Novel Mechanisms Controlling Endothelial Junctions and Vascular Permeability

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Project Summary The vascular endothelium forms a highly regulated permeability barrier between the blood stream and interstitial tissues. Excessive vascular leakiness contributes to many inflammation related disease processes, including edema, sepsis, acute respiratory distress syndrome (ARDS), ischemic stroke, and hemorrhage. To prevent tissue damage resulting from vascular leakiness during inflammation, there is a need to be able to enhance endothelial barrier function. Intercellular junctions control changes in endothelial paracellular permeability, and Vascular-endothelial cadherin (VE-cadherin, CDH5) is a major regulator of endothelial junctions and paracellular permeability. We will explore a novel hypothesis and approach to understanding its role in permeability regulation, based on our findings of allosteric regulation of other cadherins at the cell surface in response to signaling events. The main hypothesis to be examined is that allosteric regulation of VE- cadherin, in coordination with activities of the actin cytoskeleton, is a key mechanism by which it regulates endothelial permeability in response to a range of vascular factors. We will use two novel activating monoclonal antibodies (mAbs) to VE-cadherin that prevent the increase in endothelial permeability induced by thrombin, VEGF, and TNFa, as major tools to test this hypothesis both in vitro and in vivo and to study the mechanism of regulation. The specific aims are: A. Determine whether VE-cadherin cell surface regulation is a common mechanism for a range of endothelial physiological processes. In vitro experiments will be used to determine whether it is similarly regulated by other key factors, including histamine, angiopoietins, Tie2 receptors, and S1P. Endothelial cells from different microvascular beds will be examined. Collaborative experiments will be done to determine flow and shear forces affect mAb induced barrier function. We will also investigate whether activating mAbs inhibit leukocyte diapedesis or affect endothelial morphogenesis and angiogenesis. B. Elucidate the cellular and biochemical mechanisms underlying VE-cadherin cell surface regulation. We will investigate the structural and biophysical basis of its activation by mAbs and test the roles of the phosphorylation of VE-cadherin and associated catenins in activation. We’ll also explore the relationship between cell surface regulation and endocytosis as well as barrier altering cytoskeletal functions. C. Investigate whether activation of VE-cadherin by mAbs enhances barrier function in vivo in mice in leaky vascular conditions or inflammation. We’ll test the effects of activating mAbs on induced acute induced vascular leak, and leukocyte infiltration. We will also examine their effects on mouse models of inflammatory disease processes involving the vasculature, including sepsis and Inflammatory Bowel Disease. These studies will help us understand regulation of endothelial permeability and develop novel approaches to enhance endothelial barrier function to reduce inflammatory processes and diseases.