The Effect of a Novel Serine Protease Inhibitor on Inflammation and Intestinal Permeability in a Murine Colitis Transfer Model.

Background: A protease/antiprotease disbalance is observed in inflammatory bowel diseases (IBD). We therefore studied the effect of the novel serine protease inhibitor UAMC-00050 on intestinal inflammation and permeability in a chronic colitis T cell transfer mouse model to get further insight into the regulation of T cell-mediated immunopathology. Methods: Colitis was induced in severe combined immunodeficient (SCID) mice, by the adoptive transfer of CD4+CD25-CD62L+ T cells. Animals were treated intraperitoneally (i.p.) 2x/day with vehicle or UAMC-00050 (5 mg/kg) from week 2 onwards. Colonic inflammation was assessed by clinical parameters, colonoscopy, macroscopy, microscopy, myeloperoxidase activity and cytokine expression levels. At week 4, 4 kDa FITC-dextran intestinal permeability was evaluated and T helper transcription factors, protease-activated receptors and junctional proteins were quantified by RT-qPCR. Results: Adoptive transfer of CD4+CD25-CD62L+ T cells resulted in colonic inflammation and an altered intestinal permeability. The serine protease inhibitor UAMC-00050 ameliorated both the inflammatory parameters and the intestinal barrier function. Furthermore, a decrease in colonic mRNA expression of Tbet and PAR4 was observed in colitis mice after UAMC-00050 treatment. Conclusion: The beneficial effect of UAMC-00050 on inflammation was apparent via a reduction of Tbet, IFN-γ, TNF-α, IL-1ß and IL-6. Based on these results, we hypothesize a pivotal effect of serine protease inhibition on the Th1 inflammatory profile potentially mediated via PAR4.

In-Depth Study of Transmembrane Mucins in Association with Intestinal Barrier Dysfunction During the Course of T Cell Transfer and DSS-Induced Colitis.

BACKGROUND AND AIMS: There is evidence for a disturbed intestinal barrier function in inflammatory bowel diseases [IBD] but the underlying mechanisms are unclear. Because mucins represent the major components of the mucus barrier and disturbed mucin expression is reported in the colon of IBD patients, we studied the association between mucin expression, inflammation and intestinal permeability in experimental colitis. METHODS: We quantified 4-kDa FITC-dextran intestinal permeability and the expression of cytokines, mucins, junctional and polarity proteins at dedicated time points in the adoptive T cell transfer and dextran sodium sulfate [DSS]-induced colitis models. Mucin expression was also validated in biopsies from IBD patients. RESULTS: In both animal models, the course of colitis was associated with increased interleukin-1ß [IL-1ß] and tumour necrosis factor-α [TNF-α] expression and increased Muc1 and Muc13 expression. In the T cell transfer model, a gradually increasing Muc1 expression coincided with gradually increasing 4-kDa FITC-dextran intestinal permeability and correlated with enhanced IL-1ß expression. In the DSS model, Muc13 expression coincided with rapidly increased 4-kDa FITC-dextran intestinal permeability and correlated with TNF-α and Muc1 overexpression. Moreover, a significant association was observed between Muc1, Cldn1, Ocln, Par3 and aPKCζ expression in the T cell transfer model and between Muc13, Cldn1, Jam2, Tjp2, aPkcζ, Crb3 and Scrib expression in the DSS model. Additionally, MUC1 and MUC13 expression was upregulated in inflamed mucosa of IBD patients. CONCLUSIONS: Aberrantly expressed MUC1 and MUC13 might be involved in intestinal barrier dysfunction upon inflammation by affecting junctional and cell polarity proteins, indicating their potential as therapeutic targets in IBD.

Regulation of intestinal permeability: The role of proteases.

The gastrointestinal barrier is - with approximately 400 m2 - the human body's largest surface separating the external environment from the internal milieu. This barrier serves a dual function: permitting the absorption of nutrients, water and electrolytes on the one hand, while limiting host contact with noxious luminal antigens on the other hand. To maintain this selective barrier, junction protein complexes seal the intercellular space between adjacent epithelial cells and regulate the paracellular transport. Increased intestinal permeability is associated with and suggested as a player in the pathophysiology of various gastrointestinal and extra-intestinal diseases such as inflammatory bowel disease, celiac disease and type 1 diabetes. The gastrointestinal tract is exposed to high levels of endogenous and exogenous proteases, both in the lumen and in the mucosa. There is increasing evidence to suggest that a dysregulation of the protease/antiprotease balance in the gut contributes to epithelial damage and increased permeability. Excessive proteolysis leads to direct cleavage of intercellular junction proteins, or to opening of the junction proteins via activation of protease activated receptors. In addition, proteases regulate the activity and availability of cytokines and growth factors, which are also known modulators of intestinal permeability. This review aims at outlining the mechanisms by which proteases alter the intestinal permeability. More knowledge on the role of proteases in mucosal homeostasis and gastrointestinal barrier function will definitely contribute to the identification of new therapeutic targets for permeability-related diseases.

NecroX-7 reduces necrotic core formation in atherosclerotic plaques of Apoe knockout mice.

BACKGROUND AND AIMS: A large necrotic core is a key feature of atherosclerotic plaque instability. Necrotic cellular debris accumulates in the lipid-rich core and promotes inflammation, destabilization and ultimately rupture of the plaque. Although the role of necrosis in atherosclerosis is rather clear-cut, not many strategies have been performed up till now to specifically target plaque necrosis. In the present study, we tested the plaque stabilizing potential of NecroX-7, a novel compound with antioxidative and anti-necrotic properties. METHODS: Male apolipoprotein E (Apoe) knockout mice were treated with NecroX-7 (30 mg/kg) or vehicle, 3 times per week, via intraperitoneal injections for 16 weeks. Meanwhile, mice were fed a western-type diet to induce plaque formation. RESULTS: NecroX-7 reduced total plaque burden in the thoracic aorta as compared to vehicle-treated mice, without affecting total plasma cholesterol. Plaques in the aortic root of NecroX-7-treated mice showed a significant decrease in necrotic core area, 8-oxodG, iNOS and MMP13 expression, while collagen content and minimum fibrous cap thickness were increased. Moreover, NecroX-7 treatment reduced the expression of multiple inflammation markers such as TNFα, IL1ß, iNOS, HMGB1 and RAGE in a NF-κB-dependent manner. In vitro, NecroX-7 prevented tert-butyl hydroperoxide (tBHP)-induced mitochondrial ROS formation, necrosis, iNOS expression and HMGB1 release in primary macrophages. CONCLUSIONS: NecroX-7 improves features of plaque stability in Apoe knockout mice by reducing necrotic core formation, oxidative stress and inflammation, and by increasing collagen deposition and fibrous cap thickness. Therefore, NecroX-7 could be a promising pleiotropic drug for the treatment of atherosclerosis.

Visceral hypersensitivity in inflammatory bowel diseases and irritable bowel syndrome: The role of proteases.

Proteases, enzymes catalyzing the hydrolysis of peptide bonds, are present at high concentrations in the gastrointestinal tract. Besides their well-known role in the digestive process, they also function as signaling molecules through the activation of protease-activated receptors (PARs). Based on their chemical mechanism for catalysis, proteases can be classified into several classes: serine, cysteine, aspartic, metallo- and threonine proteases represent the mammalian protease families. In particular, the class of serine proteases will play a significant role in this review. In the last decades, proteases have been suggested to play a key role in the pathogenesis of visceral hypersensitivity, which is a major factor contributing to abdominal pain in patients with inflammatory bowel diseases and/or irritable bowel syndrome. So far, only a few preclinical animal studies have investigated the effect of protease inhibitors specifically on visceral sensitivity while their effect on inflammation is described in more detail. In our accompanying review we describe their effect on gastrointestinal permeability. On account of their promising results in the field of visceral hypersensitivity, further research is warranted. The aim of this review is to give an overview on the concept of visceral hypersensitivity as well as on the physiological and pathophysiological functions of proteases herein.