Role of reactive oxygen species in the transforming growth factor-beta1-induced collagen production and differentiation of cardiac fibroblasts into myofibroblasts

Abstract

The aim of the present study was to determine whether transforming growth factor-β1 (TGF-β1)-induced collagen production in cardiac fibroblasts is affected by reactive oxygen species (ROS). Cardiac fibroblasts (passage 2) from normal male adult rats were cultured to confluency and incubated in serum-free Dulbecco’s modified Eagle’s medium for 24 h. The cells were then preincubated with(out) the tested inhibitors for 1 h and further incubated with(out) TGF-β1 at various concentrations and for 1, 2, 4, 24 or 48 h. TGF-β1 induced a dose-dependent increase in the soluble collagen production in cardiac fibroblasts after 48 h of incubation. No significant effect of TGF-β1 (600 pmol/l) on collagen production and on α-smooth muscle actin (α-SMA) protein expression was found after 1, 2 and 4 h of incubation. After 24 and 48 h, TGF-β1 stimulated collagen production and α-SMA protein expression in cardiac fibroblasts. Intracellular ROS were not affected by TGF-β1 after 0.5 and 1 h of incubation, began to rise after 2 h, and reached a maximal increase after 4 h. The TGF-β1-stimulated ROS and collagen production is reduced by the ROS inhibitor diphenyleneiodonium chloride (DPI). DPI also decreased the TGF-β1-stimulated α-SMA protein expression in rat cardiac fibroblasts. Our data indicate that the TGF-β1-induced increase in ROS preceded the rise in collagen production and α-SMA protein expression and that ROS inhibition diminished the conversion of fibroblasts into myofibroblasts.