Mechanism of Myocardial Angiogenesis in Transgenic/Knockout Animals

描述(由申请人提供)：新生血管是新血管形成的自然生理过程，对于改善经历缺血应激的心脏功能极其重要。这一过程对治疗心脏和肢体缺血具有潜在的重要意义，包括毛细血管的形成(血管生成)和侧支动脉的形成。我们证明了心脏的缺血预适应(IP)可以触发血管生成。含有gp91Phox(NOX2)的NAD(P)H氧化酶产生的活性氧(ROS)参与了涉及血管内皮生长因子(VEGF)信号转导的血管生成过程。Gp91Phox衍生的ROS在组织缺血反应中的新生血管中的作用尚不清楚。为了再次确定这个氧化还原信号，我们将检查主要的氧化还原调节基因，硫氧还蛋白和谷氧还蛋白在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，对于开发靶向药物和将治疗从工作台推向床边是至关重要的。以前的研究，包括我们的研究，也表明短暂的缺血会上调心脏组织中的血管内皮生长因子蛋白。因此，本研究试图通过在几种基因敲除动物中确定潜在的候选血管内皮生长因子信号，如Flk-1/-、Flt-1/-、GP-91Phox-/-、HO-1/、Trx-1/、Grx1/、Grx1-/-，以解决一个重要的临床问题。这项研究将利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.研究GSK-3β磷酸化、β-连环蛋白转位及促血管生成分子血管内皮生长因子表达的激活及其分子机制。目的2.基因工程小鼠(如MK2-/-)将通过其受体在体内IP和缺血再灌注心肌中寻找VEGF信号的下游候选靶点。目的3.ROS介导的应激通过TRX和GRX蛋白在血管生成中发挥重要作用。目的4.利用Affymetrix基因芯片技术对gp91Phox/-纯合子小鼠血管内皮生长因子-NAD(P)H氧化酶信号转导相关候选基因进行分析。这项建议将为血管内皮生长因子通过重要候选基因提供心脏保护信号的细胞和分子机制提供并将确定重要的新见解。

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.