TGF-BETA-1 STIMULATES CULTURED HUMAN FIBROBLASTS TO PROLIFERATE AND PRODUCE TISSUE-LIKE FIBROPLASIA - A FIBRONECTIN MATRIX-DEPENDENT EVENT

During wound repair, fibroblasts accumulate in the injured area until any defect is filled with stratified layers of cells and matrix. Such fibroplasia also occurs in many fibrotic disorders. Transforming growt h factor-beta (TGF-beta), a promotor of granulation tissue in vivo and extracellular matrix production in vitro, is expressed during the act ive fibroplasia of wound healing and fibroproliferative diseases. Unde r usual tissue culture conditions, normal fibroblasts grow to confluen ce and then cease proliferation. In this study, culture conditions wit h TGF-beta 1 have been delineated that promote human fibroblasts to gr ow in stratified layers mimicking in vivo fibroplasia. When medium sup plemented with serum, ascorbate, proline, and TGF-P was added thrice w eekly to normal human dermal fibroblasts, the cells proliferated and s tratified up to 16 cell layers thick within the culture dish, producin g a tissue-like fibroplasia. TGF-beta stimulated both DNA synthesis as measured by H-3-thymidine uptake and cell proliferation as measured b y a Hoechst dye DNA assay in these postconfluent cultures. The stratif ication was dependent on fibronectin assembly, as demonstrated by anti -fibronectin antibodies which inhibited both basal and TGF-beta-stimul ated cell proliferation and stratification. Suppression of collagen ma trix assembly in cell layers with beta-amino-proprionitrile (BAPN) did not inhibit basal or TGF-beta stimulated in vitro fibroplasia. BAPN d id not interfere with fibronectin matrix assembly as judged by immunof luorescence microscopy. Thus, in concert with serum factors, TGF-beta stimulates postconfluent, fibronectin matrix-dependent, fibroblast gro wth creating a fibroplasia-like tissue in vitro.