The beta3 integrins mediate cellular functions that underlie a number of physiological and pathophysiological processes including angiogenesis which in turn are critical during atherosclerosis, myocardial injury, wound healing, tumor development and inflammatory diseases. Angiogenesis is triggered by interaction of vascular endothelial growth factors (VEGFs) with their receptors on endothelial cells which, in turn, leads to the cell activation and the changes of integrin functions. Despite an importance of the process of angiogenesis, its mechanisms are poorly understood. The overall objective of this proposal is to determine molecular mechanisms of communication between VEGFRs and alpha-v-beta3 integrin in angiogenesis and lymphangiogenesis. The major focus of this proposal is to assess the role of beta3 integrin cytoplasmic domain and its phosphorylation in the regulation of alpha-v-beta3 activity during the process of VEGF-induced angiogenesis, The elucidation of these mechanisms would create the basis for the development of novel therapeutic strategies in cardiology and vascular medicine. Proposed specific aims are:
Aim I. To evaluate the role of beta3 integrin phosphorylation in VEGF-induced angiogenesis in vivo. The angiogenic responses will be assessed in mice expressing a mutated beta3 subunit that impairs its capacity to undergo tyrosine phosphorylation (DiYF) using matrigel and tumor-induced angiogenesis models. These studies will be complemented by unique adenovirus-induced subcutaneous and muscle models developed in our previous studies. We will compare responses initiated by different sets of VEGF receptors, VEGFR-2 and VEGFR-3, and we will utilize two VEGFs, VEGF-A165 and VEGF-D. Through these analyses, we will establish a physiological role of beta3 integrin phosphorylation and dissect how different communication pathways from VEGFRs to integrins influence angiogenesis.
Aim II. To assess the molecular mechanism for the role of beta3 integrin phosphorylation in angiogenesis. In these studies we will utilize endothelial cells of different origin isolated from normal and DiYF mice. We will characterize the adhesive, migratory and proliferative responses of endothelial cells of different origin from normal and DiYF animals to VEGF as compared to other agonists. We will compare normal and DiYF endothelial cells for their ability to form capillary-like structures ex vitro.
Aim III. To further determine the molecular requirements for beta3 integrin subunit to interact with VEGF receptors. In this set of studies, we will focus on the role of beta 3 cytoplasmic domain and its phosphorylation in the VEGF-induced functional responses. We will assess whether beta3 integrin phosphorylation plays a role in VEGF-induced integrin activation. The role of specific intermediates in the signal transduction from different VEGFRs will be considered (including role of talin and skelemin in VEGFRs-induced activation).
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