Transforming growth factor-beta 1-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-stimulated 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 did 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 after 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 for 48 h. Under these conditions, alpha-SMA was increased by 3.5-fold (p < 0.001) and second-passage cultures of fibroblasts and their offspring in the next passage consisted mainly of myofibroblasts. 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 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.
Lijnen
P
P
Hypertension and Cardiovascular Rehabilitation Unit, Department of Molecular and Cardiovascular Research, Faculty of Medicine, University of Leuven, Leuven, Belgium. paul.lijnen@med.kuleuven.ac.be
Petrov
V
V
D000199:Actins / Q000096:biosynthesis; D000818:Animals; D015153:Blotting, Western; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D003094:Collagen / Q000096:biosynthesis*; D004305:Dose-Response Relationship, Drug; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D007150:Immunohistochemistry; D008297:Male; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D013997:Time Factors; D016212:Transforming Growth Factor beta / Q000378:metabolism / Q000494:pharmacology*; D053773:Transforming Growth Factor beta1
D000199:Actins; C508962:Tgfb1 protein, rat; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D003094:Collagen