Proteolysis in septic shock patients: plasma peptidomic patterns are associated with mortality.

BACKGROUND: Uncontrolled proteolysis contributes to cell injury and organ dysfunction in animal models of circulatory shock. We investigated in humans the relationship between septic shock, proteolysis, and outcome. METHODS: Intensive care patients with septic shock (n=29) or sepsis (n=6) and non-hospitalised subjects (n=9) were recruited as part of the prospective observational trial 'ShockOmics' (ClinicalTrials.gov Identifier NCT02141607). A mass spectrometry-based approach was used to analyse the plasma peptidomes and the origin of circulating peptides from proteolysis in the enrolled subjects. RESULTS: Evidence of systemic proteolysis was indicated by a larger number of circulating peptides in septic shock patients, compared with septic patients and non-hospitalised healthy subjects. The peptide count and abundance in the septic shock patients were greater in patients who died (n=6) than in survivors (n=23), suggesting an association between magnitude of proteolysis and outcome. In silico analysis of the peptide sequences and of the sites of cleavage on the proteins of origin indicated a predominant role for serine proteases, such as chymotrypsin, and matrix metalloproteases in causing the observed proteolytic degradation. CONCLUSIONS: Systemic proteolysis is a novel fundamental pathological mechanism in septic shock. Plasma peptidomics is proposed as a new tool to monitor clinical trajectory in septic shock patients. CLINICAL TRIAL REGISTRATION: NCT02141607.

Preliminary profiling of blood transcriptome in a rat model of hemorrhagic shock.

Hemorrhagic shock is a leading cause of morbidity and mortality worldwide. Significant blood loss may lead to decreased blood pressure and inadequate tissue perfusion with resultant organ failure and death, even after replacement of lost blood volume. One reason for this high acuity is that the fundamental mechanisms of shock are poorly understood. Proteomic and metabolomic approaches have been used to investigate the molecular events occurring in hemorrhagic shock but, to our knowledge, a systematic analysis of the transcriptomic profile is missing. Therefore, a pilot analysis using paired-end RNA sequencing was used to identify changes that occur in the blood transcriptome of rats subjected to hemorrhagic shock after blood reinfusion. Hemorrhagic shock was induced using a Wigger's shock model. The transcriptome of whole blood from shocked animals shows modulation of genes related to inflammation and immune response (Tlr13, Il1b, Ccl6, Lgals3), antioxidant functions (Mt2A, Mt1), tissue injury and repair pathways (Gpnmb, Trim72) and lipid mediators (Alox5ap, Ltb4r, Ptger2) compared with control animals. These findings are congruent with results obtained in hemorrhagic shock analysis by other authors using metabolomics and proteomics. The analysis of blood transcriptome may be a valuable tool to understand the biological changes occurring in hemorrhagic shock and a promising approach for the identification of novel biomarkers and therapeutic targets. Impact statement This study provides the first pilot analysis of the changes occurring in transcriptome expression of whole blood in hemorrhagic shock (HS) rats. We showed that the analysis of blood transcriptome is a useful approach to investigate pathways and functional alterations in this disease condition. This pilot study encourages the possible application of transcriptome analysis in the clinical setting, for the molecular profiling of whole blood in HS patients.

Plasma activation during splanchnic arterial occlusion shock.

During circulatory shock, activating factors for cells in the microcirculation can be detected in plasma. But the source of such activators has remained uncertain. We have demonstrated recently that homogenates derived from the pancreas but not other peritoneal organs activate naive leukocytes. Production of such activating factors can be blocked by a serine protease inhibitor. Thus, factors generated by pancreatic proteases may possibly produce cellular activation in vivo. Rats were subjected to 90 min of superior mesenteric and celiac artery occlusion followed by reperfusion (SAO shock). In addition, rats were subjected to SAO shock for 120 min, after a 60-min pretreatment prior to occlusion with either saline or the serine protease inhibitor Futhan (nafamostat mesilate, 3.3 mg/kg b.w.). A sham SAO protocol was carried out as a control. Cellular activation was tested by neutrophil pseudopod formation and NBT reduction. Plasma from SAO-shocked animals but not sham shock rats exhibited a significant increase (P < 0.001) in the activation of naive leukocytes. Futhan-treated animals subjected to SAO shock exhibited a significantly higher post-reperfusion blood pressure than non-treated animals (P < 0.005 for all time points greater than 120 minutes), as well as significantly greater survival (P < 0.001). Neutrophil pseudopod formation and plasma peroxide production, an additional index of cellular activation, were significantly lower in Futhan-treated SAO shock plasma (P < 0.05) than levels in non-treated SAO shock animals. These results demonstrate that activating factors for leukocyte are released in SAO shock and can be mitigated by pretreatment with the serine protease inhibitor Futhan. Proteolytically derived plasma factors released during SAO shock may contribute to leukocyte activation and ensuing organ dysfunction.

Mechanisms for cell activation and its consequences for biorheology and microcirculation: Multi-organ failure in shock.

Activation of cells in the vascular compartment causes profound alteration of cell rheological properties with impairment of the microcirculation and initiation of inflammatory reactions. Many cardiovascular diseases have been shown to be associated with cell activation and inflammation. While this situation offers the opportunity for new interventions against the deleterious effects of cell activation, there is the need for a better understanding of the mechanisms that lead to cell activation in the first place. We review here several mechanisms for cell activation in the circulation. We show that in shock, a condition associated with severe forms of cell activation, humoral cell activation factors can be detected in plasma. Further analysis indicates that the source of these humoral activators may be due to the action of pancreatic digestive enzymes in the intestine. Ischemia may serve to open the intestinal brush border and permit entry of pancreatic enzymes into the wall of the intestine to initiate self digestion. In this process low molecular weight but potent cell activators are produced which may escape via the intestinal circulation and the lymphatics into the general circulation. Inhibition of pancreatic enzymes in the lumen of the intestine leads to complete attenuation of humoral activator production as well as many of the deleterious sequelae that accompany shock, such as inflammation and multi-organ failure. We outline a method to carry out biochemical isolation of the cell activators derived from pancreatic enzymes. This analysis shows that there are multiple species of cell activators above and beyond currently known species, many of which have molecular weights below 3000 Da. Identification of the mechanisms that lead to cell activation is an important part to understand the mechanisms that lead to alterations of rheological properties of blood cells in disease and dysfunction of the endothelium and parenchymal cells. Our current evidence suggests that pancreatic digestive enzymes and tissue enzymes may play a central role in humoral activator production.

Limitations of volumetric indices obtained by trans-thoracic thermodilution.

Transthoracic thermodilution (TTT) measures cardiac output without the need for right heart catheterization. In addition, two volumetric hemodynamic indices have been derived from the mathematical analysis of the TTT curve: the global end diastolic volume (a quantitative measure of cardiac preload) and the extravascular lung water volume (a quantitative measure of pulmonary edema). Despite the undeniable appeal of these two novel parameters, uncertainty exists regarding both the validity of their mathematical derivation and their physiological significance. This concise review attempts to discuss such concerns.

Generation of in vivo activating factors in the ischemic intestine by pancreatic enzymes.

One of the early events in physiological shock is the generation of activators for leukocytes, endothelial cells, and other cells in the cardiovascular system. The mechanism by which these activators are produced has remained unresolved. We examine here the hypothesis that pancreatic digestive enzymes in the ischemic intestine may be involved in the generation of activators during intestinal ischemia. The lumen of the small intestine of rats was continuously perfused with saline containing a broadly acting pancreatic enzyme inhibitor (6-amidino-2-naphthyl p-guanidinobenzoate dimethanesulfate, 0.37 mM) before and during ischemia of the small intestine by splanchnic artery occlusion. This procedure inhibited activation of circulating leukocytes during occlusion and reperfusion. It also prevented the appearance of activators in portal venous and systemic artery plasma and attenuated initiating symptoms of multiple organ injury in shock. Intestinal tissue produces only low levels of activators in the absence of pancreatic enzymes, whereas in the presence of enzymes, activators are produced in a concentration- and time-dependent fashion. The results indicate that pancreatic digestive enzymes in the ischemic intestine serve as an important source for cell activation and inflammation, as well as multiple organ failure.

The pancreas as a source of cardiovascular cell activating factors.

OBJECTIVE: Physiological shock leads to elevated levels of plasma factors that activate circulating leukocytes and endothelial cells, thereby compromising microvascular functions. The nature and source of these plasma-derived activators are unknown. To examine the possible origin of these factors, we homogenized rat internal organs and measured their activity on cardiovascular cells in vivo and in vitro. METHODS: Fresh tissue samples from small intestine, spleen, heart, liver, kidney, adrenals, and pancreas were homogenized. Their ability to induce leukocyte pseudopod formation and nitroblue tetrazolium (NBT) reduction was tested and their impact in vivo on blood pressure, survival, and microvascular cell injury was examined. RESULTS: A dramatic increase (p < 0.001) in leukocyte activation compared to controls was observed with pancreas homogenate but not with homogenates from the other organs. Leukocyte activation was induced by homogenates of other tissues only after prior incubation with substimulatory concentrations of pancreatic homogenate. Pancreatic serine proteases, trypsin and chymotrypsin, which did not stimulate leukocytes, also generated activity from other tissues. Leukocyte pseudopod formation could be significantly inhibited by adding the serine protease inhibitor 6-amidino-2-naphthyl p-guanidinobenzoate dimethanesulfonate (ANGD) during tissue homogenization (p < 0.001). Injection of pancreatic homogenate into rats led to increased plasma hydrogen peroxide levels and an instantaneous drop in mean arterial pressure that was often lethal. These responses were prevented by prior infusion of ANGD (p < 0.001). Intravital microscopy of the rat mesentery confirmed that superfusion of filtered pancreatic homogenate leads to significant increases in cell death (p < 0.05), as detected by propidium iodide, and hydrogen peroxide formation (p < 0.05), as determined by dichlorofluorescein diacetate (DCFH) fluorescence. CONCLUSION: These results suggest that pancreatic enzymes attack tissue and generate cellular activators that are associated with organ dysfunction in shock.

Pancreatic enzymes and microvascular cell activation in multiorgan failure.

Cell activation in the microcirculation leads to an inflammatory cascade and is accompanied by many cardiovascular complications. There is a need to identify the trigger mechanisms that lead to the production of in vivo activating factors. We review here mechanisms for cell activation in the microcirculation and specifically the production of humoral cell activators in physiological shock. The elevated levels of activating factors in plasma could be traced to the action of pancreatic enzymes in the ischemic intestine. New interventions against the production of the activators are proposed. The evidence suggests that pancreatic enzymes in the ischemic intestine may attack several tissue components and generate cellular activators that are associated with multiorgan dysfunction in physiological shock.