Yves L. Marcel

High-density lipoprotein binding to scavenger receptor-BI activates endothelial nitric oxide synthase

Atherosclerosis is the primary cause of cardiovascular disease, and the risk for atherosclerosis is inversely proportional to circulating levels of high-density lipoprotein (HDL) cholesterol. However, the mechanisms by which HDL is atheroprotective are complex and not well understood. Here we show that HDL stimulates endothelial nitric oxide synthase (eNOS) in cultured endothelial cells. In contrast, eNOS is not activated by purified forms of the major HDL apolipoproteins ApoA-I and ApoA-II or by low-density lipoprotein. Heterologous expression experiments in Chinese hamster ovary cells reveal that scavenger receptor-BI (SR-BI) mediates the effects of HDL on the enzyme. HDL activation of eNOS is demonstrable in isolated endothelial-cell caveolae where SR-BI and eNOS are colocalized, and the response in isolated plasma membranes is blocked by antibodies to ApoA-I and SR-BI, but not by antibody to ApoA-II. HDL also enhances endothelium- and nitric-oxide–dependent relaxation in aortae from wild-type mice, but not in aortae from homozygous null SR-BI knockout mice. Thus, HDL activates eNOS via SR-BI through a process that requires ApoA-I binding. The resulting increase in nitric-oxide production might be critical to the atheroprotective properties of HDL and ApoA-I.

Molecular basis of lipid transfer protein deficiency in a family with increased high-density lipoproteins.

PLASMA high density lipoproteins (HDL) are a negative risk factor for atherosclerosis. Increased HDL is sometimes clustered in families, but a genetic basis has never been clearly documented1. The plasma cholesteryl ester transfer protein (CETP) catalyses the transfer of cholesteryl ester from HDL to other lipoproteins and therefore might influence HDL levels2. Using monoclonal antibodies, we show that CETP is absent in two Japanese siblings who have markedly increased and enlarged HDL. Furthermore, they are homozygous for a point mutation in the 5′-splice donor site of intron 14 of the gene for CETP, a change that is incompatible with normal splicing of pre-messenger RNA3. The results indicate that the family has an inherited deficiency of CETP due to a gene splicing defect, and illustrate the key role that CETP has in human HDL metabolism.

Autophagy Regulates Cholesterol Efflux from Macrophage Foam Cells via Lysosomal Acid Lipase

The lipid droplet (LD) is the major site of cholesterol storage in macrophage foam cells and is a potential therapeutic target for the treatment of atherosclerosis. Cholesterol, stored as cholesteryl esters (CEs), is liberated from this organelle and delivered to cholesterol acceptors. The current paradigm attributes all cytoplasmic CE hydrolysis to the action of neutral CE hydrolases. Here, we demonstrate an important role for lysosomes in LD CE hydrolysis in cholesterol-loaded macrophages, in addition to that mediated by neutral hydrolases. Furthermore, we demonstrate that LDs are delivered to lysosomes via autophagy, where lysosomal acid lipase (LAL) acts to hydrolyze LD CE to generate free cholesterol mainly for ABCA1-dependent efflux; this process is specifically induced upon macrophage cholesterol loading. We conclude that, in macrophage foam cells, lysosomal hydrolysis contributes to the mobilization of LD-associated cholesterol for reverse cholesterol transport.

High-Density Lipoprotein Promotes Endothelial Cell Migration and Reendothelialization via Scavenger Receptor-B Type I

Vascular disease risk is inversely related to circulating levels of high-density lipoprotein (HDL) cholesterol. However, the mechanisms by which HDL provides vascular protection are unclear. The disruption of endothelial monolayer integrity is an important contributing factor in multiple vascular disorders, and vascular lesion severity is tempered by enhanced endothelial repair. Here, we show that HDL stimulates endothelial cell migration in vitro in a nitric oxide-independent manner via scavenger receptor B type I (SR-BI)-mediated activation of Rac GTPase. This process does not require HDL cargo molecules, and it is dependent on the activation of Src kinases, phosphatidylinositol 3-kinase, and p44/42 mitogen-activated protein kinases. Rapid initial stimulation of lamellipodia formation by HDL via SR-BI, Src kinases, and Rac is also demonstrable. Paralleling the in vitro findings, carotid artery reendothelialization after perivascular electric injury is blunted in apolipoprotein A-I−/− mice, and reconstitution of apolipoprotein A-I expression rescues normal reendothelialization. Furthermore, reendothelialization is impaired in SR-BI−/− mice. Thus, HDL stimulates endothelial cell migration via SR-BI-initiated signaling, and these mechanisms promote endothelial monolayer integrity in vivo.

Distribution and concentration of cholesteryl ester transfer protein in plasma of normolipemic subjects.

A MAb (TP-2) directed against human cholesteryl ester transfer protein (CETP) has been applied to the development of a competitive solid-phase RIA. Experiments with immobilized CETP have shown that upon incubation with plasma or HDL in the presence of Tween (0.05%) apo A-I (but not apo A-II) binds to CETP while TP-2 binding to CETP is concomitantly decreased. With high detergent concentration (0.5% Triton), the interference is eliminated and a specific RIA in which all plasma CETP fractions have the same affinity can be obtained. Plasma levels of CETP, apo A-I, lipids, and lipoproteins were measured in 50 normolipemic, healthy subjects of both sexes. CETP levels varied nearly fourfold with a mean value of 1.7 micrograms/ml. CETP was positively correlated only with apo A-I (r = 0.38) and HDL-triglyceride (r = 0.39). In 29 other normolipemic subjects, where several apolipoproteins were also measured, significant correlations of CETP with apo A-I (0.41), apo E (0.43), and HDL-cholesterol (0.41) were observed, but there was no significant relationship between CETP and either apo A-II, B, or D. In other experiments CETP was shown to be present mostly in HDL3 and VHDL, to display exclusively an alpha 2-electrophoretic migration, and to occur within discrete particles ranging in size from 129 to 154 kD. In conclusion, the association of CETP with apo A-I-containing lipoproteins probably explains the correlation between CETP and apo A-I levels. The relationship between CETP and apo E suggests either a common metabolism or a specific cooperative role in cholesterol ester transport for these proteins.

Plasma concentrations of cholesteryl ester transfer protein in hyperlipoproteinemia. Relation to cholesteryl ester transfer protein activity and other lipoprotein variables.

Cholesteryl ester transfer protein (CETP) mediates an important pathway for reverse cholesterol transport. Concentrations of CETP in fasting plasma were measured by radioimmunoassay in two different groups of hyperlipoproteinemic subjects. Plasma CETP concentrations measured by radioimmunoassay correlated closely with cholesterol ester transfer activity in normal plasma (r = 0.86). In the first group of 58 patients, plasma CETP concentrations were significantly increased, as compared with those in 79 normal subjects and in hypercholesterolemic (+26%) and combined hyperlipoproteinemic (+25%) subjects but were not altered in moderately hypertriglyceridemic subjects. Marked elevations in plasma CETP levels were documented in patients with dysbetalipoproteinemia (+68%) and severe chylomicronemia (+85%). Similar results were obtained in a second population of 50 hyperlipoproteinemic subjects. Significant correlations were found between plasma CETP levels and total cholesterol (r = 0.52), very low density lipoprotein (VLDL) cholesterol (r = 0.63), and apolipoprotein E concentration (r = 0.40). Correction of the lipoprotein phenotype by dietary means resulted in significant reductions in plasma CETP concentrations in patients with chylomicronemia and dysbetalipoproteinemia. In these subjects, plasma high density lipoprotein cholesterol concentrations increased as CETP decreased. These studies indicate that CETP levels increase in association with enhanced peripheral cholesterol transport via low density lipoprotein, beta-VLDL, or chylomicron remnants.

Cholesterol binding, efflux, and a PDZ-interacting domain of scavenger receptor–BI mediate HDL-initiated signaling

The binding of HDL to scavenger receptor-BI (SR-BI) mediates cholesterol movement. HDL also induces multiple cellular signals, which in endothelium occur through SR-BI and converge to activate eNOS. To determine the molecular basis of a signaling event induced by HDL, we examined the proximal mechanisms in HDL activation of eNOS. In endothelial cells, HDL and methyl-beta-cyclodextrin caused comparable eNOS activation, whereas cholesterol-loaded methyl-beta-cyclodextrin had no effect. Phosphatidylcholine-loaded HDL caused greater stimulation than native HDL, and blocking antibody against SR-BI, which prevents cholesterol efflux, prevented eNOS activation. In a reconstitution model in COS-M6 cells, wild-type SR-BI mediated eNOS activation by both HDL and small unilamellar vesicles (SUVs), whereas the SR-BI mutant AVI, which is incapable of efflux to SUV, transmitted signal by only HDL. In addition, eNOS activation by methyl-beta-cyclodextrin was SR-BI dependent. Studies of mutant and chimeric class B scavenger receptors revealed that the C-terminal cytoplasmic PDZ-interacting domain and the C-terminal transmembrane domains of SR-BI are both necessary for HDL signaling. Furthermore, we demonstrated direct binding of cholesterol to the C-terminal transmembrane domain using a photoactivated derivative of cholesterol. Thus, HDL signaling requires cholesterol binding and efflux and C-terminal domains of SR-BI, and SR-BI serves as a cholesterol sensor on the plasma membrane.

Atherogenic diet increases cholesteryl ester transfer protein messenger RNA levels in rabbit liver.

Cholesteryl ester transfer activity is increased in plasma of cholesterol-fed rabbits. To investigate the mechanisms leading to changes in activity, we measured cholesteryl ester transfer protein (CETP) mass by RIA and CETP mRNA abundance by Northern and slot blot analysis using a human CETP cDNA probe in control (n = 8) and cholesterol-fed rabbits (n = 10). Cholesterol feeding (chow plus 0.5% cholesterol, 10% corn oil) for 30 d increased CETP mass in plasma 3.2-fold in the cholesterol-fed rabbits (12.45 +/- 0.82 micrograms/ml) compared with controls (3.86 +/- 0.38 micrograms/ml). In the hypercholesterolemic rabbit, liver CETP mRNA levels were increased 2.8 times control mRNA levels. Actin, apo E, lecithin-cholesterol acyltransferase, and albumin mRNA abundances were unchanged. In contrast to the widespread tissue distribution in humans, CETP mRNA was not detected in extrahepatic tissues of either control or cholesterol-fed animals. Using a sensitive RNase protection assay, the increase in liver CETP mRNA was detectable within 3 d of beginning the high cholesterol diet. Thus, in response to the atherogenic diet there is an early increase in liver CETP mRNA, probably causing increased CETP synthesis and secretion, and increased plasma CETP. The results indicate that the CETP gene may be regulated by diet-induced changes in lipid metabolism.

Characterization of monoclonal antibodies against human low density lipoprotein.

Seven monoclonal antibodies against human low density lipoprotein (LDL) have been characterized as to their specificity and ability to interfere with the LDL pathway in cultured human flbroblasts. The Immunoreactivity with LDL of two of the antibodies (2D8 and 4G3) was particularly sensitive to modification of lysine and arginlne residues In LDL. Cotitration experiments indicated that the antibodies 3A8 and 3A10 may react with the same determinant and that five antibodies (5E11, 3A8, 3A10, 4G3 and 3F5) recognized determinants that were grouped in the same region of the molecule. The two other antibodies (1D1 and 2D8) reacted with determinants distant from this region. When tested In molar excess relative to LDL, Fab fragments of 5E11, 3A8, 3A10,4G3 and 3F5 (but not 1D1 or 2D8) were capable of blocking the binding of 125 I-LDL to the LDL receptor and Interfering with LDL suppression of cholesterol synthesis in cultured fibroblasts. Increasing the concentration 10-fold did not change the results significantly. Based on these results we have proposed a map of the determinants as they would appear in LDL.

Regulation of Lipid Droplet Cholesterol Efflux From Macrophage Foam Cells

Cholesterol efflux from macrophages is the first and potentially most important step in reverse cholesterol transport, a process especially relevant to atherosclerosis and to the regression of atherosclerotic plaques. Increasingly, lipid droplet (LD) cholesteryl ester (CE) hydrolysis is being recognized as a rate-limiting step in cholesterol efflux. The traditional view on macrophage CE hydrolysis is that this pathway is entirely dependent on the action of neutral hydrolases, and numerous candidate CE hydrolases have been proposed to play a role in lipid hydrolysis in macrophages and atherogenesis. Although the exact identity of macrophage-specific CE hydrolases remains to be clarified, a common point to all of these studies is that enhancing LD-associated CE hydrolysis increases cholesterol efflux and is antiatherogenic. Understanding how cholesterol is mobilized from LDs offers new steps for modulating cholesterol efflux, and recently a role for autophagy and lysosomal acid lipase in macrophage lipolysis has emerged. Autophagy and lysosomal acid lipase thus represent novel therapeutic targets to enhance macrophage reverse cholesterol transport. This review discusses our current understanding of the relationship between macrophage LDs and atherosclerosis and presents recent insights into the mechanisms for LD CE hydrolysis in macrophage foam cells.