Endothelial cells, which line blood vessels, continuously interact with blood cells, but the latter do not adhere under usual circumstances. This is crucial since inappropriate activation of leukocytes and platelets would lead to vascular damage and thrombosis. We have studied the endothelial cell synthesis of lipid mediators that influence these interactions. One interest has been the regulation of synthesis of platelet-activating factor, a potent phospholipid autacoid with diverse functions, including mediation of the adhesion of leukocytes to activated endothelial cells. It also increases vascular permeability and stimulates smooth muscle cell growth. We found that its synthesis is tightly regulated, and described mechanisms at the initiating and subsequent steps. In preliminary studies we have substantially purified the enzyme responsible for the second step in the synthesis of PAF, the acetyltransferase, which we showed to be important in determining the amount of PAF synthesized by endothelial cells. We have established methods for examining the transfer of PAF to the cell surface and its expression on the cell surface. Another key lipid mediator is prostacyclin (PGI2), a metabolite of arachidonic acid, which is a potent vasodilator and prevents platelet aggregation. One component of the regulation of PGI2 synthesis is the availability of arachidonate, which requires its release from phospholipids. We found that the release of arachidonate is augmented in proliferating cells compared to confluent cells and described the mechanism for this change. The amount of PGI2 synthesized by endothelial cells is increased after exposure to cytokines such as TNF and IL- 1, and we found that both increased release of arachidonate and increased expression of cyclooxygenase (prostaglandin H synthase; PGHS) occur. We have isolated a cDNA encoding a novel form of the PGHS (Type II). We propose to complete the purification of acetyl coA:lysoPAF acetyltransferase, prepare antibodies to it and isolate a cDNA. We will use these reagents to examine the regulation of the acetyltransferase, which probably undergoes phosphorylation/dephosphorylation, and also may be regulated by increased synthesis. We will test the hypothesis that PAF can serve as an intracellular messenger. We will determine the mechanism(s) by which PAF is transferred from its site of synthesis an internal membrane to the surface of the endothelial cell. Additionally, we will characterize binding sites for PAF on the surface of endothelial cells that do not appear to be the known PAF receptor. We will determine the mechanism by which increased release of arachidonate occurs in cells exposed to cytokines, and will define the basis for increased expression of PGHS (both types). Finally, we will determine whether the increased expression of this enzyme can be regulated pharmacologically. These experiments will have important clinical implications since prostaglandin synthesis is the target for most anti-inflammatory, and some anti-thrombotic, agents.
Showing the most recent 10 out of 44 publications
