VEGF-induced microvascular hyperpermeability not only represents a physiological consequence of angiogenesis, but also contributes to the development of diabetic retinopathy, ischemic heart disease, reperfusion injury, and tumor metastasis. The precise mechanisms by which VEGF modulates microvascular barrier function have not been established. Recent experimental evidence indicates that endothelial structures, namely VE-cadherin-composed adherens junctions and integrin-mediated cell-matrix focal adhesions are essential to the maintenance of endothelial barrier integrity. However, what molecular reactions mediate the structural changes and whether they are responsible for VEGF-induced microvascular leakage remain as questions. Among the reasons given to the shortcoming of such important information is the technical difficulty in making truly quantification of the permeability in living microvessels under native environment where the endothelium is anchored to the basement membrane. The overall goal of this study is to understand, at the microvascular level, the molecular mechanisms of endothelial cell-cell and cell-matrix interactions in response to VEGF. Intact perfused venules will be used as the primary model to quantitatively assess the barrier property and its regulatory pathways. A central hypothesis states that VEGF stimulation of microvascular endothelium induces biochemical and conformational changes at cell-cell and cell-matrix contacts, characterized by VE-cadherin-dependent junctional disorganization and FAK-mediated focal adhesion redistribution. The two structural components interact with the contractile cytoskeleton resulting in anchorage-dependent gap formation and microvascular hyperpermeability. This hypothesis will be tested by accomplishing the following specific aims: 1) to study the role and molecular basis of cell-cell adhesions in VEGF-induced permeability, 2) to examine the role and molecular basis of cell-matrix adhesions in VEGF-induced permeability, 3) to evaluate the relative importance of cell-cell vs. cell-matrix adhesions in the VEGF response, and 4) to investigate the integrative role of the contractile cytoskeleton in junctional and focal responses to VEGF. The study will provide new insights into the molecular basis of microvascular hyperpermeability. Identification and characterization of structural molecules that serve as ultimate effectors in VEGF-induced barrier dysfunction are fundamental to the understanding of mechanisms underlying angiogenesis, inflammation, and ischemic diseases.
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