Activation of blood and endothelial cell a5b3 integrin

描述(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发展、血栓形成和血管生成。事实上，有几种整合素是心血管疾病和癌症的治疗靶点。然而，最近的发现表明，我们对整合素在血管生成中的作用，特别是α-β3的作用的了解是不完整的。这一建议的中心假设是，整合素αvbeta3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。αvbeta3的一个重要的生理激活机制是由血管生成生长因子家族通过其受体介导的，这种激活的结果在体内的血管生成反应中将是显而易见的。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。为了验证这一假说，提出了以下具体目标：目的1.确定血管内皮生长因子受体激活αvbeta3的分子机制。A)我们将确定是否只有VEGFR-2利用具有选择性受体活性的生长因子和表达这种VEGF受体的细胞株来介导αvbeta3的激活；b)知道VEGFR-2可以激活αvbeta3，我们将剖析VEGFR-2利用表达突变的VEGFR-2受体的细胞来传递导致αvbeta3激活的信号的需求；c)将使用Beta3整合素亚基的细胞质尾部的截短和突变形式来确定接收激活信号的结构要求；d)将评估αvbeta3与VEGFR-2之间的物理联系的要求，并将其与VEGFR-1进行比较。目的II.研究整合素Alphavbeta3在体外和体内激活的功能后果。我们将确定：a)由血管内皮生长因子激活的αvbeta3如何控制特定生理配体的识别；以及b)avb3激活是否发生在体内的损伤或治疗性血管生成部位。瞄准伊犁。目的：探讨Akt通路在体内外两种β3整合素激活中的作用。我们将：a)利用转基因细胞在体外操纵Akt-1的活性，并确定其在αvbeta3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是否是激活两种整合素β3所必需的；c)我们将表征Akt在体内αvbeta3激活中的作用，在正常和Akt-1缺失动物中，以及在体内alphavbeta3激活在血小板介导的反应中的作用。我们努力确定β3整合素激活的机制和意义，应该为整合素在生理和病理生理环境中的功能调节提供新的见解，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.