Transforming growth factor-β1-induced collagen production in cultures of cardiac fibroblasts is the result of the appearance of myofibroblasts

Transforming growth factor-beta(1) (TGF-beta(1)), which appears in high concentrations in fibrotic cardiac tissue, is a potent inductor of tissue collagen deposition and of the differentiation of fibroblasts to myofibroblasts. It is accepted that TGF-beta(1) is a potent stimulator of collagen secretion by fibroblasts. The aim of the present study was to determine which type of cells, fibroblasts and/or myofibroblasts are stimulated, in terms of collagen production, by TGF-beta(1). Therefore, using cultures of second-passage rat cardiac fibroblasts, we investigated the dose-(0.003-15 ng/ml) and time-dependence (2-48 h) of the TGF-beta(1)-induced effects on collagen production and on the appearance of myofibroblasts, as estimated by the presence of alpha-smooth muscle actin (alpha-SMA; a marker of myofibroblasts). The reversibility of the TGF-beta(1)-stilmulated effects was also studied. The dose- and time-dependent stimulation of collagen production was closely associated with the induction of alpha-SMA. TGF-beta(1) diet not change the cell phenotype or increase collagen production in rat cardiac fibroblasts cultures after a long incubation (24-28 h) at low concentrations (< 1 ng/ml), or alter a short incubation (2-4 h) at high concentrations (1-15 ng/ml). However, after a long incubation at high concentrations, TGF-beta(1) changed the cell phenotype and increased collagen production in these cultures through the differentiation of fibroblasts to myofibroblasts. A maximal increase of collagen production (two-fold, p < 0.001) was observed after incubation of fibroblasts with 15 ng/ml TGF-beta(1 f)or 48 h. Under these conditions, alpha-SMA was increased by 3.5-fold (p < 0.001) and second-passage cultures a fibroblasts and their offspring in the next passage consisted mainly of myofibroblasts lasts. The stimulation of collagen by 15 ng/ml TGF-beta(1) for 48 h was irreversible. In fact, additional incubation of these second-passage TGF-beta(1)-stimulated cultures without TGF-beta(1) for 2 days did not decrease the high activity of collagen production. Moreover, the third-passage offspring of these TGF-beta(1)-stimulated fibroblasts cultured without TGF-beta(1) also showed a higher production of collagen compared with control fibroblasts. Furthermore, the increased collagen production in the third-passage fibroblast of offspring of the second-passage TGF-beta(1)-stimulated fibroblasts could not be further stimulated by TGF-beta(1). Thus, the activity of collagen production in TGF-beta(1)-stimulated cultures and in their next passage offspring is not sensitive to TGF-beta(1). Our data suggest that TGF-beta(1)-stimulated collagen production in cultures of adult rat cardiac ventricular fibroblasts cannot be explained by a direct stimulation of collagen production, either in fibroblasts or in myofibroblasts. Instead, TGF-beta(1), induces differentiation of fibroblasts to myofibroblasts, the latter having a higher activity for collagen production than the former. (C) 2002 Prous Science. All rights reserved.