Reactive nitrogen species in shear-dependent signaling

层流切应力通过控制内皮细胞的结构和功能来保护动脉壁免受动脉粥样硬化斑块的发展。层流剪切的抗动脉粥样硬化作用涉及内皮细胞产生NO、活性氧(ROS)和活性氮(RNS)等血管活性因子。然而，1)层流剪切刺激eNOS产生NO、NADPH氧化酶产生ROS及其反应产物RNS的机制尚不清楚，2)这些自由基介导层流剪切抗动脉粥样硬化作用的机制尚不清楚。正如初步结果所详述的，我们最近的结果有力地表明，剪切力通过一种意想不到的机制调节eNOS的活性。与以前的假设不同的是，蛋白激酶B(Akt)是负责eNOS Ser1179残基磷酸化的蛋白激酶，我们证明了其他酶，如蛋白激酶A，负责eNOS-Ser1179的磷酸化。考虑到eNOS中存在多个潜在的磷酸化位点，有必要重新研究磷酸化/去磷酸化调控eNOS对剪切反应的详细机制。为了解决这些问题，我们从正常小鼠和几个eNOS和NADPH氧化酶成分(GP 91和P47)缺陷的基因敲除(KO)小鼠中培养了小鼠主动脉内皮细胞。利用这些内皮细胞以及转导突变的eNOS结构的eNOS-KO细胞，我们将检验假设1)层流剪切通过磷脂酰3-激酶和蛋白激酶A依赖的机制通过控制eNOS的关键调节位点的磷酸化来刺激NO的产生，以及2)剪切通过NO、ROS和RNS依赖的机制阻止促动脉粥样硬化因子诱导的内皮细胞死亡。这些假说将通过以下四个目标得到验证：目标1：确定调控eNOS剪切依赖的磷酸化和激活的信号通路。目的2：确定层流剪切对RNS产生的影响。目的4：明确NO和RNS在内皮细胞层流剪切依赖性抗动脉粥样硬化反应中的作用。

Laminar shear stress protects arterial wall from the development of atherosclerotic plaques by controlling the structure and function of endothelial cells. The anti-atherogenic effects of laminar shear involve production of vasoactive factors such as NO, reactive oxygen species (ROS) and reactive nitrogen species (RNS) in endothelial cells. However, the mechanisms 1) by which laminar shear stimulates production of NO from eNOS, ROS from NADPH oxidases, and their reaction products, RNS, and 2) by which these free radicals mediate the anti-atherogenic effects of laminar shear are not known. As detailed in the Preliminary results, our recent results strongly suggest that shear stress regulates eNOS activity by an unexpected mechanisms. Unlike the previous assumption that protein kinase B(Akt) would be the protein kinase responsible for phosphorylation of eNOS at Ser1179 resi8due, we show that other kinases such as protein kinase A is responsible for phosphorylation of eNOS-Ser1179. Considering the presence of multiple potential phosphorylation sites found in eNOS, it is important to res examine the detailed mechanisms by which phosphorylation/dephosphorylation regulate eNOS in response to shear. To address these questions, we have developed mouse aortic endothelial cells from normal and several knockout (KO) mice deficient in eNOS and NADPH oxidase components (gp 91 and p47). Using these endothelial cells as well as the eNOS-KO cells transfected with mutated eNOS constructs, we will examines the hypotheses 1) that laminar shear stimulates NO production by controlling by phosphorylation of key regulatory sites of eNOS by the phosphatidyl-3-kinase and protein kinase A-dependent mechanisms, and 2) that shear prevents endothelial cell death induced by pro-atherogenic factors by the NO, ROS, and RNS- dependent mechanisms. These hypotheses will be examined by following the four aims: Aim 1: Determine the signaling pathways regulating shear- dependent phosphorylation and activation of eNOS. Aim 2: Determine the effect of laminar shear on RNS productions. Aim 4: Define the role of NO and RNS in laminar shear-dependent, anti-atherogenic responses of endothelial cells.