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 I lI. 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.
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