A hallmark of atherosclerosis is the formation of foam cells in the vessel wall. Foam cells derive their name from the appearance of the cells, due to the pathologic deposition of multiple intracellular lipid droplets. This lipid is primarily cholesteryl ester and is thought to be in a dynamic state, undergoing constant turn over via hydrolysis and re- esterification. For regression of an atherosclerotic lesion to occur, this cycle must be interrupted so that the excess cholesterol can be returned to the liver by the process known as """"""""reverse cholesterol transport"""""""". Since cholesterol can leave a cell only as free cholesterol, the hydrolysis of stored cholesteryl ester is the first step in reverse cholesterol transport. In spite of the importance of this initial event, relatively little is known about the cholesteryl ester hydrolase in foam cells, although a body of evidence suggests that it is the enzyme known as hormone-sensitive lipase (HSL). HSL is an enzyme of central metabolic importance, functioning as a triglyceridase in adipose tissue and as a cholesteryl esterase in steroidogenic tissues. HSL has also been shown to be present in most.other tissues, with liver and kidney being notable exceptions. The hypothesis to be tested in this work is that HSL is the primary lipase capable of hydrolysing cholesteryl esters in macrophages, and that foam cell development may result from a lack of sufficient HSL activity to effectively hydrolyse cholesteryl esters at rates necessary to allow the clearance of cholesterylesters, due either to a failure in up- regulation of HSL activity or to a limited range of substrate specificity. The regulation of HSL, as assessed by measuring changes in HSL activity, immunoreactivity and mRNA levels, will be determined as a function of lipid load. Control studies will be carried out using mouse 3T3-L1 pre- adipocytes, where regulation of HSL is expected to occur. J774 cells will be transfected with the HSL cDNA to determine whether over-expression of HSL alters the capacity of the cells to store or mobilize stores of cholesteryl esters. HSL will be purified from an expression system, which offer the advantage of purifying relatively large amounts of this enzyme from a source which is not rich in lipid. Enzymatic properties of the purified HSL will be examined using carefully controlled substrate preparations, either in the form of monolayers or emulsion particles, to determine fatty acyl specificities for various cholesteryl esters and glycerides. The interfacial binding characteristics and influence of lipid physical state on hydrolytic activity will also be examined. Using J774 macrophage foam cells as a model system, anti-sense methods will be employed to show that, when HSL activity is abolished, cholesteryl esters are not hydrolysed. The context of this work is directed toward understanding the poor mobilization of cholesteryl esters in foam cells and specific involvement of HSL in, the hydrolysis of cholesteryl esters in macrophages. In addition, independent of foam cell physiology, investigation of the enzymatic properties of HS will provide new, basic information about a fundamentally important enzyme.
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