Abnormal extracellular matrix metabolism in chronically ischemic skin: a mechanism for dermal failure in leg ulcers.

Extracellular matrix (ECM) metabolism and homeostasis is sensitive to changes in oxygen tension manifest in ischemia. We hypothesize that in chronically ischemic limbs, abnormalities in uninjured skin, secondary to hypoxia, predispose to dermal breakdown. Paired biopsies of uninjured distal ischemic and proximal non-ischemic skin were harvested at below knee amputation from 14 patients with peripheral vascular disease following quantification of ischemia. Age- and site-matched controls were taken at total knee replacement (TKR) and varicose vein (VV) operations. Matrix metalloproteinase (MMP)-2 and -9 expression was determined using gelatin zymography, MMP-1 by western blotting and ELISA and tissue inhibitor of MMP (TIMP) by reverse zymography. Collagen content was measured by determining hydroxyproline levels, and collagen type I synthesis by ELISA. Collagen type I synthesis was upregulated in ischemic tissue compared with non-ischemic matched pairs (p<0.001) and both TKR and VV controls, however, there was no increase in collagen deposition. Levels of MMP-2 (p<0.0005) and TIMP-2 (p<0.01), were elevated in ischemic samples. MMP-9 was unaltered, signifying no inflammatory changes. Tissue ischemia was linked to elevated ECM turnover, associated with matrix failure when compounded with problems of matrix stabilization, likely in ischemia. This represents a potential mechanism for ulcer formation.

Dietary n-3 polyunsaturated fatty acids reduce disease and colonic proinflammatory cytokines in a mouse model of colitis.

BACKGROUND: n-3 polyunsaturated fatty acids (PUFAs) reduce the severity of chronic inflammatory bowel disease, probably by means of reduction of immune cell activation or enhancement of the epithelial barrier. Using the severe combined immunodeficient (SCID) mouse model of colitis, this study examined the effect of dietary n-3 PUFAs on development of colitis and on immunologic, epithelial, and matrix parameters in the intestines of control and colitic animals. METHODS: SCID mice were fed n-3-enriched or control diet for 3 weeks before colitis induction by transplantation of CD45RB T cells and maintained on the same diet for 4 to 8 weeks. Phenotype of infiltrating cells, epithelial ZO-1 protein, and mucosal type I collagen were assessed by immunohistology and tissue cytokines by ELISA. RESULTS: Transplanted n-3-fed animals had significantly reduced pathology scores, colonic tumor necrosis factor-alpha, interleukin-12, and interleukin-1beta compared with animals fed standard diet. Proinflammatory cytokines were reduced despite a similar level of immune cell infiltration by T cells, CD11c cells, and CD11b cells. Neutrophil infiltration was significantly reduced in n-3-fed control and colitic mice, and other myeloid populations were reduced in mice on the n-3 diet. Epithelial ZO-1 expression was increased, and myofibroblast activation significantly decreased in transplanted n-3-fed animals compared with standard diet mice. Submucosal collagen synthesis was enhanced in n-3-fed mice. CONCLUSIONS: Dietary n-3 PUFAs reduced clinical colitis and colonic immunopathology in this model of colonic inflammation by decreasing proinflammatory cytokine synthesis, reducing myeloid cell recruitment and activation, and enhancing epithelial barrier function and mucosal wound healing mechanisms.

Abnormal mucosal extracellular matrix deposition is associated with increased TGF-beta receptor-expressing mesenchymal cells in a mouse model of colitis.

Transforming growth factor-beta (TGF-beta) depresses mucosal inflammation and upregulates extracellular matrix (ECM) deposition. We analyzed TGF-beta receptors RI and RII as well as ECM components using the CD4(+) T-cell-transplanted SCID mouse model of colitis. The principal change in colitis was an increased proportion of TGF-beta RII(+) mucosal mesenchymal cells, predominantly alpha-smooth muscle actin (SMA)(+) myofibroblasts, co-expressing vimentin and basement membrane proteins, but not type I collagen. TGF-beta RII(+) SMA(-) fibroblasts producing type I collagen were also increased, particularly in areas of infiltration and in ulcers. Type IV collagen and laminin were distributed throughout the gut lamina propria in disease but were restricted to the basement membrane in controls. In areas of severe epithelial damage, type IV collagen was lost and increased type I collagen was observed. To examine ECM production by these cells, mucosal mesenchymal cells were isolated. Cultured cells exhibited a similar phenotype and matrix profile to those of in vivo cells. The data suggested that there were at least two populations of mesenchymal cells responsible for ECM synthesis in the mucosa and that ligation of TGF-beta receptors on these cells resulted in the disordered and increased ECM production observed in colitic mucosa.

IL-13 promotes collagen accumulation in Crohn's disease fibrosis by down-regulation of fibroblast MMP synthesis: a role for innate lymphoid cells?

BACKGROUND: Fibrosis is a serious consequence of Crohn's disease (CD), often necessitating surgical resection. We examined the hypothesis that IL-13 may promote collagen accumulation within the CD muscle microenvironment. METHODS: Factors potentially modulating collagen deposition were examined in intestinal tissue samples from fibrotic (f) CD and compared with cancer control (C), ulcerative colitis (UC) and uninvolved (u) CD. Mechanisms attributable to IL-13 were analysed using cell lines derived from uninvolved muscle tissue and tissue explants. RESULTS: In fCD muscle extracts, collagen synthesis was significantly increased compared to other groups, but MMP-2 was not co-ordinately increased. IL-13 transcripts were highest in fCD muscle compared to muscle from other groups. IL-13 receptor (R) α1 was expressed by intestinal muscle smooth muscle, nerve and KIR(+) cells. Fibroblasts from intestinal muscle expressed Rα1, phosphorylated STAT6 in response to IL-13, and subsequently down-regulated MMP-2 and TNF-α-induced MMP-1 and MMP-9 synthesis. Cells with the phenotype KIR(+)CD45(+)CD56(+/-)CD3(-) were significantly increased in fCD muscle compared to all other groups, expressed Rα1 and membrane IL-13, and transcribed high levels of IL-13. In explanted CD muscle, these cells did not phosphorylate STAT6 in response to exogenous IL-13. CONCLUSIONS: The data indicate that in fibrotic intestinal muscle of Crohn's patients, the IL-13 pathway is stimulated, involving a novel population of infiltrating IL-13Rα1(+), KIR(+) innate lymphoid cells, producing IL-13 which inhibits fibroblast MMP synthesis. Consequently, matrix degradation is down-regulated and this leads to excessive collagen deposition.