Abstract
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.
Highlights
Bosentan is an endothelin receptor antagonist that inhibits the effects of endothelin-1 (ET-1) and currently is used to treat patients with pulmonary arterial hypertension
We demonstrated that thrombin contributes to the phenotype of lung myofibroblasts [6,16]
ET-1 was measured by ELISA in supernatants from normal and scleroderma lung fibroblast cultures stimulated with various concentrations of thrombin
Summary
Bosentan is an endothelin receptor antagonist that inhibits the effects of endothelin-1 (ET-1) and currently is used to treat patients with pulmonary arterial hypertension. Using specific endothelin receptor(s) inhibitors, it has been shown that collagen matrix contraction induced by ET-1 is mediated by the ETA, but not the ETB, receptor [3] Both the ETA and the ETB receptor contribute to lung fibroblast proliferation induced by ET in scleroderma (systemic sclerosis, SSc) patients [1]. It has been shown that when both endothelin receptor subtypes coexist, selective blockade of either the ETA or the ETB receptor is ineffective or insufficient for the inhibition of collagen deposition induced by ET, indicating compensation by the other, un-antagonized ET receptor subtype, when only one receptor is antagonized [2,3]. 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
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