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

项目概述（见说明书）：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。在美国，IHD是导致死亡和发病的主要原因。血管扩张引起的冠状动脉直径增加（急性反应）和血管密度增加（延迟反应）是心肌抵御缺血性损伤的两大防御机制。虽然冠状动脉血管扩张主要依赖于内皮生成的一氧化氮（NO），但毛细血管密度的增加最初需要血管内皮细胞（ECs）的增殖和迁移。在包括IHD在内的许多心血管疾病中经常观察到活性氧（ROS）水平升高，这引起了ROS导致内皮功能障碍的概念。然而，最近使用抗氧化剂（如HOPE、ATBC）的主要干预性临床试验在降低心血管死亡和发病率的主要终点方面大多产生了负面结果。我们实验室的报告表明，ROS水平的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。初步结果还显示，c-Src响应内皮氧化还原水平的变化，促进下游Pl3K-Akt信号传导，进而激活eNOS，抑制冠状动脉内皮细胞中生长抑制转录因子FOXOi。该应用将验证一个新的假设，即内皮特异性ros的条件性增加将激活c-Src-Pl3K-Akt-eN0S通路并抑制fox01，从而导致体内心肌缺血模型的冠状动脉血管扩张和血管密度增加。我们将利用新开发的二元转基因小鼠，诱导血管内皮中Nox2的条件表达和ROS的2倍增加，确定EC-ROS是否激活体外小鼠心脏内皮细胞的c-Src-Pl3K-Akt信号、增殖和迁移（目的1）；EC-ROS是否诱导Pl3K-Akt-eNOS活化、NO合成和冠状动脉血管舒张（Aim 2）；在体内LAD结扎模型中EC-ROS是否会增加缺血心肌的血管密度（Aim 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).