Epac1 Signaling in Angiogenesis

描述(由申请人提供)：血管生成，即从先前存在的血管系统发育而来的血管，在许多疾病中是关键的。增强血管生成有利于心脏和脑缺血以及伤口愈合，而抗血管生成策略正被用于癌症和视网膜病变。腺苷通过腺苷受体(AR)作用，调节缺氧时产生的血管生成。血管生成的关键步骤是内皮细胞(EC)的增殖，而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可诱导人脐静脉内皮细胞(HUVEC)和人微血管内皮细胞(HMVEC)增殖并激活ERK1/2。此外，我们发现最近发现的Epac1蛋白是ERK1/2激活所必需的，以响应A2BAR的激活。Epac1是一种针对RapGTP酶的鸟嘌呤核苷酸交换因子，它为cAMP依赖的、蛋白激酶A非依赖的反应提供了基础。该建议的目的是描述导致ERK1/2激活的Epac1下游的信号通路，并确定Epac1及其下游蛋白的直接和A2BAR介导的激活是否导致EC增殖和管状形成。HUVEC和HMVEC，统称为ECs将在所有研究中使用。在特定的目标1中，假设Epac1通过涉及RapGTP酶(S)和B-Raf的级联作用在ECs中向ERK1/2发出信号。这将通过一种策略来探索，该策略使用Rap下拉试验，表达结构性的活性和显性的负性Rap蛋白和siRNA，以抑制特定的Rap亚型和B-Raf的表达。此外，还将分析关键蛋白激活的亚细胞位置。在特定的目标2中，假设Epac1及其相关的下游蛋白促进EC增殖。我们将确定特定的Epac1激活剂和过度表达Epac1对细胞增殖的影响。此外，siRNA和显性负结构将被用来确定Epac1和相关蛋白在A2BAR介导的增殖中的必要性。在具体目标3中，Epac1的作用将在血管生成的体外模型--内皮管形成中确定。这项建议将进一步了解Epac1在EC生物学中的功能，重点放在尚未探索的增殖和血管生成领域。 公共卫生相关性：新血管的发展对不同的疾病可能是有益的，也可能是有害的。这种发育需要内皮细胞的生长和组装，内皮细胞构成血管的内层。该项目研究了一种名为EPAC的蛋白质在这些内皮细胞的生长和有序组装中的作用。

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.