Improvement of Coronary Vascular Functions by Endothelium targeted increase in reactive oxygen species

项目总结（见说明）：缺血性心脏病（IHD）或心肌缺血是一种以心肌供血减少导致组织缺氧为特征的疾病，通常由冠状动脉疾病引起。IHD是美国死亡和发病的主要原因。通过血管扩张（急性反应）增加冠状动脉血管直径和增加血管密度（延迟反应）是心肌对缺血性损伤的两种主要防御。虽然冠状动脉血管舒张主要依赖于内皮产生的一氧化氮（NO），但毛细血管密度的增加最初需要血管内皮细胞（EC）的增殖和迁移。活性氧（ROS）水平的增加经常在许多心血管疾病中观察到，包括IHD，从而产生了ROS导致内皮功能障碍的概念。然而，最近使用抗氧化剂的主要干预性临床试验（例如HOPE、ATBC）在降低心血管死亡和发病率的主要终点方面基本上产生了负面结果。来自我们实验室的报告表明，ROS水平降低抑制了血管内皮中NO生成和冠状动脉血管舒张所必需的信号转导事件。初步结果还表明，c-Src响应于内皮细胞氧化还原水平的变化，并促进下游P13 K-Akt信号传导，这反过来激活eNOS并抑制冠状动脉血管EC中的生长抑制转录因子FOXO 1。本申请将测试一种新的假设，即内皮特异性ROS的条件性增加将激活c-Src-P13 K-Akt-eNOS途径并抑制FOXO 1，从而在体内心肌缺血模型中导致冠状血管舒张和血管密度增加。利用新开发的能诱导Nox 2条件性表达和血管内皮ROS增加2倍的双元转基因小鼠，我们将在体外确定EC-ROS是否激活小鼠心脏EC的c-Src-P13 K-Akt信号传导、增殖和迁移（Aim 1）; EC-ROS是否诱导P13 K-Akt-eNOS激活、NO合成和冠状动脉舒张（Aim 2）;以及EC-ROS是否增加体内LAD结扎模型中缺血心肌的血管密度（目的3）。

PROJECT SUMMARY (See instructions): Ischemic heart disease (IHD) or myocardial ischemia, is a disease characterized by tissue hypoxia due to reduced blood supply to the heart muscle, usually caused by coronary artery disease. IHD is the leading cause of death and morbidity in the USA. Increase in coronary vessel diameter by vasodilatation (acute response), and increase in vessel density (delayed response) are two major defenses of myocardium from ischemic insults. While coronary vasodilatation is primarily dependent on endothelium-generated nitric oxide (NO), the increase in capillary density initially requires proliferation and migration of vascular endothelial cells (ECs). Increased levels of reactive oxygen species (ROS) are often observed in many cardiovascular diseases, including IHD, giving rise to the notion that ROS cause endothelial dysfunction. However, recent major interventional clinical trials using antioxidants (e.g. HOPE, ATBC), have largely produced negative results in reducing primary endpoints of cardiovascular death and morbidity. Reports from our lab demonstrated that reduced ROS levels inhibited signal transduction events that are essential for NO generation in the vascular endothelium and for coronary vasodilatation. Preliminary Results also showed that c-Src responds to changes in endothelial redox levels and promotes downstream Pl3K-Akt signaling, which in turn activates eNOS and inhibits the growth inhibitory transcription factor, FOXOi, in coronary vascular ECs. This application will test a novel HYPOTHESIS that conditional increase in endothelium-specific-ROS will activate c-Src-Pl3K-Akt-eN0S pathway and inhibit FOXO1, and thus, will result in coronary vasodilatation and increased vessel density in a myocardial ischemia model in vivo. Utilizing a newly developed binary transgenic mice that can induce conditional expression of Nox2 and 2-fold increase in ROS in vascular endothelium, we will determine whether EC-ROS activate c-Src-Pl3K-Akt signaling, proliferation and migration of mouse heart ECs in vitro (Aim 1); whether EC-ROS induce Pl3K-Akt-eNOS activation, NO synthesis and coronary vasodilatation (Aim 2); and whether EC-ROS increase vessel density in ischemic myocardium in an LAD ligation model in vivo (Aim 3).