Fellowship/Zhu: CD36-Mediated Stimulation of Endothelial Nitric Oxide Synthase by Fatty Acids

The vascular endothelium is an import ant site of dysfunction in cardiovascular disease. There is a positive correlation between the intake of diets rich in saturated fatty acids and the development of ather osclerosis. In contrast, unsaturated fatty acids appear to have beneficial effects on vascular endothelial function. 0 iets rich in n-6 polyunsaturated fatty acid or fish-oil (n-3) fatty-acid-rich are associated with a lower rate of atherosclerosis and have a hypolipidemic effect. A rterioles are directly responsible for maintaining total peripheral resistance and are consequently directly involved in blood pressure regulation. Arteriolar compliance is under the control of both local and central regulatory mechanisms. One of the local control mechanisms is endothelial nitric oxide synthase (eNOS), which promotes vasodilation vi a nitric oxide generation. Many studies have demonstrated that eNOS is located in caveolae where it associ ates with caveolin and is held in an inactive state. ENOS stimulation can be affected by CD36, a clas s B scavenger receptor that is expressed in adipocytes, platelets, monocytes, and arteriolar endothelial cells. CD36 co-fractionates with caveol ae and co-immunoprecipitates with caveolin-1. In addition, studies with CD36 strongly suggest it functions as a long-chain fatty acid transporter. Our preliminary studies suggest that fatty acid (linol eic acid) binds to CD36 and stimulates endothelial nitric ox ide synthase (eNOS). Two specific aims will be addressed. 1) To determine the effects of fatty acid chain length, unsaturation, concentration, fatty acid/BSA ratio, and time on CD36-dependent eNOS activation. This will be achieved by using an established human microvascular endothelial (HME) cell line. This cell line expresses eNOS but not CD36. We have stably expressed human CD36 in this cell line, therefore we have the reagents required to study the CD36-dependent uptake of fatty acids in a phys iologically relevant cell type. 2) To determine the molecular mechanism(s) whereby CD36-fatty acid interaction stimulates eNO S. The same cell system used in Aim 1 will be used to determine if fatty acids stimulate eN OS by inducing an influx of calcium, activati ng Akt kinase, or increase intracellular ceramide.