Inhibitory Effects of the Dual Endothelin Receptor Blocker Bosentan on Thrombin-Activated Lung Fibroblasts Isolated from Scleroderma Patients

Background: Endothelin-1 and thrombin are elevated during lung injury and repair, as seen in scleroderma and other interstitial lung diseases, and each plays important roles in remodeling epithelium, blood vessels and connective tissue. Both factors promote lung myofibroblast differentiation, the hallmark of pulmonary fibrosis. This study was undertaken to investigate whether bosentan, the dual, specific and competitive inhibitor of endothelin, interferes with thrombin signaling in scleroderma lung fibroblasts. Methods: Endothelin-1 secretion was measured by enzyme-linked immunosorbent assay. Lung fibroblast proliferation was studied by DNA Synthesis and Quick Cell Proliferation assays. Expression of α-smooth muscle actin (α-SMA) was analyzed on Western blots. Contractile activity of lung fibroblasts was measured by a collagen gel contraction assay. Lung fibroblast migration was studied by wound-healing “scratch” assay. Results: We show that thrombin significantly induces endothelin-1 expression in human lung fibroblasts. Bosentan significantly decreases α-SMA and collagen gel contraction of human lung fibroblasts stimulated by thrombin, suggesting that thrombin-induced differentiation of fibroblasts to the myofibroblast phenotype is at least in part regulated by endothelin-1. Bosentan decreases thrombin-induced migration of normal and scleroderma lung fibroblasts and inhibits innately increased migration of scleroderma lung fibroblasts suggesting that endogenous endothelin may be in part responsible for enhanced migration of lung fibroblasts in pulmonary fibrosis. We also report that bosentan inhibits thrombin-induced thymidine incorporation and decreases lung fibroblast proliferation. Conclusions: Fibrogenic effects of thrombin in scleroderma lung fibroblasts are mediated in part by endothelin-1. The dual endothelin receptor blocker bosentan restrains profibrotic effects of endogenous and thrombin-induced endothelin-1 in scleroderma lung fibroblasts.