Mechanism of Myocardial Angiogenesis in Transgenic/Knockout Animals

描述（由申请人提供）：新生血管形成，即形成新血管的自然生理过程，对于改善经历缺血应激的心脏功能极其重要。这一过程对于治疗缺血性心脏和肢体缺血（包括毛细血管的形成（血管生成）和侧支动脉）具有潜在的重要意义。我们证明心脏的缺血预处理（IP）可以触发血管生成。研究发现，源自含 gp91phox (Nox2) 的 NAD(P)H 氧化酶的活性氧 (ROS) 参与血管生成过程，该过程涉及血管内皮生长因子 (VEGF) 信号传导。 gp91phox 衍生的 ROS 在响应组织缺血的新生血管形成中的作用尚不清楚。为了确定这种氧化还原信号传导，我们将检查IP心脏中主要氧化还原调节基因、硫氧还蛋白和谷氧还蛋白的参与情况。因此，识别可能与血管生长过程相关的无数途径对于开发靶向药物并将治疗从实验室带到临床至关重要。之前的研究，包括我们的研究，也表明短暂性缺血会上调心脏组织中的 VEGF 蛋白。因此，本研究将尝试通过鉴定几种基因敲除动物中 VEGF 信号传导的潜在候选者来解决一个重要的临床问题，例如 Flk-1 /-、Flt-1 /-、gp-91 phox-/-、HO-1 / 、Trx-1 / 、Grx1 / 、Grx1-/- 。这项研究将采用广泛的多学科方法，结合各种技术，包括最新的分子生物学技术、生理学和基因靶向。目标 1. 将研究 GSK-3beta 磷酸化和 β-catenin 易位以及促血管生成分子 VEGF 表达的激活和分子机制。目标 2. 将使用 MK2-/- 等基因工程小鼠通过 IP 中的受体以及体内缺血再灌注心肌来识别 VEGF 信号转导的下游靶标候选者。 目标 3. ROS 介导的应激在 VEGF 通过 Trx 和 Grx 蛋白诱导的血管生成中发挥重要作用。目标 4. 为了深入了解 gp91phox-/- 纯合小鼠中的机制并鉴定参与 VEGF-NAD(P)H 氧化酶信号转导的候选基因，我们将使用 Affymetrix 基因芯片分析。该提案将提供并将确定重要的新见解，以了解 VEGF 信号传导通过重要候选基因提供的心脏保护作用的细胞和分子机制。

DESCRIPTION (provided by applicant): Neovascularization, the natural physiological process of formation of new blood vessels, is extremely important to ameliorate the function of the heart that undergoes ischemic stress. This process is potentially important for the treatment of ischemic heart and limb ischemia which includes formation of capillaries (angiogenesis) and collateral arteries. We demonstrated that ischemic preconditioning (IP) of the heart can trigger angiogenesis. Reactive oxygen species (ROS) derived from gp91phox (Nox2)-containing NAD(P)H oxidase is found to be involved in angiogenic process which involves vascular endothelial growth factor (VEGF) signaling. The role of gp91phox-derived ROS in neovascularization in response to tissue ischemia is unknown. Again to determine this redox signaling we will examine the involvement of principal redox regulated genes, thioredoxin and glutaredoxin in the IP hearts. Discerning the myriad pathways that are possibly associated with this vascular growth process is therefore of utmost importance to develop targeted drugs and bring the therapy from bench to bedside. Previous studies, including ours, also have shown that transient ischemia upregulates VEGF protein in cardiac tissues. Thus, this study will attempt to address an important clinical issue by identifying potential candidates of VEGF signaling in several gene knockout animals such as Flk-1+/-, Flt-1+/-, gp-91 phox-/-, HO-1+/+, Trx-1+/+, Grx1+/+, Grx1-/-. This study will utilize a broad multidisciplinary approach that will combine various techniques, including latest molecular biology techniques, physiology and gene targeting. Aim 1. Activation and molecular mechanism of the GSK-3beta phosphorylation and beta-catenin translocation followed by pro-angiogenic molecule VEGF expression will be investigated. Aim 2. Genetically-engineered mice will be used such as MK2-/- to identify down stream target candidates of VEGF signaling through its receptors in IP as well as ischemic reperfused myocardium in vivo Aim 3. ROS mediated stress plays significant role in VEGF induced angiogenesis through Trx and Grx protein. Aim 4.To gain insights into the mechanisms and to identify candidate genes involved in VEGF- NAD(P)H oxidase signaling in gp91phox-/- homozygous mice we will use Affymetrix Gene chip Analysis. This proposal will provide and will identify important new insights into the cellular and molecular mechanisms of the cardioprotection provided by VEGF signaling through important candidate genes.