Inhibition of endothelial lipase causes increased HDL cholesterol levels in vivo

Abstract

Endothelial lipase (EL) is a recently discovered member of the triglyceride lipase gene family (1, 2).EL is highly homologous to lipoprotein lipase (LPL) and hepatic lipase (HL), both of which are known to hydrolyze lipids within lipoproteins and thereby influence their metabolism.EL has been shown to effectively hydrolyze HDL phospholipids in vitro (3), and overexpression of human EL in mouse liver markedly reduced plasma HDL cholesterol (HDL-C) levels in vivo (1).These data suggested that EL might play a physiological role in modulating HDL metabolism.However, proof of that concept requires an assessing the effect of reduction of EL activity in vivo.Antibody inhibition has been used to gain insight into the physiological roles of enzymes in vivo.Indeed, the roles of LPL and HL in lipoprotein metabolism were established in part through antibody inhibition studies in rats, chickens, and monkeys (4-9).Because overexpression of EL markedly reduced HDL-C levels in mice, we hypothesized that antibody inhibition of EL would increase HDL-C levels.Here, we report the results of several independent experiments in which we used a specific polyclonal antibody against murine EL (mEL) to inhibit EL in mice.Our results demonstrate that inhibition of EL in mice results in a significant increase in HDL-C levels and, in the absence of HL, in HDL particle size. Methods Generation of a rabbit polyclonal antibody to murine EL.A recombinant adenoviral vector containing the mEL cDNA was made using methods described previously (1) and used to immunize a rabbit.Viral particles (5 × 10 12 ) were injected into a New Zealand white rabbit (Hare-Marland, Hewitt, New Jersey, USA) through the ear vein.Sera were obtained at various intervals for analysis of antibody reactivity to mEL by Western blotting.Control serum was generated using the injection of a recombinant adenovirus that contains no transgene.Three months after injection, rabbits were anesthetized and exsanguinated.Serum was separated from blood cells and stored in aliquots at -80°C.The IgG fractions were precipitated from the sera using ammonium sulfate (10) and purified using a protein G column (Amersham Pharmacia Biotech, Uppsala, Sweden) according to the manufacturer's protocol.In vitro inhibition and immunoblotting of mEL.Transfection of expression plasmids containing mEL, murine HL (mHL), and murine LPL (mLPL) cDNAs was performed in HEK293 cells using lipofectamine (Invitrogen, Carlsbad, California, USA).Conditioned media were collected 48 hours after transfection.Aliquots of