The Role of RTEF-1 in Angiogenesis and Myocardial Ischemia

描述（由申请人提供）：我们最近证明了相关转录增强因子-1 （RTEF-1）是内皮细胞中的转录因子。我们还确定血管生成因子如血管内皮生长因子（VEGF）是体外RTEF-1的转录靶点。此外，我们发现RTEF-1在缺氧条件下表达增强。本研究的目的是进一步明确RTEF-1在内皮细胞中的作用。我们假设RTEF-1参与了缺氧相关的血管生成过程。推测RTEF-1通过其靶基因的血管生成活性在改善心肌缺血损伤中起重要作用。我们提出分子、遗传和基因组方法来确定RTEF-1在体外和体内内皮依赖事件调节中的细胞和生理作用。具体来说，在Aim1中，我们将确定内皮细胞中RTEF-1的靶基因，并试图了解这些基因启动子中常见的富含gc的区域如何与RTEF-1相互作用。我们将重点关注FGFR1和COX-2启动子通过GC富区结合的活性，并旨在通过电泳迁移量转移测定（emsa）和诱变方法定义与RTEF-1相互作用的结合元件。我们还将研究RTEF-1过表达、RTEF-1显性阴性结构和RTEF-1 siRNA的内皮细胞系，通过体外血管生成实验确定RTEF-1在血管生成中的作用。在Aim 2中，我们将确定缺氧条件下RTEF-1的调控机制，并试图确定缺氧内皮细胞中RTEF-1激活的信号通路。我们将通过体内蛋白磷酸化试验、药物抑制剂和免疫化学试验，通过MAP激酶途径确定RTEF-1表达和磷酸化在缺氧反应中的调节。此外，我们将描述HIF-1a信号对RTEF-1启动子活性的功能影响。最后，在Aim 3中，我们将通过RTEF-1靶基因的血管生成作用来研究RTEF-1在心肌缺血中的作用。我们将在心肌缺血模型中使用逆转录病毒-RTEF-1和带有ve -钙粘蛋白启动子的RTEF-1转基因小鼠来确定血管生成反应。我们的方法是转录因子研究的一种新的多学科范式，这将有助于我们理解RTEF-1在血管中的作用。

DESCRIPTION (provided by applicant): We have recently demonstrated that Related Transcription Enhance Factor-1 (RTEF-1) is a transcription factor in endothelial cells. We have also determined that angiogenic factors such as Vascular Endothelial Growth Factor (VEGF) is a transcriptional target of RTEF-1 in vitro. In addition, we established that RTEF-1 expression is enhanced under hypoxic conditions. The goal in this proposal is to further define the role played by RTEF-1 in endothelial cells. We hypothesize that RTEF-1 is involved in the hypoxia-related angiogenesis process. Presumably RTEF-1 plays an important role in improvement of myocardial ischemic injury via angiogenic activities of its target genes. We propose molecular, genetic, and genomic approaches to determine the cellular and physiologic role of RTEF-1 in regulation of endothelium-dependent events in vitro and in vivo. Specifically, in Aim1, we will identify the target genes of RTEF-1 in endothelial cells and attempt to understand how common GC-rich areas of these genes' promoter interact with RTEF-1. We will focus on FGFR1 and COX-2 promoter activity though the binding of the GC rich area, and aim to define the binding elements interacting with RTEF-1 using electrophoretic mobility shift assays (EMSAs) and mutagenesis methods. We will also study the endothelial cell lines with RTEF-1 overexpression, RTEF-1 dominant-negative construct and RTEF-1 siRNA to determine the role of RTEF-1 in angiogenesis using in vitro angiogenesis assays. In Aim 2, we will determine the mechanism of regulation of RTEF-1 in hypoxia and attempt to identify signaling pathways in RTEF-1 activation in hypoxic endothelial cells. We will determine the modulation of RTEF-1 expression and phosphorylation in response to hypoxia via MAP kinase pathway using in vivo protein phosphorylation assay, pharmacological inhibitors and immunochemical assays. Further, we will characterize the functional consequences of HIF-1a signaling on RTEF-1 promoter activity. Finally, in Aim 3, we will examine the role of RTEF-1 in myocardial ischemia through the angiogenic effects of its target genes. We will use retrovirus-RTEF-1 and RTEF-1 trangenic mice with VE-cadherin promoter in a myocardial ischemia model to determine angiogenic response. Our approach is a new, multidisciplinary paradigm for the study of transcription factors that will be of an obvious benefit in helping us understand the vascular role of RTEF-1.