Expression of transforming growth factor (TGF)-β1 mRNA and contractility of extracellular matrices in three-dimensional culture of rat peritoneal fibroblasts

Abstract

Background. In a fibrotic process the interaction between resident cells and extracellular matrices is important in the control of cell function. Our preliminary experiments indicated that concentrated peritoneal dialysis effluent caused contraction of collagen matrices by peritoneal fibroblasts. In this study we attempted to mimic the inflammatory milieu present during peritoneal inflammation and assessed its effect on fibroblast activation. Methods. Rat peritoneal fibroblasts (RPFB) were isolated by a time-elapsed differential subculture from mixtures of primary cultures of peritoneal resident cells, and then cultured in collagen matrices. Various inflammatory mediators, known to be present in the peritoneal cavity during inflammation, (i.e., interleukin-6 [IL-6], IL-1 , and tumor necrosing factor (TNF)α were added to three-dimensional-RPFB cultures (1 ϫ 10 5 /ml) prior to their incubation for either 48 h or 10 days. The contractility of collagen matrices over this time period was monitored, as was the expression of transforming growth factor (TGF)-1 mRNA. Experimental conditions were: (i) control gels, (ii) gels with IL-6, (iii) gels with IL-1 , (iv) gels with TNFα, (v) gels with each cytokine plus glucose (90 mM). Results. With 48 h stimulation, a greater than tenfold increase in TGF-1 mRNA expression (measured as the ratio to TGF-1/glyceraldehyde 3-phosphate dehydrogenase [GAPDH] mRNA) was observed compared with the control, and this was accompanied by gel contraction. With 10-day stimulation, both IL-1 and TNFα suppressed the expression of TGF-1 mRNA as well as suppressing gel contraction. There was a 30% to 40% gel contraction accompanied by elongation of RPFB morphology in the IL-6 and control groups. The addition of glucose promoted the extension of RPFB and gel contraction by up to 60% in both the IL-1 -and TNFα-supplemented groups. Conclusion. These in vitro findings suggest that a balance of inflammatory cytokines influences rat peritoneal fibroblast (RPFB) gene expression, causing changes in the interaction between extracellular matrices and peritoneal fibroblasts.

Image analysis of remesothelialization following chemical wounding of cultured human peritoneal mesothelial cells: The role of hyaluronan synthesis

“…HPMC cultures were established using a previously described method [31]. Chemicals and tissue culture plastics in the study were as described in our earlier studies [32].…”

Prolonged preservation of rat peritoneal mesothelial cells (RPMC) and rat peritoneal fibroblasts (RPFB) with neutral pH peritoneal dialysis solution depleted of glucose degradation products (GDPs)

“…38 After 30 min, 3 h, and 6 h exposure to test PD solutions, RNA was isolated from RPMC and RPFB, using an RNA isolator (T-9424; Sigma) method. 38 After 30 min, 3 h, and 6 h exposure to test PD solutions, RNA was isolated from RPMC and RPFB, using an RNA isolator (T-9424; Sigma) method.…”