Involvement of reactive oxygen species in angiotensin II-induced endothelin-1 gene expression in rat cardiac fibroblasts

OBJECTIVES: The aim of this study was to investigate the effects of angiotensin II (Ang II) on fibroblast proliferation and endothelin-1 (ET-1) gene induction, focusing especially on reactive oxygen species (ROS)-mediated signaling in cardiac fibroblasts. BACKGROUND: Angiotensin II increases ET-1 expression, which plays an important role in Ang II-induced fibroblast proliferation. Angiotensin II also stimulates ROS generation in cardiac fibroblasts. However, whether ROS are involved in Ang II-induced proliferation and ET-1 expression remains unknown. METHODS: Cultured neonatal rat cardiac fibroblasts were stimulated with Ang II, and then [(3)H]thymidine incorporation and the ET-1 gene expression were examined. We also examined the effects of antioxidants on Ang II-induced proliferation and mitogen-activated protein kinase (MAPK) phosphorylation to elucidate the redox-sensitive pathway in fibroblast proliferation and ET-1 gene expression. RESULTS: Both AT(1) receptor antagonist (losartan) and ET(A) receptor antagonist (BQ485) inhibited Ang II-increased DNA synthesis. Endothelin-1 gene was induced with Ang II as revealed by Northern blotting and promoter activity assay. Angiotensin II increased intracellular ROS levels, which were inhibited with losartan and antioxidants. Antioxidants further suppressed Ang II-induced ET-1 gene expression, DNA synthesis, and MAPK phosphorylation. PD98059, but not SB203580, fully inhibited Ang II-induced ET-1 expression. Truncation and mutational analysis of the ET-1 gene promoter showed that AP-1 binding site was an important cis-element in Ang II-induced ET-1 gene expression. CONCLUSIONS: Our data suggest that ROS are involved in Ang II-induced proliferation and ET-1 gene expression. Our findings imply that the combination of AT(I) and ET(A) receptor antagonists plus antioxidants may be beneficial in preventing the formation of excessive cardiac fibrosis.