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

Abstract

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-β1 (TGF-β 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-β1 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-β1 (10 ng/ml) for seven days. Cell characterisation was performed using light microscopy and indirect immunostaining. Presence of vimentin, desmin, factor VIII, α-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 3H-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-β1 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-β1 profoundly inhibited 3H-thymidine incorporation and the number of cells in growing cultures. The induction of α-smooth muscle actin by TGF-β 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-β 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.