CYTOKINE REGULATION OF ENDOTHELIUM PROSTANOID SYNTHESIS

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The control of endothelial cell (EC) function by cytokines such as tumor necrosis factor and interleukin 1 (IL.1)* serves to regulate inflammation and tissue repair. These cytokines are well known inducers of arachidonic acid metabolism, and in EC they bring about synthesis of prostacyclin (PGI-2). PGI-2 in combination with other signals, is a potent vasodilator that promotes leukocyte recruitment and extravasation. Several members of the intracellular, enzymatic cascade responsible for PGI-2 synthesis have recently been cloned and characterized, including cytosolic phospholipase A2 (cPLA2) and cyclooxygenases (Cox-l and Cox-2). Recent evidence indicates that both cPLA2 and Cox may have distinct constitutive and hormonally regulated isoforms, and that there are changes in expression these enzymes induced by IL-I and TNF in several cell systems including EC. However, the precise isoforms which are regulated and the changes in enzymatic activity that occur in cytokine treated EC are not clearly understood. During the period of this grant, I will pursue a systematic approach to understand the molecular aspects of regulation of these enzymes by TNF and IL-1 in human umbilical vein EC, including analysis of mRNA transcriptional rates, mRNA and protein stability and levels, and changes in enzyme activities. Techniques to be utilized include nuclear runoff, northern blotting, western blotting, immunoprecipitation and biochemical enzyme assays. These studies should provide a complete description of cytokine regulation of this critical biological pathway in EC.