Inflammation in Human Disease and the Autodigestion Hypothesis

Inflammation in Human Disease and the Autodigestion Hypothesis

The Autodigestion Hypothesis
Geert W. Schmid-Schönbein
The Microcirculation Hypertension Project
The Microcirculation Laboratory
Randy Balete
Frank A. Delano
Neema Jamshidi
Dr. Ryan Friese
Dr. Shunichi Fukuda
Daniel Katz
Dr. Nobuhiko Kobayashi
Maya T. Kailasam
Dr. Fred Lacy
Hubert Lim
Dr. Ayako Makino
Guillermo Moratorio
Dr. Lee Murfee
Dr. Alex Penn
Dr. Stephen Rodriguez
Dr. Hansworth Shin
Dr. Alan Swei
Dr. Edward Tran
Dr. Camille Vogt/Wallwork
Our Collaborators
Dr. Bernhard Babior
Dr. David Gough
Dr. D. Neil Granger
Dr. Hiromasa Ischii
Dr. Daniel T. O’Connor
Dr. Robert Palmer
Dr. Dale A. Parks
Dr. Paul Mills
Dr. Kyo Mun
Dr. John Reed
Andrew Chen
Dr. Makoto Suematsu
Jonathan
Hurng
Dr. Hidekazu Suzuki
Daniel Nunez
Bryan Sheu
Dr. Takanori Yasu
The Microcirculation Shock Project
The Microcirculation Laboratory
Our Collaborators
Dr. Jorge Barroso-Aranda
Scott A. Becker,
Marisol Chang
Dr. Ricardo Chavez- Chavez
Frank A. DeLano
Dr. Florian Fitzal
Alex Hu
Dr. Eric B. Kistler
Dr. Hiroshi Mitsuoka
Dr. Hui Miao
Dr. Alexander Penn
Dr. Henrique De Rosário
Anasthasia Sofianos
Suzanne Schimmeyer
Stephan Waldo
Corey Young
Dr. José A. Acosta
Dr. Devashish J Anjaria
Dr. Raul Coimbra
Dr. Jay Doucet
Dr. David A Frankel
Dr. David Hoyt
Dr. Toni E. Hugli
Dr. K. Ishimaru
Dr. Wolfgang G. Junger
Marlene Kawahara
Dr. William J. Kremp
Dr. Alan M. Lefer
Dr. William H. Loomis
Dr. Phillip Pfeiffer
Dr. Paul L. Wolf
Cardiovascular Disease is Accompanied
By Cell Activation and Inflammation
9
9
9
9
9
9
9
9
9
9
Infectious Diseases
Chronic Degenerative Diseases
(arthritis, retinopathy, dementia,
venous disease, …. )
Diabetes
Cardiovascular Risks (smoking, obesity)
Myocardial ischemia
Stroke
Atherosclerosis
Arterial Hypertension
Cancer
Physiological Shock
The Inflammatory Cascade
Early Responses:
Tissue Degradation:
Initial Repair:
Repair:
Trigger mechanism
Ion exchange
Cell degranulation
Production and release of inflammatory mediators
Enhancement of endothelial permeability
Upregulation of membrane adhesion molecules
Protease release and activation
Oxygen free radical formation
Cell activation pseudopod formation and actin polymerization
Neutrophil entrapment in microvessels, transvascular migration
Platelet attachment, aggregation, thrombosis, red cell aggregation
Organ dysfunction
Apoptosis
Downregulation of anti-inflammatory genes
Upregulation of pro-inflammatory genes (cytokines, etc.)
Monocyte and T-Lymphocyte infiltration
Release of growth factors
Connective tissue growth
Revascularization
Resolution of Inflammation
The Inflammatory Cascade
Early Responses:
Tissue Degradation:
Initial Repair:
Repair:
Trigger mechanism
Ion exchange
Cell degranulation
Production and release of inflammatory mediators
Enhancement of endothelial permeability
Upregulation of membrane adhesion molecules
Protease release and activation
Oxygen free radical formation
Cell activation pseudopod formation and actin polymerization
Neutrophil entrapment in microvessels, transvascular migration
Platelet attachment, aggregation, thrombosis, red cell aggregation
Organ dysfunction
Apoptosis
Downregulation of anti-inflammatory genes
Upregulation of pro-inflammatory genes (cytokines, etc.)
Monocyte and T-Lymphocyte infiltration
Release of growth factors
Connective tissue growth
Revascularization
Resolution of Inflammation
Trigger Mechanisms for Cardiovascular Cell Activation
• Inflammatory mediators (bacterial/viral/fungal sources,
endotoxins, cytokines, histamine, oxidized
products, complement fragments, LTB4, PAF, etc.)
• Depletion of anti-inflammatory mediators (nitric oxide,
IL-10, glucocorticoids, albumin, etc.)
• Fluid stress
• Transients of Gas Pressure or Temperature
• Juxtacrine Activation
• Bio-Implant Interfaces
Sepsis and Physiological Shock
Number of Failing
Mortality (%)*
Organ System
0
1
2
3
4
5
3
30
50-60
85-100
72-100
100
*Marshall, J.C., Nathens, A.B.: Multiple Organ Dysfunction Syndrome.
Chapter 95 in American College of Surgeons Surgery.
D.W. Wilmore et al. (eds), Web MD Corp., New York, 2002.
Inflammatory Mediators in Hemorrhagic Shock
l
Nonsurvivors
m
Survivors
Barroso-Aranda et al., Cardiovasc. Pharmacol., 25:S23,
1995
Leukocyte Activation by Tissue Homogenates
Kistler et al., Microcirculation 2000
Pancreatic Digestive
Proenzymes
Proteolytic Enzymes:
Trypsinogen
Chymotrypsinogen
Proelastase
Procarboxypeptidase A
Procarboxypeptidase B
Amylolytic Enzymes:
Amylase
Lipolytic Enzymes:
Lipase
Prophospholipase A1
Prophospholipase A2
Nucleases:
Deoxyribonucleases (DNAse)
Ribonuclease (RNAse)
Hydrolyse proteins, carbohydrates, lipids, nucleic acid
Waldo et al, 2003
Faculty Club Chongqing, China, 2007
What mechanism
prevents autodigestion?
The intestinal mucosal barrier
cat
Rhodin, 1975
Rollwagen et al, Am. J. Path., 156, 1177, 2000
Digestive Proteases Penetration Into the Intestinal Wall During Ischemia
Control
Ischemia Reperfusion
Rosario et al., Am. J. Pathol. 164:1707-1716, 2004
Damage to Intestinal Villi during Superior Mesentery Artery Occlusion
Sham Control
180 min after
Ischemia and
Reperfusion
Fitzal et al, 2004
Intestinal Wall Homogenate is Cytotoxic after Autodigestion due to Lipids
PBS
PBS Control Buffer
Protein
Lipid
Penn et al., in press, 2008
Intact - but not Digested - Albumin Prevents Lipid-derived Cytotoxicity
Curry et al. 1999
Penn et al., in press, 2008
Pancreatic Homogenates Cause 100% Mortality
Mean Arterial Pressure
(mm Hg)
140
Homogenate
Injection
120
100
80
60
Vehicle
Vehicle
ANGD
Futhan
40
*
*
20
0
Futhan or saline infusion
-50
0
50
Time (min)
Kistler et al., Shock, 14:30-34, 2000
(n=1)
100
150
An intervention against the action of pancreatic
digestive enzymes in the ischemic and permeable
intestine?
Intestinal Lumen Lavage
37oC
ANGD
(6-amidino-2-naphtyl p-guanidinobenzoate dimethanesulfate,
nafamostat mesilate: Potent serine protease inhibitor
Inhibits trypsin, chymotrypsin, lipase, thrombin, kallikrein, phospholipase, complement
Mitsuoka P N A S 97:1772-1777 2000
Blockade of digestive enzymes in the intestine prevents formation of
inflammatory mediators in plasma
Mitsuoka et
al., P. N.
97:1772-1777,
2000.
Mitsuoka
P. A.
N. S.,
A. S.,
97:1772-1777,
2000
MABP (mmHg)
Pancreatic Digestive Enzyme Blockade with ANGD, FOY® (Gabaxate mesilate), and Aprotinin
Mitsuoka et al., Shock:17, 205 2002
MABP (mmHg)
Mitsuoka P. N. A. S., 97:1772, 2000.
Schimmeyer , 2005
Time (min)
Fitzal et al., Arch. Surg, 139:1008, 2004
Intestinal Mucosal Injury
Non
Ischemic
Saline
ANGD
Intestinal
Perfusion Perfusion
Fluid
Mitsuoka P. N. A. S., 97:1772-1777, 2000.
Leukocyte Infiltration
Intestinal Enzyme Blockade Reduces Multiorgan Failure
Untreated
Treated
DeLano et al., unpublished
Lung Wet/Dry Weight
Acute Respiratory Distress Syndrome
6
5
4
3
Mitsuoka P. N. A. S., 97:1772-1777, 2000.
Sha
m
Intestinal Lavage
with Buffer
Intestinal Lavage
with FOY
Fitzal et al., J. Vasc. Res., 39:320-329, 2002
80 min Ischemia
80 min Reperfusion
Preclinical Hemorrhagic Shock
70
Baseline
60
MAPMAP
(mmHg
(torr))
50
Sampling times
30 ml/kg
bleed
40
30
Start resuscitation and
intestinal blockade
20
10
60 min shock, MAP
< 30 torr
0
END BASELINE END
SURGERY
BLEED
Doucet et al, J. Trauma, 56:501-510, 2004
0
30
Time (min)
60
90
150
180
Neutrophil Activation
Doucet et al, J. Trauma, 56:501-510, 2004
Whole Blood Infused (ml/kg )
35
30
Transfusion Requirements in Porcine
Hemorrhagic Shock
Controls needed
100% of shed blood
re-infused to
maintain MAP
25
20
15
Control
Treated
p = 0.002
10
5
0
Doucet et al., 2004
Conclusion:
Experimental Shock and Multiorgan Failure is due to escape of
digestive enzymes from the lumen into the wall of the intestine
and from there into the central circulation with enzymatic
autodigestion.
Arterial Blood Pressure in the Spontaneously Hypertensive Rats
Graph from Frank
Hypertension Syndrome
- Elevated arterial blood pressure
- Leukocyte membrane adhesion defect
- Type II diabetes
- Oxygen free radical production
- Apoptosis
- Capillary rarefaction
- Attenuated fluid shear stress response
-…
The SHR has a Reversed Fluid Shear Response
WKY
Fukuda et al., Circ. Res., 2004
SHR
Fukuda et al., 2004
Suppression of FPR expression in differentiated HL60 attenuates the fluid shear-induced response
HL60 Cells
Makino et al., Am. J. Physiol., 2006
Makino et al, 2005
Shin et al., unpublished, 2009
Enhanced And Unchecked Protease Activity in SHR Plasma
Casein derivative substrate cleaved by metallo-, serine,
acid and sulfhydryl proteases
(digital fluorescent unit)
Plasma proteolytic activity
*
n=6
n=6
SHR
WKY
Wistar
EDTA reduces SHR plasma protease activity 29%
Tran et al., 2009
n=6
SHR Plasma Protease Activity
Casein derivative substrate cleaved by metallo-, serine, acid
and sulfhydryl proteases
DeLano et al., in press, 2008
Enhanced MMP-1& MMP-9 Activity in the SHR
MMP-1
(collagenase-1)
MMP -9
(gelatinase B)
Tryptophane Fluorogenic
Substrate:
N-(2,4-Dinitrophenyl)-Phe-LeuGly-Leu-Trp-Ala-D-Arg amide
DeLano et al., in press,
2008
Stack and Gray, J. Biol. Chem.,
264, 4277, 1989
Elevation of MMP-9 Protein Levels on SHR Endothelium
DeLano et al., in press, 2008
Insulin Receptor Density in the SHR
DeLano et al., in press, 2008
SHR Plasma Proteases Cleave the Insulin Receptor
30 min incubation
DeLano et al., in press, 2008
SHR Plasma Blocks Transmembrane Fluorescent Glucose Transport in Naïve
Wistar Leukocytes
30 min incubation
Wistar Plasma
13.1 ± 9.5 digital units
100%
SHR Plasma
5.2 ± 3.2 digital units
39%, p<0.0001
WKY Plasma
11.4 ± 7.2 digital units
87%, p<0.0001
DeLano et al., in press, 2008
non-hydrolyzable glucose analog 6-NBD-deoxyglucose
SHR Plasma Proteases Cleave Insulin Receptor
DeLano et al., Hypertension 2008
SHR Plasma Proteases Cleave Insulin Receptor
Proteas
e
DeLano et al., Hypertension 2008
Blockade of Protease Activity Normalizes Blood Pressure and Leukocyte Count in the
SHR
DeLano et al., in press, 2008
SHR Plasma Proteases Cleave Leukocyte β2 Integrins
DeLano et al., in press, 2008
Conclusions
The SHR has an unchecked protease activity in the plasma, endothelium and
parenchymal cells, which causes proteolytic damage of several membrane
receptors.
Cleavage of the extracellular domain of the
•
β2 adrenergic receptor
(vasoconstriction)
•
insulin receptor
(type 2 diabetes mellitus)
•
β2 integrin adhesion molecule (immune suppression)
•
FPR
•VEGFR2
(shear response suppression)
(endothelial apoptosis, capillary
rarefaction)
Mechanisms for Cell Injury & Inflammation
Free Radical Hypothesis
Auto-Digestion Hypothesis
Superoxide anion
O2 ●-
Hydrogen peroxide
H2O2
Hydroxyl radical
●
Perhydroxy radical
HO2*
Lipases
Alkoxy radical
R-O*
Amylases
Peroxy radical
ROO*
HO
Serine, Cysteine, Metallo,
Aspartic, Threonine,
Glutamic Acid Proteases