Alexander H. Penn

PANCREATIC DIGESTIVE ENZYMES ARE POTENT GENERATORS OF MEDIATORS FOR LEUKOCYTE ACTIVATION AND MORTALITY

Shock is associated with a dramatic rise in the level of inflammatory mediators found in plasma. The exact source of these mediators has remained uncertain. We recently examined a previously undescribed mechanism for production of inflammatory mediators in shock involving pancreatic digestive enzymes. The current in vitro study was designed to identify particular pancreatic enzymes and organs that may potentially produce inflammatory mediators. A selection of different organs from the rat (heart, liver, brain, spleen, pancreas, intestine, diaphragm, kidney, and lung) were homogenized and incubated with purified trypsin, chymotrypsin, elastase, lipase, nuclease, or amylase and the supernatant was incubated with fresh naïve leukocytes for 15 min. The level of leukocyte activation in the form of pseudopod formation and the fraction of cell death were measured. Without the addition of purified enzymes, only the homogenate of the pancreas yielded enhanced cell activation. Organs incubated with physiological concentrations of trypsin also stimulated significantly higher levels of pseudopod formation as compared with the undigested organs or enzymatic controls. Lipase and chymotrypsin were able to elicit cellular activation from selected organs such as the heart, intestine, liver and diaphragm. Undigested pancreatic homogenates were capable of producing substantial cell death, as compared with all other undigested organs. Intestinal digests with elastase, lipase, trypsin and chymotrypsin also stimulated significant cell mortality. Lipase-treated heart, liver, intestine, diaphragm, kidney, and lung stimulated cell death as well. We conclude that the intestine, as well as several other organs, may serve as a major source of inflammatory mediators during shock if exposed to digestive enzymes.

Venous hypertension and the inflammatory cascade : Major manifestations and trigger mechanisms

Recent histologic and immunocytochemical evidence of venous leg ulcers supports the hypothesis that lesions observed at different stages of chronic venous insufficiency may be associated with, and possibly caused by, an inflammatory process. Evidence has been obtained that venous valve deficiency may be associated with leukocyte infiltration into valve leaflets; therefore, it is hypothesized that an essential event in the inflammatory cascade is the enzymatic degradation of the valve leaflets and venous wall. The metalloproteinases (MMP) in veins exposed to elevated pressures up to 6 weeks were examined in a rat femoral fistula model with venous hypertension. Zymography shows increased activity of pro-MMP-2 at 3 and 6 weeks. MMP-2 and MMP-9 activity was predominantly observed at days 7 and 21 after creation of the fistula. The degree of extracellular matrix remodeling correlates with the morphological finding of macroscopic lesions. Therefore, the MMP-2 and MMP-9 activation is already present in veins days after exposure to elevated blood pressure and coincides with periods of early alterations in the valve morphology and early forms of reflux.

The intestine as source of cytotoxic mediators in shock: free fatty acids and degradation of lipid-binding proteins

Shock and multiple organ failure remain primary causes of late-stage morbidity and mortality in victims of trauma. During shock, the intestine is subject to extensive cell death and is the source of inflammatory factors that cause multiorgan failure. We (34) showed previously that ischemic, but not nonischemic, small intestines and pancreatic protease digested homogenates of normal small intestine can generate cytotoxic factors capable of killing naive cells within minutes. Using chloroform/methanol separation of rat small intestine homogenates into lipid fractions and aqueous and sedimented protein fractions and measuring cell death caused by those fractions, we found that the cytotoxic factors are lipid in nature. Recombining the lipid fraction with protein fractions prevented cell death, except when homogenates were protease digested. Using a fluorescent substrate, we found high levels of lipase activity in intestinal homogenates and cytotoxic levels of free fatty acids. Addition of albumin, a lipid binding protein, prevented cell death, unless the albumin was previously digested with protease. Homogenization of intestinal wall in the presence of the lipase inhibitor orlistat prevented cell death after protease digestion. In vivo, orlistat plus the protease inhibitor aprotinin, administered to the intestinal lumen, significantly improved survival time compared with saline in a splanchnic arterial occlusion model of shock. These results indicate that major cytotoxic mediators derived from an intestine under in vitro conditions are free fatty acids. Breakdown of free fatty acid binding proteins by proteases causes release of free fatty acids to act as powerful cytotoxic mediators.

Digested formula but not digested fresh human milk causes death of intestinal cells in vitro: implications for necrotizing enterocolitis

Background:Premature infants fed formula are more likely to develop necrotizing enterocolitis (NEC) than those who are breastfed, but the mechanisms of intestinal necrosis in NEC and protection by breast milk are unknown. We hypothesized that after lipase digestion, formula, but not fresh breast milk, contains levels of unbound free fatty acids (FFAs) that are cytotoxic to intestinal cells.Methods:We digested multiple term and preterm infant formulas or human milk with pancreatic lipase, proteases (trypsin and chymotrypsin), lipase + proteases, or luminal fluid from a rat small intestine and tested FFA levels and cytotoxicity in vitro on intestinal epithelial cells, endothelial cells, and neutrophils.Results:Lipase digestion of formula, but not milk, caused significant death of neutrophils (ranging from 47 to 99% with formulas vs. 6% with milk) with similar results in endothelial and epithelial cells. FFAs were significantly elevated in digested formula vs. milk and death from formula was significantly decreased with lipase inhibitor pretreatment, or treatments to bind FFAs. Protease digestion significantly increased FFA binding capacity of formula and milk but only enough to decrease cytotoxicity from milk.Conclusion:FFA-induced cytotoxicity may contribute to the pathogenesis of NEC.

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.

Transmural Intestinal Wall Permeability in Severe Ischemia after Enteral Protease Inhibition

In intestinal ischemia, inflammatory mediators in the small intestines 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 intestines 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.

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.

Severe intestinal ischemia can trigger cardiovascular collapse and sudden death via a parasympathetic mechanism

Hemorrhagic shock and splanchnic arterial occlusion (SAO) followed by reperfusion are associated with high mortality. However, rapid cardiovascular failure and death may also occur before reperfusion in hemorrhagic shock and SAO. We show in a rat SAO model that, upon gut ischemia, mean arterial blood pressure transiently elevates and then drops fatally in one of two time courses: (i) gradually over ∼1 to 3 h or (ii) rapidly (often by >80 mmHg) over a period of 1 to 6 min. We hypothesize that fast fatal pressure drops (FFPDs) are due to failure of autonomic nervous system control. To test this, we treated rats with Glucose (10%) in the small intestinal lumen and intramuscularly administered xylazine to activate the parasympathetic nervous system or with a muscarinic anticholinergic (glycopyrrolate) or by total subdiaphragmatic vagotomy to attenuate parasympathetic nervous system activity. We also tested nafamostat mesilate (ANGD [6-amidino-2-naphthyl p-guanidinobenzoate dimethanesulfonate]), a protease inhibitor efficacious in preventing blood pressure loss in SAO with reperfusion, in the intestinal lumen. Fifty percent of animals receiving xylazine and Glucose died by FFPD (vs. 33% with neither, not statistically significant). Total subdiaphragmatic vagotomy or glycopyrrolate treatment significantly reduced the incidence to 0% (P < 0.008), although slow fatal pressure drops still occurred. ANGD did not prevent FFPDs, but delayed onset of slow fatal pressure drops (P < 0.013). These results suggest that gut ischemia can cause sudden death via an autonomic nervous system mechanism and that SAO with Glucose and xylazine may serve as a useful model for the study of neurogenic shock or autonomic dysregulation associated with sudden death.ABBREVIATIONS-ANGD - 6-amidino-2-naphthyl p-guanidinobenzoate dimethanesulfonate; FFPD - fast fatal pressure drop; MABP - mean arterial blood pressure; SAO - splanchnic arterial occlusion; SCD - sudden cardiac death; SIDS - sudden infant death syndrome; SFPD - slow fatal pressure drop; TSV - total subdiaphragmatic vagotomy

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 whether stored milk and milk from donor milk (DM) banks 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°C 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°C 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: Present storage practices may allow milk to become cytotoxic and contribute to intestinal damage in NEC.

An elementary analysis of physiologic shock and multi-organ failure: The Autodigestion Hypothesis

Physiological shock and subsequent multi-organ failure is one of the most important medical problems from a mortality point of view. No agreement exists for mechanisms that lead to the relative rapid cell and organ failure during this process and no effective treatment. We postulate that the digestive enzymes synthesized in the pancreas and transported in the lumen of the small intestine as requirement of normal food digestion play a central role in multi-organ failure. These powerful enzymes are usually compartmentalized in the lumen of the intestine by the mucosal barrier, but may escape into the wall of the intestine if the permeability of the mucosal lining increases. Entry of the digestive enzymes into the wall of the intestine precipitates an autodigestion process as well as an escape of pancreatic enzymes and breakdown products generated by them into the system circulation. The consequence of autodigestion is multiorgan failure. We discuss the possibility to block the digestive enzymes in acute forms of shock as a potential therapeutic intervention.