M. Müfit Kahraman

Hypoxia enhances secretion of matrix metalloproteinases in bovine dermal fibroblasts: an in vitro approach to bovine digital dermatitis

ABSTRACT Dermal fibroblasts are of great importance in skin homeostasis. They are responsible for the synthesis and remodelling of extracellular proteins by the way of synthesis of some specific kind of Matrix Metalloproteinases (MMPs). Imbalance between MMPs and their tissue inhibitors (TIMPs) are known to involve ulcerative wounds in many tissues, including skin. In veterinary medicine, bovine digital dermatitis (BDD) is an ulcerative skin disorder of dairy cattle. To date, Koch’s postulate has not yet been fulfilled for the pathogenesis. In present study, we tested in vitro role of hypoxia if it evokes a response in MMP-1, MMP-2 and MMP-13 enzymes and their inhibitors in bovine dermal fibroblasts. Dermal fibroblasts were exposed to hypoxia in various time periods (6, 12 and 24 h). Immunoblotting revealed 6-, 12- and 24-h hypoxia, which resulted in 43.5%, 75% and 65% increase in MMP-1, 76%, 61% and 55% increase in MMP-2 and 80%, 78% and 55% increase in MMP-13 enzymes levels, respectively. We observed slight increases in TIMP-1 and TIMP-2 enzymes, but differences between treatment and control groups did not show any statistical significance. Our findings indicate that hypoxia induces MMP-1, MMP-2 and MMP-13 enzymes and may contribute to pathogenesis of BDD through in the development of in vivo lesions.

Effects of choline treatment in concentrations of serum matrix metalloproteinases (MMPs), MMP tissue inhibitors (TIMPs) and immunoglobulins in an experimental model of canine sepsis

The aim of the present study was to investigate effects of intravenous (i.v.) choline treatment on serum matrix metalloproteinases (MMP), MMP tissue inhibitors (TIMP) and immunoglobulins (Igs), and to determine if there were relations between serum MMPs/TIMPs and C-reactive protein (CRP) (as a marker of the acute phase response), immunoglobulin G and M (IgG and IgM) (as a maker of the Ig responses) and markers of organ damage such as muscular damage (creatine phosphokinase, [CPK]), liver damage (alanine aminotransferase [ALT]) and renal dysfunction (blood urea nitrogen [BUN] and creatinine, [Cr]) in dogs with endotoxemia. Healthy dogs (n=24) were randomized to Saline, Choline (C), Lipopolysaccharide (LPS), and LPS+C groups and received 0.9% NaCl (5mL/i.v.), choline chloride (20mg/kg/i.v.), LPS (0.02mg/kg/i.v.) and LPS (0.02mg/kg/i.v.) plus choline chloride (20mg/kg/i.v.), respectively. Serum MMPs and TIMPs concentrations were analyzed by commercial ELISA kits. MMP and TIMP increased at 1-48h (P<0.05), whereas IgG and IgM decreased at 24-48h in LPS group, compared to their baselines. Choline treatment reduced changes in serum MMPs, TIMPs and markers of organ damage, and prevented the hypoimmunoglobulinemia in LPS+C. MMPs and TIMPs were correlated positively (P<0.05) with serum CRP, CPK, ALT, BUN and Cr, but not with serum Igs. Our findings suggest that the serum MMPs, TIMPs and Igs are involved in the pathophysiology of endotoxemia, and MMPs and TIMPs are correlated with the acute phase reaction and multi-organ failure. In addition, we demonstrated a direct effect of choline administration in decreasing serum MMPs and TIMPs, and preserving serum Igs in the course of endotoxemia.

Effects of hypoxia and hyperosmosis on the expression of matrix metalloproteinases in broiler lung fibroblasts

This study investigated the effects of hypoxic and hyperosmotic stresses on the matrix metalloproteinases 2 and 9 (MMP-2, MMP-9) and their tissue inhibitors 2 and 1 (TIMP-2, TIMP-1) respectively in cultured lung fibroblasts from broiler chickens. Isolated and sub-cultured primary lung fibroblasts from 1-day-old broilers were exposed to hypoxia and hyperosmosis. The presence of the enzymes and the effects of hypoxia and hyperosmosis on the enzymes in cultured lung fibroblasts, and in the culture media, were evaluated with the streptavidin-biotin-peroxidase method and western blotting using commercially available primary monoclonal antibodies. Short (6 hours) and long term (12 hours) hypoxia, hypoxia + hyperosmosis (6 hours) and hyperosmosis (6 hours) triggered the release of both MMP-2 and TIMP-1 in experimental groups when compared with their controls. Neither MMP-9 nor TIMP-2 antibodies stained the fibroblast cells kept in normoxic, hypoxic or hyperosmotic conditions.