Pawan Noel

Fibrosis Reduces Severity of Acute-on-Chronic Pancreatitis in Humans

BACKGROUND & AIMS Acute pancreatitis (AP) and chronic pancreatitis (CP) share etiologies, but AP can be more severe and is associated with a higher rate of mortality. We investigated features of CP that protect against severe disease. The amount of intrapancreatic fat (IPF) is increased in obese patients and fibrosis is increased in patients with CP, so we studied whether fibrosis or fat regulate severity of AP attacks in patients with CP. METHODS We reviewed records from the University of Pittsburgh Medical Center/Presbyterian Hospital Autopsy Database (1998-2008) for patients with a diagnosis of AP (n = 23), CP (n = 35), or both (AP-on-CP; n = 15). Pancreatic histology samples from these patients and 50 randomly selected controls (no pancreatic disease) were analyzed, and IPF data were correlated with computed tomography data. An adipocyte and acinar cell Transwell coculture system, with or without collagen type I, was used to study the effects of fibrosis on acinar-adipocyte interactions. We studied the effects of nonesterified fatty acids (NEFAs) and adipokines on acinar cells in culture. RESULTS Levels of IPF were significantly higher in nonobese patients with CP than in nonobese controls. In patients with CP or AP-on-CP, areas of IPF were surrounded by significantly more fibrosis than in controls or patients with AP. Fat necrosis-associated peri-fat acinar necrosis (PFAN, indicated by NEFA spillage) contributed to most of the necrosis observed in samples from patients with AP; however, findings of peri-fat acinar necrosis and total necrosis were significantly lower in samples from patients with CP or AP-on-CP. Fibrosis appeared to wall off the fat necrosis and limit peri-fat acinar necrosis, reducing acinar necrosis. In vitro, collagen I limited the lipolytic flux between acinar cells and adipocytes and prevented increases in adipokines in the acinar compartment. This was associated with reduced acinar cell necrosis. However, NEFAs, but not adipokines, caused acinar cell necrosis. CONCLUSIONS Based on analysis of pancreatic samples from patients with CP, AP, or AP-on-CP and in vitro studies, fibrosis reduces the severity of acute exacerbations of CP by reducing lipolytic flux between adipocytes and acinar cells.

Src Dependent Pancreatic Acinar Injury Can Be Initiated Independent of an Increase in Cytosolic Calcium

Several deleterious intra-acinar phenomena are simultaneously triggered on initiating acute pancreatitis. These culminate in acinar injury or inflammatory mediator generation in vitro and parenchymal damage in vivo. Supraphysiologic caerulein is one such initiator which simultaneously activates numerous signaling pathways including non-receptor tyrosine kinases such as of the Src family. It also causes a sustained increase in cytosolic calcium- a player thought to be crucial in regulating deleterious phenomena. We have shown Src to be involved in caerulein induced actin remodeling, and caerulein induced changes in the Golgi and post-Golgi trafficking to be involved in trypsinogen activation, which initiates acinar cell injury. However, it remains unclear whether an increase in cytosolic calcium is necessary to initiate acinar injury or if injury can be initiated at basal cytosolic calcium levels by an alternate pathway. To study the interplay between tyrosine kinase signaling and calcium, we treated mouse pancreatic acinar cells with the tyrosine phosphatase inhibitor pervanadate. We studied the effect of the clinically used Src inhibitor Dasatinib (BMS-354825) on pervanadate or caerulein induced changes in Src activation, trypsinogen activation, cell injury, upstream cytosolic calcium, actin and Golgi morphology. Pervanadate, like supraphysiologic caerulein, induced Src activation, redistribution of the F-actin from its normal location in the sub-apical area to the basolateral areas, and caused antegrade fragmentation of the Golgi. These changes, like those induced by supraphysiologic caerulein, were associated with trypsinogen activation and acinar injury, all of which were prevented by Dasatinib. Interestingly, however, pervanadate did not cause an increase in cytosolic calcium, and the caerulein induced increase in cytosolic calcium was not affected by Dasatinib. These findings suggest that intra-acinar deleterious phenomena may be initiated independent of an increase in cytosolic calcium. Other players resulting in acinar injury along with the Src family of tyrosine kinases remain to be explored.

ADP-ribosylation Factor 1 Protein Regulates Trypsinogen Activation via Organellar Trafficking of Procathepsin B Protein and Autophagic Maturation in Acute Pancreatitis

Background: Autophagy and cathepsin B-mediated trypsin generation may be deleterious in acute pancreatitis. The role of ARF1 in the process is unknown. Results: BFA-mediated ARF1 inhibition prevents caerulein-induced processing of procathepsin B and perturbs autophagic maturation. Conclusion: ARF1-dependent trafficking of procathepsin B and autophagic maturation result in trypsinogen activation. Significance: ARF1 plays a significant role in acute pancreatitis. Several studies have suggested that autophagy might play a deleterious role in acute pancreatitis via intra-acinar activation of digestive enzymes. The prototype for this phenomenon is cathepsin B-mediated trypsin generation. To determine the organellar basis of this process, we investigated the subcellular distribution of the cathepsin B precursor, procathepsin B. We found that procathepsin B is enriched in Golgi-containing microsomes, suggesting a role for the ADP-ribosylation (ARF)-dependent trafficking of cathepsin B. Indeed, caerulein treatment increased processing of procathepsin B, whereas a known ARF inhibitor brefeldin A (BFA) prevented this. Similar treatment did not affect processing of procathepsin L. BFA-mediated ARF1 inhibition resulted in reduced cathepsin B activity and consequently reduced trypsinogen activation. However, formation of light chain 3 (LC3-II) was not affected, suggesting that BFA did not prevent autophagy induction. Instead, sucrose density gradient centrifugation and electron microscopy showed that BFA arrested caerulein-induced autophagosomal maturation. Therefore, ARF1-dependent trafficking of procathepsin B and the maturation of autophagosomes results in cathepsin B-mediated trypsinogen activation induced by caerulein.