| 16531806 |
Lijnen P, Papparella I, Petrov V, Semplicini A, Fagard R: Angiotensin II-stimulated collagen production in cardiac fibroblasts is mediated by reactive oxygen species. J Hypertens. 2006 Apr;24(4):757-66.
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 micromol/l) for 24 h. Collagen production was measured spectrophotometrically with picrosirius red as dye and with [3H] 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. |
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