Lijnen Paul, Papparella Italia, Petrov Victor, Semplicini Andrea, Fagard Robert
Hypertension and Cardiovascular Rehabilitation Unit, Department of Cardiovascular Diseases, Katholieke Universiteit Leuven (K. U. Leuven), Belgium.
J Hypertens. 2006 Apr;24(4):757-66. doi: 10.1097/01.hjh.0000217860.04994.54.
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.
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.
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.
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.
本研究旨在确定抑制还原型烟酰胺腺嘌呤二核苷酸（磷酸）[NAD(P)H]氧化酶及各种超氧化物生成系统是否会影响对照及血管紧张素II处理的心脏成纤维细胞中胶原蛋白的产生、I型和III型胶原蛋白的mRNA及蛋白表达。
将来自正常成年雄性大鼠的第2代心脏成纤维细胞培养至汇合状态，并在无血清的杜尔贝科改良伊格尔培养基中孵育24小时。然后将细胞在有（无）测试抑制剂的情况下预孵育1小时，接着在有（无）血管紧张素II（1微摩尔/升）的情况下进一步孵育24小时。用天狼星红作为染料通过分光光度法以及通过[3H]脯氨酸掺入来测量胶原蛋白的产生；通过酶联免疫吸附测定法测量I型和III型胶原蛋白的含量，通过半定量逆转录-聚合酶链反应（RT-PCR）测量I型和III型胶原蛋白的mRNA表达。以超氧化物歧化酶可抑制的细胞色素c还原反应来测定NAD(P)H依赖性超氧阴离子的产生。用2',7'-二氯荧光素二乙酸酯作为荧光探针评估细胞内活性氧的形成。
血管紧张素II刺激心脏成纤维细胞中的胶原蛋白产生、I型和III型胶原蛋白的含量及mRNA表达，而膜NAD(P)H氧化酶抑制剂夹竹桃麻素消除了这种诱导作用。鱼藤酮、别嘌呤醇、吲哚美辛、去甲二氢愈创木酸、酮康唑和硝基-L-精氨酸（分别为线粒体NAD(P)H氧化酶、黄嘌呤氧化酶、环氧化酶、脂氧化酶、细胞色素P450加氧酶和一氧化氮合酶的抑制剂）不影响血管紧张素II诱导的胶原蛋白产生。血管紧张素II增加心脏成纤维细胞中NAD(P)H依赖性超氧阴离子的产生及细胞内活性氧的生成，夹竹桃麻素消除了这种增加。
我们的数据表明，在成年大鼠心脏成纤维细胞中，膜相关的NAD(P)H氧化酶复合物是血管紧张素II刺激的成年心脏成纤维细胞中超氧阴离子和活性氧生成的主要来源。用夹竹桃麻素抑制这种NAD(P)H氧化酶复合物可完全阻断血管紧张素II刺激的胶原蛋白产生以及I型和III型胶原蛋白的蛋白及mRNA表达。
