Scarpino Stefania, Marchitti Simona, Stanzione Rosita, Evangelista Anna, Di Castro Sara, Savoia Carmine, Quarta Giovanni, Sciarretta Sebastiano, Ruco Luigi, Volpe Massimo, Rubattu Speranza
Department of Cytology and Histology, University of Rome La Sapienza, Ospedale S. Andrea, Italy.
J Hypertens. 2009 Sep;27(9):1804-13. doi: 10.1097/hjh.0b013e32832d229f.
T2238C ANP (atrial natriuretic peptide) gene variant has been associated with increased cardiovascular risk in humans and with a significant pharmacogenomic effect on cardiovascular disease outcome in hypertensive patients. We investigated the impact of T2238C ANP gene variant on oxidative stress production, cell proliferation and migration, angiogenesis and vascular remodeling in human umbilical vein endothelial cells in vitro.
Differentially expressed genes in human umbilical vein endothelial cells exposed to either wild-type (TT2238) or mutant (CC2238) alpha-ANP were characterized by real time-PCR-macroarray analysis using human oxidative stress, angiogenesis and matrix arrays. Reactive oxygen species (ROS) production was determined by dihydroethidium and by evaluation of dichlorofluorescein content. NADPH oxidase gp91phox subunit was investigated by western blotting. Endothelial cell proliferation, migration and tube formation were characterized both in the presence and in the absence of NADPH oxidase inhibition.
Compared with TT2238, CC2238 alpha-ANP altered the redox state balance of the cells in a more significant manner, favoring ROS production and reducing antioxidative stress response. Gene expression of molecules involved in atherogenesis and vascular remodeling was enhanced. In contrast to TT2238 peptide, CC2238 was unable to stimulate cell proliferation and it markedly inhibited endothelial cell tube formation. NADPH oxidase inhibition restored the cell proliferative properties under CC2238 peptide exposure.
CC2238 alpha-ANP led to ROS accumulation and increased expression of genes related to atherosclerosis and vascular remodeling in human umbilical vein endothelial cells. As a consequence of NADPH-derived ROS, blunted endothelial cell proliferation and impaired endothelial cell tube formation were observed. These in-vitro effects may link the T2238C alpha-ANP variant to enhanced susceptibility to vascular damage in vivo.
T2238C心房利钠肽（ANP）基因变异与人类心血管风险增加相关，且对高血压患者的心血管疾病结局具有显著的药物基因组学效应。我们在体外研究了T2238C ANP基因变异对人脐静脉内皮细胞氧化应激产生、细胞增殖与迁移、血管生成及血管重塑的影响。
使用人类氧化应激、血管生成和基质芯片，通过实时PCR-宏阵列分析对暴露于野生型（TT2238）或突变型（CC2238）α-ANP的人脐静脉内皮细胞中差异表达的基因进行表征。通过二氢乙锭和二氯荧光素含量评估来测定活性氧（ROS）的产生。通过蛋白质印迹法研究NADPH氧化酶gp91phox亚基。在有和没有NADPH氧化酶抑制的情况下，对内皮细胞增殖、迁移和管形成进行表征。
与TT2238相比，CC2238α-ANP以更显著的方式改变细胞的氧化还原状态平衡，促进ROS产生并降低抗氧化应激反应。参与动脉粥样硬化和血管重塑的分子的基因表达增强。与TT2238肽相反，CC2238不能刺激细胞增殖，并且明显抑制内皮细胞管形成。NADPH氧化酶抑制在CC2238肽暴露下恢复了细胞增殖特性。
CC2238α-ANP导致人脐静脉内皮细胞中ROS积累，并增加与动脉粥样硬化和血管重塑相关的基因表达。作为NADPH衍生的ROS的结果，观察到内皮细胞增殖减弱和内皮细胞管形成受损。这些体外效应可能将T2238Cα-ANP变异与体内血管损伤易感性增加联系起来。
