In vitro models of TGF-β-induced fibrosis suitable for high-throughput screening of antifibrotic agents

Abstract

Progressive fibrosis is a cause of progressive organ dysfunction. Lack of quantitative in vitro models of fibrosis accounts, at least partially, for the slow progress in developing effective antifibrotic drugs. Here, we report two complementary in vitro models of fibrosis suitable for high-throughput screening. We found that, in mesangial cells and renal fibroblasts grown in eight-well chamber slides, transforming growth factor-β1 (TGF-β1) disrupted the cell monolayer and induced cell migration into nodules in a dose-, time- and Smad3-dependent manner. The nodules contained increased interstitial collagens and showed an increased collagen I:IV ratio. Nodules are likely a biological consequence of TGF-β1-induced matrix overexpression since they were mimicked by addition of collagen I to the cell culture medium. TGF-β1-induced nodule formation was inhibited by vacuum ionized gas treatment of the plate surface. This blockage was further enhanced by precoating plates with matrix proteins but was prevented, at least in part, by poly-l-lysine (PLL). We have established two cell-based models of TGF-β-induced fibrogenesis, using mesangial cells or fibroblasts cultured in matrix protein or PLL-coated 96-well plates, on which TGF-β1-induced two-dimensional matrix accumulation, three-dimensional nodule formation, and monolayer disruption can be quantitated either spectrophotometrically or by using a colony counter, respectively. As a proof of principle, chemical inhibitors of Alk5 and the antifibrotic compound tranilast were shown to have inhibitory activities in both assays.

Emodin protects rat liver from CCl4-induced fibrogenesis via inhibition of hepatic stellate cells activation

“…Results in this study indicated that emodin suppressed inflammation caused by CCl4, which might lead to the protection of the liver from injury. It is now widely accepted that the pro-inflammatory cytokine TGF-β1 is a major cytokine in the regulation of the production, degradation, and accumulation of ECM [38] , and it has been suggested that overexpression of TGF-β1 for a prolonged period of time after tissue damage may induce a fibroproliferative response and deposition of ECM, resulting in fibrosis in vital organs [39] . Many studies have detected the presence of TGF-β1, in the form of either protein or message, in the fibrotic tissues of animal models or human samples [40] .…”

Potential nephroprotective effects of the Chinese herbAngelica sinensisagainst cisplatin tubulotoxicity

“…Picrosirius red staining allows the collagen produced by cell cultures to be quantified (Hu et al, 2009;Xu et al, 2007). Exposure of HK-2 cells to 50 and 5 mg/ml AS for 48 h did not modify the amount of collagen produced as compared to the control condition ( Figure 6).…”

Shear stress-induced volume decrease in C11-MDCK cells by BK-α/β4

“…A second pool of four nontargeting siRNA oligos were used for negative-control experiments (Dharmacon nontargeting siGENOME SMART pool 2). C11 were transfected by electroporation according to the manufacturer's instructions as previously described (52).…”