In vitro models of TGF-beta-induced fibrosis suitable for high-throughput screening of antifibrotic agents.
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-beta1 (TGF-beta1) 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-beta1-induced matrix overexpression since they were mimicked by addition of collagen I to the cell culture medium. TGF-beta1-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-beta-induced fibrogenesis, using mesangial cells or fibroblasts cultured in matrix protein or PLL-coated 96-well plates, on which TGF-beta1-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.
Xu
Qihe
Q
Kidney Disease Section, National Institute of Diabetes and Digestive and Kidney Disases, National Institutes of Health, Bethesda, MD 20892-1268, USA.
Norman
Jill T
JT
Shrivastav
Shashi
S
Lucio-Cazana
Javier
J
Kopp
Jeffrey B
JB
D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D004396:Coloring Agents; D004285:Dogs; D004353:Drug Evaluation, Preclinical; D004847:Epithelial Cells / Q000187:drug effects; D005347:Fibroblasts / Q000187:drug effects / Q000473:pathology; D005355:Fibrosis / Q000139:chemically induced* / Q000188:drug therapy* / Q000473:pathology; D005920:Glomerular Mesangium / Q000166:cytology / Q000187:drug effects; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007668:Kidney / Q000166:cytology; D059630:Mesenchymal Stem Cells / Q000187:drug effects; D051379:Mice; D008854:Microscopy, Electron; D011107:Polylysine / Q000494:pharmacology; D051900:Smad3 Protein / Q000502:physiology; D016212:Transforming Growth Factor beta / Q000633:toxicity*
D004396:Coloring Agents; D051900:Smad3 Protein; D016212:Transforming Growth Factor beta; D011107:Polylysine