Thromboxane A2(TxA2) and related endoperoxides cause vasoconstriction and platelet aggregation following their binding to specific sites (putative TxA2 receptors) on vascular smooth muscle cells and platelets. Such sites have not been described in human endothelial cells. The overall hypothesis to be examined in these studies is that binding of endoperoxides to TxA2 receptors on human endothelial cells has potentially important functional effects. Two secondary hypotheses are that the expression of the mRNA encoding the thromboxane A2 receptor is altered by factors likely to be relevant in vascular diseases and that the variation in the molecular structure of the cytoplasmic domain might confer differences in the intracellular mediators and functional events associated with thromboxane A2 binding.
The first aim of the proposal is to determine the physiologic and functional effects of TxA2 binding human endothelial cells. The release of prostacyclin and the expression of P-selectin on the cell surface, and the production of endothelial- derived relaxing factor by cultured endothelial cells will be measured following stimulation with endoperoxide analogs. The ability of TxA2 binding to induce expression of the protooncogenes c-fos, c-myc, and c- jun/AP1 and proliferation of endothelial cells will be assessed, as will the TxA2 stimulated expression of mRNA encoding plasminogen activator inhibitors.
The second aim of this proposal is to determine the signal transduction mechanism responsible for these functional effects. Calcium rise following endoperoxide analogs will be measured, and the possibility that this rise results from internal mobilization as well as influx via a Ca2+ channel will be assessed. The effect of activation of individual protein kinase C isozymes will be assessed as will the contributions of Ca2+ and PKC activation to desensitization of the TxA2 receptor following occupancy.
The third aim s to characterize the expression of the gene encoding the human endothelial thromboxane A2 receptor in endothelial cells stimulated with cytokines and growth factors. A full length cDNA encoding this receptor has been isolated form a human endothelial cell library, and will be labelled so the expression of Txa@ receptor mRNA in human endothelial cells following stimulation with cytokines growth factors and exposure to shear stress can be studies. Finally, the full length cDNA encoding the endothelial TxA2 receptor will be overexpressed in both Chines Hamster Ovary cells and in endothelial like cell lines devoid of thromboxane A2 receptors, so the functional events and signal transduction can be assessed in autologous cells. Such information may permit development of more selective and potent therapy for thrombosis and other vascular disorders.
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