Activation of blood and endothelial cell a5b3 integrin

描述（由申请人提供）：细胞表面分子家族，整合素，介导多种生理和病理反应，包括炎症，动脉粥样硬化和癌症的发展，血栓形成和血管生成。事实上，一些整合素是心血管疾病和癌症的治疗靶点。然而，最近的研究结果表明，我们对整合素在血管生成中的作用的理解是不完整的，尤其是α - β 3。该建议的中心假设是整合素α β 3与基质配体的细胞相互作用受到严格控制，并取决于受体的激活状态。一个生理上重要的激活机制是由VEGF家族的血管生成生长因子通过其受体介导的，这种激活的后果将在体内血管生成反应中得到明显体现。Akt将在alphavbeta3的激活中发挥关键作用，通过比较Akt基因失活对alphavbeta3和alphaIIbeta3功能的影响，可以最令人信服地证明这一作用。我们提出以下具体目的来验证这一假设：目的1 .确定vegfr激活alphavbeta3的分子机制。a)我们将使用具有选择性受体活性的生长因子以及表达该VEGF受体的细胞系来确定alphavbet3激活是否仅由VEGFR-2介导；b)了解到VEGFR-2可以激活alphavbeta3，我们将分析VEGFR-2使用表达突变体VEGFR-2受体的细胞传递导致alphavbeta3激活的信号的要求；c) β a3整合素亚基接收激活信号的结构要求将通过β a3整合素亚基细胞质尾部的截短和突变形式来确定；d)将评估alphavbet3和VEGFR-2之间是否存在物理关联，并与VEGFR-1进行比较。目的二世。在体外和体内研究整合素α β 3激活的功能后果。我们将确定：a) VEGF如何激活alphavbeta3控制对特定生理配体的识别；b) avb3激活是否发生在体内损伤部位或治疗性血管生成部位。目的伊犁。建立Akt通路在体内外活化两种β a3整合素中的作用。我们将：a)使用转染的细胞在体外操纵Akt-1的活性，并确定其在alphavbeta3激活中的作用；b)结合对转染细胞和Akt-1缺失动物细胞的分析，确定Akt是否需要激活这两种β a3整合素；c)我们将描述Akt在正常和Akt-1缺失动物体内vegf刺激血管生成过程中对alphavbeta3激活的作用，以及在血小板介导的体内反应中对alphavbeta3激活的作用。我们确定β 3整合素激活的机制和意义的努力将为生理和病理生理环境下整合素功能的调节提供新的见解，并确定新的治疗靶点。

DESCRIPTION (provided by applicant): The family of cell surface molecules, the integrins, mediates multiple physiological and pathological responses, including inflammation, atherosclerosis and cancer development, thrombus formation and angiogenesis. Indeed, several integrins are therapeutic targets for cardiovascular diseases and for cancer. However, recent findings indicate that our understanding of the role of integrins in general and alphavbeta3 in particular in angiogenesis is incomplete. The central hypothesis of this proposal is that cellular interaction of integrin alphavbeta3 with matrix ligands is tightly controlled and depends on the activation state of the receptor. A physiologically important activation mechanism of alphavbeta3 is mediated by angiogenic growth factors of the VEGF family acting through their receptors, and the consequence of this activation will be evident in the angiogenic response in vivo. Akt will play a pivotal role in activation of alphavbeta3 and this role can be most convincingly demonstrated by comparing the consequences of inactivation of the Akt gene on the function of alphavbeta3 and alphaIIbeta3. The following Specific Aims are proposed to test this hypothesis: Aim I. To determine the molecular mechanisms for alphavbeta3 activation by VEGFRs. a) We will determine whether alphavbeta3 activation is mediated exclusively by VEGFR-2 using growth factors with selective receptor activity as well as cell lines expressing this VEGF receptor; b) Knowing that VEGFR-2 can activate alphavbeta3, we will dissect the requirements for VEGFR-2 to transmit a signal leading to alphavbeta3 activation using cells expressing mutant VEGFR-2 receptors; c) The structural requirements for beta3 integrin subunit to receive an activating signal will be determined using truncated and mutated forms of the cytoplasmic tail of the beta3 integrin subunit; d) the requirement for a physical association between alphavbeta3 and VEGFR-2 will be assessed and compared to that of VEGFR-1. Aim II. To characterize the functional consequences of activation of integrin alphavbeta3 in vitro and in vivo. We will determine: a) how alphavbeta3 activation by VEGF controls the recognition of specific physiological ligands; and b) whether avb3 activation occurs in sites of injury or therapeutic angiogenesis in vivo. Aim IlI. To establish the role of the Akt pathway in activation of the two beta3 integrins in vitro and in vivo. We will: a) manipulate the activity of Akt-1 in vitro using transfected cells and determine its role in alphavbeta3 activation; b) combine an analysis of transfected cells and cells from Akt-1 null animals to determine if Akt is necessary for the activation of both beta3 integrins; c) We will characterize the role of Akt in alphavbeta3 activation in vivo in the process of VEGF-stimulated angiogenesis in normal and Akt-1 null animals and in alphavbeta3 activation in platelet mediated responses in vivo. Our efforts to determine the mechanisms and significance of beta3 integrin activation should provide new insights into regulation of integrin functions in physiological and pathophysiological settings and identify new targets for therapy.