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 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.
Hypertension and Cardiovascular Rehabilitation Unit, Department of Cardiovascular Diseases, Katholieke Universiteit Leuven (K. U. Leuven), Belgium. paul.lijnen@med.kuleuven.be
Paul
P
Lijnen
Italia
I
Papparella
Victor
V
Petrov
Andrea
A
Semplicini
Robert
R
Fagard
D000098:Acetophenones / Q000494:pharmacology; D000804:Angiotensin II / Q000494:pharmacology*; D000818:Animals; D002478:Cells, Cultured; D003094:Collagen / Q000235:genetics / Q000378:metabolism*; D024042:Collagen Type I / Q000235:genetics / Q000378:metabolism; D024061:Collagen Type III / Q000235:genetics / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D008297:Male; D020781:Matrix Metalloproteinase 1 / Q000378:metabolism; D009206:Myocardium / Q000166:cytology*; D009249:NADP / Q000378:metabolism; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000378:metabolism; D009861:Onium Compounds / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000378:metabolism
D000098:Acetophenones; D024042:Collagen Type I; D024061:Collagen Type III; D004791:Enzyme Inhibitors; D009861:Onium Compounds; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D019715:Tissue Inhibitor of Metalloproteinase-1; D000804:Angiotensin II; D009249:NADP; C007517:diphenyleneiodonium; D003094:Collagen; C056165:acetovanillone; D019255:NADPH Oxidases; D020781:Matrix Metalloproteinase 1