgram
μL
Microliter
ACOG
The American College of Obstetricians and Gynecologists
ALT
Alanine aminotransferase
amp
Ampicillin
AST
Aspartate aminotransferase
ATCC
American Type Culture Collection
BeWo
Choriocarcinome cells
Blast
Basic local alignment search tool
BMI
Body mass index
bp
Base pairs
cDNA
Complementary Deoxyribonucleic acid
CT
Threshold cycle
D-MEM
Dulbecco’s modified eagle media
DNA
Deoxyribonucleic acid
DNAfree
DNase treatment and removal reagent kit
DNase
Enzymatic treatment to digest residual DNA residual
dNTP’s
Deoxynucleotides
EDTA
Ethylenedyaminetetraacetic acid
F12
Hank’s F12 media
FBS
Fetal bovine serum
G
Gram
G6PD
Glucose-6-Phosphate Dehydrogenase
HEPES
2-[4-(2-hydroxyethyl)piperazin-1-yl]ethane sulfonic acid
HGD
Hospital General de Durango
INEGI
Instituto Nacional de Estadistica, Geografia e Informatica
Kn
Kanamycin
VIII
LB
Luria-broth culture media
LUC
Luciferase
MAP
Media arterial pressure
Mg
Milligram
Min
Minute
mL
Milliliter
mm3
Cubic millimeter
mmHg
Millimeters of mercury
mRNA
Messenger ribonucleic acid
Mw
Molecular weight
NEaa
Non-essential aminoacids
NEBcutter
Program to identify restrictions sites in DNA
NEP
Normo-evolutive pregnancy
Ng
Nano gram
Nm
Nanometer
OTR
Osteotendinous reflex
PBS
Phosphate buffer saline
PCR
Polymerase chain reaction
pDNA
Plasmidic DNA
PEE
Preeclampsia
pH
Potential of hydrogen
qPCR
Semi-quantitative polymerase chain reaction
RLU
Relative light unit
RNA
Ribonucleic acid
Rpm
revolution per minute
rt
Room temperature
RT-PCR
Retro transcriptase reverse of polymerase chain reaction
S
Seconds
SSD
Servicios de Salud de Durango
TAE
Tris-Acetate-EDTA buffer
TE
Tris-EDTA buffer
IX
Tm
Alignment temperature
TSAP
Thermosensitive alkaline phosphate
UV
Ultraviolet
USA
United States of America
V
Voltage
Vent-pol
Vent® High effiency Polymerase
VEGF
Vascular endothelial growht factor
WHO
World Health Organization
SCT
Syncytiotrophoblast
CT
Cytotrophoblast
EVT
Extravillous trophoblast
X
FIGURES INDEX
Figure 1 Schematic representation of a human term placenta .................................... 4
Figure 2 Schematic representation of a blastocyst approaching the receptive
endometrium ............................................................................................................... 7
Figure 3 Maternal death causes ................................................................................ 11
Figure 4 Maternal deaths in Mexico from 2002 to 2008. ........................................... 13
Figure 5 Maternal death rates in Durango from 2002 to 2008. .................................. 13
Figure 6 INEGI maternal death calculated by each 100,000 newborn live ................ 14
Figure 7 Trophoblast invasion into the spiral arteries in the placental bed ................ 16
Figure 8 Susceptibility regions for preeclampsia and their chromosomal localization.
.................................................................................................................................. 24
Figure 9 Scheme of pathophysiological relevant factors in preeclampsia and
corresponding candidate genes ............................................................................... 26
Figure 10 Schematic representation of interstitial endovascular trophoblast invasion in
human pregnancy...................................................................................................... 28
Figure 11 Eukaryotic transcription complex and its components............................... 40
Figure 12 pCR®-Blunt II-TOPO PCR cloning vector .................................................. 54
Figure 13 The family of pGL4 Luciferase vectors ...................................................... 57
Figure 14 Construct B ............................................................................................... 58
XI
Figure 15 Construct “C”............................................................................................. 58
Figure 16 Construct “D”............................................................................................. 59
Figure 17 Construct "E" ............................................................................................. 59
Figure 18 The Control vector pGL4.13 ...................................................................... 60
Figure 19 Bioluminescent reaction catalyzed by firefly luciferase.............................. 62
Figure 20 pSV-ȕ-galactosidase vector w................................................................... 63
Figure 21 Different species of RNA after extraction and purification with DNasa. ..... 65
Figure 22 Amplification plots ..................................................................................... 66
Figure 23 The different values in amplification efficiency (90-100%) and slope-values
.................................................................................................................................. 67
Figure 24 The slope and R2 values calculated to get a efficiency amplification percent
.................................................................................................................................. 68
Figure 25 The expression of KiSS-1 gene................................................................. 69
Figure 26 The Relative expression for REN gene ..................................................... 69
Figure 27 Comparative expression analyses for KiSS1 and REN genes .................. 70
Figure 28 Cluster diagram for PEE and NEP women................................................ 71
Figure 29 REN gene expression levels analysis for PEE subgroups ........................ 72
Figure 30 Fragments PEK, CHE and KE with specific end tails restriction enzymes. 74
Figure 31 Workflow design constructs....................................................................... 75
XII
Figure 32 Fragments PEK, ChE and KE from pCR®-Blunt II-TOPO vector............... 75
Figure 33 Different constructs from pGL4 plus PEK plus ChE and/or KE with correct
orientation 5’-3’.......................................................................................................... 76
Figure 34 Differents fragment ChE, KE and PEK can be appreciate on this gel later
restriction with specific enzymes KpnI, SacI and XhoI at pGL4 plasmid ................... 77
Figure 35 Blast of fragment PEK ............................................................................... 78
Figure 36 Blast of fragment ChE ............................................................................... 78
Figure 37 Blast analysis KE....................................................................................... 79
Figure 38 Standardization of transfection conditions in.pGL4.13 .............................. 81
Figure 39 pSV-ȕ-gal stain and ȕ-galactosidase expressed in BeWo cells................. 82
Figure 40 Expresion of Luciferase in pGL4.10 and 4.11 with all constructs and their
combinations. ............................................................................................................ 83
XIII
TABLES INDEX
Table 1 ACOG criteria for mild and severe preeclampsia ………………………….… 21
Table 2 Principal candidate genes related with preeclampsia ……………….……… 25
Table 3 Bacterian strain used in this experiment ……………………………………… 52
Table 4 Plasmids used in this research ………………………………………………… 53
Table 5 Clinical characteristic for PEE and NEP women ………………………..…… 64
Table 6 Correlation test for distinct analyzed variables with gene expression ……... 73
XIV
ABSTRACT
Preeclampsia is the main and most important complication of pregnancy-specific
disorders, the identification of markers for preeclampsia development has become a
very important area of biomedical research. The objective was to identify a common
transcriptional regulatory element for the expression of KiSS-1 and REN genes in
placenta from women with preeclampsia-eclampsia in comparison with women with
normo-evolutive pregnancy. This experimental and case control study involved 27
preeclamptic women and 27 normoevolutive pregnant women. We compared KiSS-1
and REN expression gene levels in the studied groups. Transient transfection of
constructs with minimal regions of KiSS-1 promoter plus chorionic and/or kidney
enhancers on BeWo cells was done to know if these elements regulate co-expression
for both genes. Higher expression for KiSS-1 gene in PEE vs. control woman
(p=0.001) was observed, whereas no difference was observed for REN gene
expression (p=0.300). However, REN gene expression was significantly higher when
the samples were stratified according to preeclampsia severity and treatment
scheme. The Luc expression assays did not show differences with the different
constructs assayed. Our results suggested that the observed KiSS-1 gene high
expression seen in preeclamptic patients is in accordance with its role as an inhibitor
of trophoblast invasiveness. On the other hand, aggressive therapeutic management
and/or severity status of patients have a direct effect on placental REN gene
expression levels. The finding of common transcriptional regulatory element(s) for
KiSS-1 and REN genes was not possible may be because small constructs were
used for transfections assays.
Keywords: Preeclampsia; KiSS-1; REN; expression gene; regulatory elements.
XV
Fernando Vazquez Alaniz
CIIDIR IPN Unidad Durango
Doctorado en Ciencias en Biotecnología
RESUMEN
La preeclampsia es la principal complicación de los trastornos específicos del
embarazo. La identificación de marcadores para su desarrollo se ha convertido en un
área importante de la investigación biomédica. El objetivo fue identificar algún
elemento de regulación transcripcional común para la expresión de los genes KiSS-1
y REN en tejido placentario de mujeres con preeclampsia-eclampsia, comparándolo
con mujeres con embarazo normo-evolutivo. En este trabajo experimental de casos y
controles utilizamos 27 mujeres preeclámpticas y 27 con embarazo normoevolutivo.
Nosotros comparamos los niveles de expresión génica de los genes KiSS-1 y REN
en ambos grupos. Realizamos transfección transitoria en células BeWo de las
regiones mínimas del promotor de KiSS-1 y los potenciadores renal y coriónico para
determinar la co-expresión de ambos genes. Se observó una sobre-expresión de gen
KISS-1 (p= 0,001) en mujeres con PEE vs. control, mientras que no se observó
diferencia significativa en la expresión del gene REN (p = 0,300). La sobre-expresión
de REN fue significativa cuando las muestras fueron estratificadas de acuerdo a la
severidad de la preeclampsia y al esquema de tratamiento. Los ensayos de
expresión Luc no mostraron diferencias en las diferentes construcciones analizadas.
Nuestros resultados sugieren que la sobreexpresión del gen KISS-1 en pacientes con
preeclampsia, está relacionada con la función de KISS-1 como inhibidor de la
invasión trofoblastica. Por otro lado, un tratamiento terapéutico agresivo y/o el estado
de gravedad de las pacientes tuvieron un efecto directo sobre los niveles de
expresión del gen REN en tejido placentario. La búsqueda del(os) elementos de
regulación transcripcional común para la expresión de los genes KiSS-1 y REN no
fue posible establecerse, tal vez debido a que los constructos usados en los ensayos
de transfección fueron demasiado cortos.
Palabras clave: Preeclampsia, KiSS-1, REN, Expresión génica y elementos
reguladores.
XVI
BACKGROUND
Pregnancy is a wonderful and natural phenomenon which permits the creation of life
and human evolution. However it sometimes involves health risks to mother and
developing fetus. Pregnancy is the result of thousands of years of evolution that
requires a specialized body structure named the placenta to come to fruition. The
placenta has multiple functions that are vital for a successful pregnancy. Among the
major functions of the placenta are: (1) regulation of the flow and waste of nutrients
between maternal and fetal compartments and (2) modification of maternal and fetal
environment by secreting hormones, cytoquines, and growth factors as well as others
proteins that modify the activities of these ligands. All these functions are mainly
achieved by trophoblast cells, although some are accomplished by placental stromal
cells, which include fibroblasts, macrophages, and endothelial cells. Alterations in the
function of these cells can produce diseases which put on risk the mother and fetus
when there are not attended opportunely. During pregnancy, diseases related to
placental malfunction are by far the most dangerous for the mother and the fetus.
Examples of such placental diseases are: preeclampsia, eclampsia, hydatidiform
mole and ectopic pregnancy. The first two conditions are the aim of the current thesis.
The preeclampsia and eclampsia represent the main causes of mortality and comorbidity in the pregnant mother and the fetus. They are present in 4-8% of
pregnancies, principally in developing countries. Preeclampsia is currently considered
as a defective implantation process with two principal theories accepted about the
physiopathology of the diseases. The first theory considers a deficient trophoblast
invasiveness process; meanwhile the second is a generalized endothelial
dysfunction. These two phenomena are regulated by different molecular mechanisms
engaged in regulating gene expression. Here we hypothesize that gene regulatory
elements located in between KiSS1 and REN genes drive their co-expression,
explaining some pathophysiology events taking part on the development of this
disease.
XVII
I. ANTECEDENTS
1.1
Pregnancy
Pregnancy is the basic reproductive biological processes required for woman to
successfully achieve life. The ability of mother and fetus to coexist as two distinct
immunological systems results from endocrine, paracrine, and immunological
modification of fetal and maternal tissues in a manner not seen elsewhere. The
placenta mediates a unique fetal–maternal communication system, which creates a
hormonal environment that helps initially to maintain pregnancy and eventually
initiates the events leading to parturition (Cunningham, Leveno et al. 2010).
Since thousands years, human populations characterized by a natural balance
between fertility and mortality, the excess of births over deaths was very modest in
the light of population growth was seen periodically interrupted by episodes of crisis
epidemics, famines, and wars. The arrival of the twentieth century represents a
quantum leap in technology, men and women contributing to better health and
longevity and a significant decline in infant mortality and for the first time women are no longer entirely linked to reproduction role (Goberna 2002). Currently, both
pregnancy and childbirth are very safe process. However, arise in life expectancy and
reproductive age in women put them at high risk for developing pregnancy
complications (Jolly, Sebire et al. 2000).
1.2
Placental Anatomy
The placenta is basically the “tree of life”. The word placenta arises from the Latin
word for pie (placenta); from Greek language meaning foe flat, slab-like
(plakóenta/plakoúnta) and from the German word for mother cake (mutterkuchen).
1
All words refer to the round, flat appearance of the human placenta. Structurally, the
placenta is the hemochorial villous organ. Functionally, the placenta is a highly
complex organ that provides: (1) the exchange of oxygen and CO2; (2) the absorption
of all necessary nutrients for fetal development and growth; (3) the remotion of waste;
and (4) the prevention of an immune maternal system attack to the growing fetus. The
placenta is also an important endocrine organ producing many hormones and growth
factors that regulate the course of pregnancy, support and promote fetal grow, and
initiate parturition. However, all these tasks depend on a normal implantation process
and a successful vascular development within the placenta. Normal placental
vascular development ensures a healthy pregnancy outcome, whereas insufficient or
abnormal placental vascular development will compromise pregnancy outcomes for
both, the mother and the fetus. The alterations in placental function are mainly
represented by preeclampsia and intrauterine growth restriction syndrome. The
functional unit of the placenta is the chorionic villous, which contains the layers of
SCT/CT, villous stromal, and fetal vascular endothelium that separate maternal blood
from the fetal circulation (Wang and Zhao 2010).
The placenta is a highly specialized organ, specific to pregnancy that develops
simultaneously with the developing embryo and fetus. From an evolutionary
perspective, the placenta was the essential factor in permitting viviparity, a
reproductive strategy in which fetal development proceeds within the female
reproductive tract. Viviparous species are able to provide greater protection from
environmental risks and can more precisely control the development of their progeny
while they reside in utero. The placenta is comprised of numerous cell types. Among
these, the specialized epithelioid cells, called trophoblast, possess several important
functions enabling viviparous development (Soares and Hunt 2006).
At the end of pregnancy, normal placenta measured 15 to 20 cm in diameter; 1.5 to 3
cm thick and weighs from 450 to 600 g. The main components of placenta are the
umbilical cord, the amnion and chorion membranes, the villous parenchyma, and the
maternal decidual tissue (Figure 1). The umbilical cord connects the mother and
2
fetus; at term it measures 55 to 65 cm in length. It has an outer layer of amniotic
epithelium, which becomes stratified at its fetal end. The bulk of the cord is made up
of highly mucoid connective tissue know as Wharton’s jelly. Embedded within this
substance are the umbilical vessels, represented by two arteries and a single vein
(Rosai 2004). The placental membranes consist of the amnion and chorion. The
amnion, which represents the inner most covering of the amniotic cavity, is lined by a
single layer of flat epithelial cells resting on a basement membrane. The chorion is
composed of a connective tissue membrane that carries the fetal vasculature. Some
of the trophoblast located in the chorion has a characteristic vacuolated appearance.
The trophoblast villi constitute the functional unit of the placenta; they are composed
of an outer syncytiotrophoblast (SCT) layer and inner cytotrophoblast (CT) layer. The
CT is composed of multinucleated giant cells and is the progenitor of the SCT. In the
term placenta, the CT is inconspicuous, and the SCT is clumped in the form of
syncytial knots. The third trophoblastic cell type is represented by the intermediate
trophoblast, also known as interstitial extravillous trophoblast and X cells. This cell
type is present in the villi and the membranes but is particularly numerous in the
extravillous region that form the implantation site (Langston, Kaplan et al. 1997). The
decidua is present both in the placental disk and on the chorionic side of the
membranes. Decidual tissue is thought to contain a special type of dendritic cells,
which may play a role in the tolerance of maternal immune system toward the
allogenic embryo (Tagliani and Erlebacher 2011).
3
Figure 1 Schematic representation of a human term placenta (Sood, Zehnder et al. 2006)
Placental-related disorders of pregnancy are almost unique to the human species.
These disorders, which affect around a third of human pregnancies, primarily include
miscarriage and preeclampsia. In other mammalian species, the incidence of both
disorders is extremely low. Although epidemiological data on animals living in the
wild, such monkeys, are limited, laboratory rodents are known to have postimplantation pregnancy loss rate of less than 10%. In humans, both disorders have
been reported in to medical scence since ancient times with clear descriptions
recorded in ancient Egypt and Greece (Jauniaux, Poston et al. 2006).
4
1.3
Cell types of the placenta
Placenta villi are composed of three layers of components with different cell types in
each: (1) SCT/CT that cover the entire surface of the villus tree and are bathed in
maternal blood within the intravellous space; (2) mesenchymal cells, mesenchymal
derived macrophages (Hofbauer cells), and trophoblast cells. Hofbauer cells
synthesize VEGF and other proangiogenic factors that initiate vasculogenesis in the
placenta; and (3) fetal vascular cells that include vascular smooth muscle cells,
perivascular cells (pericytes), and endothelial cells (Wang and Zhao 2010). The
trophoblast rapidly differentiates into two layers: the SCT and the CT. The
multinucleated SCT develops lacunae, between which the cytotrophoblastic layer
projects villi. The SCT becomes progressively compressed to form a discontinuous
layer covering each villus and separate it from the lacunae, which merge to form
intervilluos spaces. Trophoblastic enzymes erode about 100 spiral arteries and veins
of the uterine wall, and so the branched villi become bathed in approximately 150 mL
of maternal blood, which is replaced three-four times a minute. Fetal mesenchymal
cells invade the villi and generate vascular networks connecting the umbilical arteries
and vein (Donnelly and Campling 2008). Therefore, the fetal and maternal circulations
become separated, in transfer areas between CT’s cells by endothelial cells lining the
microvessels of the fetal villi, associated basement membrane, and thin areas of
SCT. These areas may constitute 5-10% of the 16m2 surface within the term
placenta. The apices of the villi tend not to develop a mesenchymal core, but remain
as solid SCT cell columns. While others remain free-floating in the invervillous space,
some villi take on an anchoring function by abutting onto the maternal decidual cells
(pijnenborg, vercruysse et al. 2009). Moreover, the CTS spread over the decidua
basalis to form a complete layer – the cytotrophoblastic shell. Specialized SCT further
invade the spiral arteries and cause them to become remodeled. So that blood enters
the intervillous space at a lower than normal arterial pressure(Blankenship and
Enders 1997).
5
1.4
Implantation and Placentation
Normal implantation and placentation are critical for successful pregnancy. A better
understanding of the molecular mechanisms responsible for these processes will
improve clinician’s ability to treat related disorders such as infertility, pregnancy loss,
and preeclampsia-eclampsia (PEE). Human reproduction entails a fundamental
paradox: although it is critical to the survival of the species, the process is relatively
inefficient. The highest fertility rate is 30% with only 30-50% of conceptions
progressing beyond 20 weeks of gestation, being implantation failure the most
frequent cause of pregnancy loss (75%) which is not clinically recognized (Wilcox,
Weinberg et al. 1988).
There is little information regarding the events that occur during the first weeks of
gestation in humans. In some cases, information about a particular stage of
development comes from a single specimen. Crucial events on early human
development, such as the initial adhesion of the blastocyst to maternal decidua are
inferred from studies in animals (Norwitz, Schust et al. 2001). Implantation occurs
approximately six or seven days after conception, with similar events to those
occurring in other species of primates. In a preimplantation stage the blastocyst is
oriented with its embryonic pole toward the uterine epithelium (Hertig, Rock et al.
1959)(Figure 2).
After the zona-pellucida hatching is done, implantation in human takes place and
includes three stages: 1) loose initial adhesion of the blastocyst to the uterine wall
called apposition; 2) trophoblast adhesion of the blastocyst to the endometrium
pinopodes where strong receptor-ligand communication is established and 3)
Invasion of the trophoblast and differentiation to syncitiotrophoblast.
6
Figure 2 Schematic representation of a blastocyst approaching the receptive
endometrium, defined by the integrin profile and appearance of
pinopodes. Early signaling between the blastocyst and the endometrium
precedes the attachment (Staun-Ram and Shalev 2005).
Shortly after SCT differentiation is accomplished, it penetrates the uterine epithelium
through proteolytic degradation and apoptosis stimulation of endometrial epithelial
cells.
By the 10th day after conception, the blastocyst is completely embedded in the stromal
tissue of the uterus; the uterine epithelium grown again to cover the site of
implantation and mononuclear CT stream out of the trophoblast layer. Eventually CT
invades the entire endometrium and the third of the myometrium (interstitial invasion)
as well as the uterine vasculature (endovascular invasion) and it establishes the
7
uteroplacental circulation, places trophoblast in direct contact with maternal blood
(Dey, Lim et al. 2004).
1.5
The trophoblast invasion and spiral artery remodeling
In human pregnancy fetal trophoblast invades into the wall of the uterus in a tightly
regulated manner. CT cells columns from the tips of anchoring villi and a shell
develops from which extravillous trophoblast (EVT) invades into the decidua. The
blood supplied to the uterus comprises a branched structure with successive
decreases in vessel diameter as they move through the myometrium and
endometrium. Spiral arteries, supply to the endometrial layer and, in the pregnant
uterus, span the inner myometrium and the decidua. During pregnancy, spiral arteries
change transforming from a high to low resistance vascular system allowing an
increase in blood flow (Burton, Woods et al. 2009). In pregnancies complicated by the
common pregnancy disorder PEE, remodeling is restricted to the decidual regions of
the spiral arteries (Brosens, Robertson et al. 1972; Pijnenborg, Vercruysse et al.
1998). The altered uteroplacental hemodynamics, as a consequence of impaired
vessel remodeling, has been implicated as a contributing factor to the pathology of
this syndrome. Similar deficiencies have also been described in spiral arteries of
women with small-for-gestational-age-infants (Sebire, Jain et al. 2004). Two different
types of trophoblast invasiveness exist: endovascular and interstitial and, their
impairments likely to be a component of PEE. Interstitial invasion did not appear
altered in initial studies (Brosens, Robertson et al. 1972), which suggested that
defects may exist in the endovascular trophoblast invasion pathway. Subsequent
studies demonstrated also a lesser degree in interstitial invasion (Pijnenborg, Brosens
et al. 2010).
In some cases there is an adequate trophoblast invasion in preeclampsia, so there
must be a primary defect in the way as the trophoblast interacts and modify blood
vessels (Whitley and Cartwright 2010).
8
1.6
Placental disorders
The role of the placental bed in normal pregnancy and its complications has been
intensively investigated for 50 years, following the introduction of a technique for
placental bed biopsy. It is now recognized that disorders of the maternal-fetal
interface in humans have been complicated in a broad range of pathological
conditions, including spontaneous abortion, preterm labor, premature rupture of
membranes, preeclampsia, intrauterine growth restriction, abruption placentae, and
fetal death. These clinical disorders are the major complications of pregnancy and
leading causes of perinatal and maternal morbidity and mortality. Moreover, recent
evidence indicates that these disorders have a potential to reprogram the endocrine,
metabolic, vascular and immune response of the human fetus, and predispose to
adult diseases. Thus premature death from cardiovascular diseases (myocardial
infarction, or stroke), diabetes mellitus, obesity, and hypertension may have their
origins in abnormal placental development (Pijnenborg, Brosens et al. 2010).
1.7
Preeclampsia
Between 5 and 10% of pregnancies are complicated by hypertensive disorders.
These along with bleeding and perinatal infections contribute to the high rates of
morbidity and mortality in the maternal-fetal binomial triad (Cunningham, Leveno et al.
2010). PEE is the main and most important complication of these pregnancy-specific
disorders and characterized clinically by new onset hypertension and proteinuria after
20 weeks of gestation. It is the most frequent obstetric complication during
pregnancy, affecting 3-5% of pregnant women worldwide (WHO. 2005)
PEE often affects young and nulliparous women, whereas older women are in greater
risk for chronic hypertension with superimposed preeclampsia. Also the incidence is
markedly influenced by race and ethnicity-and thus by genetic predisposition. Other
factors include environmental, socioeconomic and even seasonal influences
9
(Palmer, Moore et al. 1999; Lawlor, Morton et al. 2005; Wellington and Mulla 2012).
The exact incidence of PEE is unknown but it has been reported to be approximately
3 to 10 percent principally in nulliparous women (Lindheimer, Taler et al. 2008), In
developing countries where access to health care is limited, the hypertensive
disorders in pregnancy (including PEE) is a second-leading cause of maternal
mortality, with estimates of 60,000 maternal deaths each year (WHO. 2005) (figure 3).
Women who die from this complication are generally those who lack access to
treatment both globally and in the United States. Despite this, 90 countries have
achieved a significant reduction in maternal mortality rates, Latin America has seen a
steady decline in the number of maternal, fetal and postnatal deaths, reflecting an
improvement in obstetric care and a ~40% decrease in fertility rates. (Paxton and
Wardlaw 2011). Furthermore, the overall downward trends is insufficient to achieve
the Millennium Development Goal of 75% reduction in maternal mortality rates
between 1990 and 2015.(Paxton and Wardlaw 2011). In rich countries, the burden of
this disease falls on the neonates because of premature deliveries performed to
preserve the health of the mother.
Worldwide, PEE is associated with a perinatal and neonatal mortality rate of 10%
(Altman, Carroli et al. 2002), The most common neonatal complications are the low
gestational age (35±3 weeks) (Gruslin and Lemyre 2011) fetal growth restriction
(28%) and death (2.6%) (Ferrazzani, Luciano et al. 2011). All maternal and neonatal
complications are related to PEE classification.
10
Other
causes, 28%
Post-partum
hemorrhage
25%
Infections, 15%
Unsafe
abortions, 13%
Obstructed
labor, 8%
Pre-eclampsia
and
eclampsia, 12
%
Figure 3 Maternal death causes. PEE is situated as principals causes of maternal
death worldwide (WHO. 2005).
In Mexico the prevalence has been described around 4-8%, similar to other countries
which reported that risk factors associated with PEE were primigravity, alcoholism,
low socioeconomic level and previous pregnancy with preeclampsia (Morgan-Ortiz,
Calderon-Lara et al. 2010). In Durango City a prevalence of 4% was found in the
Hospital General (Torres-Felix, Vazquez-Alaniz et al. 2008). The PEE has been
reported as one of the three main causes of maternal mortality and morbidity in
Mexico, PEE syndrome is the main cause of perinatal morbidity and mortality with 1530% all maternal death (Duran Nah and Couoh Noh 1999; Gonzalez-Rosales, AyalaLeal et al. 2010; Morgan-Ortiz, Calderon-Lara et al. 2010).(Roiz Hernandez and
Jimenez Lopez 2001).
According to reports from National Institute of Statistics, Geography and Informatics
(INEGI), maternal death rates in Mexico vary from 57 to 63 per 10,000 live births
newborns from 2002 to 2008 with a tendency to decrease last years. Despite the
11
latter, a total of 1,119 maternal deaths due to preventable causes, are reported in
Mexico. The most common causes of maternal death are hypertensive disorders of
pregnancy (31%), bleeding in pregnancy and childbirth (26%) and the main
complication are preeclampsia and eclampsia with 76% of all cases. However, the
factors associated with these deaths are different and depend among other on the
place of residence. In urban areas almost half of maternal deaths are due to
complications of pregnancy, while in rural areas the same proportion correspond to
complications in childbirth (Avila, Cahuana et al. 2010). In Durango, the rates are
between 41 to 86 maternal deaths per 100,000 live newborns with no decreases in
the same period from 2002 to 2008 (Figures 4 and 5) (National Institute of Geography
Statistical and Informatics 2010).
Recently, the National meeting “Arranque Parejo de la Vida”, celebrated in
December, 2010 published a decrease in maternal deaths in Durango state to 45 in
100,000 newborn lives. Such tendency is not observed in Chiapas, Guerrero and
Oaxaca states which remain at the top of the list.
The highest rates of maternal death are registered among 20-24 years in women
without any health care service. However during the event 90% they have health
service meaning that, the prenatal care is the best way to prevent the maternal
deaths in Mexico (Sistema Nacional de Informacion en Salud/SINAIS 2010).
12
Figure 4 Maternal deaths in Mexico from 2002 to 2008.
Figure 5 Maternal death rates in Durango from 2002 to 2008.
13
In agreement with these data from INEGI, the Durango state ranked fourth under
Chiapas, Guerrero and Oaxaca, states considered as highly marginated (figure 6)
(National Institute of Geography Statistical and Informatics 2010).
Figure 6 INEGI reported that the main entities with highest rates of maternal death
are Chiapas, Guererro, Oaxaca, Durango and Veracruz meanwhile lowest
rations are seen in Tlaxcala and Nuevo León. This data are calculated by
each 100,000 newborn live
1.8
Etiology and classification of preeclampsia.
The etiology of PEE is still unclear. Pregnancy at early age displays a suboptimal
placentation, and an inadequate hemodynamic adaptation. Maternal constitutional
14
factors such as genetic predisposition, immunological maladaptation to pregnancy
and pre-existing vascular diseases seem to be involved in early vascular dysfunction
(VanWijk, Kublickiene et al. 2000).The disturbed placentation leads to hypoperfusion
of the placenta and ischemia, resulting in the release of one or more factors that
cause the late vascular dysfunction in PEE. This is mainly due to the presence of
generalized endothelial dysfunction, resulting in vasoconstriction, activation of the
coagulation system and redistribution of fluids, and eventually the appearance of PEE
symptoms and fetal growth restriction as major complications in early pregnancy,
trophoblast cells invade the placental bed, leading to remodeling of the spiral arteries
into maximally dilated low resistance vascular channels, unable to constrict to
maternal vasoactive stimuli, ensuring a high flow volume to the utero-placental bed. In
PEE such invasion is confined to the decidual part of the spiral arteries, with about
one third of these arteries which evades trophoblast invasion (Pijnenborg, Anthony et
al. 1991; Meekins, Pijnenborg et al. 1994) (Figure 7).
Perturbation of trophoblast functions may result in a range of adverse pregnancy
outcomes such as malformations, fetal growth retardation, spontaneous abortion and
stillbirth. For instance, a limited trophoblast invasion of maternal vessels has been
correlated to both PEE and fetal growth restriction, whereas an excessive trophoblast
invasion is associated with invasive mole, placenta accrete and choriocarcinoma
(Lunghi, Ferreti et al. 2007).
15
Figure 7 Trophoblast invasion into the spiral arteries in the placental bed in
normal pregnancy and preeclampsia. Normal placentation (A), and
poor placentation (B), at 15-16 weeks of pregnancy. Then placenta is
linked to the maternal decidua by anchoring villi.
Recent advances have demonstrated that severity of PEE depends upon a profound
dialogue between the decidua and trophoblast; such a dialogue is sustained by
cytotrophoblast differentiation into syncitiotrophoblast as well as extravillious
trophoblast, in order to invade maternal spiral arteries for optimal fetal oxygenation
and nutrition (Kharfi, Giguere et al. 2003).
Although PEE may develop at any time after 20 weeks of gestation, early onset
disease is more severe and characterized by a higher rate of small size for
16
gestational age neonates as well as a higher recurrence rate than later onset disease
(Rasmussen and Irgens 2008; Proctor, Toal et al. 2009). It is generally agreed that
preeclampsia results from the presence of a placenta and, in particular, the
trophoblast cells that are found only in this tissue (Karumanchi and Lindheimer 2008;
Akhlaq, Nagi et al. 2012).
1.9
Classification of preeclampsia-eclampsia
There are different organizations that have established the diagnosis criteria of
preeclampsia such as The International code of Diseases promoted by World Health
Organization (WHO). However this is not the best way to classify the preeclampsia
because the ICD-9 vary greatly in their accuracy of diagnosis with a positive
predictive value of 84.8% for severe preeclampsia, 45.5% for mild preeclampsia and
41.7% for eclampsia (Geller, Ahmed et al. 2004). On this sense, different
gynecological and obstetric worldwide organizations have established their own
criteria under WHO guidelines and adapted for each particular population.
For example, in USA, The National High Blood Pressure Education program
(NHBPEP) of Working Group Report on High Blood Pressure in Pregnancy
(WGRHBPP) provides a guidance to practicing clinicians on managing (1) patients
with hypertension who become pregnant and (2) patients who develop hypertensive
disorders during gestation.
The WGRHBPP proposed the following classification and criteria for hypertensive
disorders in pregnancy:
1. Chronic hypertension.
2. Preeclampsia-eclampsia.
3. Preeclampsia superimposed on chronic hypertension.
4. Gestational hypertension.
5. Transient hypertension.
17
1.
Chronic Hypertension is defined as hypertension present before pregnancy or
diagnosed before the 20th week of gestation. Hypertension is defined as a blood
pressure equal to or greater than 140 mm Hg systolic or 90 mm Hg diastolic.
2.
Preeclampsia-eclampsia is a pregnancy-specific syndrome which usually
occurs after 20th week of gestation (or earlier in the presence of trophoblastic
diseases such as hydatidiform mole or hydrops fetalis). It is determined by increased
blood pressure (greater than 140 mm Hg systolic or 90 mm Hg diastolic in a woman
normotensive before 20 weeks) accompanied by new onset proteinuria. The disease
is highly suspected when increased blood pressure appears accompanied by the
symptoms of headache, blurred vision, and abdominal pain, or with abnormal
laboratory tests, specifically, low platelet counts, and abnormal liver enzymes.
3.
Preeclampsia superimposed on chronic hypertension. There is evidence that
PEE may occur in already hypertensive women, being the prognosis for mother and
fetus much worse than in the presence of either condition alone.
The diagnosis of superimposed preeclampsia is highly suspected in the presence of
the following findings:
x A previously diagnosed hypertensive women with no proteinuria early in pregnancy
(<20 weeks).
x New-onset proteinuria, defined as the urinary excretion of 0.3 g protein or greater in
a 24h specimen.
x A women with hypertension and proteinuria appearing before 20 weeks gestation
x Sudden increase in proteinuria
x A sudden increase in blood pressure in a woman whose hypertension has
previously been well controlled
x Thrombocytopenia (platelet count >100,000 cells/mm3)
x An increase in ALT or AST to abnormal levels.
18
4.
Gestational hypertension. A woman who has blood pressure elevation
detected for the first time after mid-pregnancy, without proteinuria. This unspecific
condition, which will influence management and, the final differentiation that the
woman does not have the PEE syndrome is made only postpartum. It includes
women with the PEE syndrome who have not manifested proteinuria as well as
women who do not have the syndrome.
5.
Transient hypertension. Transient hypertension of pregnancy is considered if
PEE is not present at the time of delivery and blood pressure returns to normal by 12
weeks postpartum (a retrospective diagnosis.
As there is a large variation between criteria established by the international
classification of diseases as well as the accuracy of their criteria for PE diagnosis.
The ACOG based on WHO and the WGRHBPP classification for hypertensive
disorders, proposed the following classification for the disease: mild preeclampsia,
severe preeclampsia, and eclampsia with clinical features and laboratory values
showed in table 1 (Geller, Ahmed et al. 2004). In Mexico the Federacion Mexicana de
Ginecologia y Obstetricia (FEMEGO) recommends the guideline proposed by SS
through “Lineamiento Técnico para la prevención, Diagnóstico y Manejo de la
Preeclampsia/Eclampsia” applicable to all heath atention Centers in public, social and
private sectors with maternal heath attention (Secretaria de Salud 2007).
The so-called triad of preeclampsia includes hypertension, proteinuria and edema.,
Currently a general agreement exist that edema should not be considered as part of
the diagnosis of preeclampsia (NIH. 2000; ACOG. 2001; Sibai 2003; Sibai 2005; Sibai
and Stella 2009). Edema is neither sufficient nor necessary to confirm the diagnosis
of PEE, because it is a normal finding in normal pregnancy, and approximately one
third of women with eclampsia never demonstrate the presence of edema (Mattar and
Sibai 2000). Despite de above, sudden or generalized edema should be considered
as a concerning clinical sign because may precede the appearance of seizures and
pulmonary edema (Sibai 2005).
19
The syndrome of hypertension and proteinuria that heralds the seizures of eclampsia
remains one of the great mysteries on the field of obstetrics. Despite the latter, our
understanding of the pathophysiology of PEE has increased over the past 50 years
(Karumanchi, Maynard et al. 2005). The eclampsia is defined as the development of
convulsions and/or unexplained coma during pregnancy or postpartum in patients
with or without signs and symptoms of preeclampsia. In the western world the
incidence of eclampsia is estimated to be about 5–7 cases per 10,000 deliveries
ranges, or 1 in 2000 to 1 in 3448 pregnancies (Osungbade and Ige 2011). Some
studies (Rudra, Sorensen et al. 2008; Yeo 2010; Fortner, Pekow et al. 2011), suggest
that recreational activity in the year before pregnancy is associated with reduced risk
of PEE. The same is true for an adjustment for body mass index (BMI) in pregnancy.
This suggests that beneficial impacts of activity may arise via mechanisms
independent of reducing maternal adiposity. Physical activity may favorably affect
several pathophysiology’s associated with PEE including hypertension, inflammation,
elevated leptin, dyslipidemia, and reduced placental vascular volume (Dempsey,
Butler et al. 2005; Pivarnik, Chambliss et al. 2006; Rudra, Sorensen et al. 2008).
20
Table 1 ACOG criteria for mild and severe preeclampsia
Diagnosis
Mild preeclampsia
Severe preeclampsia
Eclampsia
Criteria
Hypertension with
o Edema (RUERWKDIWHUth week of gestation
o Blood pressure PP +J RQ occasions 6
hr apart.
1. Blood pressure
Systolic PP+JEORRGSUHVVXUHGLDVWROLF
mm Hg (with patents at rest, 2 measurements at
least 6 h apart
2. Proteinuria (JKRU proteinuria) occurring for
the first time in pregnancy and regressing after
delivery
3. Increased in serum creatinine level

( PJG/
unless know to be elevated previously)
4. Platelet count <100,000 (thrombocytopenia) or
evidence of microangiopathic hemolytic anemia (with
elevated lactate dehydrogenase).
5. Intrauterine growth retardation or oligohydramnios
6. Elevated
hepatic
enzymes
(alanine
aminotransferase, aspartate aminotransferase)
7. Microangiophatic hemolysis
8. Symptoms that suggest significant end-organ
involvement:
Headache,
visual
disturbances,
epigastric or right upper quadrant pain, retinal
hemorrhage, exudates or papilledema.
9. Pulmonary edema
10. Oliguria <500 mL/24h
Mild or severe preeclampsia AND seizures or coma
21
1.10
Complications of preeclampsia
The control of time of birth in humans is the greatest unresolved question in
reproductive biology, and preterm birth is the most important medical issue in
maternal and child health. Our understanding of pathways involved in the time of birth
in humans is limited, yet its elucidation remains one of the most important issues in
biology and medicine (Plunkett, Doniger et al. 2011). The pregnancy-associated
hypertension appeared to have little effect on a woman’s risk of this disorder. In
woman who remained with the same sexual partner and conceives, there is a slightly
lower risk of pregnancy-associated hypertension than women pregnant for the first
time or change sexual partner. Some mild variation in the age-stratified odds ratios
occurred among women who had a prior abortion with a different father (Saftlas,
Levine et al. 2003).
1.11 Genetics of preeclampsia
Some evidence that preeclampsia is a genetic disease comes from observations like
those of Esplin et al. who found that an increase in the risk to develop preeclampsia is
seen in both men and women who were the product of a pregnancy complicated by
preeclampsia (Esplin, Fausett et al. 2001).
There is no report which demonstrates that PEE is a genetic disease in the classical
sense, because no single gene has been absolutely linked to it. Instead, complex
inheritance patterns could be expected because of the maternal and fetal genetic
components that affect the incidence of the disease.
The PEE is a disease of pregnancy suggesting that the fetus and/or placenta interact
with maternal factors contributing to the disease. The requirement for a fetal-maternal
interaction has lead to speculation that development of PEE may require both fetoplacental defects as well maternal susceptibility to hypertensive and renal diseases
(Roberts and Lain 2002). Several studies indicate that PEE tends to have a familial
22
predisposition. However, most of the recent data rule out simple genetic mechanisms
such as a single acting dominant gene (Arngrímsson, Björnsson et al. 1995;
Lachmeijer, Arngrimsson et al. 2001; Roberts and Cooper 2001; Lachmeijer, Dekker
et al. 2002). On the other side, Berends et al. in 2008 suggested an autosomal
recessive inheritance because of the high rate of preeclampsia in a high
consanguineous isolated population (Berends, Steegers et al. 2008).
Different genome-wide screens performed on preeclamptic patients have found
strong linkage to different loci as 2p13, 2p25 and 9p13. Other suggestive loci have
been linked on chromosomes 2q, 9p, 10q, 11q, and 22q. Close examination on those
chromosomal regions revealed the presence of relevant genes for pathophysiological
events
in
preeclampsia
as
hemodynamic,
thrombophilia,
oxidative
stress,
immunogenetics/placenta development and mitochondrial processes (Lachmeijer,
Dekker et al. 2002; Mutze, Rudnik-Schoneborn et al. 2008) (Table 2 & figure 8).
Based on these data it has been suggested that PEE may be a multigenic disease. It
has been noted that the typical clinical presentation of the disease differs
geographically, making clear the influence of distinct interacting genetic with
environmental factors (Redman 1991). From the results on the genetics of PEE
obtained up to now, it seems likely that, excluding familial cases, no single gene can
account for the liability to PEE and its variants. Until 2007, more than 50 candidate
genes related with this syndrome have been reported (Oudejans, van Dijk et al.
2007). With the advent of new methodologies such as microarrays, now it is possible
to scan a great number (thousands) of candidate genes. Different authors have found
more than 140 genes differentially expressed between normal and preeclamptic
pregnancies (Reimer, Koczan et al. 2002; Nishizawa, Pryor-Koishi et al. 2007; Sun,
Zhang et al. 2008; Rajakumar, Chu et al. 2011), however results are frequently
inconsistent genetic predisposition may be involved in any of the three etiologic
concepts of preeclampsia: immune maladaptation, placental ischemia and oxidative
stress as proposed by Wilson et al. (Wilson, Goodwin et al. 2003).
23
Figure 8 Susceptibility regions for preeclampsia and their chromosomal localization.
The candidate genes can be subdivided into groups according to their assumed roles
in
pathophysiology
for
example:
vasoactive
proteins/vascular
remodeling,
thrombophilia and hypofibrinolysis, oxidative stress, lipid metabolism, endothelial
injury, immunogenetics, placentation/imprinting and growth factors (Mutze, RudnikSchoneborn et al. 2008) (figure 9).
24
Table 2 Principal candidate genes related with preeclampsia.
Chromosome location
1q13.3
1q23
1q32
1q32.1
1p36.3
1q42-q43
1q42.1
1q42.2
2p13
2q14-q14.2
2p23
3q21-q25
6p12
6p21.3
6p21.3
6p21.3
6p24-p23
6q25.1
7q21.3
7q21.3-q12
7q36
8p22
9
10p15p14
10q22.1
Symbol Gene
GSTM1
F5
REN
KiSS1
MTHFR
AGT
EPHX
AGT
MXD1
IL-1 IL1RN
HADHA
AGTR1A/B1
VEGF
TNF
HLA/HLA-DR
HLA-G
EDN1
ESR1
SLC25A13
PAI-I
NSO3
LPL
Trisomy 9
AKR1C3
STOX1
Chromosome location
11p11-q12
11q13
11p15.5
11p15.5
11q22-q23
11q22.3
13
13q12
14q11.1
14q21-q24
14q23.2
14q23.3
14p24.3
16p13.3
17q23.3
17q32
19q13.1
19q13.2
19p13.3
19q13.4
20p-11.2
21q22.3
Xq12q13.3
Xp22.2-p22.1
Xp26
Symbol gene
F2
GSTP1
CDKN1C
IGF-2
MMP-1
MMP-8
Trisomy 13
FLT-1
ANG
HIF1
HIF1A
CHURC1
PGF
FOX-1
PSMC5
ACE
TGFB1
apoE
CNN2
KIR2DL4R
THBD
CBS
VSIG4
PDHA
PLAC1
25
Figure 9 Scheme of pathophysiological relevant factors in preeclampsia and
corresponding candidate genes (Mutze, Rudnik-Schoneborn et al. 2008).
Another possible model to explain the genetic basis of preeclampsia is that fetoplacental rather than maternal acting genes carry an associated risk. These could be
genes encoding factors associated with CT invasion or placental products that
regulate maternal physiological changes, and if this model is correct, genes from the
partner (as inherited by the baby), would also contribute to the disease (Apps,
Sharkey et al. 2011; Clifton, Stark et al. 2012).
The risk of PEE increases with partner change, implying the importance of paternal
genes (Treloar, Cooper et al. 2001). However, the data were not strong enough to
support a model for paternal gene contribution because one of the difficulties in
interpreting the linkage data is that potential disease gene could contribute to disease
26
either by actions in the mother herself (maternal effect) or as a result of inheritance
through the baby (feto-placental effect). Therefore, in families in which the disease
gene is inherited from the mother, the relative risk needs to be compared with
pregnancies in which the baby does or does not carry the suspected allele (Cross
2003). The complexities of the pathological findings in PEE, the epidemiology and
genetics left the field with a complex model of how the disease may develop. The
essence of model is that the pathogenesis is initiated by an altered placenta
implantation which, when combined with maternal risk factors, result in preeclampsia,
however a fundamental question remain to be answered. While there is a link
between an abnormal invasion of EVT cells and PEE, it is unclear whether this
change is sufficient to initiate the cascade of pathophysiological events or whether
maternal susceptibility to hypertension or vascular disease are also required (Cross
2003).
As it was previously mentioned, vascular disease and trophoblast invasiveness are
the principal theories related with the preeclampsia syndrome. On this context it is
important to mention the role of the adjacent genes KiSS-1 and REN on the
regulation of trophoblastic invasiveness and the pathophysiology of hypertension and
vascular diseases respectively (Padmanabhan, Padmanabhan et al. 2000; Bilban,
Ghaffari-Tabrizi et al. 2004).
1.12 Trophoblastic invasion
It has been known for a long time that the occurrence of PEE is somehow linked to
the presence of placenta (Redman 1991) During the first half of human pregnancy,
uteroplacental arteries undergo a series of pregnancy-specific changes that include 1)
apparent replacement of endothelium and media smooth muscle cells by invasive
trophoblast, 2) loss of elasticity, 3) dilation to wide, in contractile tubes, and 4) loss of
vasomotor control (Figure 10).
27
Figure 10 Schematic representation of interstitial endovascular trophoblast invasion in
human pregnancy before week 6th week of gestation and hypothetical
routes of endovascular trophoblast invasion(Kaufmann, Black et al. 2003).
During placentation the mother comes in close contact with semi-allogenic fetal
trophoblastic cells which play a key role in maternal-fetal pathophysiological
exchange. Because of the reduced number of layers separating the two circulations,
the most intimate association between mother and fetus occurs in species with
hemochorial placentation as humans, apes, monkeys and rodents in which fetal
trophoblast is directly exposed to circulating maternal blood (Burton, Barker et al.
2011).
1.13 Renin-Angiotensin System
The renin-angiotensin system (RAS) was among the first hormone systems to be
studied when Tigerstedt and Bergman in 1898 described how intravenously injected
saline extracts of kidney caused pronounced hypertension in rabbits (Phillips and
Schmidt-Ott 1999), and it is a vasoconstrictor system that has largely been regarded
even since. This is, however, only one aspect of the chameleon-like RAS (Lyall and
28
Belfort 2007). During normal pregnancy, the RAS is activated due to increased
estrogen levels, which consequently causes high levels of angiotensinogen and renin
(Hsueh, Luetscher et al. 1982). The circulating renin-angiotensin system is a signaling
cascade that plays a key role in regulating blood pressure and electrolyte balance. It
is classically described in the kidney. However, RAS exists in tissues that have the
capacity for both the local generation and action of Angiotensin II (Ang-II). All
components of the RAS can be found in brain (Bader, Peters et al. 2001; Morimoto
and Sigmund 2002), kidney and heart, vasculature (Bader, Peters et al. 2001),
adipose tissue (Engeli, Negrel et al. 2000), gonads (Speth, Daubert et al. 1999),
pancreas (Sernia 2001) and placenta (Nielsen, Schauser et al. 2000; Anton and
Brosnihan 2008). During pregnancy one of the major extra-renal of RAS production is
the placenta. As early as 1967, Hodari et al. described a placental RAS and identified
a renin-like substance in human placental tissue (Hodari, Smeby et al. 1967). Renin
expression in cultured chorionic cells was first reported by Symonds in 1968
(Symonds,
Stanley
et
al.
1968).
Since
then,
pro-renin,
angiotensinogen,
angiotensinogen converting enzyme (ACE), angiotensin I (ANG I) and ANG II have all
been identified in fetal placental tissues (Shaw, Do et al. 1989; Marques, Pringle et al.
2011; Pringle, Zakar et al. 2011). Angiotensinogen I (AT1) receptor expression in fetal
placental vasculature has also been demonstrated (Li, Shams et al. 1998). Many
other experiments using first-trimester human decidua showed expression of renin,
angiotensinogen, ACE and AT1 receptors (Sasa, Umekage et al. 1997; Pringle,
Tadros et al. 2011). More recent studies using human third trimester decidual cells
also indicate the presence of angiotensinogen and renin in human uterine decidual
cells (Li, Ansari et al. 2000). Localization studies around the decidual spiral arteries
show expression of angiotensinogen, renin, ACE, and AT1 receptors(Morgan, Craven
et al. 1998). Thus in the gravid woman, the maternal decidua and the fetal placental
tissues each contain all necessary components for a functional RAS.
In kidney the enzyme renin is synthetized and released by juxtaglomerular cells of the
efferent renal arterioles in response to low blood pressure and low sodium chloride.
29
Renin release is mediated in part by prostaglandins produced by cells of the kidney’s
macula dense (Jensen, Schmid et al. 1996).
There are two major types of angiotensin receptors: AT1 and AT2. They belong to the
seven trans membrane G-protein-coupled receptor family. They share thirty-four
percent sequence identity and have similar affinities for ANG II (Chung, Kuhl et al.
1998). Most of the effects of ANG II are mediated through activation of AT1 receptors
which are expressed on the surface of vascular smooth muscle cells and adrenal
glands, among others. The AT1 receptor is coupled to the Gq protein that functions in
a signaling pathway to increase intracellular calcium. Its activation promotes
vasoconstriction, sympathetic activity and aldosterone release. The AT2 receptor is
highly expressed in the fetal kidney and its expression decreases during the neonatal
period (Ozono, Wang et al. 1997). In the adult kidney AT2 is much less abundant
than AT1 (Chung, Kuhl et al. 1998); AT2 stimulation can inhibit cell growth, increase
apoptosis, cause vasodilation and is involved in fetal tissue development (Grishko,
Pastukh et al. 2003).
It should be noted that ACE, made by endothelial cells and others, such as smooth
muscle cells, is not the only enzyme that can generate ANG II from ANG I. Chymase,
a chymotrypsin-like serine protease, is a non-ACE angiotensin generating enzyme
that is produced by villous syncytiotrophoblasts (Grishko, Pastukh et al. 2003).
Chymase is also found in great quantities in mast cells, as well as in the skin, heart
and arteries and is a major contributor to the pool of ANG II found in these tissues
(Urata, Kinoshita et al. 1990; Caughey, Schaumberg et al. 1993).
In the humans the RAS undergoes major changes in response to pregnancy. There is
an early increase in renin due to extra-renal local release by the ovaries and maternal
decidua (Hsueh, Luetscher et al. 1982). Angiotensinogen synthesis by the liver is
increased by circulating estrogen produced by the growing placenta. This leads to
increased serum ANG II and aldosterone levels. ACE is the only RAS component that
decreases during pregnancy (Merrill, Karoly et al. 2002). Although ANG II levels
increase during pregnancy, normotensive pregnant women are actually refractory to
30
its vasopressor effects. This is thought to be due to the presence of increased
progesterone and prostacyclin’s which can decrease ANG II sensitivity. RAS is
believed to play a critical regulatory role in feto-placental circulation, facilitating
adequate placental blood flow for fetal oxygenation and maturation. Recent studies
suggest that decidual tissue serves as a source of ANG II production and trophoblast
serve as paracrine targets of ANG II signaling through AT1 receptor activation
(Takimoto, Ishida et al. 1996).
The changes observed in the uteroplacental RAS expression in healthy pregnant
women are different from those in the circulation in preeclampsia. Recent studies by
Herse et al. demonstrate that the only change in placental or decidual RAS
component expression in preeclampsia is the up-regulation of the AT1 receptor in the
maternal decidua (Herse, Dechend et al. 2007).
1.14 REN gene
Renin is an endocrine hormone catalyzing the first step in a cascade of factors that
modulate arteriole blood pressure. It hydrolyzes a single peptide bond in the
circulating globulin, angiotensinogen, releasing the amino-terminal decapeptide
angiotensin I. The absolute specificity of renin is in contrast to other aspartyl
proteases, which act on a broad range of substrates (Hobart, Fogliano et al. 1984).
The renin-angiotensin system plays an integral role in the homeostatic control of
arterial pressure, tissue perfusion, extracellular volume and electrolyte balance (Atlas
2007). Activation of the system is initiated by the release of the enzyme renin from the
kidney into the bloodstream, where it acts on its substrate to produce angiotensin I.
The decapeptide angiotensin I is subsequently converted to angiotensin II, an
octapeptide that causes marked vasoconstriction of arteriolar smooth muscle and
stimulates aldosterone secretion in the adrenal cortex (Imai, Miyazaki et al. 1983).
Analysis of the organization and structure of the renin gene (REN) revealed that it
31
exists in a single copy in 1q32.1, spans roughly 11.7 kb, and consists of 10 exons and
9 introns. It codes for a polypeptide chain of 406 amino acids (Miyazaki, Fukamizu et
al. 1984).
Renin is involved in the activation of RAS through the release of the enzyme by the
kidney into the bloodstream. Once there, renin acts on the generation of ANG l which
is a decapeptide and subsequently converted to an octapeptide ANG ll responsible
for vasoconstriction. ANG II stimulates aldosterone secretion by renal cortex (Imai,
Miyazaki et al. 1983), which is closely linked to the phenomenon of hypertension and
renal injury, two of the main manifestations of PEE (Herse, Dechend et al. 2007). The
renin plays an important role in placentation and pregnancy; it is also involved in the
pathophysiology of PEE. Normal pregnancy is subjected to tight regulation by RAS
which is lost in preeclampsia. In this sense, the main role of the placenta is the
production of soluble fragments of tyrosine kinase-1 (Maynard, Min et al. 2003), and
ANG-ll type l receptor agonist autoantibodies (AT1-AA).These can enhance the RAS
cascade synergistically with ANG-II, which were observed in women with PEE,
suggesting also an immune origin for PEE (Irani and Xia 2008).
The Renin-Angiotensin-Systems decreased up to three-times in PEE, compared to
normal pregnancy; the same behavior was also noted in ANG-ll which is decreased
only in women with PEE despite the increased sodium and water. On the other side,
aldosterone is decreased in this syndrome (Herse, Dechend et al. 2007). The
hemodynamic maladaptation of women with PEE includes increasing rigidity of the
abdominal aorta and rise of resistance of the peripheral arteries and arterioles, with a
direct relationship between a progressive increase in aortic stiffness index and
severity of PEE (Uchida, Mori et al. 2007).These altered mechanisms have been
found in PEE patients but not in women with healthy pregnancy (Farina, Sekizawa et
al. 2006).
1.15 KiSS-1 gene
32
KiSS-1 gene was identified in malignant melanoma cells and located on chromosome
1q32-q41, its regulation by elements located on chromosome 6 was demonstrated by
analysis of the hybrid cell line Neo6/C8161 (Lee, Miele et al. 1996; Lee and Welch
1997). Furthermore, KiSS-1 can suppress metastasis without affecting tumorogenicity
in lung cancer cells, squamous epithelial cells, breast and renal cells transfected into
MDA-MB-435 cell line in athymic mice(Lee and Welch 1997; Ikeguchi, Yamaguchi et
al. 2004; Shoji, Tang et al. 2008). In metastatic breast cancer, Starks et al. found a
significantly decrease in mRNA expression of KiSS-1 and some other genes involved
in metastatic suppression, by PCR-RT assays (Stark, Tongers et al. 2005). KiSS-1
gene with 6151 bp of genomic sequence and is composed of four exons, where the
first two exons are not translated, the third exon contains 38 bp embedded in noncoding 5’ region, followed by a translational initiator site and other translated 100 bp;
the terminal exon contains 760 bp that include a 332 bp translated region, the stop
codon and the sequence of poly-A signal (West, Vojta et al. 1998). It has been shown
that expression of KiSS-1 is regulated by both the transcription factor Sp1 within the
promoter region (-100 pb) and a GC rich sequence located between -93 to -58 bp
(Mitchell, Stafford et al. 2007).
The gene KiSS-1 codes for a proteolytic peptide included in the family of kisspeptins
which have the highest content of aminated aminoacids. Kisspeptin is the most
abundant protein. It is also known as AXOR12, hOT7T175, Metastin or protein
binding to receptor GPR54. Kisspeptins inhibit chemotaxis, and affect the growth of
cancer cells through metastasis and endocrine actions (Makri, Pissimissis et al.
2008).
Kisspeptin protein is strongly expressed in placenta and others organs (Muir,
Chamberlain et al. 2001; Seminara and Kaiser 2005), it is highly expressed in various
tissues including the placenta that can be activated by KiSS1 (Muir, Chamberlain et
al. 2001). KiSS-1 expression is significantly associated with histological appearance
of some types of cancer and has a predictive value for patients with metastatic
cancer. However, these results are not conclusive (Sanchez-Carbayo, Capodieci et
al. 2003). The loss of expression of KiSS-1 and its receptor GPR54 are associated
33
with a high incidence of squamous cell carcinoma of the esophagus and liver
(Ikeguchi, Hirooka et al. 2003; Ikeguchi, Yamaguchi et al. 2004). The GPR54 or
Metastin is a 54 aminoacid peptide, structurally related to neuropeptides receptors
and particularly to the galanin receptor, peptide derived from KiSS1 and, also isolated
from placental tissue and other organs (Kotani, Detheux et al. 2001).
Metastin concentration in pregnancy is increased more than twice in regard to the
reference values for non-pregnant women; this increase is proportional according to
both the progression of pregnancy and, the size of the placenta. Detection of metastin
is carried out by enzyme immunoassay (EIA) during pregnancy and postpartum with a
concentration six fold higher concentration (9590 fmol/mL) than in non-pregnant
women (Horikoshi, Matsumoto et al. 2003). Given its role as a marker of trophoblastic
invasion it could also be useful as a tumor metastasis predictor (Horikoshi,
Matsumoto et al. 2003).
GPR54 has provided additional clues about metastin and its receptors; they are
involved in MAP kinase pathways and also suggest the potential for autocrine,
paracrine and endocrine roles. It also impacts on motility, chemotaxis, adhesion and
invasion each documented in different cell lines, however conflicting observations
require resolution. Nevertheless, clinical evidence demonstrate the loss of KiSS-1 in
metastases; a correlation exists among, KISS-1 and metastin receptor expression
with human tumor progression (Harms, Welch et al. 2003).
KiSS-1 secretion is necessary for metastasis suppression in various organs and
tissues through the GPR54 receptor.KiSS-1 has the potential role to be a good
cancer prognostic indicator and a therapeutic target for the management of metastatic
disease (Nash and Welch 2006),
The primary translation product of KiSS-1 is a 145 amino acid polypeptide (Kp-145),
but shorter kisspeptins (KP) with 10, 13, 14 or 54 amino acid residues also may be
produced. Kp-10, a decapeptide derived from the primary translation product, was
identified in conditioned medium of first trimester cultured human trophoblast. Kp-10,
34
but not other kisspeptins, increased intracellular Ca(+2) levels in isolated first trimester
trophoblast. Kp-10 inhibited trophoblast migration in explants as well as in cell
migration/invasion assay without affecting proliferation. Suppressed motility was
paralleled with suppressed gelatinolytic activity of isolated trophoblast. Identified Kp10 with paracrine/endocrine functions, regulates the fine-tuning trophoblast invasion
generated by the trophoblast itself (Bilban, Ghaffari-Tabrizi et al. 2004). In conjunction
with the matrix metalloproteinase (MMP-9), Kp-10 acts synergistically blocking cell
migration which could be used as antimetastasic agent while the expression of the
invasion-related gene, MMP-9, is positively related with, invasiveness process, the
invasion suppressor gene, KiSS-1, is negatively related with the invasive ability of
trophoblast. The interaction of these two genes plays an important role in regulation
of the invasion of trophoblast (Qiao, Cheng et al. 2005).An imbalance in the
expression of MMP-9 and KiSS-1 in trophoblast may play an important role in the
pathogenesis of PEE. On the opposite side decreased expression of MMP-2 and
MMP-9 in placentae may lead to impaired invasion of trophoblast cells, causing
abnormal placentation and occurrence of preeclampsia (Li, Xiong et al. 2007).
Another function of KiSS-1 and GPR54 receptor is related to vasoconstriction of the
coronary endothelium and umbilical vein endothelium-independent in humans,
suggesting a previously undescribed role for GPR54 and KP in the cardiovascular
system. So its presence in pregnancy is more complex than just inhibiting trophoblast
invasion (Mead, Maguire et al. 2007).
On the other hand, there is evidence that the hypothalamic KiSS-1/GPR54 system is
a pivotal factor in central regulation of the gonadotropic axis at puberty and in
adulthood (Navarro, Castellano et al. 2004). Functional evidence demonstrated the
KiSS1 ability to activate reproduction in immature females, with an increase in uterine
weight, levels of luteinizing hormone (LH), estrogen and possibly prolactin in serum,
however, ovulation process has not been well monitorized. These markers strongly
support the hypothesis that administration of KiSS1 at the hypothalamus is sufficient
to trigger the onset of puberty in young rats and young adult rats (Navarro, Castellano
et al. 2004). Also, an increase of LH was observed 15-60 min after subcutaneous
35
administration of Kisspeptin at doses of 300 mmol although kisspeptin levels
decreased more rapidly in subcutaneous administration compared with central
administration, Thus reducing their effect on LH (Thompson, Patterson et al. 2004).
The loss of GPR-54 function causes hypogonadism hypogodadotropic by impairing
the secretion of follicle stimulating hormone (FSH) and LH, both of which impact on
the maturation of puberty. It has been shown that the genes of GnRH (de Roux,
Genin et al. 2003; de Roux 2006), are involved in sexual maturation and GnRH are
related with PROK2 gene and his receptorPROKR2 (Corowley, Pitteloud et al. 2008).
This opens new possibilities in the treatment of reproductive disorders such as
delayed or advanced puberty, hormone-dependent cancers and infertility (Messager
2005). Studies in experimental models on the administration of KiSS-1 peptide has
revealed a significant increase in the activation of reproduction by increasing
luteinizing hormone, estrogen in serum and increased uterine weight (Navarro,
Fernandez-Fernandez et al. 2004).
In 2004, Nistala et al, demonstrated the presence of a conserved stretch of five genes
in a P1-derived artificial chromosome (PAC) containing part of chromosome 1
(Nistala, Zhang et al. 2004). Two of these genes, KiSS-1 and REN, are contiguous
each other and involved in two of the main process related to the pathophysiology of
the disease. In this sense it is important to know their expression profile in the
placenta of women with PEE vs. those placentas from women with normal pregnancy,
likewise to determine if there is a common regulatory element for both genes.
1.16 Transcriptional control of gene expression
The development of multicellular organisms is, to a large extent, dictated by a
carefully choreographed progression of domain and tissue-specific gene expression.
To understand development, it is therefore necessary to understand the logic and
mechanisms of the transcriptional network. Much of the information that determines
when and where genes will be expressed is encoded in an organism’s genome
sequence (Berman, Nibu et al. 2002). Although we now have human genome
36
sequences, our understanding of how and where this information is used is extremely
limited.
Organisms ranging in complexity from bacteria to higher eukaryotes are able to react
and adapt to environmental and cellular signals. These responses are often encoded
as complex gene regulatory networks. In these networks the expression of a gene’s
product is regulated by the activity of other genes. Although regulation can occur at
many levels as: promotors and enhancers sequences, TF’s, and enhanceosomes and
TF’s complex. Tthe transcriptional regulation is one of the most important and
dominant level of regulation in eukaryotes.
Transcription involves synthesis of RNA chain representing the coding strand of DNA
duplex (Lewin, Aguilera Lopez et al. 2010). The initial step of the synthesis of
messenger RNA (mRNA), ribosomal RNA (rRNA) and transfer RNA (tRNA) is the
finding of location of the RNA polymerases with a DNA sequence called promoter of
the gene to be transcribed by RNA polymerase. RNA polymerase type l synthesizes
rRNA (except 5S), the type II synthesizes mRNA, some mRNA involved in the
splicing of the primary transcript while the RNA polymerase III synthesizes rRNA 5S
and tRNA. The more complex regulatory processes encompass the genes encoding
class II or mRNA (Janky, Helden et al. 2009).
Gene transcription includes various elements such as the promoter located at the 5´
end of the coding region and containing TFBSs, the presence of enhancer regulatory
sequences (enhancers), and the interaction between multiple activating or inhibitory
proteins that act by binding to specific sequences of DNA recognition (Bradshaw and
Dennis 2003). Transcriptional regulation involves the participation of transcription
factor (TF), that bind to DNA at specific transcription factor binding sites (TFBSs) in
order to regulate the transcription of a gene through modulation of RNA polymerase
complex activity. TFBSs often appear in clusters or cis-regulatory modules,
presumably to enable interactions between TFs bound to them. In fact, TF do not
work isolated from each other. Particularly in higher organisms, the formation of
complexes of various TF bound to DNA is often necessary to induce the response of
37
a cell to external stimuli or developmental programs. Such a response is frequently
implemented as a transcriptional switch where a combination of presence or absence
of certain TFs regulates the expression of a certain gene (Reid, Ott et al. 2009).
The organization of the transcriptional regulation network includes a few global
regulatory transcription factors and many fine turners, regulatory cascades and dense
overlapping regions, in which several transcription factors jointly regulate several
sites. TFs can be classified into 10 broad functional classes, and in certain of these,
there are global regulators that control many genes. The global regulators amplify
their effect by regulating other transcriptional factors (Madan Babu and Teichmann
2003). Regulation of gene expression at the transcriptional level is a fundamental
mechanism which is evolutionarily conserved in all cellular systems. Transcriptional
regulation is mediated by the physical interaction between TFs and specific cis-acting
regulatory elements in promoter regions of target genes (TGs) (Browning and Busby
2004). Graphs have been largely used in biology to represent study and integrate
such regulatory information at several levels (Babu, Luscombe et al. 2004;
Luscombe, Babu et al. 2004; Deplancke, Mukhopadhyay et al. 2006). In a typical
transcriptional regulatory network TFs and TGs are represented as nodes, and their
regulatory interactions are depicted as a directed edge from TFs to their TGs. Other
representation of transcriptional regulation is the co-regulation network (CRN) where
each node is either a TG or a TF only and undirected arcs link pairs of genes that are
regulated by the same TF or are regulating the same gene (Balaji, Babu et al. 2006;
Balaji, Iyer et al. 2006). This undirected network representation is also useful to link
pairs of genes that are co-expressed (which show similar expression pattern across
multiple conditions) in co-expression networks (Stuart, Segal et al. 2003). However,
the grammar of the cis-regulatory code is clearly more complex than simply the
sensitivity of transcription factor binding sites (Pennacchio and Rubin 2001).
The promoter contains a group of sequence known as
that defines the site of
initiation of RNA transcription (Lewin, Aguilera Lopez et al. 2010). The most common
sequence present in proximal promoter is the TATA box with is highly conserved
throughout evolution.The TATA box is located 20-30 bp upstream from the start site
38
of transcription and is bound by the TATA box binding protein (TBP). However, other
transcriptional factors are involved in transcription initiation: numerous proteins
identified asTFIIA, B, C, D, and so (regulatory factors of RNA polymerase II) which
interact with TBP bound to TATA box. The promoter contains other specific DNA
sequences that are recognized by TF. For example, some promoters contain the
CCAAT box which resides 50-130 bp upstream of the transcriptional start site. The
protein called C/EBP (CCAAT-box/enhancer/binding/protein) binds to this sequence.
Another regulatory sequence includes the GC box. The number and type of
transcriptional regulatory elements vary with each gene. The nature of the promoters
and the combination of proteins interacting with them is one of the main mechanisms
of gene expression regulation (figure 11). The elements and their functions in
transcriptional process are following:
x
Activators- Proteins that bind to Enhancer sites which helps the RNA
polymerase to position properly to begin transcription
x
Repressor- Protein that interferes with the binding of activators. This action will
slow on prevent transcription
x
Basal factors- Proteins which position RNA polymerase at the beginning of the
gene. Once positioned, the transcription begins.
x
TATA box- Highly conserved DNA sequence (part of the promoter) upstream
of the structural gene. A TATA binding proteins attaches to the site, which is
essentially the first step in the binding of the entire transcription complex.
x
Transcription factor.- Can sense the correct positioning of activators. If aligned
properly, transcriptional factors will release RNA polymerase and transcription
begins.
While most regulatory sequences are preferably located upstream (5') of the
transcriptional starting point, some may be located downstream (3') or even be in
intragenic regions.
39
Figure 11 Eukaryotic transcription complex and its components
1.17
Enhancer elements
Since their identification in the early 1980s, transcriptional enhancers have been the
subjects of numerous studies because of their ubiquitous roles in higher eukaryotic
gene regulation (Arnosti and Kulkarni 2005).
Enhancers are DNA sequences that need not need to be located near to the
transcription initiation site to affect transcription, they can be located in several
hundreds or thousands of base pairs upstream or downstream of a gene and its
promoter, and be able to regulate gene expression. Enhancers do not act on the
promoter region itself, but are bound by activator proteins.that are able to activate
transcription through their interaction with the other TF bound to promoter. In general,
enhancers have the ability to greatly increase the expression of genes in their vicinity
(Brown 1981). Typical enhancers span 200-1000 bp, and bind to dozens of
sequence-specific proteins upwards (Carroll, Grenier et al. 2001).
40
Extensive analyses of enhancers have evidenced several specific features. First,
enhancers are functional over a large distance. For example, an enhancer that was
placed 3000 bp away from the gene, was still able to increase gene expression.
Second, enhancers are orientation independent. This means that the element can be
inverted and still affect gene expression. Another feature of enhancers is that they are
often associated with a gene that is abundantly expressed; moreover, is they show
tissue specificity (Yamaguchi and Matsukage 1990; Palacios DeBeer and Fox 1999).
At a general level, there are two basic structural features that apply across the
spectrum of enhancers found in multicellular organisms: transcription factor binding
sites are typically within 100 kbp of a gene, and they are usually clustered.
(enhancers are generally liked in cis-to their targets, although examples of trans
regulation generally via pairing of sister chromosomes are documented (Duncan
2002). The clustering of binding sites seems to be a common feature of enhancers;
single factor binding sites are general insufficient to drive gene expression, a
mechanistic feature that appears to prevent unwanted activation by randomly
occurring sites. Instead, multiple, clustered sites are a hallmark of enhancers, and
presumably reflect the synergy required for important , but weak, protein-protein
interactions to occur (Droge and Muller-Hill 2001).
The specificity of transcription may be controlled by either a promoter or an enhancer.
A promoter may be specifically regulated, and a nearby enhancer used to increase
the efficiency of initiation; or a promoter may lack specific regulation, but become
active only when a nearby enhancer is specifically activated (Lewin, Aguilera Lopez et
al. 2010). A difference between enhancers and promoters may be that an enhancer
shows greater cooperatively between the bindings of factors. A complex that
assembles at the enhancer that responds to IFN-Ȗ (gamma interferon) acting
cooperatively to form a functional structure called the “enhanceosome”. An enhancer
contains several structural motifs, and mutations on these motifs reduce enhancer
function to <75% of wild type. In contrast with the constructions of promoters all
components are required to create an active structure at the enhancer and these
components do not themselves directly bind to RNA polymerase, but they create a
41
surface that bind a co-activating complex and this complex helps the pre-initiation
complex of basal TFs that is assembling at the promoter to recruit RNA polymerase
(Arnosti and Kulkarni 2005).
1.18 Enhancers related with REN gene expression
Regulatory elements controlling human REN gene expression have been identified in
the proximal and the distal promoter region, the complexity of renin gene regulation in
humans and rodents is evidenced by the abundance of TFs involved (Pan and Gross
2005). Binding of several TFs have been reported in the proximal promoter that have
either stimulatory or inhibitory effects on renin expression (Pan, Jones et al. 2004;
Tamura, Chen et al. 2004; Pan, Glenn et al. 2005; Todorov, Desch et al. 2007).
Additional regulatory elements have also been described in distal promoter.
Itani et al. described two critical regions identified in the 5-flanking region of the REN
gene, that are required for its transcriptional regulation the proximal promoter and an
enhancer element located at -2866 to -2625 bp (Itani, Liu et al. 2007). The proximal
promoter was originally reported to consist in binding sites for cAMP response
element-binding protein. The enhancer was first called mouse renin enhancer now
known as Kidney Enhancer (KE), it was identified by deletion mutagenesis and
transient transfection analysis in mouse kidney renin-expressing As4.1 cells and is
now recognized to be a highly conserved transcriptional regulatory element required
for high-level expression in As4.1 cells (Petrovic, Black et al. 1996; Shi, Black et al.
1999). In addition, to the kidney enhancer located at 2.6 kb upstream of the mouse
and 11 kb upstream of human renin gene. (Shi, Gross et al. 2001; Pan, Glenn et al.
2003; Liu, Shi et al. 2006). There was reported a second enhancer highly acti ve
enhancer was reported in choriodecidual cells (Germain, Bonnet et al. 1998). This
enhancer termed the Chorionic enhancer (ChE) derived from human chorion tissue
that have been used to study renin gene transcription despite the fact they are of
42
extra-renal origin. Recent studies demonstrate that human chorionic cells are suitable
for studying the transcriptional regulatory elements in renin gene expression
(Borensztein, Germain et al. 1994). The production of renin, which catalysis the ratelimiting step of the renin-angiotensin system, is also strongly stimulated by the novel
500 bp enhancer element in the distal region of the promoter of the human gene (5777 to -5312) (Fuchs, Philippe et al. 2002). This fragment had full enhancer activity
with 56-fold higher transcriptional activity in chorionic cells. (Fuchs, Philippe et al.
2002).
Sigmund and colleagues showed that deletion of the chorionic enhancer (ChE) had
no qualitative or quantitative effect on the tissue-specific expression of human renin,
or on the cellular localization of human renin in the kidney or placenta. Combined
deletion of both the ChE and KE caused a decreased in the level of renal renin
expression consistent with the established role of the KE (Zhou and Sigmund 2008).
They also considered the possibility that the ChE is a downstream enhancer on the
KiSS1 gene, which lies directly upstream of renin and is also expressed in the
placenta. However the deletion of the ChE alone or deletion of ChE and KE together,
had no effect on the levels of KiSS1 expression in the placenta. However, these data
provide convincing evidence that the ChE is silent in vivo, at least in the mouse.
Despite the latter we consider necessary to study this phenomena in human genome
as gene regulation could be different among species.
43
II.
JUSTIFICATION
Preeclampsia/eclampsia is a disease with high morbidity and mortality rates in
the maternal-fetal binomial around the world including Mexico. Until now, there is
not a completely accepted theory about its origin, although many hypotheses
have been proposed which are not totally conclusive. Currently two main theories
are well recognized as pathophysiologically relevant: a deficient trophoblast
invasiveness driving to insufficient vascularization utero-placentary or a
generalized endotheliosis in the mother, responsible of the clinical syndrome. So,
the role of KiSS-1 and REN genes as inhibiting agent for trophoblast
invasiveness and hypertensive agent, respectively, as well as their physical
proximity in chromosome 1 suggests a probable synergistic role in the genesis of
preeclampsia-eclampsia through their co-activation by a common regulatory
element.
44
III
3.1
OBJECTIVES
General objective:
Identify of a common transcriptional regulatory element for the expression of KiSS-1
and REN genes in placenta from women with preeclampsia-eclampsia in comparison
with women with normo-evolutive pregnancy.
3.2.1 Specific objectives:
3.2.1
To evaluate and compare the expression levels of KiSS-1 &REN
genes in women´s placenta with preeclampsia-eclampsia compared
with the expression levels of women with normo-evolutive
pregnancy
3.2.2
To identify the possible common regulator element for the
expression of KiSS-1 through luciferase expression assay in
transient transfected BeWo cells.
45
3.3
Hypothesis
The expression levels of KiSS-1 and REN genes are increased in woman´s
placenta with Preeclampsia-Eclampsia compared with expression levels in
woman´s placenta with normo-evolutive pregnancy and these differences are
driven by a common transcriptional regulatory element.
46
IV.
4.1
MATERIAL AND METHODS
KiSS-1 and REN gene expression in placental tissues from preeclamptic
womenma
4.1.1 Selection of patients
This case-control study was approved by an investigation ethical committee in the
Hospital General de Durango, México; in accordance with The Code of Ethics of the
Declaration of Helsinki (Annex I). Twenty seven preeclamptic women under antihypertensive treatment and the same number of healthy pregnant women, agreed to
donate their placenta for the study after signing an informed consent letter.
Pairing was based on chronological age, gestational age and the number of
pregnancies, as they are known to affect placental gene expression (Shah 2005;
Reynolds, Logie et al. 2009).
Inclusion criteria were those established by the Working Group Report on High Blood
Pressure in Pregnancy and patients were classified according to severity status in
mild preeclampsia (BP PP+J PHDVXUHG WZLFH ZLWKLQ D WZR KRXUV LQWHUYDO
and proteinury 300 mg/dL or 1+ inlab stick urine analysis) or severe preeclampsia (BP
PP+JRQWZRRUPRUHRFFDVLRQVKRXUs apart and proteinury 2 g or more
in 24h or 2+ qualitatively) with new onset hypertension after 20 weeks gestation (NIH.
2000).
All patients under treatment with glucocorticoids affected by gestational hypertension,
gestational diabetes, diabetes type 1 and 2 and hepatic or kidney diseases were
47
excluded. A questionnaire with clinical relevant data was also obtained from all
participants.
4.1.2 RNA extraction and cDNA synthesis
Within 30 minutes after delivery, placentas were washed with cold PBS at pH 7.4.
Twelve different 5 X 5 mm biopsies were taken from maternal side of the placenta.
Fifty to 100 mg of the sample was kept in RNAlater® (Ambion™) at 4°C until RNA
extraction with TRIpure isolation reagent® kit (ROCHE™). RNA was treated with
DNA-free® (Ambion™) and its integrity was verified by 1% electrophoresis agarose
gel. Samples were stored at -72°C until cDNA synthesis, which was carried out using
the High Capacity cDNA Reverse Transcription® (Applied Biosystems™) kit,
according to the manufacturer protocol; quantity and purity were evaluated through
spectrophotometry. The synthesized cDNA was kept at -20°C until further analysis.
4.1.3 qPCR
Gene expression was analyzed by semi-quantitative Polymerase Chain Reaction
(qPCR) in SteptOne™ Applied Biosystems equipment, using FAM™ dye-labeled
TaqMan® MGB probes (Applied Biosystems). KiSS-1 probe was based on RefSeq
NM_002256.3; assay ID Hs00158486_m, which targets exons 2-3. REN probe was
based on RefSeq NM_000537.3, assay ID Hs00166915_m1, targeting exons 1-2.
The relative expression (RQ) of KiSS-1 and REN genes was normalized to the
constitutive and endogenous control human gene beta-2-microglobulin (B2M) (FAM /
MGB Probe, Non-Primer Limited RefSeq NM_ 004048.2), (Applied Biosystems™).
This probe targets exons 2-3. Amplification efficiencies with values between 90 and
110% allowed establishing an optimal working concentration 30 ng/μL. An expression
test was also randomly run for B2M gene in five patients and five control samples in
48
triplicate, to estimate significant differences between groups and determine the
optimal PCR cycle at which fluorescence gave the better exponential cycle. The
qPCR conditions were: 10 minutes of initial hold at 95°C, followed by 48 cycles of 15
seconds at 95°C for denaturing and 1 minute at 60°C for both annealing and
extension. Cycle threshold values were obtained by triplicate and each sample was
normalized with B2M endogenous control gene to calculate RQ values or (2-ǻǻ&W) for
KiSS-1 and REN.
4.2
Searching for possible regulato elements
The expression of the gene REN is regulated through enhancer elements: ChE and
KE. So, in order to find if such elements also regulate the expression of the
contiguous
gene
KiSS-1,
constructs
were
developed
containing
different
combinations of enhancer(s) and KiSS-1 promoter regions. These constructs were
cloned
into
expression
vectors
pGL4
and
were
transfected
into
BeWo
choriocarcinoma cell line to evaluate the expression of luciferase.
4.2.1
PCR amplification and cloning
PEK, ChE and KE fragments were amplified using genomic DNA from a healthy
donor. The sequences of fragments were analyzed by molecular tool NEBcutter
disposable freeware Online in http://tools.neb.com/NEBcutter2/index.php to find a
similar restriction site that was also present in vector pCR®-Blunt II-TOPO by
Invitrogen™ and pGL4 expression vectors.
49
PEK primers sequences with XhoI and SacI enzyme restriction sites (bold) to further
cloning into the polylinker region of expression vector pGL4 were:
Forward primer 5’-GAGCTCGACATTTCACACTTCTCCCC-3’ and
Reverse primer 5’-CTCGAGCCTCCAGAGCATCCCCAGGTCAG-3’.
ChE fragment with enzymatic restriction sites for KpnI (bold) at both ends for cloning
in both directions was amplified with:
Forward primer 5’-GGTACCGGGCTTAGTTTGGGGGAAAGAGCTG-3’
Reverse primer 5’-GGTACCCAAACCTTAGAACACCAAAGCAGG-3’.
KE fragment with enzymatic restriction for KpnI and SacI (bold) was amplified using,
Forward primer 5’-GGTACCCTGATGTGGACACTGGGAGAAGAC-3’
Reverse primer 5’-CTCGAGCAAGTAGGACGTGGCTGTGGATAG-3’.
PCR amplifications were performed in Eppendorf Mastercycler DNA Engine Thermal
cycler PCR.
The amplification conditions for PEK and KE fragments were: initial denaturation 5
min at 95°C, followed by 35 cycles with denaturing 45 sec at 94°C, annealing 45 sec
60°C and extension 45 sec 72°C and final extension 5 min 72°C and hold 4°C.
The ChE fragment was amplified under the following conditions: an initial
denaturation of 1 min at 95°C, then 35 cycles of denaturation 45 sec at 94°C,
annealing 45 sec at 60°C and extension 45 sec at 72°C, final extension 5 min at 72°C
and hold 4°C.
A high efficiency VentR® DNA polymerase by New England Biolabs was used as well
as DMSO (6%) in both PCR reactions in a final volume of 15 μL. The amplified
fragments were evaluated by 1% electrophoresis agarose gel and stained with
ethidium bromide.
50
4.2.2 Bacterial strain and plasmids culture media
Escherichia coli strain as well plasmids used in this thesis are listed in table 3 and 4
repectively.
4.2.3 Escherichia coli and culture media.
Growth and spread of Escherichia coli DH5-Į was carried out on Luria-Bertani media
(LB) (Miller 1972), which contains (w/v) 0.5% yeast extract, 1.0% peptone 1.0%
Sodium Chloride and in the case of solid medium 2.0% bacteriologic agar. When
solid media was required, media was supplemented with the following antibiotics:
ampicillin (amp) at final concentration 100 μg/mL and kanamycin (Km) a final
concentration 100 μg/mL. E. coli was grown at 37°C in solid medium and/or liquid
medium with vigorous stirring in a shaker (MaxQmini4000) with circular agitation (200
rpm). Culture density was determined in spectrophotometer (6405 UV / Vis, Jen Way)
at 600 nm.
51
Table 3 Bacterial strains used in this experiment
Strain
Escherichia coli DH5-Į
Description
Reference
fhuA2 ǻ(argF-lacZ)U169
Culture
phoA glnV44 ĭ80
Laboratory
ǻ(lacZ)M15 gyrA96 recA1
Research
relA1 endA1 thi-1 hsdR17
UJED.
(Taylor, Walker et al.
Pacheco.
collection
of
Scientific
Institute
PhD.
of
from
Salas-
1993).
PEK
E. coli DH5-Į transformed
This research
with KiSS-1 promotor and
exon 1 KnR
ChE
This research
with Chorionic enhancer
KnR
KE
This research
with Kidney enhancer KnR
B1and B2
This research
with PEK fragment ampR
C1 and C2
This research
with PEK+ChE fragments
ampR
D1 and D2
This research
with PEK+KE fragments
ampR
E1 and E2
This research
with PEK+CHE+KE
fragments ampR
52
Table 4 Plasmids used in this research.
Plasmid
Description
Reference
pCR®-Blunt II- Vector to cloning PCR fragments in E. coli
Invitrogen
R
TOPO
breaking letal gene ccdB Kn
pGL4.10
Vector(a–d) encodes the luciferase reporter gene
Promega
luc2 (Photinus pyralis) and designed for high
expression and reduced anomalous transcription.
pGL4.11
Vector(a-d) encodes the luciferase reporter gene
Promega
luc2P (Photinus pyralis) and designed for high
expression and reduced anomalous transcription
pGL4.13
Vector(a-d) encodes the luciferase reporter gene
Promega
luc2 (Photinus pyralis) and designed for high
expression and reduced anomalous transcription
and used as an expression control or a co-reporter
vector.
PEK
Vector pCR®-Blunt II-TOPO containing fragment
This research
PEK with specific XhoI and SacI restriction tail KnR
ChE
ChE with specific restriction tail Kn
KE
This research
R
This research
KE with specific restriction tail KnR
B1 and B2
Vectors
pGL4.10
and
4.11
containing
PEK This research
fragment AmpR
C1 and C2
Vectors pGL4.10 and 4.11 containing PEK+ChE This research
fragments AmpR
D1 and D2
Vectors pGL4.10 and 4.11 containing PEK+KE This research
fragments AmpR
E1 and E2
Vectors
pGL4.10
and
PEK+ChE+KE fragments Amp
4.11
containing This research
R
53
The PEK, ChE and KE fragments were cloned into vector pCR®-Blunt II-TOPO by
Invitrogen™ (Figure 12) using compatible enzymatic restriction sites to generate a
ODUJH QXPEHU RI FRSLHV LQ '+Į Escherichia coli competent cells. The E. coli cells
were transformed using Calcium Chloride and treated by heath-shock at 42°C to
become competent cells (Sambrook and Russel 2001). The selection of transformed
cells was done by evaluation of resistance to kanamycin due to the gene, included in
pCR®-Blunt II-TOPO vector. The plasmidic DNA (pDNA) was recovered by alkaline
extraction method (Birnboim and Doly 1979) and modified to our laboratory
conditions.
The PEK, ChE and KE fragments were released from the recombinant pCR®-Blunt IITOPO cloning vector by digestion with specific restriction enzymes
Figure 12 pCR®-Blunt II-TOPO PCR cloning vector showing restriction poli-linker site
and topoisomerase on each extreme 5’and 3’ poli-linker site for efficient
ligation of interest fragments.
54
4.2.4 Development of constructs to assess regulation on promoter KiSS-1
gene
In order to search for a common regulatory element(s) for REN and KiSS-1 genes,
constructs were created including combinations of the following fragments, basal
promoter plus Exon 1 from KiSS-1 gene (PEK) of 512 bp, chorionic enhancer (ChE)
of 600 bp and kidney enhancer (KE) of 300 bp (Minimal regions). Each construct
containing one or both enhancers participate in cis-trans with promoter of KiSS-1
gene to regulate the expression of luciferase gene.
We consider cloning into the basic vector pGL4.10 [luc2] vector (1) without driving
expression elements, into pGL4.11 [Luc2P] vector lacking promoter (2) and, into
pGL4.13 [luc2/SV40]. The last vector contains the luc2 reporter gene and the SV40
early enhancer/promoter. All the vectors were provided by Promega® (figure 16).
4.2.5 Construction of expression plasmids
Different constructs were created using pGL4.10 (1) and pGL4.11 (2) vectors. A
construct containing PEK fragment herein after called B1 and B2 (Figures 13), the
PEK+ChE fragments called C1 and C2 (Figure 14), a construct with PEK+KE
fragments called D1 and D2 (figure 15) and a construct with PEK+ChE+KE
denominated E1 and E2, according to compatible flanking restriction sites. Before
ligation step, pGL4 vectors were dephosphorylated with thermosensitive alkaline
phosphatase (TSAP®) following the manufacturer’s instructions (Promega™). All
ligations were catalyzed with T4 DNA ligase by Promega™ in agreement with
supplier’s instructions and ligated to vector. All constructs were cloned and multiplied
into competent DH5-ĮE. coli to later extract pDNA and purify it using the WizardPlus
SV Miniprep DNA purification System. The success of ligation and verification of 5´-3´
insertions were confirmed by 1% agarose gel electrophoresis and staining with
ethidium bromide.
55
Constructs were also sequenced in Perkin Elmer/Applied Biosystems equipment Mod
3730 by method Taq FS Dye Terminator cycle Sequencing Fluorescence-Based
Sequencing using pGL4 vector primers.
The sequences obtained were analyzed with “BioEdit” sequence alignment editor
freeware software (available in: http://www.mbio.ncsu.edu/bioedit/bioedit.html) and
analyzed
with
Bio-Web
tools
freeware
available
in
web
site:
(http://www.cellbiol.com/scripts/complement/reverse_complement_sequence.html) to
verify amplification step and that the sequences in fragments have not errors regard
to NCBI genome database (http://www.ncbi.nlm.nih.gov/genome/guide/human/).
The obtained pDNA were treated with RNase solution 1 U/μL and purified with Wizard
SV Gel and PCR clean Up System by Promega. They were kept at -20°C until
transfection.
56
Figure 13 The family of pGL4 Luciferase vectors incorporates a variety of additional
features such as a choice of luciferase genes, rapid response and vector
with or without promoter and response elements
57
Construct B
SacI
Figure 14 Construct B versions 4.10 and 4.11 only contain PEK fragment
Construct C
Figure 15 Construct “C”
58
Construct D
Figure 16 Construct “D” in versions pGL4.10 and 4.11.
Construct E
Figure 17 Construct E in pGL4.11 version.
59
Figure 18 The Control vector pGL4.13
4.2.6 BeWo culture cells
The funcionality of regulatory regions in the constructs is evaluated in a cell system
simulating an in-vivo condition. For this reason, BeWo cells line from choriocarcinoma
were chosen. The BeWo cell line was kindly donated by PhD Omar Arroyo from
Universidad Veracruzana and obtained from ATCC (No.CCL-98). To confirm the
identity of the cells both karyotype and G6PD quantitation were carried out.
Mycoplasma absence contamination was evaluated by PCR (Annex VIII).
The culture conditions were established with DMEM/F12 medium and enriched with
4.5 g/L glucose, FBS 10%, HEPES 15mM, L-glutamine 2 mM, MEM-Non essentials
amino acids 1% and antibiotics 1% of Ampicillin 10,000 U/Streptomycin 10 mg/mL
The minimum cells concentration to seed in P100 dish and achieve a 85-95% of
confluency in the day 4-5 was 8X106 at 37°C, 5% CO2 and humidity. The passage or
transference of cells was done by removing the culture medium, washing with 2 mL of
60
PBS/EDTA, adding 0.25% trypsin/0.53mM EDTA sterile solution and incubating at
growth conditions by 5 min. Dissociation is done by pipetting with culture media/FBS
to stopped trypsination and centrifuge at 1400 rpm by 5 min to get a cellular bottom,
this is diluted with 1 mL completed culture medium.
4.2.7 Viability and cellular count
The cells were diluted 1:10 with Trypan blue solution 0.4% reposed by 5 min and
counted with a hemocytometer in 4 quadrants applied the hemocytometer general
formula:
=
( ) ( , ) ( )
VC= viable cells per mL
CC = total viable cell count of four corner squares
Dilution= correction for the trypan blue dilution (counting dilution /trypan blue)
Quadrants = correction to give mean cells per corner square (4)
10,000= conversion factor for counting chamber
The viability was done counting the cells stained blue vs. non stained cells, reporting
in percentage and accept more than 85% viability. (Annex VII)
61
4.2.8
Transfection and luciferase assays
Firefly luciferase is widely used as a reporter gene for the following reasons: (a)
reporter activity is available immediately upon translation since the protein does not
require post-translational processing; (b) the assay is very sensitive because its light
production has the highest quantum efficiency known for any chemiluminescent
reaction and no background luminescence is found in the host cells or the assay
chemistry; (c) the assay is rapid, requiring only few seconds per sample procedure
(Figure 17) (Shcharbin, Pedziwiatr et al. 2010). The formation of transfection
complexes (dendriplexes) were formed by adding 750 μg of pDNA and 4 μL of
Superfect® reagent by Qiagen™ completing 50 μL final volume with medium (serum
and antibiotics free) and incubated 15 min at room temperature (RT). The day of
transfection, the cells were washed twice with medium. After dendriplexes are formed
they are mixed with growth medium (serum and antibiotics free) and 10 μL added
directly to BeWo cells. After this step, cells are incubated for 4 h at 37°C, followed by
further incubation in serum* and medium containing antibiotics for 48 h with a change
of medium at 24 h of incubation. After the incubation, the cells were treated with 75
μL of lysis buffer incubated by 5 min RT. The lysate was centrifuged at 10,000 rpm for
2 min. The supernatant was separated, then 20 μL was added to 50 μL luciferase
assay reagent, mixed and measured on a FlexStation II luminometer by Molecular
Devices™ for 10 s at 562 nm. The luciferase activity was expressed in relative light
units (RLU’s) and, the assays were normalized with pGL4.13 control vector and of B1
and B2 constructs RLU’s.
O-
HO
S
N
ATP O2
N
-O
COOH
S
S
N
N
S
Firefly luciferase
Mg2
$03
33L &2
/LJKW
Figure 19 Bioluminescent reaction catalyzed by firefly luciferase.
4.2.9
Transfection efficiency
62
We used pSV-beta-galactosidase control vector® (Figure 20) from Promega to
demonstrate evidence of transfection efficiency in same transfections conditions, with
10 μL of dendriplexes in a co-transfected pGL4 vectors, as indicated by blue staining
of BeWo cells in over 30% of cells per microscopic field. The cells were incubated for
48 h after transfection, then washed again twice with PBS, fixed with 2%
paraformaldehyde prior to incubation (15 min, RT) and washed again twice with PBS
priori to incubation in the X-gal staining solution (1 mg/mL X-gal, 2 mMMgCl2, 20 mM
K3Fe(CN)6, 20 mM K4Fe(CN6) 3H2O), and incubated DW & RYHUQLJKW ȕgalactosidase
cleaves
X-gal
to
form
the
blue-colored
3,5’-dichromo-4,4’-
dichloroindigo in transfected cells. After staining, the cells were washed with PBS and
photographed with a digital camera under the optical microscope, and the percentage
of blue-colored cells was counted from the photographs.
Figure 20 pSV-ȕ-galactosidase vector with the different sites and lacZ gene.
V. RESULTS
63
5.1
Sample selection
A total of 54 placental samples of which, twenty seven belonging to preeclamptic
patients and 27 to healthy pregnant women were studied. Firstly, the normal
distribution our nominal datas was analysed in both groups calculating with Skewness
and kurtosis values. The media and standar error for chronological age was 22.2.0
±1.2 years. The media and standar error value for gestational age was 38.3±0.2
weeks. Nulliparity was found in 22 cases and 5 were multiparous. Preeclamptic (PEE)
patients displayed higher values for media arterial pressure (MAP) (120.9±9.9 vs.
86.4±4.6 mmHg p=0.000) and higher values for uric acid blood (354.9±16.4 vs.
154.9±6.9 mmol/L (p=0.000) than normo evolutive pregnant (NEP) women. All pvalues were obtained with the Student’s t-test one way for independent data
significance p-value DQG 95% of confidence interval) (Table 5).
Table 5 Clinical characteristic for PEE and NEP women with standard error
Clinical characteristics
Age (years)
Gestational age (weeks)
PEE group
(n=27)
a
22.2±1.2
38.3±0.2
NEP group
(n=27)
a
NA
a
38.3±0.2
NA
22.2±1.2
a
p-value
PEE antecedent (%)
22%
3%
0.000
b
Primiparity/multiparity cases
19/8
17-10
0.300
b
Delivery form (cesarean/vaginal) cases
22/5
3/24
0.001
b
Median arterial pressure in mmHg
PEE classification (mild/severe) cases
a
b
86.4±4.6
0.000
8/19
NEP
NA
a
5.88±0.2
354.5±16.4
Treatment rate until delivery (hr)
a
120.9±9.9
5.5±0.4
a
a
6.77±0.1
a
a
b
0.001
b
154.9±6.9
0.000
a
0.300
4.8±0.4
b
Significant differences were appreciated for PEE antecedent, delivery form, MAP,
newborn weight and serum uric acid. Using p-value is statistically significant and
b
Student’s t-test
95% confidence interval. a Media ± standar error
5.2
Expression of KiSS-1 and REN genes
64
Extraction RNA from placental tissues homogenized was successful and three
species (28S, 18S and 5.8S) were observed in 1% electrophoresis agarose gel
without any degradation all samples after of treatment with DNsa (Figure 21).
KRP-4
KRP-12
KRP-17
KRP-24
KRC-4
KRC-12
KRC-17
KRP-24
28S
18S
5.8S
Figure 21 Different species of RNA after extraction and purification with DNasa.
RNA extraction was carried out, obtaining an average RNA concentration of 380±20
ng/μL and purity values above to 1.70 in all samples. Later cDNA was synthetized
with High-Capacity cDNA Reverse Transcription Kit by Applied Biosystems™ using 1
μg of RNA and getting a cDNA media concentration 278 ng/μL. All concentrations
were measured by spectrophotometry. (Annex V)
The calibration and standardization of qPCR was done by triplicate with standard
serial dilutions, using the first concentration 400 ng/μL and finishing in 6.25 ng/μL of
cDNA to know the optimum concentration. In our system the ideal concentration was
30 ng/μL for the two problem (REN and KiSS-1) and housekeeping genes (B2M);
showing efficiency amplification between 90-110 percent and slope values around to
3.32 (Figures 22 and 23).
65
Standarization amplification plot for KiSS-1, REN and B2M
Figure 22 Amplification plots show different amplification cycles (CT), depending
concentrations of cDNA in genes tested.
66
Figure 23 The different values in amplification efficiency (90-100%) and slope-values
(-3.34±0.5) are ideals to ensure optimal results a cDNA concentration of 30 ng/μL in
our system which was considered as ideal to work.
The regression line and the standar curve were calculated to each gene KiSS-1 REN
and B2M to generate r2 and the amplification efficiency is calculated using the slope
of the regression line in the standard curve. (Slope near -3.35 indicates an efficiency
of PCR amplification optimum of 100%). (Figure 24).
67
Figure 24 The slope and R2 values calculated to get a efficiency amplification percent
We also measured the expression of B2M gene in both groups PEE and NEP
respectively, in five random samples placental tissues from PEE woman and five
samples placental tissues from NEP and we did not find differences (p=0.591) using
student’s t-test between both groups which corroborate the constitutive expression of
B2M.
KiSS-1 gene showed uniform over-expression almost of all samples (89%) with PEE
compared with NEP samples where only 11% was observed over-expression (Figure
25) with it we can say that KiSS-1 is expressed in PEE placental tissues and some
tissues had a a relative expression up-to 3-4 times compared to control tissue.
68
Relative expression
PEE patients
NEP patients
Samples
Figure 25 The expression of KiSS-1gene was observed almost 24 of 27 placental
tissues with PEE compared with tissues from woman with NEP
Respect to REN expression in placental tissues with PEE, the over-expression of this
gene was not consistent respect to placental tissues from NEP women due to only 9
of 27 PEE tissues was observed over-expression and 18 samples have a subexpression. (Figure 26).
PEE patients
NEP patients
Relative expression
Figure 26 The Relative expression for REN gene was not consistent for all samples
69
The comparison of KiSS-1 and REN placental gene expression using student t-test
one way for independent data revealed higher KiSS-1 expression levels in
preeclamptic (1.99 ± 0.80 and 0.99 ± 0.47; p=0.001 ), than in healthy women. In
contrast, no significant differences (p=0.300) were found between PEE (1.25 ± 0.51)
and NEP (1.06 ± 0.46) for REN gene relative expression analysis were found (Figure
27).
PEE Patients
p-- value= 0.001
n=27
NEP Patients
p-- value= 0.300
n=27
Figure 27 Comparative expression analyses for KiSS1 and REN genes using
Student’s t-test (p=0.001 and p=0.300 and significance p-value DQG
CI 95%).
REN expression gene levels did not differ between PEE and NEP patients although
the function of renin is involved in regulating blood pressure would suppose that in
pathological cases as this should be altered in PEE due to us regulatory role as
vasoconstrictor. So we developed a more detailed cluster analysis. The considered
variables for this analysis were PEE classification, MAP, uric acid, proteinuria, PEE
history, creatinine, urea, edema, gestational age, drugs used for hypertension control
as well as management time. All those revealed the presence of two clearly
distinguishable subgroups n=9 (circularized) and n=18 (Figure 28).
70
Figure 28 Cluster diagram for PEE and NEP women. Dendogram grouped the whole
sample in two major groups; PEE sub-group was further divided; circle
denotes the subgroup of PEE patients with MAP PP+JDQGWKHXVH
of anti-hypertensive drugs as common variables.
A new comparative analysis of the resulted subgroups demonstrated that significant
differences (p=0.001) were found for REN gene high expression in 18 mild
preeclamptic women (1.44 ± 0.51) vs. their corresponding controls (0.86 ± 0.39) (Fig.
26). The high expression on this group correlated with MAP, low proteins, urea and
uric acid high concentrations in serum, newborn low weight, PEE history, Increase of
osteo-tendinous reflexes (OTR) and the number of pregnancies (p
Sub-expression was found to be statistically significant (p=0.001) using a new student
t-test one way for independent data in the remaining 9 severe preeclamptic patients
(0.87 ± 0.51 and 1.46 ± 0.30,) compared to their controls (figure 29). These 9 affected
patients were analyzed for all clinical and biochemical variables through neural
network analysis feed forward, obtaining a classification of 100% for training data and
80% for test data. The analysis revealed that the 5 most important variables were:
71
vomit, use of at least 3 hypertension control drugs, hepatalgy, alkaline phosphatase
and treatment time before pregnancy resolution. All these data agree with those
obtained in dendrogram.
PEE Patients
n=18
p-- value =0.001
NEP Patients
n=9
p-- value =0.001
Figure 29 REN gene expression levels analysis for PEE subgroups. Over-expression
was observed in 18 mild preeclampsia affected patient’s subgroup (left).
Sub-expression was advertised in the remaining 9 severe patients treated
with at least three drugs to control blood pressure and
We also found a correlation between KiSS-1 high expression in PEE patients and
serum uric acid (r2=0.399, p-value=0.039), newborn low weight (r2=0.412,
p=value=0.035), PEE history (r2=0.386, p-value=0.05) and number of pregnancies
(r2=0.387, p-value=0.042). REN gene high expression, correlated to median arterial
pressure (MAP) (r2=0.454, p-value=0.017), used anti-hypertensive drugs (r2=0.490, pvalue=0.009) and newborn weight (r2=0.421, p-value=0.030) using a Pearson’s
correlation test. with a p-value 0.05 as statistically significant and 95% CI (Table 6).
Differences for gene expression were also evaluated in both groups (PEE and NEP)
in regard to pregnancy way resolution (cesarean section vs. vaginal delivery). PEE
72
group displayed the next p-values, for KiSS1 p=0.062 and REN p=0.387. Control
group showed the corresponding p-values, KiSS-1 p=0.276 and REN p=0.582. These
results demonstrate no significant differences for gene expression in both groups
related to pregnancy way resolution using student t-test.
Table 6. Correlation test for distinct analyzed variables with gene expression
Variable
2
p-value
Gene
expression
KiSS1
REN
Correlation coefficient r
-0.137
0.454
0.490
0.017
KiSS1
REN
0.399
-0.067
0.039
0.730
PEE history (presence or absence)
KiSS1
REN
0.386
0.071
0.05
0.72
Pregnancies number 1 to 12)
KiSS1
REN
0.387
-0.230
0.042
0.574
Used anti-hypertensive drugs (1 to 4)
KiSS1
REN
-0.172
0.490
0.390
0.009
KiSS1
REN
0.412
0.421
0.035
0.030
OTR (Presence or absence)
KiSS1
REN
0.217
0.402
0.25
0.03
Median arterial pressure (mmHg)
Pearso’n Correlation test for clinical variables and gene expression in PEE women.
As can be appreciated in bold REN gene expression correlates tightly with the use of
antihypertensive medications (significance p-value DQG&,
73
5.3 Construction of recombinant plasmids containing ChE and KE enhancers
With regard to the search for the common regulatory sequences for to KiSS-1 and
REN genes expression, we studied the possibility that minimal regions from enhancer
ChE and KE act on the promoter of KiSS-1,since such regulation has been
demonstrated for REN gene Because, the participation of its in regulation on
promoter from REN gene have been demonstrated previously. We searched at the
minimal regions reported and amplified them elements with specific primers
containing compatible specific restriction sites compatibles with the vectors pCR®Blunt II-TOPO and pGL4. Standardization was done by temperature gradient PCR
and getting the optimous temperature of annealing 45 sec 60°C, extension 45 sec
72°C and final extension 5 min 72°C and hold 4°C. to PEK and KE fragments and
ChE fragment an initial denaturation of 1 min at 95°C, then 35 cycles of denaturation
45 sec at 94°C, annealing 45 sec at 60°C and extension 45 sec at 72°C, final
extension 5 min at 72°C and hold 4°C. as are demonstrated in the figure 30
Figure 30 Fragments PEK, CHE and KE with specific end tails restriction enzymes.
Amplified by gradient PCR and optimous temperatures of annealing and
extension
Previously, the fragments were purified and ligated in pCR®-Blunt II-TOPO cloning
vector by Invitrogen® and liberated by restriction with specific enzymes XhoI, SacI
74
and KpnI respectively in agree with our workflow design (Figure 31) All fragments
obtained were purified before ligation with pGL4 (Figure 32).
Figure 31 Workflow design constructs to demonstrate the regulatory capacity of ChE
and KE enhancers on KiSS-1 promoter PEK region
Figure 32 Fragments PEK, ChE and KE from pCR®-Blunt II-TOPO vector and ready
to be linked in pGL4
After we isolated and purified the fragments from pCR®-Blunt II-TOPO vector, they
were linked into pGL4.10 and 4.11 expression vectors. First, PEK fragment was
75
linked to ChE to form construct C ; PEK and KE fragments were joined together to
form construct D and finally PEK, KE and ChE fragments were put together to create
construct D in agree with workflow pre-design. All constructs were evaluated for the
correct direction of insertion (5’-3’) by specific digestion with a single cut restriction
enzyme (HindIII) contained in extreme 5’ from ChE fragment, and, within plasmid. So,
when the afore mentioned restriction enzyme cut and linearize the vector pGL4, the
fragment ChE is in position 5’-3’ because it has the same restriction site KpnI in both
ends. (Note: other restriction site is located in 3’region) on pGL4 vector (Figure 33).
Other specific enzymes (KpnI, SacI and XhoI) corroborate the ligation of fragments
KE
and
PEK
Figure34.
Figure 33 Different constructs from pGL4 plus PEK plus ChE and/or KE with correct
orientation 5’-3’ the fragment ChE is number three, fragment KE with
restriction KpnI is number two and 3, the fragment KE with restriction SacI
is two and three fragments, fragment KE with restriction SacI were two and
three samples respectibely
76
Figure 34 Differents fragment ChE, KE and PEK can be appreciate on this gel later
restriction with specific enzymes KpnI, SacI and XhoI at pGL4 plasmid
The correct sequence from different fragments was verified by sequencing to ensure
that errors during fragment amplifications and ligation the expression of Luc on pGL4
were not affected. The result of these sequencing (Query and subject) were matched
with NCBI genome databases and both sequences in (Basic Local alignment Search
Tool)
BLAST
by
NCBI
(http://blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=Web&PAGE_TYPE=BlastHome)
The result from sequencing shows a total concordance between fragments and NCBI
databases sequences. (Figure 35, 36 and 37). This ensures that no errors will
interfere with the expression of LUC
77
Query
79
Sbjct
1
Query
139
Sbjct
61
Query
199
Sbjct
121
Query
259
Sbjct
181
Query
319
Sbjct
241
Query
379
Sbjct
301
Query
438
Sbjct
361
Query
498
Sbjct
421
Query
558
Sbjct
481
GACATTTCACACTGACACTTCTCCCCACCCTCCTCAGATAGCCCATTTCCACGTTGTTTG
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
GACATTTCACACTGACACTTCTCCCCACCCTCCTCAGATAGCCCATTTCCACGTTGTTTG
138
GGGGCAGGATGGGTCCTCTGGGTGGGAAGAGGGTGTGGAGGATGGAAAGAGCCGGTAAGA
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
GGGGCAGGATGGGTCCTCTGGGTGGGAAGAGGGTGTGGAGGATGGAAAGAGCCGGTAAGA
198
AGGAGAAAGTCCCGCCTCGGAGGGCTCGGGTGGGGCAGGAGGAGAGGCTGCCTCCAGTCA
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
AGGAGAAAGTCCCGCCTCGGAGGGCTCGGGTGGGGCAGGAGGAGAGGCTGCCTCCAGTCA
258
CAGAGCCCGGAGGCAGAGGCCTCCCAAGGTGCCATGCTCTTCAGGAGGGTCTGAGGAGGA
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CAGAGCCCGGAGGCAGAGGCCTCCCAAGGTGCCATGCTCTTCAGGAGGGTCTGAGGAGGA
318
GGGAGGGGCGGCAGAGTGGGCCTGTGCTTGGAGACGTCACCCCGGGCTTTATAAAAGGGA
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
GGGAGGGGCGGCAGAGTGGGCCTGTGCTTGGAGACGTCACCCCGGGCTTTATAAAAGGGA
378
60
120
180
240
300
TGTGATCAGGGAGCTGGGGAGAACTCTTGAGACCGGGAGCCCAGCTGCCCACCCTCTGGA
|||||||||||||||||||||||||| |||||||||||||||||||||||||||||||||
TGTGATCAGGGAGCTGGGGAGAACTCTTGAGACCGGGAGCCCAGCTGCCCACCCTCTGGA
437
CATTCACCCAGCCAGGTGGTCTCGTCACCTCAGAGGCTCCGCCAGACTCCTGCCCAGGCC
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CATTCACCCAGCCAGGTGGTCTCGTCACCTCAGAGGCTCCGCCAGACTCCTGCCCAGGCC
497
AGGACTGAGGCAAGGTAGGCACACTGCAGTGTCCACCCCTGGGAAGGGGGTCTGCCCTGA
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
AGGACTGAGGCAAGGTAGGCACACTGCAGTGTCCACCCCTGGGAAGGGGGTCTGCCCTGA
CCTGGGGATGCTCTGGAGG
|||||||||||||||||||
CCTGGGGATGCTCTGGAGG
360
420
557
480
576
499
Figure 35 Blast of fragment PEK where Query is the PEK sequence and subject is
sequence from database
Query
73
Sbjct
1
Query
133
Sbjct
61
Query
193
Sbjct
121
Query
253
Sbjct
181
CTGATGTGGACACTGGGAGAAGACCCTCTTCCCTTCAGGCCCTGCTTTTTTGCCTTGGAC
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CTGATGTGGACACTGGGAGAAGACCCTCTTCCCTTCAGGCCCTGCTTTTTTGCCTTGGAC
132
CCTCCACACACTCCCTGCTGTCATTACTGGACTAGCAGTTCCCGCCCTGTCATTTATCAA
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CCTCCACACACTCCCTGCTGTCATTACTGGACTAGCAGTTCCCGCCCTGTCATTTATCAA
192
AGACTTTGGCTCCTGATTTACAGACTTCCTCCACCCCTTGCCCTCTGCAACCTCCTGATG
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
AGACTTTGGCTCCTGATTTACAGACTTCCTCCACCCCTTGCCCTCTGCAACCTCCTGATG
252
ACATCACCAACCACGCAGATGGTGACCTGGCCATACTGGCCTCTCAGATCCTTGGGGCCC
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
ACATCACCAACCACGCAGATGGTGACCTGGCCATACTGGCCTCTCAGATCCTTGGGGCCC
312
Query 313 CATTTCCAACAACCTCACCCCTCCTCTACTTCAGCTATCCACAGCCACGTCCTACTTG
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 241 CATTTCCAACAACCTCACCCCTCCTCTACTTCAGCTATCCACAGCCACGTCCTACTTG
60
120
180
240
370
298
Figure 36 Blast of fragment ChE where Query is the PEK sequence and subject is
sequence from database
78
Fragment ChE
Query
60
Sbjct
1
Query
120
Sbjct
61
Query
180
Sbjct
121
Query
240
Sbjct
181
Query
300
Sbjct
241
Query
360
Sbjct
301
Query
420
Sbjct
361
Query
480
Sbjct
421
Query
540
Sbjct
481
Query
600
Sbjct
541
GGGCTTAGTTTGGGGGAAAGAGCTGTTATCAtttttttAAGTTGAACTGCAAGCTAATCT
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
GGGCTTAGTTTGGGGGAAAGAGCTGTTATCATTTTTTTAAGTTGAACTGCAAGCTAATCT
119
60
ATAATTAGCTGGTCTGTGTACAGAGCTAAGCAGAAGCTTTTAACCTAAAAGATATTACCC
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
ATAATTAGCTGGTCTGTGTACAGAGCTAAGCAGAAGCTTTTAACCTAAAAGATATTACCC
179
CTGGGGGTCAGAGGCAAAATGGAGTCAGTCATGCTAAGCCTCCCTCCACTATTTCAATTT
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CTGGGGGTCAGAGGCAAAATGGAGTCAGTCATGCTAAGCCTCCCTCCACTATTTCAATTT
239
CCCCATTGTCAAAGGTTCATCCCACAGTCTTGTGGGATTGGGGAAAATGAGACATCATTA
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CCCCATTGTCAAAGGTTCATCCCACAGTCTTGTGGGATTGGGGAAAATGAGACATCATTA
299
CTCACCTGGCTACTTCCTGCCAAACAGGGTTGACAAGGGGTGACTATGGAATGAAACCAT
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CTCACCTGGCTACTTCCTGCCAAACAGGGTTGACAAGGGGTGACTATGGAATGAAACCAT
359
TTGACCTGGCTAAGGAAATATTTCGTAGTGCCATTTTTAGGAACAATGATCATCGGCATT
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
TTGACCTGGCTAAGGAAATATTTCGTAGTGCCATTTTTAGGAACAATGATCATCGGCATT
419
TCCCTTTTTGCTTTGATAGTTTTAGCGAGACTTGCACAACCTTTGTTTACACAGTACATA
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
TCCCTTTTTGCTTTGATAGTTTTAGCGAGACTTGCACAACCTTTGTTTACACAGTACATA
479
ATAAGTTTTACTAGAATGTAGGCCACCATGACTAAAAGAAGAACATCAAGGCTGAATTTA
|||||||||||||||| |||||||||||||||||||||||||||||||||||||||||||
ATAAGTTTTACTAGAACGTAGGCCACCATGACTAAAAGAAGAACATCAAGGCTGAATTTA
539
AGAATGCCTCCCAAGATTGATGGAATTACACTAGGAATCCAGGAGAATAGGTCTTTAAAG
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
AGAATGCCTCCCAAGATTGATGGAATTACACTAGGAATCCAGGAGAATAGGTCTTTAAAG
599
CAGTCTCTGTAAGTGCCCTCAGATTAAGCCTGCTTTGGTGTTCTAAGGTTTG
|||||||||||||||||| |||||||||||||||||||||||||||||||||
CAGTCTCTGTAAGTGCCCCCAGATTAAGCCTGCTTTGGTGTTCTAAGGTTTG
120
180
240-
300
360
420
480
540
651
592
Figure 37 Blast analysis KE fragment and those inserted in pGL4.10 showing total
sequence similarities vs. NCBI databases.were “Query” is sequence of the
fragments and “Subject” is sequence in NCBI data base
5.4
Transfection and luciferase expression analysis
Once the constructs were ready to be transfected we standardized the transfection
protocol by doing three experiments with three repetitions in optimal working
laboratory conditions to eliminate any error and ensure the transfection efficiency.
The most important variables to be controlled were the plasmid concentration in
ng/μL, the concentration of transfection reagent (Superfect), and the pDNA/Superfect
reagent ratio as well as cells number. The lastest variables because an excess pDNA
can be toxic to cells or a low quantity of transfection reagent can be insufficient to
79
achieve the complexes pDNA/dendrimer so can not cross the cellular membrane and
have a low transfection efficiency
With these variables the experiments were runned out to get the optimum transfection
onditions for BeWo cell line. Optimal conditions were pDNA concentration: 100 ng/μL,
transfection reagent: 1 ng/μL, relation pDNA/Superfect 1:4 and cell concentration:
20,000 cells/well.
pGL4.13 vector was used as internal control; it was evaluated for repeatability and
reproducibility (three times, three repetitions). There were also tested different
reaction volumes. The most appropriate reaction pDNA/Superfect reagent was 1:4
and the concentration of pDNA was 100 ng/μL with 20,000 cells/well. (Figure 36)
Standardization for co-transfection with pSV-ȕ-gal by Promega® was also carried out.
In this experiment different ratios of pGL4.13/pSV-ȕ-gal in different pDNA
concentrations were assayed and the best combination was for pDNA 125 ng/μL and
pGL4.13 100 ng/μL (Figure 37). Which shows the optimum working conditions of
transfection in the second experiment with 20,000 BeWo cells, optimizing reagents
and pDNA with a good luminescence signal measured in Relative light units (RLU’s).
The transfection effiency also was measured with X-gal staining (figure 38 annex IX),
however this method was not the best way to measure transfection because of low
intensity readings.
80
Figure 38 Standardization of transfection conditions in.pGL4.13
Figure 39 Standardization conditions of co-transfection in BeWo cells with 125/100
ng/μL pSV-ȕ-gal and pGL4.13 result most convenient with relation
Transfection reagent/pDNA 1:4
81
Figure 39 pSV-ȕ-gal stain and ȕ-galactosidase expressed in BeWo cells
Once transfection was standardized, experiments were performed with construct C, D
and E with the fragments PEK, ChE and KE within pGL4.10 and pGL4.11 vectors in
BeWo cell line culture to measure the regulation of LUC expression through
quantitation of Luciferase in each case. We done three experiments with three
replicates each but no expression was found in the constructs under study using
pGL4.13 as expression control (Figure 39).
We also standardized with constructs pGL4.10 and 4.11 containing PEK fragment
assuming that Luciferase expressed in this construct has a basal expression and the
other constructs should be normalized in regard to them. However no differences
between different construct and standard were found.
82
Luciferase expression in BeWo cells with
KiSS-1 promoter and Exon 1, Chorionic & kidney enhancers
9000
Exp1
Exp2
Exp3
8000
7000
6000
RLU's
5000
4000
3000
2000
1000
0
Constructs
Figure 40 Expresion of Luciferase in pGL4.10 and 4.11 with all constructs and their
combinations.
Luciferase expression was not detected in any studied construct, the values are
similar with negative control compared with pGL4.13 positive control So, the
luciferase expression was not modified with our fragments of interest PEK, ChE and
KE. In this sense, we can say that the minimal promoter region from KiSS-1 is not
stimulated neither by the chorionic enhancer nor kidney enhancer as occurs with REN
gene. These results suggest that there is not a co-expression regulation of both
genes by ChE nor KE enhancer.
83
VI DISCUSSION
Some examples are known of genes clustered (Berman, Nibu et al. 2002; Lee and
Sonnhammer 2003; Stuart, Segal et al. 2003; Chen, de Meaux et al. 2010) in same
chromosomal regions. Those genes used to be linked throughout evolution sharing
expression
and
relationship
in
certain
organs
or
(http://www.genecards.org/cgi-bin/carddisp.pl?gene=KISS1&search=KiSS1#
tissues
2010;
http://www.genecards.org/cgi-bin/carddisp.pl?gene=REN 2010). The identifications of
markers for preeclampsia development has become a very important area of
biomedical research. In this study gene profiles for two contiguous genes REN and
KiSS-1were evaluated between preeclamptic and healthy pregnant women. Then a
search for possible regulatory elements that potentially could direct the co-expression
of both genes was done by using as target the minimal region of chorionic and kidney
enhancers which were demonstrated that regulate the expression of REN gene (Shi,
Gross et al. 2001; Liu, Shi et al. 2006; Zhou and Sigmund 2008). The main clinical
features in our population agree with previous reports (Liu, He et al. 2005; Sibai 2005;
Raymond and Peterson 2011), with regard to pregnancy at early age (16-22 years
66.6%, 23-30 years 25.9% and 31-42 years 7.5%), the number of previous
pregnancies and MAP values (120.9±12 mmHg p-value=0.000). However in our
studied patients, most preeclamptic women delivered by cesarean section whereas
majority of healthy controls delivered through normal labor. Although pregnancy
resolution way was not strictly concordant between cases and controls and is known
to affects placental gene expression (Sitras, Paulssen et al. 2008; Lee, Shim et al.
2010) we assessed the relative expression of KiSS-1 and REN genes and found
significant differences between PEE and NEP subjects. As far as we know, this is the
first study performed in placental tissue, relating KiSS-1 and REN genes with
preeclampsia. KiSS-1 gene is a metastatic inhibitor for many different types of cancer
(Makri, Pissimissis et al. 2008) without affecting its tumorigenicity. We observed
higher expression of KiSS-1 gene in placental tissues in 24 among 27 preeclamptic
women vs. normoevolutive pregnant women (1.99 ± 0.80 and 0.99 ± 0.47; p=0.001).
In-vitro analysis by matrigel (Athanassiades and Lala 1998) demonstrated altered
84
migration pattern related to KiSS-1 and PIGF high expression (Janneau, MaldonadoEstrada et al. 2002) in normal trophoblast derived cells and choriocarcinoma cells.
Further, an increase of Kisspeptins in serum of patients with PEE (Mead, Maguire et
al. 2007)has also been reported. So, there is a distinct possibility that the observed
KiSS-1 high expression could be related to impaired trophoblast migration during
early stages of preeclamptic syndrome and is continued throughout pregnancy.
On the other hand, it has been reported (Tewksbury, Pan et al. 2003) the presence of
a placenta-specific renin-angiotensin system and differences of gene expression
(Irani and Xia 2008)between PEE women and NEP are known. In our study, we did
not find statistically significant differences for REN gene expression between PEE
and NEP groups as a whole. However REN gene higher expression was found in 18
among 27 patients with regard to their respective controls. This agree with the
observed higher expression for REN gene reported in uterine placental beds on
preeclamptic vs. normotensive women(Anton and Brosnihan 2008) and also with
others studies which reported high expression of REN (Shah, Banu et al. 2000;
Herse, Dechend et al. 2007)in maternal decidua of PEE woman.
It is important to note that our sampling technique removed all decidual tissue, which
implies that REN gene higher expression can be not only found on uterine placental
beds but also on chorionic tissue. Interestingly, the observed high expression
coincided with both, a mild preeclampsia status and the use of one or two of the
following drugs: alpha methyl dopamine and hydralazine. This suggests that neither
alpha methyl dopamine nor hydralazine alone or in combination, affects the
expression of REN gene at least in placenta.
All preeclamptic patients were subjected to different anti-hypertensive drugs regimes
depending on severity status mild/severe. KiSS-1 gene expression remains high in 24
among 27 PE women; independently of degree of severity and corresponding
treatment. This could be expected as anti-hypertensive drugs are not aimed to affect
TI driven by KiSS-1 gene.
85
On the other side, differential behaviors for REN gene expression were advertised in
preeclamptic patients depending on severity status and concomitant treatment. A
higher REN gene expression was noticed in 18 mild preeclamptic patients compared
to the remaining 9 severe affected patients. The last were treated with three (alpha
methyl dopamine, hydralazine and magnesium sulfate) or 4 (alpha methyl dopamine,
hydralazine, magnesium sulfate and nifedipine) antihypertensive drugs. In opposition
to the 18 lightly affected patients, these 9 severe preeclamptic patients presented
REN gene sub-expression.
So, for this particular sub group, the observed expression pattern could be explained
by two main possibilities: 1) the lowering of REN gene expression is due to the
combined actions of the 3 or 4 antihypertensive drugs or 2) the severity of the disease
explains such decrease in expression. No reports on the literature exist that support
the first assumption. However, nine differentially expressed genes were reported
(Nishizawa, Pryor-Koishi et al. 2007) between severe early onset vs. late onset
preeclampsia. KISS-1 and REN gene high expression were also found in close
association(Rampello, Frigerio et al. 2009)to high MAP values and newborn low
weight, events commonly seen in preeclampsia. This is in direct relationship with the
functional mechanism of these genes on deficient uterus/placental blood perfusion,
avoiding the proper transfer of nutrients from the mother to growing fetus(Herse,
Dechend et al. 2007; Lunghi, Ferreti et al. 2007).
One limitation of this kind of studies is that the observed changes in gene expression
could be considered more a consequence than a cause of the disease. However we
cannot avoid this, as placental recollection is performed until resolution of pregnancy
and preeclampsia is diagnosed as early as 20 weeks of gestation
KiSS-1 and REN genes are both involved in two of the main phenomena related to
preeclampsia: trophoblast invasiveness impairment and hypertension respectively.
So, it is possible to suggest the existence of a common regulatory mechanism for
both genes based on 1) the physical contiguity for both genes on chromosome
1q32.1 (Nistala
R, et al., 2004) and 2) the presence of a couple of functional
86
enhancers directing the expression of REN gene in kidney and placenta (Galea,
Barigye et al. 2008). Then high expression levels for both genes would be expected in
this pathology.
On the other hand, we raised the possibility that the co-expression of both genes
KiSS-1 and REN is regulated by the same transcriptionally active sites such as
enhancers. The regulation by common elements has been demonstrated in other
clusters of contiguous genes (Lercher, Blumenthal et al. 2003; Chen, de Meaux et al.
2010) and even on genes located on in different other chromosomes but are still
regulated by the same elements (Lercher and Hurst 2006). An example is
Housekeeping genes which are expressed and co-regulated in many tissues tend to
cluster in humans (Lercher, Urrutia et al. 2002).
In this sense, the possible identification of minimal region regulatory elements to ChE
and KE showed not inference in co-expression with minimal promoter region of KiSS1 and REN genes. The role of chorionic and kidney enhancers on REN gene has
been demonstrated by Zhou X and Sigmund CD in 2008, using a BAC containing
REN, KiSS-1 and PEPP3, FLJ10761 and SOX3 between other genes (Zhou and
Sigmund 2008). Extrapolation of the results to human is limited because they
examined the regulatory mechanism in a mouse model.
We suggest the possible co-participation of REN and KiSS-1 genes in the
pathophysiology of preeclampsia through common regulatory elements. In order to
evaluate the last, we make different combinations of regulatory elements and clone
them on pGL4 Luciferase expression vectors. The obtained constructs were transfect
in a choriocarcinoma cell line (Fang, Antico et al. 2008; Liu, Li et al. 2009; Chuang,
Lieu et al. 2010; Yan, Zhang et al. 2012).
As the results did not h ave not shown differences in expression of luciferase gene
reporter, this suggest that that others regions could participate in the regulation KiSS1 gene expression. Another possibility is studied regions are too small. Additional
87
experiments using longer and/or different genome regions should be performed to
confirm this hypothesis.
88
VII. CONCLUSIONS.
We did not provide definitive evidence for a high parallel expression of both KiSS-1
and REN genes in preeclamptic patients. KISS-1 gene was over-expressed in almost
all placental tissues analyzed from PEE woman. In opposition the expression profile
for REN gene in preeclamptic placental tissues was not uniform, because we found
two groups of patients with high and low expression levels.
We demonstrated that a less aggressive hypertension management in these patients
is coincident with a higher expression of REN gene in placenta. Nevertheless we
cannot discard the expression parallelism for both genes because of the presence of
two variables: severity status and concomitant treatment affecting the real expression
profile for REN gene in PEE patients
We established that the minimal region from chorionic and kidney enhancers did not
regulate the minimal promoter of KiSS-1 gene in BeWo culture cell line, eventhought
over regulation of ChE and KE enhancers on REN gene expression has been
demonstrated in others cell lines. So, we could not provide evidence for a relationship
between both KiSS-1 and REN genes despite their contiguity and significant role in
the pathophysiology of PEE, however the minimal regions studied here had not effect
on the regulation of KiSS-1 and REN genes.
The lack of positive results regarding the co-expression of KiSS-1 and REN genes in
our in-vitro system, could be due to the possible presence of distinct transcription
factors, activator proteins, or silencers whose transcriptional activity change
depending among others on tissue specificity and response to developmental and
environmental signals.
Further studies should be performed to evaluate longer or different regions of ChE
and KE enhancers, KiSS-1 gene promoter, as well as other genomic sequences
located between both genes.
89
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Acknowledgements
This thesis is a result of consistency, inspiration, and enthusiasm; values imbibed to
me by my parents and my family. They both share two common characteristics:
integrity and a warm heart. I am privileged to have PhD. Jose Salas as my “guru”; he
is a role model as a researcher and a human being, but most of all he is “a friend for
life”. I would like to thank PhD. Laurence A. Marchat, my supervisor, for teaching me
“simple complexity of cellular transcription processes”. I thank her for the criticisms
and comments and for adding a “woman’s touch” in this project. I would like to thank
PhD. Ismael Lares Assef who trusted me and opened me his laboratory doors when I
began to study master science postgrad. I would also like to thank the staff at the
Biochemistry lab in Medicine School from UASLP in San Luis Potosi, Mex., Arlene
Salazar and Hilda M Gonzalez, for their excellent technical assistance and especially,
PhD. Claudia Castillo to accept me in her laboratory. Also, I wish a give a warm thank
to PhD. Marcelo Barraza, Maricela Aguilar, and clinical laboratory staff from Scientific
Research Institute from UJED, who with their smile, positive attitude and kindness
made my working days a “family business”. I am grateful to MD. Jesus M Guijarro, the
“leader-team” of the Department of Obstetrics and Gynecology, in General Hospital of
Durango, Mex. for giving me the opportunity to combine research with clinical training.
Likewise also, many thanks for my laboratory colleagues and nurses from General
Hospital that supported me and helped recruiting the study participants. Special
thanks. MD. Alberto Mireles my mentor during the early stage of my clinical training,
PhD Carlos Galaviz, my supervisor, for his support and stimulating discussions on
interesting aspects of preeclampsia-eclampsia. Thanks to my parents, Fernando and
Martha, because my optimistic attitude in life is a result of a wonderful childhood they
gave me. I thank Carmen Ramirez, my parent in law, accepted me as “a son” and I
am deeply grateful for their love and support of my daughters and also thanks my
sister on law Fatima Perez her helpful lovable care for my daugthers when work
overtook my role as father. My eternal and heartfelt thanks to my wife Carmen, is the
ideal partner. Our journey in life is filled with love, mutual respect and happiness. She
113
is critical when needed but always understanding and caring. She is the perfect
colleague, friend and mother. Together we make a “dream team”.
Finally, I wish to thank the financially support for this thesis. The grant FONSEC of
CONACyT (Grant No. 2009-01-111870), and SIP-IPN (Grant No. 20090313).
Sincerely
114
ANNEXES I
Annex 1 Approbation letter of ethical comittee from General Hospital from Durango
Annex II Consent letter
115
Fecha ____________
Folio ________
Titulo y número del proyecto
Evaluación de los niveles de expresión de los
genes KiSS1 y Renina en placentas de mujeres
con preeclampsia-eclampsia comparados con los
de las placentas de mujeres con embarazo normoevolutivo
Investigador principal
Nombre de la institución
Dirección
Teléfono
Núm. de identificación del sujeto
Iníciales del sujeto
CIIDIR-IPN Unidad Durango
Calla Paloma 104-3 Ote
618-122-6434
Naturaleza y propósito del estudio.
Se le invita a participar, de manera voluntaria, en un estudio de investigación sobre
Evaluación de los niveles de expresión de los genes KiSS1 y Renina en placentas de
mujeres con preeclampsia-eclampsia comparado con los de las placentas de mujeres
con embarazo normo-evolutivo
Para que usted pueda decidir si participa o no en este estudio, es importante que
conozca y entienda los posibles riesgos y beneficios que le permitan tomar una
decisión informada.
La persona responsable de la realización del estudio en la institución es el
M en C Fernando Vazquez Alaniz y Dr. en C. Carlos Galaviz Hernández
El propósito del estudio es:
Determinar si existe diferencia en los niveles de expresión de los genes KiSS1 y REN
en placentas de mujeres con preeclampsia-eclampsia comparados con los de las
placentas de mujeres con embarazo normo-evolutivo.
Procedimientos del estudio.
El procedimiento consistirá en la obtención de su placenta al momento del
nacimiento del producto y la toma de una muestra de sangre venosa obtenida
mediante venopunción, la cual servirá para obtener su material genético DNA y RNA.
También es posible que esta misma muestra sirva para realizar algún otro estudio de
investigación que nos permita aportar mayor conocimiento sobre su enfermedad; con
el entendido que su confidencialidad siempre será respetada.
.
Molestias y riesgos.
En la obtención de la placenta no existen riesgos potenciales ya que la obtención de
la misma es parte del procedimiento fisiológico o quirúrgico habitual del
116
alumbramiento y será obtenida por su médico tratante bajo condiciones controladas.
Las molestias para la obtención de la muestra venosa son mínimas ya que la
obtención de la muestra se realiza de forma habitual por personal capacitado y con
experiencia.
Beneficios.
Conocer los niveles de expresión de los genes KiSS1 y REN permitirá conocer si
están implicados en el desarrollo de la Preeclampsia-eclampsia y en futuro próximo
conocer más sobre la enfermedad o su tratamiento; sin embargo también es posible
que usted no tuviera ningún beneficio, pero contribuirá a la generación de
información científica acerca de la genética de la PEE que pueda ayudar a otros
pacientes en un futuro, que como usted sufran de Preeclampsia-eclampsia
Confidencialidad de sus registros.
Usted tiene derecho a su privacidad y toda la información recopilada durante este
estudio es confidencial, en conformidad con la ley. El comité de Ética de su
institución y las autoridades de salud pueden examinar su expediente médico en
relación con este estudio. Si se publican los resultados de este estudio, su identidad
permanecerá secreta. Esta información puede divulgarse a las autoridades oficiales
de los Estados Unidos Mexicanos.
Tratamiento médico en caso de lesiones.
Si usted llegará a sufrir una lesión como resultado DIRECTO de los procedimientos
de obtención de la muestra para este estudio, se le brindará atención médica
necesaria en la institución participante. El investigador asume la responsabilidad del
costo derivado de dicha atención médica, siempre y cuando exista una relación
DIRECTA entre el daño y los procedimientos del estudio. No habrá otra forma de
compensación.
Obtención de información adicional.
Se le anima a que usted haga preguntas en cualquier momento del estudio sobre sus
derechos como sujeto participante, sobre la evolución o resultados del estudio por
favor póngase en contacto con el QFB Fernando Vazquez Alaniz al teléfono 044618-122-6434 o al 618-813-4317 o con el Dr. en C Carlos Galaviz Hernández al
618-129-4111 o al 044-618-148-0200.
Si usted tiene alguna pregunta con respecto a sus derechos como participante en el
estudio de investigación, también puede ponerse en contacto con una persona no
involucrada con el equipo de investigadores de este estudio el C. Dr. Luis Ángel
Ruano Calderón representante del Comité de Ética de esta institución participante al
teléfono 618-8130011 Ext 1113
Bases para la participación
Su participación en este estudio es totalmente voluntaria y puede interrumpirla en
cualquier momento. Su decisión de participar o no en el estudio no afectará la
117
atención médica que recibirá y su médico le ofrecerá las mejores opciones
terapéuticas disponibles. En caso de existir cualquier información nueva que pudiera
influir en su voluntad de seguir participando en el estudio esta se le comunicará
oportunamente.
Declaración y firma del paciente.
He leído este consentimiento informado y he tenido la oportunidad de discutir el
contenido con el M en C Fernando Vazquez Alaniz y/o el Dr. en C. Carlos Galaviz
Hernández. He tenido la oportunidad de hacer preguntas acerca de los
procedimientos del estudio, sus inconveniencias, riesgos y posibles efectos
secundarios. Se ha dado respuesta a todas mis preguntas a mi completa
satisfacción. Toda la información verbal y por escrito y las pláticas sobre el estudio se
me han proporcionado y han sido en mi propio idioma (Español). Mi firma indica que
voluntariamente consiento en participar en este estudio después de haber leído
detenidamente, entendido y recibido una explicación completa de la anterior
información. Entiendo que puedo decidir retirarme en cualquier momento sin que
esto afecte mi atención médica futura en la institución participante. He recibido una
copia completa de este documento
Nombre y firma del paciente (o su representante legal en caso necesario):
Nombre completo ________________________________________________
Fecha _________ Teléfono _____________ Firma ______________________
Dirección del paciente _____________________________________________
_______________________________________________________________
El suscrito ha explicado ampliamente los detalles importantes de este estudio al
paciente cuyo nombre aparece arriba (o a su representante legal en caso de que
aplique).
Nombre y firma del investigador que haya explicado el consentimiento.
Nombre Completo ________________________________________________
Fecha_________________________ Firma ___________________________
Nombre y firma de un primer testigo.
Nombre completo ___________________________________________________
118
Fecha_________________________ Firma ___________________________
Dirección del testigo _________________________________________________
Relación o parentesco con el paciente ________________________________
Nombre y firma de un Segundo testigo
Nombre completo ____________________________________________________
Fecha _______________________ Firma _________________________________
Dirección del testigo __________________________________________________
Relación o parentesco con el paciente _________________________________
119
Annex III Data sheet to clinical, anthropometric
and socio-
demographic antecedents from PEE and NEP women
Fecha:______________
Folio:______________
PACIENTE
Expediente
Nombre:
Edad:
Teléfono:
Estado civil
Ocupación:
Lugar de Origen:
Domicilio:
Referencia de vecino o familiar
Dirección
Gestas:
Partos:
Abortos:
Cesáreas:
Semanas de Gestación:
AHF:
AHF de Preeclampsia:
APP:
Uso de drogas
Cigarrillos dia ( )
alcohol ( )
Drogas ( )
Peso Placenta
Peso RN
T/A al momento del diagnóstico:
mm/Hg
PAM:
mm/Hg
Proteinuria
+
++
+++ y/o
__________ g/24h
Edema en: Piernas
Manos
Cara
+
++
+++
Cefalea
Si
No
Hiperreflexia
Si
No
Acúfenos
Si
Albúmina:
g/dl
Proteínas Totales:
mg/dl
3
Plaquetas:
mm
DHL:
U/l
Fosfatasa Alcalina
U/l
Ác Úrico:
mg/dl
AST (TGO):
U/l
ALT(TGP):
U/l
Tratamiento para HTA:
Dosis:
Alfametildopamina
( )
Hidralazina
( )
Sulfato de Magnesio
( )
Nifedipino
( )
Dexametazona
( )
Betametazona
( )
Muestras Recolectadas Sangre ( )
Orina ( )
Placenta ( )
Saliva (
Tel
No
)
Marque el tipo de Preeclampsia-Eclampsia de la Paciente:
Preeclampsia Leve
Preeclampsia Severa
Eclampsia
HELLP
_______________________________
Nombre y firma del Entrevistador
120
Annex IV Data Sheet control for subculture cells
Date____________ Time___________ Researcher_____________________
Date
Cell line
Designation
Generation pass No.
Phase of growth cycle
Appearance of cells
Status before subculture
Density of cells
pH of medium (approx.)
Clarity of medium
Prewash PBS / EDTA
Trypsin
Dissociation agent
EDTA
Other
Mechanical
Concentration after resuspension (C1)
Cell count
Volume (VI)
Yield (Y=CI x VI)
Yield per flask or dish
Number (N) & type of vessel (flask, dish)
Seeding
Final concentration (CF)
Volume per flask, dish or wells (VF)
Split ratio (Y/CF X VF x N
Type
Batch No.
Serum type and concentration
Medium serum
Batch No.
HEPE’s
L-glutamine
Antibiotics
Amino acids
Others parameters
Feralaniz©
121
0.014
0.016
0.016
0.016
0.017
0.017
0.018
0.021
0.018
0.017
0.019
0.017
0.022
0.019
0.017
0.022
0.023
0.021
0.021
0.021
0.020
0.020
0.016
0.021
0.019
0.016
0.016
KRP-1
KRP-2
KRP-3
KRP-4
KRP-5
KRP-6
KRP-7
KRP-8
KRP-9
KRP-10
KRP-11
KRP-12
KRP-13
KRP-14
KRP-15
KRP-16
KRP-17
KRP-18
KRP-19
KRP-20
KRP-21
KRP-22
KRP-23
KRP-24
KRP-25
KRP-26
KRP-27
0.007
0.008
0.008
0.008
0.009
0.009
0.010
0.012
0.010
0.009
0.011
0.009
0.014
0.011
0.010
0.014
0.014
0.012
0.013
0.012
0.012
0.011
0.009
0.012
0.010
0.008
0.008
Abs. 280 nm
2.0
2.0
2.0
2.0
1.88
1.88
1.80
1.75
1.80
1.88
1.72
1.88
1.68
1.72
1.7
1.60
1.64
1.75
1.61
1.75
1.66
1.81
1.77
1.75
1.90
2
2
Purity
280/260
231.0 ng/µL
264.0 ng/µL
264.0 ng/µL
264.0 ng/µL
280.5 ng/µL
280.5 ng/µL
297.0 ng/µL
346.5 ng/µL
297.0 ng/µL
280.5 ng/µL
313.5 ng/µL
280.5 ng/µL
363.0 ng/µL
313.5 ng/µL
280.5 ng/µL
363.0 ng/µL
379.5 ng/µL
346.5 ng/µL
346.5 ng/µL
346.5 ng/µL
330.0 ng/µL
330.0 ng/µL
264.0 ng/µL
346.5 ng/µL
313.5 ng/µL
264.0 ng/µL
264.0 ng/µL
Concentration
(260)(33)(500)
*KRC-21 Se utilizó como calibrador Concentración de 396 ng/uL
Abs. 260 nm
Samples
2.59+37.41
2.27+37.63
2.27+37.63
2.27+37.63
2.13+37.87
2.27+37.63
2.02+37.98
1.73+38.27
2.02+37.98
2.27+37.63
1.91+38.09
2.27+37.63
1.65+38.35
1.91+38.09
2.27+37.63
1.65+38.35
1.58+38.42
1.73+38.27
1.73+38.27
1.73+38.27
1.81+38.19
1.81+38.19
2.27+37.63
1.73+38.27
1.91+38.09
2.27+37.63
2.27+37.63
Dilution
15 ng/uL
KRC-1
KRC-2
KRC-3
KRC-4
KRC-5
KRC-6
KRC-7
KRC-8
KRC-9
KRC-10
KRC-11
KRC-12
KRC-13
KRC-14
KRC-15
KRC-16
KRC-17
KRC-18
KRC-19
KRC-20
*KRC-21
KRC-22
KRC-23
KRC-24
KRC-25
KRC-26
KRC-27
Samples
0.015
0.016
0.014
0.016
0.016
0.014
0.013
0.013
0.016
0.014
0.016
0.014
0.016
0.014
0.014
0.015
0.015
0.015
0.015
0.016
0.024
0.016
0.013
0.013
0.018
0.013
0.015
Abs. 260mn
0.007
0.008
0.007
0.009
0.009
0.007
0.006
0.006
0.008
0.007
0.008
0.007
0.008
0.007
0.007
0.008
0.007
0.008
0.008
0.008
0.012
0.007
0.007
0.006
0.009
0.006
0.007
Abs. 280 nm
2.14
2.0
2.0
1.77
1.77
2.0
2.16
2.16
2.0
2.0
2.0
2.0
2.0
2.0
2.0
1.87
2.14
1.87
1.87
2.0
2.0
2.4
1.85
2.16
2.0
2.16
2.14
Purity
280/260
247.5 ng/µL
264.0 ng/µL
231.0 ng/µL
264.0 ng/µL
264.0 ng/µL
231.0 ng/µL
214.5 ng/µL
214.5 ng/µL
264.0 ng/µL
231.0 ng/µL
264.0 ng/µL
231.0 ng/µL
264.0 ng/µL
231.0 ng/µL
231.0 ng/µL
247.5 ng/µL
247.5 ng/µL
247.5 ng/µL
247.5 ng/µL
264.0 ng/µL
396.0 ng/ µL
264.0 ng/µL
214.5 ng/µL
214.5 ng/µL
297.0 ng/µL
214.5 ng/µL
247.5 ng/µL
Concentration
(260)(33)(500)
2.42+37.58
2.27+37.63
2.59+37.41
2.27+37.63
2.27+37.63
2.59+37.41
2.79+37.21
2.79+37.21
2.27+37.63
2.59+37.41
2.27+37.63
2.59+37.41
2.27+37.63
2.59+37.41
2.59+37.41
2.42+37.58
2.42+37.58
2.42+37.58
2.42+37.58
2.27+37.63
1.51+38.49
2.27+37.63
2.79+37.21
2.79+37.21
2.02+37.98
2.79+37.21
2.42+37.58
Dilution
15 ng/uL
122
Annex V Purity and concentration of cDNA placental tissues from PEE and NEP women also work dilutions for
RT-PCR
Annex VI Cryopreservation Cell Line Record
Cell line
Location F/C
BeWo Pass/date T-16 / Set 12 2011
G/3 Color code
Yellow
Concentration
Condition
Mycoplasma Method: PCR
Species
Normal
Adult
Date of test: JUL 21, 2011
Neoplasic
Fetal/NB
Result: NEGATIVE
Authentication:
Date of test:
Tissue
Site: PLACENTA
Pass: T-9
Gen No. CCL-98
Author Ref: ATCC
Mode of growth
DMEM/F12 with:
HEPES 1.5%,
L-GLUTAMINE 1%
ANTIBIOTIC 1%,
SFB 10%
MEM NON ESSENTIAL AA 1%.
Special stored characteristics:
Kept in vapor phase N2
2.1x106 Viability
-70°C No. tubes
92%
6
Freeze instructions:
Rate: 1.2X106 cells in Fetal Bovine
serum
with
10%
DMSO
as
Cryoprotectant. Vials cover paper at 70°C until 2 months after N2 vapor
phase indefinitely.
Thaw conditions:
Thaw rapidly to 37°C
Dilute to:
5 mL
20 mL
10 mL
50 mL
Method:
Media: Normal DMEM/F12
Normal maintenance
Subculture Frequency: 6-7 DAYS
Seeding Conc. 8X105 in P-100
Agent: Trypsin
0.25%/EDTA 0.0.53mM
5 min 37°C
Centrifuge to Remove Cryoprotectant
Yes / No
Special requirements:
Medium Change Frequency: 2-3
DAYS
Biohazard precautions:
Type: DMEM/F12 WITH
HEPES 1.5%,
L-GLUTAMINE 1%
ANTIBIOTIC 1%,
SFB 10% AND MEM NON
ESSENTIAL AMINOACIDS 1%
Habitual
Person completing card
Any other special condition
Name: Fernando Vazquez Alaniz
Viability: 92%
Signature:
123
Annex VII Cell counting
Accurate numbers in a cell suspension can be calculated by counting the cells in a
haemocytometer improved Neubauer; it is important to disperse the cells thoroughly
by pipetting up and down. A typical method for enumerating cell concentration using
“improved Neubauer” haemocytometers is given below.
1. Dilute 0.2 ml of the cell suspension in 0.2 ml of trypan blue (N.B. use 0.1% w/v
trypan blue in PBS solution); non-viable cells are stained blue.
2. Immediately mix well with a fine Pasteur pipette and aspirate sufficient volume
to fill both sides of the haemocytometer chamber.
3. Count viable cells in each of the four corner squares bordered by triple lines,
omitting cells lying on these lines.(see figure 8.1) This is repeated for the
second side of the chamber. N.B. cell counts of less than fifty cells are unlikely
to be reliable.
4. If a marked degree of cell “clumping” (aggregation) is observed, discard and
re-suspend the original cell suspension.
5. Calculate the mean count of the total viable cells per four corner squares
(N.B. viable cells are not stained by Trypan blue).
6. Count and calculate the mean count of the other half of the counting chamber.
For a valid test, the results of the two counts should be within 20% of the mean
value.
Calculate the viable cell concentration per ml using the following formula:
=
( ) ( , ) ( )
124
VC= viable cells per mL
CC = total viable cell count of four corner squares
Dilution= correction for the trypan blue dilution (counting dilution /trypan blue)
Quadrants = correction to give mean cells per corner square
10,000= conversion factor for counting chamber.
Important variables in these counting chambers are:
a. the depth of the chamber. In the example above this is 0.1 mm; in some
counting chamber types, however, this is 0.2 mm.
b. the number of smallest squares per cm2. In the example above, there are
25 squares per cm2, each 0.2 mm long and 0.2 mm wide; in some counting
chambers, however, there are 16 squares per cm2, each 0.25 mm long and
0.25 mm wide.
125
Figure 8.1 Cell counting using a haemocytometer (based on Freshney).
When counting chambers with different specifications are used, different algorithms
have to be followed for the correct calculation of the number of cells per ml. It is
important to check the specifications of the counting chamber in use and follow the
calculation instructions that go with individual counting chambers.
126
Annex VIII Determination of Mycoplasma species in culture cell lines.
Mycoplasma is a contaminant of primary cell cultures and human cell lines. The
oligonucleotides are based in those described by Hopert A. et al 1993 (Hopert, Uphoff
et al. 1993). In a nested approach is not as described by Uphoff CC. et al 2002
(Uphoff and Drexler 1999). Primers recognize sequences of 16S rRNA encoding
gene that are conserved in at least 25 different species of Mollicutes, including these
commonly implicated in the contamination of cellular cultures (Drexler and Uphoff
2002). The specificity of the oligos was confirmed form M. arginine, M. fermentans, M.
M. hyorhinis, M. laidlawii, M. orale, M. hominis and M. bovis. Depending on the
species of Mycoplasma present, the amplicón ranging between 510 and 520 pb.
Sequence of primers used
Oligos
Secuencia
pb
%GC
FMYC1
FMYC2
FMYC3
FMYC4
FMYC5
FMYC6
RMYC1
RMYC2
RMYC3
CGCCTGAGTAGTACGTTCGC
CGCCTGAGTAGTACGTACGC
TGCCTGAGTAGTACATTCGC
TGCCTGGGTAGTACATTCGC
CGCCTGGGTAGTACATTCGC
CGCCTGAGTAGTATGCTCGC
GCGGTGTGTACAAGACCCGA
GCGGTGTGTACAAAACCCGA
GCGGTGTGTACAAACCCCGA
20
20
20
20
20
20
20
20
20
60
60
50
55
60
60
60
55
60
Tm
en °C
57.8
57.3
54.4
57.1
58.1
57.8
59.7
58.1
60.3
T. alinean
en °C
60
60
60
60
60
60
60
60
60
127
PCR components
1
2.375
1.25
0.625
0.125
5
0.125
9
21.37
11.25
5.625
1.125
45
1.125
18
42.75
22.5
11.25
2.25
90
2.25
26
61.75
32.5
16.25
3.25
130
3.25
38
90.25
47.5
23.75
4.75
190
4.75
50
118.75
62.5
31.25
6.25
250
6.25
58
137.75
72.5
36.25
7.25
290
7.25
66
156.75
82.5
41.25
8.25
330
8.25
74
175.75
92.5
46.25
9.25
370
9.25
Volumen de master mix
9.5
85.5
171
247
361
475
551
627
703
Volumen para disp. En tira 8
Volumen para disp. Por tubo
-.9.5
-.9.5
21.37
9.5
30.87
9.5
45.12
9.5
59.37
9.5
68.87
9.5
78.37
9.5
87.87
9.5
500ng/μL
3
3
3
3
3
3
3
3
3
Vol final μL
12.5
12.5
12.5
12.5
12.5
12.5
12.5
12.5
12.5
dH2O μL
10x Buffer
MgCl2 50mM
4x dNTP’s 10mM
2x Oligos 0.5μM
Taq Pol 5U/μL
DNA
cf
1x
2.5mM
200μM
0.2μM
0.02U/μL
Note: Control positive a negative should be assayed to verify the method and his
components.
Thermocycler conditions
Temperatura
Tiempo
Ciclos
95°C
30seg
58°C
2min
1x
72°C
10min
95°C
10seg
60°C
10seg
30x
72°C
30seg
72°C
10min
1x
4°C

1% Electrophoresis agarose gel should be done to identify the presence of bands
between 520 and 540 pb.
128
Annex IX ȕ-gal staining of eukaryotic cells in vitro
(Modification of methods of Dr. Seong-Seng Tan and Promega's)
Description: 7KLVPHWKRGLVXVHGIRUWKHGHWHFWLRQRIȕ-galactosidase on formalinfixed, frozen sections, and it is NOT suitable for formalin-fixed, paraffin embedded
WLVVXHVHFWLRQV7KHVLWHVRIȕ-galactosidase activity will be stained blue and nuclei
will be stained pink
&HOOVSUHYLRXVO\WUDQVIHFWHGZLWKDODF=FRQVWUXFWFDQEHVWDLQHGIRUȕ-galactosidase
activity.
1. Wash cells 2-3 x with PBS (2 ml for 35 mm dish; I use room temp. tissue
culture PBS without Ca+2 or Mg+2) - beware that some cell types.
2. Aspirate.
3. Fix cells with 0.2% glutaraldehyde/PBS for 5 min at RT (1 mL/35 mm dish).
* Note that glutaraldehyde vapor is quite toxic and should be diluted in fume
hood.
We use EM grade glutaraldehyde (Merck #4239, 25%) but lesser grade may
be OK. Over fixing (longer times or higher % will reduce the signal).
4. Aspirate after 5 min and then wash 2x2 ml PBS. Aspirate and add b-gal.
stain.
Prepare just before use.
Recipe for X-gal solution (made up in PBS)
Stock solution final concentration to make 2 ml
X-gal (20 mg/ml in dimethylformamide) 1 mg/ml 100ul
potassium ferricyanide (0.5 M made in sterile and distilled H20) 5 mM 20ul
potassium ferrocyanide (0.5 M made in sterile and distilled H20 5 mM 20ul
MgCl2 (1 M) 2 mM 4ul
PBS 1.856 ml
129
7. Incubate 37°C, humidified incubator (T/C) for 2h –over night.
NOTE: The plate will dry out quickly if incubated in normal oven.
8. All cells should stain blue (shades of from deep, royal, to light) within 2h,
background shows up 3-4 days incubation in X-gal soln (should include a no DNA
transfected control).
Summary of 7 quick steps to get the Blues.
1. Wash cells 2-3 x 2 ml PBS
2. Aspirate off supernatant
3. Fix cells at RT with 0.2% glutaraldehyde 5 min (1:125 of a 25% solution).
4. Aspirate off glutaraldehyde
5. Wash 2 x 2 ml PBS. Aspirate
6. Stain for b-gal with X-gal solution.
7. Incubate 37°C, 5% CO2 2h – over night.
130
Annex X Cryopreservation Cell Line Record
Cell line
BeWo
Pass/date
Location
Species
1/Jul 14
C6/E5-A1
Mycoplasma: Method: PCR
Frezze instructions:
Rate: DMSO as Cryoprotectant
5 %
Normal
Adult
Date of test JUL 21, 2011
Neoplasic
Fetal/NB
Result:
TissueSite
Pass:
PLACENTA
5
NEGATIVO
Authentication:
Thaw conditions:
Date of test:
Thaw rapidly to 37°C
Method: PCR
Dilute to:
mL
Gen No. CCL-98
5 mL
20 mL
Normal maintenance
10
50
mL
Subculture Frequency: 6 DAYS
Seeding Conc. 4.2X10
4
Centrifuge to Remove
Cryoprotectant
Author
Ref.
ATCC
Mode of growth
DMEN/F12 with: HEPES
1.5%, L-GLUTAMINE 1%
ANTIBIOTIC 1%, SFB 10%
AND MEM NON
ESSENTIAL AA 1%.
Special characteristics:
Yes / No
Agent: Trypsin 0.25% 4 min
37°C
Medium Change Frequency: 3
DAYS
Type: DMEM/F12 WITH
HEPES 1.5%, L-GLUTAMINE
1% ANTIBIOTIC 1%, SFB
10% AND MEM NON
ESSENTIAL AMINOACIDS
1%
Gas phase
7.4
Special requirements:
Biohazard precautions:
Habituals
Buffer PBS Ph
Kept in vapor phase N2
Any other special condition
Person completing card
Name: Fernando Vazquez
Alaniz
Signature
131
ACHIEVEMENTS AND AWARDS RECEIVED DURING THIS THESIS
Paper published
Vazquez-Alaniz F, Galaviz-Hernandez C, Marchat LA, Salas-Pacheco JM, ChairezHernandez I, Guijarro-Bustillos JJ, et al. Comparative expression profiles for KiSS-1
and REN genes in preeclamptic and healthy placental tissues. Eur J Obstet Gynecol
Reprod Biol. 2011 Nov;159(1):67-71.
Meetings
Participations in cartel expositions
1. VI Encuentro participación de la mujer en la ciencia, Mayo 2009 Gto, Mexico
2. “5º Congreso Nacional estudiantil de investigación y 5º congreso de
investigación Politecnica” Nov 2009, Queretaro, Mex.
3. 7º Encuentro Nacional de Biotecnología del Instituto Politécnico Nacional,
Octubre 2010, Mazatlán Sin; Mex.
4. XXXV Congreso Nacional de Genética Humana Nov, 2010, Puebla Pue.,
Mexico.
5. 30ht Symposium of the German Society for Magnesium research Oct 2010.
Herne, Germany.
6. XI Congreso Nacional de Química Clínica y Medicina de Laboratorio Expo-Lab
León 2011 May 2011; León Gto, Mex.
7. VII International congress, XVIII National congress of Biochemical engineering
and X Biomedicine and Molecular Biothecnology meeting. March 2012; Ixtapa
Zihuatanejo Gro., Mex.
8. XI Congreso Internacional y XVII Congreso Nacional de Ciencias Ambientales,
de la “Academia Nacional América Central Caribe, México, Mazatlan”
Mazatlán Sin., Mex.
132
Evaluation Committee for abstracts
5º Congreso Nacional estudiantil de Investigación y 5º Congreso de Investigacion
politécnica. Oct 2011 Qro., Mex.
Research stay
Biochemistry laboratory of Medicine Faculty from Universidad Autonoma de San Luis
Potosi, april 2011 and jun-oct. 2011 in San Luis Potosí, Mex.
133