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Figure 3 Angiotensin II (Ang II)-induced proliferation and endothelin-1 (ET-1) gene expression were mediated by reactive oxygen species (ROS) in cardiac fibroblasts. (A) Angiotensin II increased intracellular ROS in cardiac fibroblasts. Cardiac fibroblasts were loaded with dichlorofluorescin diacetate for 30 min and stimulated with Ang II for 30 min. Intracellular ROS levels were measured by laser-confocal microscopy. Angiotensin II (100 nM) increased ROS levels in cardiac fibroblasts, and these increases were abolished by losartan (Losa; 1 µM), the NAD(P)H oxidase inhibitor diphenyleneiodonium (DPI) (1 µM), catalase (350 U/ml), or N-acetylcysteine (NAC) (10 mM). Cells treated with H₂O₂ are used as positive control. In each experiment, the densitometric analysis was performed on at least 20 cells. (B) Effect of antioxidants on Ang II-induced DNA synthesis in cardiac fibroblasts. Cells were preincubated with NAD(P)H oxidase inhibitor DPI (1 µM), catalase (350 U/ml), or NAC (10 mM) for 30 min followed by incubation with 100 nM Ang II for 24 h. Increases in [³H]thymidine incorporation are each expressed relative to the [³H] content (100%) in the respective control (C). (C) Effect of antioxidants on Ang II-induced ET-1 messenger RNA in cardiac fibroblasts. Cells were preincubated with either the catalase (350 U/ml) or NAC (10 mM) for 30 min followed by an incubation with 100 nM Ang II for 30 min. (D) Effect of antioxidants on Ang II-increased ET-1 promoter activity in cardiac fibroblasts. Cells were preincubated with either the catalase (350 U/ml) or NAC (10 mM) for 30 min followed by an incubation with 100 nM Ang II for 24 h. The results are shown as mean ± SEM (n = 3 per group). *p < 0.05 vs. control (Student t test); #p < 0.05 vs. Ang II alone (analysis of variance).