Endothelial lipase is a critical determinant of HDL-stimulated sphingosine 1-phosphate-dependent signaling in vascular endothelium

Plasma levels of high-density lipoprotein cholesterol (HDL-C) are inversely associated with the risk of cardiovascular diseases.1 The anti-atherogenic effect of HDL is attributed at least in part to reverse cholesterol transport.2 In addition, multiple lines of evidence indicate that other salutary effects of HDL on endothelial cells include anti-oxidative, anti-inflammatory, anti-thrombotic and pro-angiogenic effects.3–5 HDL is the most quantitatively important plasma carrier of sphingosine-1-phosphate (S1P), which is a bioactive lipid molecule. HDL-associated S1P has been demonstrated to be involved in a broad range of vascular responses6–9 modulated by a family of at least five different S1P receptor subtypes identified in mammals; endothelial cells express most prominently the S1P1 and S1P3 receptor isoforms.9, 10 It was recently reported that HDL-induced Akt activation and endothelial responses are mediated by S1P1 in human umbilical vein endothelial cells.11 In contrast, other studies suggest that HDL-induced Akt activation is mediated by S1P3 receptors.12 The identity of the S1P receptor subtype that modulates HDL responses in the endothelium remains unsettled, and the mechanisms whereby S1P is released from HDL is unclear. The studies presented here explore the roles of endothelial lipase (EL) and the identity of the S1P receptor subtype in determining HDL-dependent responses in the vascular wall. EL is a member of the triglyceride lipase family, which includes lipoprotein lipase and hepatic lipase. EL is principally a phospholipase, with nominal triglyceride lipase activity.13, 14 Endothelial lipase is synthesized principally by EC15 and hydrolyzes HDL much more efficiently than other lipoproteins.16 Plasma HDL levels are increased in EL knockout (EL−/−) mice and are decreased in EL transgenic mice.17 Furthermore, the N396S variant in the human EL gene (LIPG) shows reduced lipase activity and is associated with in elevated HDL-C levels.18 Another EL variant, G26S EL shows reduced plasma levels of EL protein.19 Yet the role of EL in atherosclerosis remains controversial. Inactivation of EL increases the plasma HDL-C levels and inhibits atherosclerosis in ApoE−/− mice.20 In contrast, another study reported that a deficiency of EL expression does not affect atherosclerosis in either ApoE−/− mice or LDLR−/− mice, even though plasma HDL-C levels are elevated.21 Broedl et al. have shown that EL overexpression results in reduced VLDL/LDL cholesterol and phospholipid levels.22 In addition, we have previously reported that HDL hydrolysis by EL activates PPARα and represses VCAM1 expression in endothelial cells, which may contribute to the anti-inflammatory effects of HDL.23 These multiple lines of evidence suggest EL action might limit atherosclerosis. Several genetic association studies have shown conflicting results regarding association between common genetic variants in LIPG gene and the risk of cardiovascular diseases. Vergeer et al. reported that the T111I variant in the LIPG gene is associated with higher HDL-C levels but is not associated with increased cardiovascular disease risk.24 Moreover, recent Mendelian randomization analysis studies in 20,913 myocardial infarction (MI) cases versus 95,407 controls found that a single nucleotide polymorphism in the EL gene significantly increased HDL-C levels but conferred no protection against myocardial infarction. These findings raise questions about the connection between plasma HDL-C levels per se and protection against atherosclerosis and the connection between EL’s enzymatic function and HDL’s beneficial effects.25 Sphingosine 1-phosphate (S1P) is a bioactive lipid that binds to family of G protein-coupled receptors that modulate signaling responses in a broad range of cells and tissues.26 S1P1 receptors in the vascular endothelium are reversibly targeted to plasmalemmal caveolae and promote the activation of kinase Akt and of the endothelial isoform of nitric oxide synthase (eNOS), leading to vasorelaxation.27 The EC50 for S1P-promoted eNOS phosphorylation is at least one order of magnitude lower than the plasma concentration of S1P, reflecting the fact that plasma S1P is mostly bound to plasma proteins. HDL particles represent the predominant S1P binding proteins in plasma, with recent studies revealing apolipoprotein M (apoM) in HDL as a specific S1P binding protein.28, 29 The roles of EL in modulating HDL-dependent signaling responses via S1P have not been well characterized. The current studies use experiments in ex vivo vascular preparations as well as in cultured endothelial cells to test the hypothesis that HDL hydrolysis by EL induces angiogenesis and stimulates endothelial signaling responses via S1P1 receptors in the vascular endothelium.