aDepartment of Applied Clinical Sciences and Biotechnology, University of L'Aquila bDepartment MeSVA, University of L'Aquila, L'Aquila, Italy cDepartment of Molecular Immunology, Virology, and Medical Genetics, The Ohio State University, Columbus, Ohio, USA dPreclinical Development Department, Menarini Ricerche, Firenze, Italy *Francesco Marampon and Giovanni L. Gravina contributed equally to the writing of this article.
J Hypertens. 2013 Oct;31(10):1972-83. doi: 10.1097/HJH.0b013e3283638b32.
Oxidative stress has been linked to endothelial dysfunction and angiotensin II stimulates the reactive oxygen species production contributing to several cardiovascular diseases. We have studied the chain of events induced by angiotensin-converting-enzyme (ACE) activation in vascular umbilical vein endothelial cells (HUVECs) by using an ACE inhibitor such as zofenoprilat.
We used specific assay to measure the superoxide anion production, tetrazolium bromide (MTT) assay for cell viability, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay for cell apoptosis, and western blot for protein analysis in the study.
Zofenoprilat counteracts the superoxide anion production and cell apoptosis induced by angiotensin I treatment by blocking the extrinsic caspase cascade, NF-kB and p38 activation. p38 inhibitor SB203580 reverted the angiotensin II oxidant effects while the p38 constitutively activation, by MKK6 transfection, abrogated the zofenoprilat effects. Characterizing the zofenoprilat downstream effector we found that zofenoprilat reverted the SirT-1 downregulation induced by angiotensin II. p38 activation by angiotensin II was strictly correlated with SirT1 protein downregulation; SB203580 significantly prevented SirT1 downregulation induced by angiotensin II while the p38 constitutive activation abolished SIRT1 protein basal levels. p38 directly bound SirT1 sequestering it in the cytoplasm. SirT1 inhibition by sirtinol annulled zofenoprilat action while SirT1 overexpression reverted the cytotoxic effects of angiotensin II. Finally, zofenoprilat negatively controlled angiotensin I receptor protein expression through SirT1.
The p38-SirT1 axis is found markedly relevant in modulating the cardiovascular benefit deriving from ACE-inhibitors and might represent a novel target for innovative drugs in cardiovascular prevention.
氧化应激与血管内皮功能障碍有关，血管紧张素Ⅱ刺激活性氧的产生，导致多种心血管疾病。我们使用血管紧张素转换酶（ACE）抑制剂佐芬普利拉研究了血管脐静脉内皮细胞（HUVEC）中 ACE 激活诱导的一系列事件。
我们使用特定的测定法来测量超氧阴离子的产生、四唑溴盐（MTT）测定法来测量细胞活力、末端脱氧核苷酸转移酶介导的 dUTP 缺口末端标记（TUNEL）测定法来测量细胞凋亡，以及蛋白质分析的 Western blot。
佐芬普利拉通过阻断外源性半胱天冬酶级联、NF-κB 和 p38 激活来拮抗血管紧张素 I 处理诱导的超氧阴离子产生和细胞凋亡。p38 抑制剂 SB203580 逆转了血管紧张素 II 的氧化作用，而 MKK6 转染导致 p38 的组成性激活，则消除了佐芬普利拉的作用。在确定佐芬普利拉的下游效应物时，我们发现佐芬普利拉逆转了血管紧张素 II 诱导的 SirT-1 下调。血管紧张素 II 对 p38 的激活与 SirT1 蛋白下调严格相关；SB203580 显著阻止了血管紧张素 II 诱导的 SirT1 下调，而 p38 的组成性激活则消除了 SirT1 蛋白的基础水平。p38 直接结合 SirT1，将其隔离在细胞质中。SirT1 抑制剂 sirtinol 消除了佐芬普利拉的作用，而 SirT1 的过表达则逆转了血管紧张素 II 的细胞毒性作用。最后，佐芬普利拉通过 SirT1 负调控血管紧张素 I 受体蛋白的表达。
p38-SirT1 轴在调节 ACE 抑制剂带来的心血管益处方面具有重要意义，可能成为心血管预防中新型药物的新靶点。
