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INNOVATIVE METHODOLOGY

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

¹Kidney Disease Section, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland; ²Division of Medicine, Royal Free and University College Medical School, University College London, London, United Kingdom; and ³Department of Physiology, University of Alcala, Alcala de Henares, Madrid, Spain

Submitted 22 September 2006 ; accepted in final form 7 May 2007

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-1 (TGF-1) 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-1-induced matrix overexpression since they were mimicked by addition of collagen I to the cell culture medium. TGF-1-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--induced fibrogenesis, using mesangial cells or fibroblasts cultured in matrix protein or PLL-coated 96-well plates, on which TGF-1-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.

collagen; fibronectin; cell migration; Smad3; tranilast