Diabetes mellitus affects 1-5% of the world's population and is the major cause of new blindness. Diabetic blindness is frequently characterized by proliferative retinopathy, which is associated with increased vascular permeability and neovascularization. Vascular endothelial growth factor (VEGF), an endothelial cell (CE) specific mitogen, is a major contributor to this disease process. VEGF initiates cell proliferation by binding to VEGF receptors -1 and -2 and activating a phospholipase C- /protein kinase C (PLC /PKC) signal transduction cascade. However, the exact intracellular signals that modulate the chronic increases in vascular permeability induced by VEGF have not been determined. The long-term goals of this research program are to decipher the cellular and molecular mechanisms by which VEGF directly signals chronic alterations of endothelial structure leading to increased monolayer permeability to macromolecules.
The specific aims of this experimental program are to test whether VEGF-induced chronic barrier dysfunction and EC growth are dependent on 1) the activation of the mitogen-activated protein kinase cascade, 2) the activation of protein kinase a and bII isoforms, 3) the activation of phosphatidylinositol 3-kinase and/or Rho A. This study will use the following methods: 1) a size-dependent solute (20-200 A) permeability assay developed in this laboratory to measure the """"""""pore"""""""" size of the cell-cell junctional space in large and microvessel EC monolayers, 2) immunoprecipitation and Western blotting to examine the signal transduction events initiated by VEGF, 3) digital immunofluorescent microscopy using a -25 degrees C CCD camera that permits sensitive detection of compartmentalization of intracellular signaling, cytoskeletal and cell-cell adhesion molecules, and 4) an EC proliferation assay to measure the effects of inhibitors on VEGF's mitogenic properties. These studies will allow us to identify the VEGF-induced signal transduction pathways that cause chronic endothelial barrier dysfunction and suggest new targets for therapeutic interventions.
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