John A. Krawiec

A novel endothelial-derived lipase that modulates HDL metabolism.

High-density lipoprotein (HDL) cholesterol levels are inversely associated with risk of atherosclerotic cardiovascular disease. At least 50% of the variation in HDL cholesterol levels is genetically determined, but the genes responsible for variation in HDL levels have not been fully elucidated. Lipoprotein lipase (LPL) and hepatic lipase (HL), two members of the triacylglyerol (TG) lipase family, both influence HDL metabolism and the HL (LIPC) locus has been associated with variation in HDL cholesterol levels in humans. We describe here the cloning and in vivo functional analysis of a new member of the TG lipase family. In contrast to other family members, this new lipase is synthesized by endothelial cells in vitro and thus has been termed endothelial lipase (encoded by the LIPG gene). EL is expressed in vivo in organs including liver, lung, kidney and placenta, but not in skeletal muscle. In contrast to LPL and HL, EL has a lid of only 19 residues. EL has substantial phospholipase activity, but less triglyceride lipase activity. Overexpression of EL in mice reduced plasma concentrations of HDL cholesterol and its major protein apolipoprotein A-I. The endothelial expression, enzymatic profile and in vivo effects of EL suggest that it may have a role in lipoprotein metabolism and vascular biology.

Endothelial lipase and HDL metabolism.

Purpose of review In the past year, several laboratories taking independent approaches have provided compelling evidence that endothelial lipase, a relatively recent addition to the triglyceride lipase gene family, is a major determinant of HDL metabolism. This review summarizes recent findings from experiments in mice with altered levels of endothelial lipase, from an examination of endothelial lipase catalytic and non-catalytic functions in vitro, and from human genetic studies. Recent findings An analysis of lipids and lipoproteins in endothelial lipase knockout and transgenic mice and in mice with adenovirus-driven hepatic overexpression of endothelial lipase shows, without exception, that total cholesterol, phospholipid and HDL-cholesterol all vary inversely with the endothelial lipase gene dosage, and primarily depend on endothelial lipase catalytic activity. Endothelial lipase participates in HDL metabolism by promoting the turnover of HDL components and increasing the catabolism of apolipoprotein A-I. The measurement of lipase activity on lipoprotein substrates in vitro demonstrates that endothelial lipase is distinct from other triglyceride lipases in showing the highest activity on HDL. Endothelial lipase gene polymorphisms in humans appear to be associated with HDL-cholesterol or HDL3-cholesterol concentrations. Summary A low HDL-cholesterol level in humans is a risk factor for coronary heart disease. Although not yet demonstrated, it is possible that the action of endothelial lipase on HDL may promote atherogenesis, in which case endothelial lipase may represent an attractive target for pharmaceutical intervention.

A High-Throughput Screen for Endothelial Lipase Using HDL as Substrate

Endothelial lipase (EL) is a 482-amino-acid protein from the triglyceride lipase gene family that uses a Ser-His-Asp triad for catalysis. Its expression in endothelial cells and preference for phospholipids rather than triglycerides are unique. Animal models in which it is overexpressed or knocked out indicate EL levels are inversely correlated with high-density lipoprotein cholesterol (HDL-C). HDL-C is commonly referred to as the good form of cholesterol because it is involved in the reverse cholesterol transport pathway, in which excess cholesterol is effluxed from peripheral tissues for excretion or reabsorption. Thus, EL inhibition in humans is expected to lead to increases in HDL levels and possibly a decrease in cardiovascular disease. To discover inhibitors of EL, a coupled assay for EL has been developed, using its native substrate, HDL. Hydrolysis of HDL by EL yields free fatty acids, which are coupled through acyl-CoA synthetase, acyl-CoA oxidase, and horseradish peroxidase to produce the fluorescent species resorufin. This assay was developed into a 5-µL, 1536-well assay format, and a high-throughput screen was executed against the GSK collection. In addition to describing the screening results, novel post-HTS mechanism-of-action studies were developed for EL and applied to 1 of the screening hits as an example. (Journal of Biomolecular Screening 2008:468-475)