Endothelium serves a vital homeostatic function by preserving the fluidity of blood and preventing escape of blood constituents from the intravascular space. This proposal examines the effects of hypoxia on endothelial cell coagulant and barrier function. Preliminary studies indicate that hypoxia down-regulates two endothelial cell anticoagulant properties, fibrinolysis and the protein C/protein S pathway. The mechanisms underlying these observations will be investigated, as well as the effects of hypoxia to modulate other endothelial coagulant functions. Because hypoxia also appears to perturb endothelial monolayer barrier properties, the integrity of the monolayer under hypoxic conditions will be determined by examining permeability to a variety of macromolecules and lower molecular weight solutes. Permeability changes will be correlated with changes in cell morphology (shape, cytoskeleton and junctions). A hypoxic vascular microenvironment occurs in the setting of a variety of diseases in{ which host response mediators, such as norepinephrine and TNF, are elaborated. Studies will examine the endothelial cell response to these agents in hypoxia. Preliminary data indicating that the number of endothelial alpha1-adrenergic receptors and an alpha1-adrenergic response increases following hypoxic exposure provide a rationale for performing a detailed examination of transduction proteins and effector mechanisms (phosphoinositide hydrolysis, calcium mobilization, eicosanoid production) activated by alpha1-adrenergic receptors and the role of these intracellular signalling mechanisms in the control of cellular coagulant and barrier function. Similar studies investigating the effect of TNF on endothelial cell function, the cellular mechanisms involved and their modulation by hypoxia are proposed. Exposure of cultured cells to low levels of oxygen increases the rate of synthesis of a specific class of oxygen-regulated proteins. Our preliminary data indicate that endothelium labelled metabolically with 35S-methionine and exposed to low concentrations oxygen demonstrates new/enhanced bands on autoradiograms from SDS- PAGE of culture supernatants and lysates. The final objective of this proposal is to produce monoclonal antibodies relatively selective for hypoxic endothelium. These immunologic reagents will be used to characterize the evolution of the hypoxic state in vitro and, in the future, hypoxic vascular injury in vivo These specific plans are directed toward our long-term objective: understanding the role of endothelium in the pathogenesis of vascular injury.
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