Transforming growth factor-β1 induces angiotensin-converting enzyme synthesis in rat cardiac fibroblasts during their differentiation to myofibroblasts

OBJECTIVES: Appearance of angiotensin-converting enzyme (ACE) in fibrotic tissue can be the result of the action either of one particular growth factor or of cross-talk between multiple factors. Transforming growth factor-beta1 (TGF-beta(1)) is an effective inducor of the differentiation of cultured fibroblasts to myofibroblasts, which are heterogeneous cells with different phenotypes. The present study investigated whether TGF-beta(1) is able to induce, in vitro, the differentiation of cultured fibroblasts to myofibroblasts with a phenotype containing ACE. DESIGN: Adult rat cardiac ventricular fibroblasts were obtained from hearts perfused with collagenase. Cells from second passage were always used. Rat cardiac ventricular fibroblasts were incubated with TGF-beta(1) (10 ng/ml) for seven days. Cell characterisation was performed using light microscopy and indirect immunostaining. Presence of vimentin, desmin, factor VIII, alpha-smooth muscle actin, and ACE was checked with both immunostaining and Western blotting. ACE activity was measured fluorometrically with hippuryl-histidyl-leucine as substrate. Synthesis of DNA was measured as (3)H-thymidine incorporation. RESULTS: Fibroblasts contained two types of activity of hip-his-leu degradation, namely a lisinopril-dependent activity (ACE activity) and a lisinopril-independent activity ('ACE-like' activity) which is performed by peptidase(s) other than ACE. The ACE activity constituted approximately 30% of the total activity. TGF-beta(1) induced an increase in both ACE activity and ACE protein (detected by immunoblotting) by 4.5 +/- 0.9- and 6.9 +/- 2.0-fold, respectively (p<0.05). This induction of ACE was accompanied by a profound modification of the fibroblasts phenotype, which consisted of a change in cell morphology, an enlargement of cell volume and an increase in cell protein content. TGF-beta(1) profoundly inhibited (3)H-thymidine incorporation and the number of cells in growing cultures. The induction of alpha-smooth muscle actin by TGF-beta(1) (6.8 +/- 2.8-fold increase, p<0.05) simultaneously with these modifications in cell morphology and proliferation indicates the appearance of myofibroblasts. These myofibroblasts did not contain desmin. CONCLUSION: TGF-beta(1) is able to induce the appearance of ACE in cultures of adult rat cardiac ventricular fibroblasts. The appearance of the enzyme is accompanied by the differentiation of fibroblasts to myofibroblasts.