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，一定数量的活性氧和一定范围内的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，即氧化还原状态向氧化性更强的环境转变，会损害冠状动脉侧枝的生长。先前的研究未能确定细胞类型（或类型），其中氧化还原状态的改变很重要，并且氧化还原信号在其中至关重要。这项建议的总体目标是确定心脏中负责重复缺血时冠状动脉侧枝循环生长中的氧化还原信号的细胞类型或类型。这一目标的一个必然结果是，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细胞类型。为解决这些问题，我们提出以下具体目标：确定哪种细胞类型（或哪种细胞类型）氧化应激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-Cadherin、SM22和心肌蛋白重链（CMHC）诱导冠状动脉内皮、平滑肌细胞和心肌细胞的氧化应激。细胞将被质粒转染（使用体内电穿孔）或用表达iNOS突变体（E371A）的腺病毒转导，该突变体不结合精氨酸，因此只产生O2。目标2。确定氧化还原敏感的p38 MAPK对冠状动脉侧枝生长至关重要的细胞类型。我们将使用表达显性/阴性p38 MAPK （DNp38）的载体，使用针对Aim 1描述的细胞特异性启动子转染或转导冠状动脉内皮细胞、平滑肌细胞和心肌细胞。在确定心脏中对氧化应激和氧化还原信号在冠状动脉侧枝生长中最敏感的特定细胞类型后，我们将把我们的发现扩展到血管病理动物模型（JCR大鼠：代谢综合征模型），该模型显示冠状动脉侧枝生长不良。特别是在最后两个目标中，我们将：目标3。确定通过在JCR大鼠（冠状动脉侧枝生长和氧化应激减少的模型）中过度表达Nrf2来减少氧化应激会恢复侧枝生长的细胞类型。目标4。确定组成活性p38 MAP激酶的表达将恢复JCR大鼠侧枝生长的细胞类型。拟议的研究采用多方面的方法来解决体内细胞特异性信号，通过采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，并最终将这些测量结果与冠状动脉侧枝生长联系起来。这些研究将深入了解ROS和氧化还原信号调节冠状动脉侧枝生长的细胞特异性位置。公共卫生相关性：缺血性心脏病（IHD）仍然是美国死亡的主要原因之一，并且代表了美国医疗保健系统的主要成本。心脏侧支循环发育良好对IHD的发病率和死亡率有巨大影响。侧络发育良好的患者猝死发生率较低，闭塞时梗死灶较小，预后优于侧络不良的患者（低传导或侧络生长的证据）。同样值得注意的是，大约40%的IHD患者很少或没有冠状动脉旁支。人们也认识到，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，发生腐蚀的细胞类型，例如，氧化应激是否破坏心肌细胞生长因子的产生，或者它是否破坏内皮细胞对生长因子的反应，仍然未知。这项建议的目的是描述心脏中氧化还原信号和氧化应激对冠状动脉侧枝生长有影响的细胞类型。该项目的最终目标是为更有针对性的治疗提供基础，以刺激IHD患者冠状动脉侧枝的生长。

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.