The Role of RTEF-1 in Angiogenesis and Myocardial Ischemia

描述（由申请人提供）：我们最近证明相关转录增强因子-1（RTEF-1）是内皮细胞中的转录因子。我们还确定了血管生成因子如血管内皮生长因子（VEGF）是RTEF-1在体外的转录靶点。此外，我们确定RTEF-1表达在缺氧条件下增强。该提案的目标是进一步确定RTEF-1在内皮细胞中发挥的作用。我们推测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。我们提出了分子，遗传和基因组的方法来确定细胞和生理作用的RTEF-1在调节内皮依赖性事件在体外和体内。具体而言，在Aim 1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的常见GC丰富区域如何与RTEF-1相互作用。我们将通过GC富集区的结合来关注FGFR 1和考克斯-2启动子的活性，并旨在使用电泳迁移率变动分析（EMSA）和诱变方法来确定与RTEF-1相互作用的结合元件。我们还将研究具有RTEF-1过表达、RTEF-1显性阴性构建体和RTEF-1 siRNA的内皮细胞系，以使用体外血管生成测定来确定RTEF-1在血管生成中的作用。目的2：研究缺氧条件下RTEF-1的调控机制，并尝试识别缺氧内皮细胞中RTEF-1活化的信号通路。我们将使用体内蛋白磷酸化测定、药理学抑制剂和免疫化学测定，通过MAP激酶途径确定RTEF-1表达和磷酸化对缺氧的调节。此外，我们将表征HIF-1a信号对RTEF-1启动子活性的功能后果。最后，在目标3中，我们将通过其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和具有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。我们的方法是一种新的，多学科的转录因子研究范式，这将有助于我们了解RTEF-1的血管作用。

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.