Removal of luminal content protects the small intestine during hemorrhagic shock but is not sufficient to prevent lung injury.

The small intestine plays a key role in the pathogenesis of multiple organ failure following circulatory shock. Current results show that reduced perfusion of the small intestine compromises the mucosal epithelial barrier, and the intestinal contents (including pancreatic digestive enzymes and partially digested food) can enter the intestinal wall and transport through the circulation or mesenteric lymph to other organs such as the lung. The extent to which the luminal contents of the small intestine mediate tissue damage in the intestine and lung is poorly understood in shock. Therefore, rats were assigned to three groups: No-hemorrhagic shock (HS) control and HS with or without a flushed intestine. HS was induced by reducing the mean arterial pressure (30 mmHg; 90 min) followed by return of shed blood and observation (3 h). The small intestine and lung were analyzed for hemorrhage, neutrophil accumulation, and cellular membrane protein degradation. After HS, animals with luminal contents had increased neutrophil accumulation, bleeding, and destruction of E-cadherin in the intestine. Serine protease activity was elevated in mesenteric lymph fluid collected from a separate group of animals subjected to intestinal ischemia/reperfusion. Serine protease activity was elevated in the plasma after HS but was detected in lungs only in animals with nonflushed lumens. Despite removal of the luminal contents, lung injury occurred in both groups as determined by elevated neutrophil accumulation, permeability, and lung protein destruction. In conclusion, luminal contents significantly increase intestinal damage during experimental HS, suggesting transport of luminal contents across the intestinal wall should be minimized

Transmural intestinal wall permeability in severe ischemia after enteral protease inhibition.

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

Protease Activity Increases in Plasma, Peritoneal Fluid, and Vital Organs after Hemorrhagic Shock in Rats

Hemorrhagic shock (HS) is associated with high mortality. A severe decrease in blood pressure causes the intestine, a major site of digestive enzymes, to become permeable – possibly releasing those enzymes into the circulation and peritoneal space, where they may in turn activate other enzymes, e.g. matrix metalloproteinases (MMPs). If uncontrolled, these enzymes may result in pathophysiologic cleavage of receptors or plasma proteins. Our first objective was to determine, in compartments outside of the intestine (plasma, peritoneal fluid, brain, heart, liver, and lung) protease activities and select protease concentrations after hemorrhagic shock (2 hours ischemia, 2 hours reperfusion). Our second objective was to determine whether inhibition of proteases in the intestinal lumen with a serine protease inhibitor (ANGD), a process that improves survival after shock in rats, reduces the protease activities distant from the intestine. To determine the protease activity, plasma and peritoneal fluid were incubated with small peptide substrates for trypsin-, chymotrypsin-, and elastase-like activities or with casein, a substrate cleaved by multiple proteases. Gelatinase activities were determined by gelatin gel zymography and a specific MMP-9 substrate. Immunoblotting was used to confirm elevated pancreatic trypsin in plasma, peritoneal fluid, and lung and MMP-9 concentrations in all samples after hemorrhagic shock. Caseinolytic, trypsin-, chymotrypsin-, elastase-like, and MMP-9 activities were all significantly (p<0.05) upregulated after hemorrhagic shock regardless of enteral pretreatment with ANGD. Pancreatic trypsin was detected by immunoblot in the plasma, peritoneal space, and lungs after hemorrhagic shock. MMP-9 concentrations and activities were significantly upregulated after hemorrhagic shock in plasma, peritoneal fluid, heart, liver, and lung. These results indicate that protease activities, including that of trypsin, increase in sites distant from the intestine after hemorrhagic shock. Proteases, including pancreatic proteases, may be shock mediators and potential targets for therapy in shock

Proteolytic receptor cleavage and attenuated endothelial cell response to fluid shear stress in a model for autodigestion in shock

In physiological shock, a leading cause of death, cell and tissue function are compromised. Once proteases enter into the circulation during shock, there may be proteolytic damage to the extracellular components of cells including cleavage of surface receptors and the glycocalyx of the endothelial cells (ECs). The endothelial cell is sensitive to physiological shear stress (typically 10-15 dyn/cm²) and aligns in the direction of the fluid flow. Sheared endothelial cells exhibited a higher tolerance to protease exposure than static cells. Extracellular damage to the endothelial cell by active proteases causes the ECs to not align in the direction of the fluid shear stress; however when the proteases were inhibited, ECs exposed to shear stress could realign with the direction of the flow. Receptor intensity of the mechanosensors VEGFR-2 and junctional PECAM-1 was reduced on both sheared and unsheared ECs. The insulin receptor surface density was also reduced for cells exposed to proteases. In conclusion, proteases present in the circulation during shock may damage extracellular molecular membrane components on the cell and interfere with one of the most basic mechanotransduction mechanisms in endothelial cells, i.e. aligning in the presence of physiological shear stres

Proteolytic receptor cleavage and attenuated endothelial cell response to fluid shear stress in a model for autodigestion in shock

In physiological shock, a leading cause of death, cell and tissue function are compromised. Once proteases enter into the circulation during shock, there may be proteolytic damage to the extracellular components of cells including cleavage of surface receptors and the glycocalyx of the endothelial cells (ECs). The endothelial cell is sensitive to physiological shear stress (typically 10-15 dyn/cm²) and aligns in the direction of the fluid flow. Sheared endothelial cells exhibited a higher tolerance to protease exposure than static cells. Extracellular damage to the endothelial cell by active proteases causes the ECs to not align in the direction of the fluid shear stress; however when the proteases were inhibited, ECs exposed to shear stress could realign with the direction of the flow. Receptor intensity of the mechanosensors VEGFR-2 and junctional PECAM-1 was reduced on both sheared and unsheared ECs. The insulin receptor surface density was also reduced for cells exposed to proteases. In conclusion, proteases present in the circulation during shock may damage extracellular molecular membrane components on the cell and interfere with one of the most basic mechanotransduction mechanisms in endothelial cells, i.e. aligning in the presence of physiological shear stres

Degrading Proteases and Organ Failure during Physiological Shock

Circulatory shock is a frequent cause of death and one of the most important unresolved medical problems. Reduction of blood supply to the small intestine during ischemia disrupts the mucosal epithelial barrier, allowing inflammatory materials in the lumen of the intestine, including digestive enzymes, to cross into the intestinal wall. If digestive enzymes are transported into the periphery, they can activate other proteases and degrade extracellular structures. Understanding the contribution of degrading proteases to circulatory shock may be essential to interfere with its lethal course. My objective is to determine which proteases are involved in the progression of shock and their contribution to intestinal degradation and peripheral organ failure. I hypothesize that during shock, the intestine is proteolytically degraded, accelerating leakage of proteases into the plasma, lymphatics, and peritoneal space, which may cause subsequent activation of proteases in peripheral organs and degradation of extracellular structures on endothelial and epithelial cells. To determine degradative mechanisms inherent to the intestinal wall and independent of luminal contents, I will use a model of severe intestinal ischemia to study barrier failure, digestive and wall proteases, and epithelial protein degradation. I will study the activities and transport of digestive proteases from the lumen of the intestine during hemorrhagic shock. I will determine the contribution of luminal contents to intestine and lung injury after hemorrhagic shock. Lastly, I will test methods to prevent breakdown of the intestinal barrier to reduce penetration of luminal contents past the mucosal barrier and reduce peripheral organ injury. I obtained evidence that the intestinal tissue degrades in severe ischemia even without luminal digestive enzymes. In hemorrhagic shock, I show that luminal contents are responsible for intestinal injury but not lung injury as determined by neutrophil accumulation and endothelial protein degradation. Protease activity and levels are elevated in the peritoneal space, lymph, blood, and vital organs after hemorrhagic shock. Protease inhibition to the gut reduces intestinal injury and protein degradation in the lung. These results suggest that proteases play a critical role in the pathophysiology of shock and may be important targets to reduce organ injur

Autodigestion: Proteolytic Degradation and Multiple Organ Failure in Shock.

There is currently no effective treatment for multiorgan failure following shock other than supportive care. A better understanding of the pathogenesis of these sequelae to shock is required. The intestine plays a central role in multiorgan failure. It was previously suggested that bacteria and their toxins are responsible for the organ failure seen in circulatory shock, but clinical trials in septic patients have not confirmed this hypothesis. Instead, we review here evidence that the digestive enzymes, synthesized in the pancreas and discharged into the small intestine as requirement for normal digestion, may play a role in multiorgan failure. These powerful enzymes are nonspecific, highly concentrated, and fully activated in the lumen of the intestine. During normal digestion they are compartmentalized in the lumen of the intestine by the mucosal epithelial barrier. However, if this barrier becomes permeable, e.g. in an ischemic state, the digestive enzymes escape into the wall of the intestine. They digest tissues in the mucosa and generate small molecular weight cytotoxic fragments such as unbound free fatty acids. Digestive enzymes may also escape into the systemic circulation and activate other degrading proteases. These proteases have the ability to clip the ectodomain of surface receptors and compromise their function, for example cleaving the insulin receptor causing insulin resistance. The combination of digestive enzymes and cytotoxic fragments leaking into the central circulation causes cell and organ dysfunction, and ultimately may lead to complete organ failure and death. We summarize current evidence suggesting that enteral blockade of digestive enzymes inside the lumen of the intestine may serve to reduce acute cell and organ damage and improve survival in experimental shock