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

项目摘要(见说明)：缺血性心脏病(IHD)或心肌缺血，是一种由于心肌供血减少而导致组织缺氧的疾病，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。血管扩张导致的冠状动脉内径增加(急性反应)和血管密度增加(延迟反应)是心肌对缺血损伤的两大防御措施。冠状动脉扩张主要依赖于内皮产生的一氧化氮(NO)，而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，经常观察到活性氧簇(ROS)水平的升高，这导致了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减少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。我们实验室的报告显示，ROS水平的降低抑制了信号转导事件，而信号转导事件对于血管内皮细胞中NO的产生和冠状动脉血管扩张是必不可少的。初步结果还表明，c-Src响应内皮细胞氧化还原水平的变化，促进下游的Pl3K-Akt信号转导，进而激活eNOS，抑制冠状动脉血管内皮细胞中的生长抑制转录因子FOXOi。这一应用将检验一种新的假说，即有条件地增加内皮特异性-ROS将激活c-Src-Pl3K-Akt-eN0S途径并抑制FOXO1，从而在活体心肌缺血模型中导致冠状动脉扩张和血管密度增加。利用新开发的可诱导NOX2有条件表达和血管内皮细胞ROS增加2倍的二元转基因小鼠，我们将确定EC-ROS是否在体外激活c-Src-Pl3K-Akt信号转导，促进小鼠心脏内皮细胞的增殖和迁移(目标1)；EC-ROS是否诱导Pl3K-Akt-eNOS激活、NO合成和冠状动脉血管扩张(目标2)；以及EC-ROS是否在体内LAD结扎模型(LAD)中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).