Epac1 Signaling in Angiogenesis

描述（由申请人提供）：血管生成，即从已有的血管系统中发展血管，在许多疾病中都是至关重要的。增强血管生成有利于心脏和脑缺血和伤口愈合，而抗血管生成策略正在研究癌症和视网膜病变。通过腺苷受体（AR），腺苷调节血管生成，因为它是在缺氧时产生的。血管生成的一个关键步骤是内皮细胞（EC）的增殖和细胞外信号调节激酶（ERK1/2）的激活被认为是一个关键的潜在事件。我们的研究表明，A2BAR诱导人脐静脉内皮细胞（HUVEC）和人微血管内皮细胞（HMVEC）增殖和ERK1/2活化。此外，我们发现最近发现的Epac1蛋白是响应A2BAR激活的ERK1/2刺激所必需的。Epac1是RapGTPases的鸟嘌呤核苷酸交换因子，为camp依赖性、蛋白激酶a依赖性应答提供了基础。本提案的目的是描述导致ERK1/2激活的Epac1下游信号通路，并确定直接和a2bar介导的Epac1及其下游蛋白的激活是否导致EC增殖和管形成。HUVEC和HMVEC，统称为ec，将在所有研究中使用。在Specific Aim 1中，假设Epac1通过涉及RapGTPase(s)和B-Raf的级联向ECs中的ERK1/2发出信号。这将通过采用Rap下拉试验，表达构成活性和显性负Rap蛋白和siRNA来敲低特定Rap异构体和B-Raf的表达来探索。此外，将分析关键蛋白激活的亚细胞位置。在Specific Aim 2中，假设Epac1和相关的下游蛋白促进EC增殖。我们将确定Epac1特异性激活因子和Epac1过表达对细胞增殖的影响。此外，siRNA和显性阴性结构将用于确定Epac1和相关蛋白在a2bar介导的增殖中的必要性。在Specific Aim 3中，Epac1的作用将在内皮管形成（一种体外血管生成模型）中被确定。该提案将进一步了解Epac1在EC生物学中的功能，重点关注未开发的增殖和血管生成领域。

DESCRIPTION (provided by applicant): Angiogenesis, the development of blood vessels from pre-existing vasculature, is critical in many diseases. Enhancement of angiogenesis is beneficial in cardiac and cerebral ischemia and wound healing, while anti-angiogenic strategies are being examined in cancer and retinopathies. Acting via adenosine receptors (AR), adenosine regulates angiogenesis as it is produced during hypoxia. A critical step in angiogenesis is endothelial cell (EC) proliferation and activation of extracellular signal regulated kinase (ERK1/2) is considered to be a key underlying event. Our studies show that the A2BAR elicits proliferation and ERK1/2 activation in human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells (HMVEC). Furthermore, we found the recently discovered Epac1 protein is required for ERK1/2 stimulation in response to A2BAR activation. Epac1 is a guanine nucleotide exchange factor for RapGTPases and provides a basis for cAMP-dependent, Protein Kinase A independent responses. The goal of this proposal is to delineate the signaling pathway downstream of Epac1 that results in ERK1/2 activation and to determine if direct and A2BAR-mediated activation of Epac1 and downstream proteins results in EC proliferation and tube formation. Both HUVEC and HMVEC, collectively referred to as ECs will be used in all studies. In Specific Aim 1, it is hypothesized that Epac1 signals to ERK1/2 in ECs via a cascade involving RapGTPase(s) and B-Raf. This will be explored by a strategy employing Rap pull-down assays, expression of constitutively active and dominant negative Rap proteins and siRNA to knockdown expression of specific Rap isoforms and B-Raf. Additionally, the subcellular location of activation of key proteins will be analyzed. In Specific Aim 2, it is hypothesized that Epac1 and associated downstream proteins promote EC proliferation. We will determine the effects of a specific Epac1 activator and overexpression of Epac1 on proliferation. Additionally, siRNA and dominant negative constructs will be used to determine the necessity of Epac1 and associated proteins in A2BAR-mediated proliferation. In Specific Aim 3, the effects of Epac1 will be determined in endothelial tube formation, an in vitro model of angiogenesis. This proposal will further an understanding of Epac1 function in EC biology with a focus on the unexplored areas of proliferation and angiogenesis. PUBLIC HEALTH RELEVANCE: The development of new blood vessels may be beneficial or detrimental in different diseases. This development requires the growth and assembly of endothelial cells which form the inner lining of blood vessels. This project examines the role of a protein known as EPAC in growth and ordered assembly of these endothelial cells.