Role of the renin angiotensin system in oxidative stress-induced endothelial cell apoptosis

为了探讨肾素血管紧张素系统（RAS）在氧化应激诱导的内皮细胞（EC）凋亡中的作用，我们使用大鼠模型和培养的EC研究RAS调节的作用及其机制。用过氧化氢（H2 O2）处理大鼠颈动脉5分钟，诱导EC凋亡。动脉匀浆中的ACE活性没有增加H2 O2处理，而在24小时与EC剥脱平行下降。H2 O2处理后24小时，通过评估用Hoechst 33342荧光染料染色的enface标本的染色质染色来计数凋亡EC。在H2 O2处理前3天给予ACE抑制剂替莫普利或AT 1受体阻断剂奥美沙坦可抑制EC凋亡。相反，血管紧张素II管理增强H2 O2诱导的EC凋亡。此外，在H2 O2治疗前给予替莫普利抑制2周后发生的新生内膜形成，表明EC凋亡对新生内膜形成的因果影响。接着，用H2 O2处理来源于牛颈动脉的培养EC以诱导凋亡。此外，EC凋亡被抑制，通过添加替莫普利或奥美沙坦，但不受AT 2受体阻滞剂，PD 123319。一氧化氮合酶抑制剂L-NAME不影响替莫普利诱导EC凋亡的作用。H_2O_2刺激ERK、JNK、p38 MAP激酶和Akt活性，30 min达高峰。Temocapril抑制促凋亡丝氨酸/苏氨酸激酶，p38 MAP激酶的活性，但不是其他的。两者合计，它的结论是，血管紧张素II-AT 1受体信号增强EC凋亡和由此产生的血管病变的形成过程中的氧化应激诱导的血管损伤。

To investigate the role of the renin angiotensin system (RAS) in oxidative stress-induced endothelial cell (EC) apoptosis, we examined the effects of RAS modulation and the underlying mechanism using the rat model and cultured EC. EC apoptosis was induced by a 5-minute exposure of hydrogen peroxide (H2O2) into the rat carotid artery. ACE activities in arterial homogenates were not increased by H2O2 treatment, rather were decreased at 24 hours in parallel with EC denudation. At 24 hours after H2O2 treatment, apoptotic EC were counted by evaluating the chromatin staining of enface specimens with Hoechst33342 fluorescent dye. Administration of an ACE inhibitor, temocapril or an AT1 receptor blocker, olmesartan for 3 days before H2O2 treatment inhibited EC apoptosis. Conversely, angiotensin II administration augmented H2O2-induced EC apoptosis. Furthermore, administration of temocapril before H2O2 treatment inhibited neointima formation that occurred 2 weeks later, suggesting the causal influence of EC apoptosis on neointima formation. Next, cultured EC derived from the bovine carotid artery were treated with H2O2 to induce apoptosis. Again, EC apoptosis was inhibited by addition of temocapril or olmesartan, but was not affected by an AT2 receptor blocker, PD123319. Nitric oxide synthase inhibitor, L-NAME did not influence the effects of temocapril on EC apoptosis. H2O2 treatment stimulated the activities of ERK, JNK, p38 MAP kinase and Akt peaking at 30min. Temocapril inhibited the activities of a proapoptotic serin/ threonine kinase, p38 MAP kinase but not of others. Taken together, it is concluded that angiotensin II-AT1 receptor signaling augments EC apoptosis and the resulting vascular lesion formation in the process of oxidative stress-induced vascular injury.