No abstract

Background Premature infants fed formula are more likely to develop necrotizing enterocolitis (NEC) than if fed breast milk, 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 47–99% with formulas vs. 6% with milk) with similar results in endothelial and epithelial cells. FFAs were significantly elevated in digested formula versus 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.

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.

Recent evidence indicates that shock is accompanied by a failure of the mucosal barrier in the intestine and entry of pancreatic digestive enzymes into the wall of the intestine. To investigate the formation of cytotoxic mediators produced by enzymatic digestion of the intestine, we applied homogenates of rat small intestinal wall to human neutrophils and used flow cytometry measurements of propidium iodide uptake to determine cytotoxicity. We show that homogenates of the small intestine after ischemia by occlusion of the superior mesenteric and celiac arteries for 3 h, but not without ischemia, are cytotoxic. Digestion of homogenates of nonischemic intestinal wall with purified trypsin, chymotrypsin, or elastase, proteases normally present in the intestinal lumen, yielded cytotoxic mediators. Before cell death, we saw cell damage in the form of bleb formation and flow cytometry measurements of cell size changes due to blebbing. Cytotoxicity was prevented by serine protease inhibition with phenylmethylsulfonyl fluoride (PMSF) before, but not after proteolytic digestion of the wall homogenates, indicating that enzymatic action of proteases on the homogenate is necessary for cytotoxicity. Cytotoxicity of wall homogenates digested by enzymes in the fluid collected from the lumen of the intestine was greater than digests by the individual purified proteases. Cytotoxicity is undetectable if digestive enzymes in the luminal fluid are inhibited with a combination of enzyme inhibitors PMSF and 6-amidino-2-naphthyl p-guanidinobenzoate dimethanesulfonate before addition of wall homogenates. Passage of digested intestinal wall homogenates across a hydrophobic glass-fiber filter reduced cytotoxicity. Furthermore, we found that luminal fluid itself may be cytotoxic, possibly because of digestion of ingested food. To test whether digested food can be cytotoxic, we homogenized rat food and digested it in vitro with chymotrypsin or endogenous enzymes in luminal fluid. Cytotoxicity was significantly increased after digestion of food by luminal fluid compared with luminal fluid or undigested food. These results indicate the presence of a previously unknown mechanism for hemorrhagic necrosis in shock.

Hemorrhagic shock (HS) is a major cause of death after trauma. During HS, perfusion to the intestine and pancreas are reduced, resulting in increased intestinal mucosal permeability, pancreatitis, and potential leakage of serine proteases (i.e. trypsin‐a potent matrix metalloproteinase [MMP] activator) into the circulation. We hypothesize that after HS, both trypsin and MMP‐9 enter the circulation, peritoneal space, and appear in peripheral organs. Male Wistar rats were grouped into No‐HS or HS (N=5). Mean arterial blood pressure of HS animals was reduced to 35 mmHg (2 hr) followed by resuscitation of shed blood (2 hr). Plasma and peritoneal lavage fluid were obtained before and after HS. The heart, lung, liver, and brain were collected. Protease activities in plasma and peritoneal fluid were measured by plate zymography (trypsin‐like and MMP‐9 specific substrates) or gelatin gel zymography. Pancreatic trypsin and MMP‐9 levels were identified by immunoblot. Trypsin‐like activities were elevated in plasma and peritoneal space fluid after HS (p<0.05). Immunoblot for pancreatic trypsin detected elevated levels in the plasma, peritoneal fluid, and lungs. MMP‐9 activity and protein levels were elevated in all compartments after HS (p<0.05). In HS, trypsin may be transported from the permeable intestine and/or pancreas into the periphery and may contribute to MMP‐9 activation.

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.

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.

Hemorrhagic shock (HS) and splanchnic arterial occlusion (SAO) followed by reperfusion are associated with high mortality. However, rapid cardiovascular failure and death may also occur prior to reperfusion in HS 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 hours or (ii) rapidly (often by more than 80 mmHg) over a period of 1 to 6 minutes. We hypothesize that fast fatal pressure drops (FFPD) are due to failure of autonomic nervous system control. To test this, we treated rats with glucose (10%) in the small intestinal lumen and intramuscular xylazine to activate the parasympathetic nervous system, or with a muscarinic anti-cholinergic (glycopyrrolate) or by total subdiaphragmatic vagotomy (TSV) to attenuate parasympathetic nervous system activity. We also tested nafamostat mesilate (ANGD), a protease inhibitor efficacious in preventing blood pressure loss in SAO with reperfusion, in the intestinal lumen. 50% of animals receiving xylazine and glucose died by FFPD (vs. 33% with neither, NS). TSV or glycopyrrolate treatment significantly reduced the incidence to 0% (P<0.008), though slow fatal pressure drops (SFPDs) still occurred. ANGD did not prevent FFPDs, but delayed onset of SFPDs (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 study of neurogenic shock or autonomic dysregulation associated with sudden death.