Reactive Oxygen Species in Coronary Collateral Growth

描述(由申请人提供)：我们和其他实验室的结果表明，在特定边界内的临界量的ROS和氧化还原状态对冠状动脉侧枝生长至关重要；然而，氧化应激，即从氧化还原状态转变到更氧化的环境，会损害冠脉侧枝生长。以前的研究没有确定细胞类型，在这些类型中，氧化还原状态的改变是重要的，而氧化还原信号是关键的。这项建议的首要目标是确定心脏中负责氧化还原信号的一个或多个细胞类型，这些细胞在冠脉侧支循环的增长中对反复缺血做出反应。这一目标的必然结果是，我们还将确定氧化应激对冠状动脉侧枝生长产生负面影响的一个或多个细胞类型。为了解决这些问题，我们提出了以下具体目标：目的1.确定氧化应激在哪种细胞类型(或哪几种类型)中破坏冠状动脉侧枝生长。我们将分别使用细胞特异性启动子VE-钙粘附素、SM22和心肌肌球蛋白重链(CMHC)诱导冠状动脉内皮细胞、平滑肌细胞和心肌细胞的氧化应激。细胞将被导入(使用体内电穿孔)质粒或转导表达iNOS突变体(E371a)的腺病毒，该突变体不结合精氨酸，因此只产生O2。目的2.确定氧化还原敏感的p38MAPK在哪种细胞类型(或几种类型)中对冠状动脉侧枝生长至关重要。我们将使用针对目标1的细胞特异性启动子，利用表达显性/阴性p38MAPK(DNp38)的载体，对冠状动脉内皮细胞、平滑肌细胞和心肌细胞进行转基因或转导。在确定了心脏中对氧化应激和氧化还原信号在冠脉侧支生长中的影响最敏感的特定细胞类型之后，我们将把我们的发现扩展到血管病理的动物模型(JCR大鼠：代谢综合征的模型)，该模型显示冠脉侧支生长不良。具体地说，在最后两个目标中，我们将：目标3.确定通过过度表达Nrf2在JCR大鼠(冠状动脉侧支生长和氧化应激减少的模型)中减少氧化应激将恢复侧支生长的细胞类型。目的4.确定在JCR大鼠中，表达具有结构性活性的p38MAPK将恢复侧枝生长的细胞类型。这项拟议的研究采用了一种多方面的方法来解决体内细胞特异的信号传递问题，方法是使用技术来确定蛋白质表达、硫醇氧化和ROS产生的细胞特异性变化，并最终将这些测量与冠状动脉侧支生长联系起来。这些研究将深入了解ROS和氧化还原信号调节冠脉侧支生长的细胞特异性位置。公共卫生相关性：缺血性心脏病(IHD)仍然是美国主要的死亡原因之一，也是美国医疗保健系统的主要成本。心脏有发达的侧支循环对IHD的发病率和死亡率有很大影响。侧支发育良好的患者猝死的发生率较低，在闭塞的情况下梗塞较小，与侧支循环不良(低电导或侧支生长的证据)的患者相比，预后更好。同样值得注意的是，大约40%的IHD患者几乎没有冠脉侧支。人们还认识到，许多IHD的危险因素，如高血压、肥胖、血脂异常，也会产生氧化应激，对冠状动脉侧枝生长有负面影响。然而，发生腐败的细胞类型仍不清楚，例如，氧化应激是否破坏了心肌细胞产生生长因子，或者它是否破坏了内皮细胞对生长因子的反应。这项建议的目标是描绘心脏中氧化还原信号和氧化应激对冠状动脉侧枝生长起作用的细胞类型。该项目的最终目标是为更多的定向治疗提供基础，以刺激IHD患者的冠状动脉侧支生长。

DESCRIPTION (provided by applicant): Results from our and other laboratories demonstrate that a critical amount of ROS and a redox state within a certain boundary is critical for coronary collateral growth; however, oxidative stress, i.e., a shift in the redox state to a more oxidative environment, impairs coronary collateral growth. Previous investigations fall short of ascertaining the cell type (or types), in which alterations of redox state matter, and where redox signaling is critical. The overarching goal of this proposal is to determine the cell type or types in the heart responsible for redox signaling in the growth of the coronary collateral circulation in response to repetitive ischemia. A corollary to this aim is that we will also determine the cell type or types in which oxidative stress confers negative influences on coronary collateral growth. To solve these problems we propose the following specific aims: Aim 1. Determine in which cell type (or types) does oxidative stress corrupt coronary collateral growth. We will induce oxidative stress in the coronary endothelium, smooth muscle cells and cardiac myocytes using the cell-specific promoters VE-Cadherin, SM22, and cardiac myosin heavy chain (CMHC), respectively. Cells will be transfected with a plasmid (using in vivo electroporation) or transduced with an adenovirus expressing an iNOS mutant (E371A) that does not bind arginine, and therefore, only produces O2. Aim 2. Determine in which cell type (or types) the redox sensitive p38 MAPK is critical for coronary collateral growth. We will transfect or transduce coronary endothelium, smooth muscle cells and cardiac myocytes using cell-specific promoters described for Aim 1 using a vector expressing a dominant/negative p38 MAPK (DNp38). After determining the particular cell type in the heart most sensitive to the effects of oxidative stress and redox signaling in coronary collateral growth, we will extend our findings to an animal model of vascular pathology (the JCR rat: a model of the metabolic syndrome), which demonstrates poor coronary collateral growth. Specifically in the final two aims we will: Aim 3. Determine the cell type where reducing oxidative stress by over expressing Nrf2 in the JCR rat (a model of reduced coronary collateral growth and oxidative stress) will restore collateral growth. Aim 4. Determine the cell type where expression of a constitutively active p38 MAP kinase will restore collateral growth in the JCR rat. The proposed studies employ a multifaceted approach to solving cell-specific signaling in vivo by employing techniques to determine cell specific changes in protein expression, thiol oxidation and ROS production and ultimately linking these measurements to coronary collateral growth. These studies will provide insight into the cell-specific locations where ROS and redox signaling modulate coronary collateral growth. PUBLIC HEALTH RELEVANCE: Ischemic heart disease (IHD) continues to be one of the leading causes of death in the United States, and represents a major cost in the US healthcare system. The presence of a well developed collateral circulation in the heart has a tremendous impact on the morbidity and mortality of IHD. Patients with well developed collaterals show a lower incidence of sudden death, have smaller infarcts in the event of an occlusion, and demonstrate a better prognosis than patients with poor collaterals (low conductance or evidence of collateral growth). It is also worth noting that about 40% of patients with IHD demonstrate very little to no coronary collaterals. It is also recognized that many risk factors for IHD, e.g., hypertension, obesity, dyslipidemia, which also produce oxidative stress, have a negative influence on coronary collateral growth. However, the cell type where the corruption occurs, e.g., does oxidative stress corrupt the production of growth factors by cardiac myocytes, or does it corrupt endothelial responsiveness to growth factors, remains unknown. The goal of this proposal is to delineate the cell type in the heart where redox signaling and oxidative stress matter for coronary collateral growth. The ultimate goal of this project is to provide the foundation for more directed therapies to stimulate the growth of coronary collaterals in patients with IHD.