Mechanism of Myocardial Angiogenesis in Transgenic/Knockout Animals

描述（申请人提供）：新生血管形成是新血管形成的自然生理过程，对改善缺血应激心脏的功能至关重要。这一过程对缺血性心脏和肢体缺血的治疗具有潜在的重要意义，包括毛细血管（血管生成）和侧支动脉的形成。我们证明了心脏缺血预处理（IP）可以触发血管生成。由含gp91phox （Nox2）的NAD(P)H氧化酶产生的活性氧（ROS）被发现参与血管生成过程，涉及血管内皮生长因子（VEGF）信号传导。gp91phox来源的ROS在组织缺血时新生血管形成中的作用尚不清楚。同样，为了确定这种氧化还原信号，我们将检查主要的氧化还原调节基因，硫氧还蛋白和glutaredoxin在IP心脏中的参与。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床边至关重要。先前的研究，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，本研究将试图通过在几种基因敲除动物（如Flk-1+/-， Flt-1+/-, gp-91 phox-/-, HO-1+/+, Trx-1+/+, Grx1+/+, Grx1-/-）中识别VEGF信号的潜在候选者来解决一个重要的临床问题。本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。目的1。我们将研究gsk -3 β磷酸化和β -连环蛋白易位的激活和分子机制，以及促血管生成分子VEGF的表达。目标2。基因工程小鼠如MK2-/-将被用于在体内通过其受体在IP和缺血再灌注心肌中识别VEGF信号的下游靶标候选物。ROS介导的应激通过Trx和Grx蛋白在VEGF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VEGF- NAD(P)H氧化酶信号传导的机制和候选基因，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信号通过重要的候选基因提供的心脏保护的细胞和分子机制。

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.