Angiotensin II-stimulated collagen production in cardiac fibroblasts is mediated by reactive oxygen species

Objective The aim of the present study was to determine whether inhibition of reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] oxidase and of various superoxide generating systems could affect the collagen production, the mRNA and protein expression of collagen types I and III in control and angiotensin II-treated cardiac fibroblasts. Methods Cardiac fibroblasts from 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 then further incubated with(out) angiotensin II (1 mu mol/l) for 24 h. Collagen production was measured spectrophotometrically with picrosirius red as dye and with [H-3]proline incorporation; collagen type I and III content by enzyme-linked immunosorbent assay and collagen type I and III mRNA expression by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), NAD(P) H-dependent superoxide anion production was assayed as superoxide dismutase-inhibitable cytochrome c reduction. Intracellular formation of reactive oxygen species was assessed with 2',7'-dichlorofluorescein diacetate as fluorescent probe. Results Angiotensin II stimulated the collagen production, the collagen I and III content and mRNA expression in cardiac fibroblasts, and apocynin, a membrane NAD(P)H oxidase inhibitor, abolished this induction. Rotenone, allopurinol, indomethacin, nordihydroguiaretic acid, ketoconazole and nitro-L-arginine (inhibitors of mitochondrial NAD(P)H oxidase, xanthine oxidase, cyclooxygenase, lipoxygenase, cytochrome P450 oxygenase and nitric oxide synthase, respectively) did not affect the angiotensin II-induced collagen production. Angiotensin II increased the NAD(P)H -dependent superoxide anion production and the intracellular generation of reactive oxygen species in cardiac fibroblasts, and apocynin abrogated this rise. Conclusions Our data show that in adult rat cardiac fibroblasts the membrane-associated NAD(P)H oxidase complex is the predominant source of superoxide anion and reactive oxygen species generation in angiotensin II-stimulated adult cardiac fibroblasts. Inhibition of this NAD(P)H oxidase complex with apocynin completely blocked the angiotensin II-stimulated collagen production, and collagen I and III protein and mRNA expression.