Pai Wan-Yu, Lo Wan-Yu, Hsu Todd, Peng Ching-Tien, Wang Huang-Joe
Department of Bioscience and Biotechnology, Center of Excellence for the Oceans, National Taiwan Ocean University, Keelung, Taiwan.
Cardiovascular and Translational Medicine Laboratory, Department of Biotechnology, Hungkuang University, Taichung, Taiwan.
Front Physiol. 2017 Dec 8;8:994. doi: 10.3389/fphys.2017.00994. eCollection 2017.
The angiotensin-(1-7)/angiotensin-converting enzyme 2/Mas receptor axis counter-regulates the detrimental effects of angiotensin II. Beneficial effects of angiotensin-(1-7), including anti-inflammation, oxidative stress reduction, and anti-thrombosis, have been reported. Previous studies documented that ramipril decreased thrombin generation in human hypertension and that the anti-thrombotic effects of captopril and losartan were angiotensin-(1-7)-dependent, suggesting an interaction between thrombin and angiotensin-(1-7). However, it is not clear whether angiotensin-(1-7) can alleviate the endothelial phenotypic changes induced by thrombin. We have previously documented cytoskeleton remodeling, cell adhesion, and cell migration as dominant altered phenotypes in thrombin-stimulated human aortic endothelial cells (HAECs). In this study, we investigated whether angiotensin-(1-7) can modulate thrombin-induced phenotypic changes. Furthermore, we investigated whether NAPDH oxidase 5 (Nox5)-produced reactive oxygen species (ROS) play a significant role in angiotensin-(1-7)-mediated phenotypic changes. HAECs were pretreated with 100 nM angiotensin-(1-7) for 1 h, followed by stimulation with 2 units/mL thrombin for different times. Immunofluorescent assay, monocyte adhesion assay, wound-healing assay, ROS assay, real-time PCR, Western blotting, and Nox5 siRNA transfection were conducted. HAECs were pretreated with the ROS scavenger N-acetylcysteine (NAC) to determine whether thrombin-induced phenotypic changes depended on ROS production. Angiotensin-(1-7) prevented thrombin-induced actin cytoskeleton derangements, monocyte adhesion, and migratory impairment. Nox5 siRNA transfection confirmed that thrombin-induced Nox5 expression stimulated ROS production and increased HO-1/NQO-1/ICAM-1/VCAM-1 gene expression, all of which were decreased by angiotensin-(1-7). Phenotypic changes induced by thrombin were prevented by NAC pretreatment. Angiotensin-(1-7) prevents actin cytoskeleton derangement, monocyte adhesion, and migration impairment induced by thrombin via downregulation of ROS production. In addition, thrombin-induced Nox5 expression is involved in the production of ROS, and angiotensin-(1-7) decreases ROS through its inhibitory effect on Nox5 expression.
血管紧张素 -（1 - 7）/血管紧张素转换酶2/ Mas受体轴可对抗血管紧张素II的有害作用。据报道，血管紧张素 -（1 - 7）具有有益作用，包括抗炎、减轻氧化应激和抗血栓形成。先前的研究表明，雷米普利可降低人类高血压患者的凝血酶生成，且卡托普利和氯沙坦的抗血栓作用依赖于血管紧张素 -（1 - 7），这表明凝血酶与血管紧张素 -（1 - 7）之间存在相互作用。然而，尚不清楚血管紧张素 -（1 - 7）是否能减轻凝血酶诱导的内皮细胞表型变化。我们之前记录了细胞骨架重塑、细胞黏附和细胞迁移是凝血酶刺激的人主动脉内皮细胞（HAECs）中主要的改变表型。在本研究中，我们调查了血管紧张素 -（1 - 7）是否能调节凝血酶诱导的表型变化。此外，我们还研究了NADPH氧化酶5（Nox5）产生的活性氧（ROS）在血管紧张素 -（1 - 7）介导的表型变化中是否起重要作用。将HAECs用100 nM血管紧张素 -（1 - 7）预处理1小时，然后用不同时间的2单位/ mL凝血酶刺激。进行了免疫荧光分析、单核细胞黏附分析、伤口愈合分析、ROS分析、实时PCR、蛋白质印迹和Nox5 siRNA转染。用ROS清除剂N - 乙酰半胱氨酸（NAC）预处理HAECs，以确定凝血酶诱导的表型变化是否依赖于ROS的产生。血管紧张素 -（1 - 7）可防止凝血酶诱导的肌动蛋白细胞骨架紊乱、单核细胞黏附和迁移障碍。Nox5 siRNA转染证实，凝血酶诱导的Nox5表达刺激了ROS的产生，并增加了HO - 1/ NQO - 1/ ICAM - 1/ VCAM - 1基因的表达，而血管紧张素 -（1 - 7）均可使其降低。NAC预处理可防止凝血酶诱导的表型变化。血管紧张素 -（1 - 7）通过下调ROS的产生，防止凝血酶诱导的肌动蛋白细胞骨架紊乱、单核细胞黏附和迁移障碍。此外，凝血酶诱导的Nox5表达参与了ROS的产生，而血管紧张素 -（1 - 7）通过对Nox5表达的抑制作用降低了ROS水平。
