Corneal keratin aggresome (CKAGG) formation and clearance by proteasome activation.

PURPOSE: To understand the mechanism of corneal keratin expression and clearance in corneal epithelium with Limbal Stem Cell Deficiency (LSCD). The hypothesis is that LSCD-induced proteasome dysfunction is a contributing factor to keratin aggregation, causing corneal keratin aggresome (CKAGG) formation. METHOD: LSCD was surgically induced in rabbit corneas. LSCD corneal epithelial cells (D-CEC) were collected to investigate keratin K4 and K13 expression and CKAGG formation. Oral mucosal epithelial cells (OMECS) were isolated and cultured to study K4 and K13 expression. Cultured cells were treated with proteasome inhibitor to induce CKAGG formation. RESULTS: K4 and K13 were strongly expressed in D-CEC, with additional higher molecular weight bands of K4 and K13, suggesting CKAGG formation. Double staining of K4/K13 and ubiquitin showed co-localization of these keratins with ubiquitin in D-CEC. Proteasome inhibition also showed K4/K13 modification and accumulation in cultured OMECS, similar to D-CEC. Proteasome activation was then performed in cultured OMEC. There was no accumulation of keratins, and levels of unmodified keratins were found significantly reduced. CONCLUSION: Results showed an abnormal expression of K4 and K13 after LSCD-induced proteasome dysfunction, which coalesce to form CKAGG in Corneal Epithelial Cells (CEC). We propose that CKAGG formation may be one of the causative factors of morphological alterations in the injured corneal epithelium, and that CKAGG could potentially be cleared by enhancing proteasome activity.

Early endothelial damage and increased colonic vascular permeability in the development of experimental ulcerative colitis in rats and mice.

The role of endothelial damage and increased vascular permeability (VP) in the pathogenesis of ulcerative colitis (UC) has not been investigated. We examined using functional, morphologic, and molecular biologic studies whether and to what extent the endothelial barrier dysfunction precedes enhanced epithelial permeability (EP) and the development of mucosal lesions during the early stages of experimental UC. We showed that in rats with iodoacetamide (IA)-induced UC increased colonic VP occurs early (ie, 2.6-fold increase at 15 min, P<0.01) preceding changes in epithelial barrier permeability. EP was unchanged at 15 and 30 min after IA administration and was increased 1.9-fold at 1 h and 6.7-fold at 2 h (both P<0.001) after IA. In the dextran sodium sulfate-induced slowly developing UC, colonic VP was significantly increased in 2 days (P<0.05) and EP only in 4 days (P<0.05). Mucosal endothelial injury led to hypoxia (P<0.05) of colonic surface epithelial cells 30 min after IA administration that was associated with increased expressions of transcription factors hypoxia-inducible factor-1α and early growth response-1. Electron and light microscopy demonstrated areas of colonic mucosa with perivascular edema covered by intact layer of surface epithelial cells in both rat and mouse models of UC. This is the first demonstration in four models of UC that endothelial damage, increased colonic VP, perivascular edema, and epithelial hypoxia precede epithelial barrier dysfunction that is followed by erosions, ulceration, and inflammation in UC.

A rat model reproducing key pathological responses of alcoholic chronic pancreatitis.

Although alcohol abuse is the major cause of chronic pancreatitis, the pathogenesis of alcoholic chronic pancreatitis (ACP) remains obscure. A critical obstacle to understanding the mechanism of ACP is lack of animal models. Our objective was to develop one such model. Rats were pair-fed for 8 wk ethanol or control Lieber-DeCarli liquid diet. For the last 2 wk, they received cyclosporin A (CsA; 20 mg/kg once daily) or vehicle. After 1 wk on CsA, one episode of acute pancreatitis was induced by four 20 microg/kg injections of cerulein (Cer); controls received saline. Pancreas was analyzed 1 wk after the acute pancreatitis. CsA or Cer treatments alone did not result in pancreatic injury in either control (C)- or ethanol (E)-fed rats. We found, however, that alcohol dramatically aggravated pathological effect of the combined CsA+Cer treatment on pancreas, resulting in massive loss of acinar cells, persistent inflammatory infiltration, and fibrosis. Macrophages were prominent in the inflammatory infiltrate. Compared with control-fed C+CsA+Cer rats, their ethanol-fed E+CsA+Cer counterparts showed marked increases in pancreatic NF-kappaB activation and cytokine/chemokine mRNA expression, collagen and fibronectin, the expression and activities of matrix metalloproteinase-2 and -9, and activation of pancreatic stellate cells. Thus we have developed a model of alcohol-mediated postacute pancreatitis that reproduces three key responses of human ACP: loss of parenchyma, sustained inflammation, and fibrosis. The results indicate that alcohol impairs recovery from acute pancreatitis, suggesting a mechanism by which alcohol sensitizes pancreas to chronic injury.