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1.  PANCREATIC DIGESTIVE ENZYME BLOCKADE IN THE SMALL INTESTINE PREVENTS INSULIN RESISTANCE IN HEMORRHAGIC SHOCK 
Shock (Augusta, Ga.)  2014;41(1):10.1097/SHK.0000000000000048.
Hemorrhagic shock is associated with metabolic defects, including hyperglycemia and insulin resistance but the mechanisms are unknown. We recently demonstrated that reduction of the extracellular domain of the insulin receptor by degrading proteases may lead to a reduced ability to maintain normal plasma glucose values. In shock, transfer of digestive enzymes from the lumen of the intestine into the systemic circulation after breakdown of the intestinal mucosal barrier causes inflammation and organ dysfunction. Suppression of the digestive enzymes in the lumen of the intestine with protease inhibitors is effective in reducing the level of the inflammatory reactions. To determine the degree to which blockade of digestive enzymes affects insulin resistance in shock, rats were exposed to acute hemorrhagic shock (mean arterial pressure of 30 mmHg for 2 hours) at which time all shed blood volume was returned. Digestive proteases in the intestine were blocked with a serine protease inhibitor (tranexamic acid in polyethylene glycol and physiological electrolyte solution) and the density of the insulin receptor was measured with immunohistochemistry in the mesentery microcirculation. The untreated rat without enzyme blockade had significantly attenuated levels of insulin receptor density as compared to control and treated rats. Blockade of the digestive proteases after 60 min of hypotension in the lumen of the small intestine lead to a lesser decrease in insulin receptor density compared to controls without protease blockade. Glucose tolerance test indicates a significant increase in plasma glucose levels two hours after hemorrhagic shock, which are reduced to control values in the presence of protease inhibition in the lumen of the intestine. The transient reduction of the plasma glucose levels after an insulin bolus is significantly attenuated after shock, but is restored in when digestive enzymes in the lumen of the intestine are blocked. These results suggest that in hemorrhagic shock elevated microvascular extracellular digestive enzyme activity causes insulin receptor dysfunction, hyperglycemia and reduced ability to regulate blood glucose values.
doi:10.1097/SHK.0000000000000048
PMCID: PMC3884675  PMID: 24088998
Glucose tolerance; pancreatic enzymes; proteases; auto-digestion; mesentery microvessels; leukocytes; Wistar rat
2.  The Autodigestion Hypothesis in Shock and Multi-Organ Failure: Degrading Protease Activity 
Shock and multi-organ failure have one of the highest levels of inflammatory markers, morbidities and mortality. The underlying mechanisms are currently unknown and no effective intervention exists. We present evidence for a previously untested mechanism due to autodigestion by the digestive enzymes synthesized in the pancreas and transported in the lumen of the intestine as normal part of food digestion. We summarize experimental evidence in support of the autodigestion hypothesis and a new approach for possible intervention against multi-organ failure that is currently entering clinical trials.
PMCID: PMC4180238  PMID: 25284955
Intestine; digestive pancreas; inflammation; trypsin; chymotrypsin; elastase; intestinal mucosa; hemorrhagic shock; sepsis
3.  Matrix Metalloproteinases Activities in Hypertension: Emerging Opportunities 
Hypertension  2010;57(1):24-25.
doi:10.1161/HYPERTENSIONAHA.110.162032
PMCID: PMC4123869  PMID: 21079046
4.  Proteolytic Cleavage of the Red Blood Cell Glycocalyx in a Genetic Form of Hypertension 
Recent evidence suggests that the spontaneously hypertensive rat (SHR) has an elevated level of proteases, including matrix metalloproteinases (MMPs), involved in cell membrane receptor cleavage. We hypothesize that SHR red blood cells (RBCs) may be subject to an enhanced glycocalyx cleavage compared to the RBCs of the normotensive Wistar-Kyoto (WKY) rats. By direct observation of RBC rouleaux, we found no significant difference in RBC aggregation for unseparated SHR and WKY RBCs. However, lighter SHR RBCs have a greater tendency to aggregate than WKY RBCs when separated by centrifugation. When SHR plasma was mixed with WKY RBCs, SHR plasma proteases cleaved the glycocalyx of WKY RBCs, a process that can be blocked by MMP inhibition. When treated with MMPs, WKY RBCs showed strong aggregation in dextran but not in fibrinogen, indicating that RBC membrane glycoproteins from the inner core of the glycocalyx were cleaved and that dextran was able to bind to the lipid portion of the RBC membrane. In contrast, treatment with amylases produced fibrinogen-induced aggregation with fibrinogen binding to the protein core. MMP cleavage of RBC glycocalyx reduces RBC adhesion to macrophages as a mechanism to remove old RBCs from the circulation.
doi:10.1007/s12195-011-0180-0
PMCID: PMC3711591  PMID: 23864910
Spontaneously hypertensive rat; matrix metalloproteinases; red blood cell aggregation; glycocalyx cleavage; dextran; fibrinogen
5.  Disruption of the Mucosal Barrier during Gut Ischemia Allows Entry of Digestive Enzymes into the Intestinal Wall 
Shock (Augusta, Ga.)  2012;37(3):297-305.
Intestinal ischemia is associated with high morbidity and mortality but the underlying mechanisms are uncertain. We hypothesize that during ischemia the intestinal mucosal barrier becomes disrupted, allowing digestive enzymes access into the intestinal wall initiating autodigestion. We used a rat model of splanchnic ischemia by occlusion of the superior mesenteric and celiac arteries up to 30 min with and without luminal injection of tranexamic acid as a trypsin inhibitor. We determined the location and activity of digestive proteases on intestinal sections with in-situ zymography and we examined the disruption of two components of the mucosal barrier: mucin isoforms and the extra- and intracellular domains of E-cadherin with immunohistochemistry and western blot techniques. The results indicate that non-ischemic intestine has low levels of protease activity in its wall. After 15 min ischemia protease activity was visible at the tip of the villi and after 30 min enhanced activity was seen across the full thickness of the intestinal wall. This activity was accompanied by disruption of the mucin layer and loss of both intra- and extracellular domains of E-cadherin. Digestive protease inhibition in the intestinal lumen with tranexamic acid reduced morphological damage and entry of digestive enzymes into the intestinal wall. This study demonstrates that disruption of the mucosal epithelial barrier within minutes of intestinal ischemia allows entry of fully activated pancreatic digestive proteases across the intestinal barrier triggering autodigestion.
doi:10.1097/SHK.0b013e318240b59b
PMCID: PMC3288241  PMID: 22089198
Shock; mucin; serine proteases; e-cadherin
6.  Proteolytic Activity Attenuates the Response of Endothelial Cells to Fluid Shear Stress 
Recent evidence indicates that several experimental pathophysiological conditions are associated with elevated protease activity in plasma, which impacts endothelial function. We hypothesize that extracellular structures bound to the endothelial cell (EC) membrane may be degraded by proteolytic activity and cause the cells to respond abnormally to physiological shear stress (12 dyn/cm2). To test this hypothesis, cultured bovine aortic endothelial cells (BAECs) were exposed to low levels of a serine protease, trypsin. Extracellular mechanosensor densities of the glycocalyx and vascular endothelial growth factor receptor 2 (VEGFR-2) were determined. Metabolic dysfunction was tested by examining insulin receptor and glucose uptake levels. Protease treatment impaired the cells’ ability to align in the direction of fluid flow after 12 hours of shear stress; however, cells realigned after an additional 12 hours of shear stress with protease inhibition. Proteases caused reduction in the densities of glycocalyx, VEGFR-2, and insulin receptor in static and shear conditions, except for static VEGFR-2 cells. Under static conditions, protease-treated endothelial cells had reduced glucose uptake compared to untreated controls. Under shear, however, glucose uptake for protease-treated BAECs was greater than untreated controls. In conclusion, protease activity in plasma alters the exofacial membrane components of ECs and may interfere with mechanotransduction.
doi:10.1007/s12195-011-0207-6
PMCID: PMC3337682  PMID: 22545072
Mechanotransduction; VEGFR-2; insulin resistance; lectin; glycocalyx; autodigestion
7.  Constitutive Expression and Enzymatic Cleavage of ICAM-1 in the Spontaneously Hypertensive Rat 
Journal of Vascular Research  2011;48(5):386-396.
Background/Aims:
Leukocyte adhesion to the endothelium is abnormal in hypertension. We have recently shown that spontaneously hypertensive rats (SHRs) have circulating leukocytes with enhanced CD18 receptor cleavage. In the current study, we investigate expression levels of its counter receptor, intercellular adhesion molecule (ICAM-1), and its possible proteolytic cleavage in the SHR and control Wistar rat.
Methods
ICAM-1 was labeled on tissue sections with two antibodies targeting its extracellular and intracellular domains and evaluated by light absorption measurements. The in situ cleavage of ICAM-1 was assessed by treating vessel sections with matrix metalloproteinase (MMP)-7, MMP-9 and elastase.
Results
SHRs showed a significant increase in ICAM-1 expression in liver and kidney compared with Wistar rats. The liver and kidney glomeruli exhibit a discrepancy in label density between intra- and extracellular antibodies, which suggests that enzymatic cleavage may be a factor determining ICAM-1 distribution. MMP-7 and MMP-9, which are elevated in SHR plasma, and elastase, which has elevated activity in SHR neutrophils, cleave the extracellular domain of ICAM-1 when applied to the tissue.
Conclusion
ICAM-1 expression in SHRs is upregulated in a tissue-specific manner. Proteolytic cleavage of the extracellular domain of ICAM-1 and accumulation in kidney glomeruli may play a role in the renal involvement of inflammation.
doi:10.1159/000323474
PMCID: PMC3080588  PMID: 21464573
Leukocyte adhesion; Endothelium; Receptor cleavage; Arterioles; Venules
8.  Structure of Microvascular Networks in Genetic Hypertension 
Methods in Enzymology  2008;444:271-284.
Microvascular rarefaction, defined by a loss of terminal arterioles, small venules and/or capillaries, is a common characteristic of the hypertension syndrome. While rarefaction has been associated with vessel specific free radical production, deficient leukocyte adhesion, and cellular apoptosis, the relationships of rarefaction with structural alterations at the network and cellular level remain largely unexplored. The objective of this study was to examine the architecture and perivascular cell phenotypes along microvascular networks in hypertensive versus normotensive controls in the context of imbalanced angiogenesis. Mesenteric tissues from age-matched adult male spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats were harvested and immnolabeled for PECAM and neuron-glia antigen 2 (NG2). Evaluation of intact rat mesenteric microvascular networks rats suggests that network alterations associated with hypertension are more complex than just a loss of vessels. Typical SHR versus WKY networks demonstrate a reduced branching architecture marked by more proximal arteriole/venous anastomoses and an absence of NG2 labeling along arterioles. Although less frequent, larger SHR microvascular networks display regions of dramatically increased vascular density. SHR and WKY lymphatic networks demonstrate increased vessel diameters and vascular density compared to networks in normotensive Wistar rats (the strain from which both the SHR and WKY originated). These observations provide a rationale for investigating the presence of local angiogenic factors and response of microvascular networks to therapies aimed at reversing rarefaction in genetic hypertension.
doi:10.1016/S0076-6879(08)02812-7
PMCID: PMC3325543  PMID: 19007669
9.  Matrix Metalloproteinase Activity Causes VEGFR-2 Cleavage and Microvascular Rarefaction in Rat Mesentery 
A complication of the spontaneously hypertensive rat (SHR) is microvascular rarefaction, defined by the loss of microvessels. However, the molecular mechanisms involved in this process remain incompletely identified. Recent work in our laboratory suggests that matrix metalloproteinases (MMPs) may play a role by cleavage of the vascular endothelial growth factor receptor 2 (VEGFR-2). In order to further delineate the role for MMPs in microvascular rarefaction, the objective of the current study was to examine the relationship in the same tissue between MMP activity, VEGFR-2 cleavage and rarefaction. Using an in-vivo microzymographic technique we show significantly enhanced levels of MMP-1, -1/-9, -7, and -8 activities, but not MMP-2 and-3 activities, along mesenteric microvessels of the SHR compared to its normotensive control, Wistar Kyoto (WKY) rat. Based on immunohistochemical methods, the SHR exhibited a decreased labeling of the extracellular, but not the intracellular, domain of VEGFR-2 along mesenteric microvessels. Chronic MMP inhibition served to attenuate VEGFR-2 cleavage and microvascular network rarefaction in the SHR mesentery. These results spatially link MMP-induced VEGFR-2 cleavage and rarefaction in the mesentery of the SHR and thus support the hypothesis that MMPs serve as regulators of microvascular dysfunction in hypertension.
doi:10.1111/j.1549-8719.2011.00082.x
PMCID: PMC3081385  PMID: 21418372
Spontaneously Hypertensive Rat; Wister Kyoto rat; capillary; arteriole; venule; microzymography; matrix metalloproteinase inhibition
10.  NF kappa B and Matrix Metalloproteinase induced Receptor Cleavage in the Spontaneously Hypertensive Rat 
Hypertension  2011;57(2):261-268.
Recent evidence suggests that inflammation in the spontaneously hypertensive rat (SHR) is associated with an uncontrolled matrix metalloproteinase (MMP) activity. We hypothesize that the transcription factor nuclear factor kappa B (NF–κB) is overexpressed in the SHR, enhancing its MMP activity and enzymatic cleavage of the beta-2 adrenergic receptor (β2AR), thereby diminishing catecholamine-mediated arteriolar vasodilation. NF-κB expression level and translocation were compared between Wistar Kyoto rat (WKY) and SHR kidney, heart and brain. The animals were treated with a NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC), for ten weeks and correlations between NF-κB and MMP activity were determined. Immunohistochemistry showed that NF-κB expression is increased in untreated SHR kidney (~ 14%) and brain hypothalamus (~ 22%) compared to that in WKY (p <0.05), but not in myocardium and cerebral cortex. After PDTC treatment, the SHR systolic blood pressure was reduced close to WKY levels. NF-κB expression level in treated-SHR was also decreased in kidney and hypothalamus compared to non-treated animals (p <0.05). Furthermore, MMP-2 and -9 activities in SHR plasma were significantly reduced (~41%) by PDTC treatment. Additionally, zymographic analyses and in situ zymography showed decreased MMP-2 activity in kidney homogenates and decreased MMP-1,-9 activities in brain. The level of the β2AR extracellular, but not intracellular, domain density was found reduced in kidney showing a receptor cleavage process that can be blocked by PDTC treatment. These results suggest NF-κB is an important transcription factor in the SHR and may be involved in the enhanced MMP activity and consequently receptor cleavage.
doi:10.1161/HYPERTENSIONAHA.110.158709
PMCID: PMC3077933  PMID: 21220710
Microcirculation; matrix metalloproteinases; beta-2 adrenergic receptor; receptor cleavage; NF-κB inhibitor; pyrrolidine dithiocarbamate
11.  Acute Venous Occlusion Enhances Matrix Metalloprotease Activity. Implications on Endothelial Dysfunction 
Microvascular research  2010;81(1):108-116.
Venous hypertension is associated with microvascular inflammation, restructuring, and apoptosis, but the cellular and molecular mechanisms underlying these events remain uncertain. In the present study we tested the hypothesis that elevated venous pressure and reduction of shear stress induces elevated enzymatic activity. This activity in turn may affect endothelial surface receptors and promote their dysfunction. Using a rodent model for venous hypertension using acute venular occlusion, microzymographic techniques for enzyme detection, and immunohistochemistry for receptor labeling, we found increased activity of the matrix metalloproteases (MMPs) -1, -8 and -9 and tissue inhibitors of metalloproteases (TIMPs) -1,-2 in both high and low-pressure regions. In this short time frame we also observed that elevated venule pressure led to two different fates for the vascular endothelial growth factor receptor-2 (VEGFR2); in higher-pressure upstream regions some animals exhibited higher VEGFR2 expression, while others displayed lower levels upstream compared to their downstream counterparts with lower pressure. VEGFR2 expression was, on average, more pronounced upon application of MMP inhibitor, suggesting possible cleavage of the receptor by activated enzymes in this model. We conclude that venous pressure elevation increases enzymatic activity which may contribute to inflammation and endothelial dysfunction associated with this disease by influencing critical surface receptors.
doi:10.1016/j.mvr.2010.09.010
PMCID: PMC3021174  PMID: 20923679
matrix metalloproteases; inflammation; vascular endothelial growth factor-2; occlusion; mesentery; venule; endothelial cell
12.  ShockOmics: multiscale approach to the identification of molecular biomarkers in acute heart failure induced by shock 
Background
The ShockOmics study (ClinicalTrials.gov identifier NCT02141607) is a multicenter prospective observational trial aimed at identifying new biomarkers of acute heart failure in circulatory shock, by means of a multiscale analysis of blood samples and hemodynamic data from subjects with circulatory shock.
Methods and Design
Ninety septic shock and cardiogenic shock patients will be recruited in three intensive care units (ICU) (Hôpital Erasme, Université Libre de Bruxelles, Belgium; Hospital Universitari Mutua Terrassa, Spain; Hôpitaux Universitaires de Genève, Switzerland). Hemodynamic signals will be recorded every day for up to seven days from shock diagnosis (time T0). Clinical data and blood samples will be collected for analysis at: i) T1 < 16 h from T0; ii) T2 = 48 h after T0; iii) T3 = day 7 or before discharge or before discontinuation of therapy in case of fatal outcome; iv) T4 = day 100.
The inclusion criteria are: shock, Sequential Organ Failure Assessment (SOFA) score > 5 and lactate levels ≥ 2 mmol/L. The exclusion criteria are: expected death within 24 h since ICU admission; > 4 units of red blood cells or >1 fresh frozen plasma transfused; active hematological malignancy; metastatic cancer; chronic immunodepression; pre-existing end stage renal disease requiring renal replacement therapy; recent cardiac surgery; Child-Pugh C cirrhosis; terminal illness. Enrollment will be preceded by the signature of the Informed Consent by the patient or his/her relatives and by the physician in charge.
Three non-shock control groups will be included in the study: a) healthy blood donors (n = 5); b) septic patients (n = 10); c) acute myocardial infarction or patients with prolonged acute arrhythmia (n = 10).
The hemodynamic data will be downloaded from the ICU monitors by means of dedicated software. The blood samples will be utilized for transcriptomics, proteomics and metabolomics (“-omics”) analyses.
Discussion
ShockOmics will provide new insights into the pathophysiological mechanisms underlying shock as well as new biomarkers for the timely diagnosis of cardiac dysfunction in shock and quantitative indices for assisting the therapeutic management of shock patients.
doi:10.1186/s13049-016-0197-4
PMCID: PMC4730667  PMID: 26822963
Shock; Acute heart failure; Biomarkers; Transcriptomics; Proteomics; Metabolomics; Hemodynamics; Multiscale modeling
13.  A New Hypothesis for Insulin Resistance in Hypertension Due to Receptor Cleavage 
Background
One of the most important unresolved issues in diabetes is the mechanism for the attenuated response to insulin, i.e. insulin resistance.
Aims and methods
We hypothesize that the mechanism for the insulin resistance is due to uncontrolled protease activity in the plasma, on endothelial cells and in the tissue parenchyma. To examine this hypothesis we use of microzymographic techniques in the microcirculation, plasma zymography, and receptor labeling techniques with antibodies against an extracellular domain of the insulin receptor α.
Results
The spontaneously hypertensive rat has an enhanced proteolytic activity and significant cleavage of the receptor with attenuated glucose transport. We present evidence for insulin receptor cleavage in a high fat diet and a transgenic model of diabetes.
Conclusion
These results suggest that cleavage of the extracellular domain of the insulin receptor, a situation that interferes with the ability for insulin to bind and provide an intracellular signal for glucose transport, may be involved in insulin resistance.
PMCID: PMC2995254  PMID: 21132054
Protease activity; matrix metalloproteases; insulin resistance; receptor cleavage; leukocyte; adhesion; integrin
14.  Inflammation and the Autodigestion Hypothesis 
Although long recognized in microvascular research, an increasing body of evidence suggests that inflammatory markers are present in human diseases. Since the inflammatory cascade serves as a repair mechanism, the presence of inflammatory markers in patient groups has raised an important question about the mechanisms that initiate the inflammatory cascade, i.e. the mechanisms that cause tissue injury. Using a severe forms of inflammation, shock and multi-organ failure, for which there is no accepted injury mechanism, we summarize studies which suggest that the powerful pancreatic digestive enzymes play a central role in destruction of the intestine and other tissues if their compartmentalization in the lumen of the intestine and in the pancreas is compromised. Furthermore, we summarize evidence that uncontrolled degrading enzyme activity in plasma causes proteolytic cleavage of the extracellular domain of membrane receptors and loss of associated cell functions. For example, in a model of metabolic disease with Type II diabetes proteolytic cleavage of the insulin receptor causes the inability of insulin to signal glucose transport across membranes. The evidence suggests that uncontrolled proteolytic and lipolytic enzyme activity may trigger mechanism for tissue injury. The significance of such mechanisms remain to be explored in human diseases.
doi:10.1080/10739680902801949
PMCID: PMC2677689  PMID: 19384726
Microcirculation; inflammation; pancreatic enzymes; matrix metalloproteinases; shock; multi-organ failure; hypertension
15.  Biomechanical Aspects of the Auto-digestion Theory 
Increasing evidence suggests that most cardiovascular diseases, tumors and other ailments are associated with an inflammatory cascade. The inflammation is accompanied by activation of cells in the circulation and fundamental changes in the mechanics of the microcirculation, expression of pro-inflammatory genes and downregulation of anti-inflammatory genes, attachment of leukocytes to the endothelium, elevated permeability of the endothelium, and many other events. The evidence has opened great opportunities for medicine to develop new anti-inflammatory interventions. But it also raises a fundamental question: What is the origin of inflammation? I will discuss a basic series of studies that was designed to explore trigger mechanisms for inflammation in shock and multi-organ failure, an important clinical problem associated with high mortality. We traced the source of the inflammatory mediators to the powerful digestive enzymes in the intestine. Synthesized in the pancreas as part of normal digestion, they have the ability to degrade almost all biological tissues and molecules. In the lumen of the intestine, digestive enzymes are fully activated and self-digestion of the intestine is prevented by compartmentalization in the lumen of the intestine facilitated by the mucosal epithelial barrier. Under conditions of intestinal ischemia, however, the mucosal barrier becomes permeable to pancreatic enzymes allowing their entry into the wall of the intestine. The process leads to auto-digestion of the intestinal wall and production of inflammatory mediators. The hypothesis that multi-organ failure in shock may be due an auto-digestion process by pancreatic enzymes is ready to be tested in a variety of shock conditions.
PMCID: PMC2671552  PMID: 18589497
shock; intestinal ischemia; digestive enzymes; transport; epithelial barrier
16.  PROTEINASE ACTIVITY AND RECEPTOR CLEAVAGE: MECHANISM FOR INSULIN RESISTANCE IN SPONTANEOUSLY HYPERTENSIVE RAT 
Hypertension  2008;52(2):415-423.
Arterial hypertension is associated with organ dysfunctions, but the mechanisms are uncertain. We hypothesize that enhanced proteolytic activity in the microcirculation of spontaneously hypertensive rats (SHRs) may be a pathophysiological mechanism causing cell membrane receptors cleavage and examine this for two different receptors. Immunohistochemistry of matrix-degrading metalloproteinases (MMP-9) protein shows enhanced levels in SHR microvessels, mast cells, and leukocytes compared to normotensive Wistar-Kyoto (WKY) rats. In-vivo micro-zymography shows cleavage by MMP-1,9 in SHRs that co-localizes with MMP-9 and is blocked by metal chelation. SHR plasma also has enhanced protease activity. We demonstrate with an antibody against the extracellular domain that the insulin receptor-α density is reduced in SHR, in line with elevated blood glucose levels and glycated hemoglobin. There is also cleavage of the binding domain of the leukocyte integrin receptor CD18 in line with previously reported reduced leukocyte adhesion. Blockade of MMPs with broad acting inhibitor (doxycycline, 5.4mg/kg/day) reduces protease activity in plasma and microvessels, blocks the proteolytic cleavage of the insulin receptor, the reduced glucose transport, normalizes blood glucose levels and glycated hemoglobin levels, as well as reduces blood pressure and enhanced microvascular oxidative stress of SHRs. The results suggest that elevated MMP activity leads to proteolytic cleavage of membrane receptors in the SHR, e.g. cleavage of the insulin receptor-binding domain associated with insulin resistance.
doi:10.1161/HYPERTENSIONAHA.107.104356
PMCID: PMC2677556  PMID: 18606910
Microcirculation; matrix metalloproteinases; insulin receptor; integrin; receptor cleavage; oxygen free radical
17.  A Journey with Tony Hugli up the Inflammatory Cascade towards the Auto-Digestion Hypothesis 
International immunopharmacology  2007;7(14):1845-1851.
My association with Tony Hugli, long-term editor of Immunopharmacology and International Immunopharmacology, came about by a specific and long-standing problem in inflammation research. What is the trigger mechanism of inflammation in physiological shock? This is an important clinical problem due to the high mortality associated with physiological shock. We joined forces in the search of the answer to this question for more than a decade. Our journey eventually led to development of the hypothesis that shock may be associated with pancreatic enzymes, a set of powerful digestive enzymes that are an integral part of human digestion. The digestive enzymes need to be compartmentalized in the lumen of the intestine where they break down a broad spectrum of biological molecules into their building blocks, suitable for molecular transport across the mucosal epithelium into the circulation. The mucosal epithelial barrier is the key element for compartmentalization of the digestive enzymes. But under conditions when the mucosal barrier is compromised, the fully activated digestive enzymes in the lumen of the intestine are transported into the wall of the intestine, starting an auto-digestion process. In the process several classes of mediators are generated that by themselves have inflammatory activity and upon entry into the central circulation generate the hallmarks of inflammation and eventually cause multi-organ failure. Thus, our journey led to a new hypothesis, which is potentially of fundamental importance for death by multi-organ failure. The auto-digestion hypothesis is in line with the century old observation that the intestine plays a special role on shock - indeed it is the organ for digestion. Auto-digestion may be the prize to pay for life-long nutrition.
doi:10.1016/j.intimp.2007.07.015
PMCID: PMC2174519  PMID: 18039521
Auto-digestion; shock; inflammation; cytokines; leukocytes; microcirculation; pancreatic enzymes; trypsin; chymotrypsin; elastase
18.  Systematic polymorphism discovery after genome-wide identification of potential susceptibility loci in a hereditary rodent model of human hypertension 
Blood pressure  2011;20(4):222-231.
Genetic strategies such as linkage analysis and quantitative trait locus (QTL) mapping have identified a multitude of loci implicated in the pathogenesis of hypertension in the spontaneously hypertensive rat (SHR). While several candidate genetic regions have been identified in the SHR and its control, the Wistar–Kyoto rat (WKY), systematic follow-up of candidate identification with polymorphism discovery has not been widespread. In the current report, we develop a data-mining strategy to identify candidate genes for hypertension in the SHR, and then sequence each gene in the SHR and WKY strains. We integrate blood pressure QTL data, microarray data and data-mining methods. First, we determined the set of genes differentially expressed in SHR and WKY adrenal glands. Next, the chromosomal position of all differentially expressed genes was compared with peak marker position of all reported SHR blood pressure QTLs. We also identified the set of differentially expressed genes with the most extreme fold-change. Finally, the QTL positional candidates and the genes with extreme differential expression were proposed as candidate genes if they had biologically plausible roles in hypertensive pathology. We identified seven candidate genes that merit resequencing (catechol-O-methyltransferase [Comt], chromogranin A [Chga], dopamine beta-hydroxylase [Dbh], electron transferring flavoprotein dehydrogenase [Etfdh], endothelin receptor type B [Ednrb], neuropeptide Y [Npy] and phenylethanolamine-N-methyltransferase [Pnmt]), and then discovered polymorphism in four of these seven candidate genes. Chga is proposed as the strongest candidate for additional functional investigation. Our method for candidate gene identification is portable and can be applied to microarray data from any tissue, in any disease model with a QTL database.
doi:10.3109/08037051.2011.566012
PMCID: PMC4648345  PMID: 21428728
Essential hypertension; polymorphism; sequencing; SHR rat strain; WKY rat strain
19.  Pancreatic Digestive Enzyme Blockade in the Intestine Increases Survival After Experimental Shock 
Science translational medicine  2013;5(169):169ra11.
Shock, sepsis, and multiorgan failure are associated with inflammation, morbidity, and high mortality. The underlying pathophysiological mechanism is unknown, but evidence suggests that pancreatic enzymes in the intestinal lumen autodigest the intestine and generate systemic inflammation. Blocking these enzymes in the intestine reduces inflammation and multiorgan dysfunction. We investigated whether enzymatic blockade also reduces mortality after shock. Three rat shock models were used here: hemorrhagic shock, peritonitis shock induced by placement of cecal material into the peritoneum, and endotoxin shock. One hour after initiation of hemorrhagic, peritonitis, or endotoxin shock, animals were administered one of three different pancreatic enzyme inhibitors—6-amidino-2-naphtyl p-guanidinobenzoate di-methanesulfate, tranexamic acid, or aprotinin—into the lumen of the small intestine. In all forms of shock, blockade of digestive proteases with protease inhibitor attenuated entry of digestive enzymes into the wall of the intestine and subsequent autodigestion and morphological damage to the intestine, lung, and heart. Animals treated with protease inhibitors also survived in larger numbers than untreated controls over a period of 12 weeks. Surviving animals recovered completely and returned to normal weight within 14 days after shock. The results suggest that the active and concentrated digestive enzymes in the lumen of the intestine play a central role in shock and multi-organ failure, which can be treated with protease inhibitors that are currently available for use in the clinic.
doi:10.1126/scitranslmed.3005046
PMCID: PMC4643000  PMID: 23345609
20.  Cleavage and reduced CD36 ectodomain density on heart and spleen macrophages in the Spontaneously Hypertensive Rat 
Microvascular research  2014;95:131-142.
Metabolic disease is accompanied by a range of cellular defects (“comorbidities”) whose origin is uncertain. To investigate this pathophysiological phenomenon we used the Spontaneously Hypertensive Rat (SHR), which besides an elevated arterial blood pressure also has many other comorbidities, including a defective glucose and lipid metabolism. We have shown that this model of metabolic disease has elevated plasma matrix metalloproteinase (MMP) activity, which cleaves the extracellular domain of membrane receptors. We hypothesize here that the increased MMP activity also leads to abnormal cleavage of the scavenger receptor and fatty acid transporter CD36. To test this idea, chronic pharmaceutical MMP inhibition (CGS27023A) of the SHR and its normotensive control, the Wistar Kyoto Rat (WKY), was used to determine if inhibition of MMP activity serves to maintain CD36 receptor density and function. Surface density of CD36 on macrophages from the heart, spleen, and liver was determined in WKY, SHR, CGS-treated WKY (CGS WKY), and CGS-treated SHR (CGS SHR) by immunohistochemistry with an antibody against the CD36 ectodomain. The extracellular CD36 density was lower in SHR heart and spleen macrophages compared to that in the WKY. MMP inhibition by CGS served to restore the reduced CD36 density on SHR cardiac and splanchnic macrophages to levels of the WKY. To examine CD36 function, culture assays with murine macrophages (RAW 264.7) after incubation in fresh WKY or SHR plasma were used to test for adhesion of light-weight donor red blood cell (RBC) by CD36. This form of RBC adhesion to macrophages was reduced after incubation in SHR compared WKY plasma. Analysis of the supernatant macrophage media by Western blot shows a higher level of CD36 extracellular protein fragments following exposure to SHR plasma compared to WKY. MMP inhibition in the SHR plasma compared to untreated plasma, served to increase the RBC adhesion to macrophages and decrease the number of receptor fragments in the macrophage media. In conclusion, these studies bring to light that plasma in the SHR model of metabolic disease has an unchecked MMP degrading activity which causes cleavage of a variety of membrane receptors, including CD36, which attenuates several cellular functions typical for the metabolic disease, including RBC adhesion to the scavenger receptor CD36. In addition to other cell dysfunctions chronic MMP inhibition restores CD36 in the SHR.
doi:10.1016/j.mvr.2014.08.004
PMCID: PMC4642997  PMID: 25172177
CD36; MMP; TIMP; Spontaneously Hypertensive Rat (SHR); MMP inhibitor CGS27023A
21.  The oxygen free radicals control MMP-9 and transcription factors expression in the spontaneously hypertensive rat 
Microvascular research  2013;90:154-161.
Oxygen free radical and matrix metalloproteinases-9 (MMP-9) play an important pathophysiological role in the development of chronic hypertension. MMP-9 activities are regulated at different levels. We hypothesize that as mediators of the expression of MMP-9 the transcription factors like nuclear factor kappa B (NF-κB), c-fos and retinoic acid receptors-α (RAR-α) with binding sites to the MMP-9 promoter are overexpressed in the spontaneously hypertensive rat (SHR) in a process that is regulated by oxygen free radicals. Transcription factor NF-κB, c-fos and RAR-α expression levels were determined by immunohistochemistry in renal, cardiac and mesentery micro-circulation of the SHR and its normotensive control, the Wistar Kyoto (WKY) rat. The animals were treated with a superoxide scavenger (Tempol) for eight weeks. The elevated plasma levels of thiobarbituric acid reactive substances and MMP-9 levels in the SHR were significantly decreased by Tempol treatment (P < 0.05). The NF-κB, c-fos and RAR-α expression levels in renal glomerular, heart and mesentery microvessels were enhanced in the SHR and could also be reduced by Tempol compared to untreated animals (P < 0.05). The enhanced MMP-9 levels in SHR microvessels co-express with transcription factors. These results suggest that elevated NF-κB, c-fos and RAR-α expressions and MMP-9 activity in the SHR are superoxide-dependent.
doi:10.1016/j.mvr.2013.09.003
PMCID: PMC4642998  PMID: 24060804
22.  Effect of Digestion and Storage of Human Milk on Free Fatty Acid Concentration and Cytotoxicity 
Objectives
Fat is digested in the intestine into free fatty acids (FFAs), which are detergents and therefore toxic to cells at micromolar concentration. The mucosal barrier protects cells in the adult intestine, but this barrier may not be fully developed in premature infants. Lipase-digested infant formula, but not fresh human milk, has elevated FFAs and is cytotoxic to intestinal cells, and therefore could contribute to intestinal injury in necrotizing enterocolitis (NEC). But even infants exclusively fed breast milk may develop NEC. Our objective was to determine if stored milk and milk from donor milk banks (DM) could also become cytotoxic, especially after digestion.
Methods
We exposed cultured rat intestinal epithelial cells or human neutrophils to DM and milk collected fresh and stored at 4 or −20 °C for up to 12 weeks and then treated for 2 hours (37°C) with 0.1 or 1 mg/ml pancreatic lipase and/or trypsin and chymotrypsin.
Results
DM and milk stored 3 days (at 4 or −20 °C) and then digested were cytotoxic. Storage at −20 °C for 8 and 12 weeks resulted in an additional increase in cytotoxicity. Protease digestion decreased, but did not eliminate cell death.
Conclusions
Current storage practices may allow milk to become cytotoxic and contribute to intestinal damage in NEC.
doi:10.1097/MPG.0000000000000441
PMCID: PMC4140960  PMID: 24840512
Lipase; necrotizing enterocolitis; breast milk donor banks; necrosis; intestinal cell damage
23.  Cellular and molecular basis of Venous insufficiency 
Vascular Cell  2014;6:24.
Chronic venous disease (CVD) has a range of clinical presentations, including tortuous, distended veins in lower extremities, increasing skin pigmentation, and in severe cases ulceration of the affected skin. Venous insufficiency, a precursor to CVD characterized by improper return of blood from the lower extremities to the heart, must be studied in its earliest stages at a time when preventative measures could be applied in man. This underscores the need for basic research into biomarkers and genetic predisposing factors affecting the progression of venous disease. Investigation over the past decade has yielded insight into these specific genetic, cellular and molecular mechanisms underlying the development of venous disease. Among the many advances include the elucidation of an increasing role for matrix metalloproteinases as important mediators of the degenerative process involved with venous insufficiency. This may be preceded by an inflammatory process which further contributes to venular degeneration and endothelial dysfunction seen in advanced presentation of disease. Furthermore, genomic analyses have shed light upon temporal expression patterns of matrix remodeling proteins in diseased tissue samples. In this review we examine some of the current findings surrounding cellular, molecular and genetic advances in delineating the etiology of chronic venous disease.
doi:10.1186/s13221-014-0024-5
PMCID: PMC4268799  PMID: 25520775
Vein; Venous disease; Molecular; Genetics; Valves; Endothelial; Blood vessel
24.  MicroRNA-22 and promoter motif polymorphisms at the Chga locus in genetic hypertension: functional and therapeutic implications for gene expression and the pathogenesis of hypertension 
Human Molecular Genetics  2013;22(18):3624-3640.
Hypertension is a common hereditary syndrome with unclear pathogenesis. Chromogranin A (Chga), which catalyzes formation and cargo storage of regulated secretory granules in neuroendocrine cells, contributes to blood pressure homeostasis centrally and peripherally. Elevated Chga occurs in spontaneously hypertensive rat (SHR) adrenal glands and plasma, but central expression is unexplored. In this report, we measured SHR and Wistar–Kyoto rat (control) Chga expression in central and peripheral nervous systems, and found Chga protein to be decreased in the SHR brainstem, yet increased in the adrenal and the plasma. By re-sequencing, we systematically identified five promoter, two coding and one 3′-untranslated region (3′-UTR) polymorphism at the SHR (versus WKY or BN) Chga locus. Using HXB/BXH recombinant inbred (RI) strain linkage and correlations, we demonstrated genetic determination of Chga expression in SHR, including a cis-quantitative trait loci (QTLs) (i.e. at the Chga locus), and such expression influenced biochemical determinants of blood pressure, including a cascade of catecholamine biosynthetic enzymes, catecholamines themselves and steroids. Luciferase reporter assays demonstrated that the 3′-UTR polymorphism (which disrupts a microRNA miR-22 motif) and promoter polymorphisms altered gene expression consistent with the decline in SHR central Chga expression. Coding region polymorphisms did not account for changes in Chga expression or function. Thus, we hypothesized that the 3′-UTR and promoter mutations lead to dysregulation (diminution) of Chga in brainstem cardiovascular control nuclei, ultimately contributing to the pathogenesis of hypertension in SHR. Accordingly, we demonstrated that in vivo administration of miR-22 antagomir to SHR causes substantial (∼18 mmHg) reductions in blood pressure, opening a novel therapeutic avenue for hypertension.
doi:10.1093/hmg/ddt213
PMCID: PMC3749858  PMID: 23674521
25.  Transmural Intestinal Wall Permeability in Severe Ischemia after Enteral Protease Inhibition 
PLoS ONE  2014;9(5):e96655.
In intestinal ischemia, inflammatory mediators in the small intestine's lumen such as food byproducts, bacteria, and digestive enzymes leak into the peritoneal space, lymph, and circulation, but the mechanisms by which the intestinal wall permeability initially increases are not well defined. We hypothesize that wall protease activity (independent of luminal proteases) and apoptosis contribute to the increased transmural permeability of the intestine's wall in an acutely ischemic small intestine. To model intestinal ischemia, the proximal jejunum to the distal ileum in the rat was excised, the lumen was rapidly flushed with saline to remove luminal contents, sectioned into equal length segments, and filled with a tracer (fluorescein) in saline, glucose, or protease inhibitors. The transmural fluorescein transport was determined over 2 hours. Villi structure and epithelial junctional proteins were analyzed. After ischemia, there was increased transmural permeability, loss of villi structure, and destruction of epithelial proteins. Supplementation with luminal glucose preserved the epithelium and significantly attenuated permeability and villi damage. Matrix metalloproteinase (MMP) inhibitors (doxycycline, GM 6001), and serine protease inhibitor (tranexamic acid) in the lumen, significantly reduced the fluorescein transport compared to saline for 90 min of ischemia. Based on these results, we tested in an in-vivo model of hemorrhagic shock (90 min 30 mmHg, 3 hours observation) for intestinal lesion formation. Single enteral interventions (saline, glucose, tranexamic acid) did not prevent intestinal lesions, while the combination of enteral glucose and tranexamic acid prevented lesion formation after hemorrhagic shock. The results suggest that apoptotic and protease mediated breakdown cause increased permeability and damage to the intestinal wall. Metabolic support in the lumen of an ischemic intestine with glucose reduces the transport from the lumen across the wall and enteral proteolytic inhibition attenuates tissue breakdown. These combined interventions ameliorate lesion formation in the small intestine after hemorrhagic shock.
doi:10.1371/journal.pone.0096655
PMCID: PMC4013012  PMID: 24805256

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