High-density Lipoprotein Cholesterol Efflux Research Articles

HDL Cholesterol Efflux Capacity and Incident Cardiovascular Events

BackgroundIt is unclear whether high-density lipoprotein (HDL) cholesterol concentration plays a causal role in atherosclerosis. A more important factor may be HDL cholesterol efflux capacity, the ability of HDL to accept cholesterol from macrophages, which is a key step in reverse cholesterol transport. We investigated the epidemiology of cholesterol efflux capacity and its association with incident atherosclerotic cardiovascular disease outcomes in a large, multiethnic population cohort.MethodsWe measured HDL cholesterol level, HDL particle concentration, and cholesterol efflux capacity at baseline in 2924 adults free from cardiovascular disease who were participants in the Dallas Heart Study, a probability-based population sample. The primary end point was atherosclerotic cardiovascular disease, defined as a first nonfatal myocardial infarction, nonfatal stroke, or coronary revascularization or death from cardiovascular causes. The median follow-up period was 9.4 years.ResultsIn contrast to HDL cholesterol level, which was associated with multiple traditional risk factors and metabolic variables, cholesterol efflux capacity had minimal association with these factors. Baseline HDL cholesterol level was not associated with cardiovascular events in an adjusted analysis (hazard ratio, 1.08; 95% confidence interval [CI], 0.59 to 1.99). In a fully adjusted model that included traditional risk factors, HDL cholesterol level, and HDL particle concentration, there was a 67% reduction in cardiovascular risk in the highest quartile of cholesterol efflux capacity versus the lowest quartile (hazard ratio, 0.33; 95% CI, 0.19 to 0.55). Adding cholesterol efflux capacity to traditional risk factors was associated with improvement in discrimination and reclassification indexes.ConclusionsCholesterol efflux capacity, a new biomarker that characterizes a key step in reverse cholesterol transport, was inversely associated with the incidence of cardiovascular events in a population-based cohort. (Funded by the Donald W. Reynolds Foundation and others.)

Large scale analysis of determinants of HDL efflux capacity

High-density lipoprotein (HDL) may provide cardiovascular protection by promoting reverse cholesterol transport (RCT). HDL cholesterol efflux is a measure of RCT. Although plasma HDL level is an important determinant of HDL efflux capacity (EC) the correlation is not perfect. No large scale study of additional determinants of HDL EC has been undertaken. Here we sought to identify determinants of HDL EC efflux including clinical characteristics as well as features of the HDL particle. We also determined the heritability of HDL efflux.

High-Density Lipoprotein Mediated Cellular Cholesterol Efflux in Acute Coronary Syndromes

Systemic inflammation at the development of an acute coronary syndrome (ACS) might alter the high-density lipoprotein (HDL) components and function. One of the major functions of HDL particles is their ability to remove cellular cholesterol from macrophages. The aim of the present study was to characterize the HDL efflux capacity in patients with ACS. We analyzed the cholesterol efflux in those ACS (within 72 hours of symptoms [ACS1]) and, again, 3 months later (ACS2). As controls, we used normal subjects without coronary artery disease (CAD) and patients with chronic, stable CAD. The 4 groups were matched for age and HDL cholesterol levels. We used a cell-based efflux system in (3)[H]-cholesterol-labeled J774 macrophages to measure cholesterol efflux from apolipoprotein B-depleted plasma. The present study included 20 patients with ACS. Their mean age was 58 ± 9 years, and the mean HDL cholesterol level was 1.06 ± 0.22 mmol/L (41 ± 9 mg/dl). The ACS1 group showed a marked increase in high-sensitivity C-reactive protein and serum amyloid A, reflecting systemic inflammation. The HDL cholesterol efflux capacity was reduced in ACS1 subjects and remained reduced 12 weeks later and in those with stable CAD. These results suggest that the acute presence of serum amyloid A does not account for the impairment of HDL-mediated cholesterol efflux capacity in the ACS1 group. Little correlation was found between HDL cholesterol and HDL efflux capacity (r = 0.233; p = 0.049), suggesting that HDL cholesterol is a poor marker of HDL function in inflammatory states and CAD. In conclusion, our data support the concept that atherogenic HDL dysfunction and impaired efflux occur in ACS, independent of changes in plasma HDL cholesterol and apolipoprotein A-I levels.

Evidence for a role of CETP in HDL remodeling and cholesterol efflux: Role of cysteine 13 of CETP

Cholesteryl ester transfer protein (CETP), a key regulator of high-density lipoprotein (HDL) metabolism, induces HDL remodeling by transferring lipids between apolipoprotein B-containing lipoproteins and HDL, and/or by promoting lipid transfer between HDL subparticles. In this study, we investigated the mechanism as to how CETP induces the generation of lipid-poor particles (pre-β-HDL) from HDL, which increases ATP-binding cassette transporter 1-mediated cholesterol efflux. This CETP-dependent HDL remodeling is enhanced by the CETP modulator dalcetrapib both in plasma and isolated HDL. The interaction of dalcetrapib with cysteine 13 of CETP is required, since this effect was abolished when using mutant CETP in which cysteine 13 was substituted for a serine residue. Other thiol-containing compounds were identified as CETP modulators interacting with cysteine 13 of CETP. In order to mimic dalcetrapib-bound CETP, mutant CETP proteins were prepared by replacing cysteine 13 with the bulky amino acid tyrosine or tryptophan. The resultant mutants showed virtually no CETP-dependent lipid transfer activity but demonstrated preserved CETP-dependent pre-β-HDL generation. Overall, these data demonstrate that the two functions of CETP i.e., cholesteryl ester transfer and HDL remodeling can be uncoupled by interaction of thiol-containing compounds with cysteine 13 of CETP or by introducing large amino acid residues in place of cysteine 13.

Differential effects of fenofibrate and extended-release niacin on high-density lipoprotein particle size distribution and cholesterol efflux capacity in dyslipidemic patients

The effectiveness of therapies that raise high-density lipoprotein cholesterol (HDL-C) to lower cardiovascular disease risk is currently under debate, and further research into the relationship between HDL-C and function is required. o investigate whether 2 established HDL-C-raising therapies had differential effects on parameters of high-density lipoprotein (HDL) quality and function, such as HDL particle profile and cholesterol efflux capacity (CEC), in patients with dyslipidemia. Sixty-six patients with dyslipidemia, 24 with low HDL-C levels (<40 mg/dL) and 42 with normal HDL-C levels (40-59 mg/dL), were treated for 6 weeks with fenofibrate (160 mg/d) or extended-release (ER) niacin (0.5 g/d for 3 weeks, then 1 g/d) with 4 weeks of washout between treatments. Lipoprotein particle size distribution was determined using nuclear magnetic resonance, and pathway-specific serum CECs were assessed in J774 macrophages, hepatoma, and Chinese hamster ovary-human adenosine triphosphate-binding cassette transporter G1 cells. Comparable increases in HDL-C and apolipoprotein A-I levels were seen with fenofibrate and ER niacin. There was a shift toward larger HDL, predominantly to medium-size HDL particles for fenofibrate (+209%) and to large HDL particles for ER niacin (+221%). Minor changes in serum CECs were observed with fenofibrate and ER niacin for all the efflux pathways measured. Small increases in plasma cholesteryl ester transfer protein and lecithin: cholesterol acyltransferase concentrations, and decreases in cholesteryl ester transfer protein activity were seen with both drugs. Fenofibrate and ER niacin increased plasma HDL-C level similarly, but modulated HDL particle size distribution differently; however, these changes did not result in differential effects on serum CECs.

Abstract 274: HDL-Mediated Cellular Cholesterol Efflux in Patients with Severe Carotid Atherosclerosis

The objective of this study was to examine the function of high-density lipoproteins (HDL) in patients with severe carotid atherosclerosis undergoing carotid endarterectomy (CEA) and in patients with coronary artery disease (CAD). We examined clinical, biochemical and lipoprotein profiles and HDL cholesterol efflux capacity (CEC) in 155 patients undergoing CEA and compared the results with patients with an acute coronary syndrome (ACS time 0 n=26) measured within 48 hours of the event, and again at 12 weeks (ACS recovery, n=26), in patients with chronic, stable CAD (sCAD) n=27 and in healthy controls. There were 110 men (71%) and 45 women (29%) with a mean age 69±10 years in the CEA group. Mean HDL-C was 0.96±0.26 mmol/L (38±10 mg/dL). There were no significant differences in HDL-C between CEA, ACS and sCAD subjects. HDL was obtained after depletion of apo B-containing lipoproteins with PEG precipitation. Cellular efflux capacity was determined by incubating HDL in cAMP-stimulated J774 mouse peritoneal macrophages for 6 hours. Specific cholesterol efflux was obtained by subtracting total efflux from efflux in non-cAMP stimulated cells. The procedure was standardized to enable medium throughput. Coefficient of variability was approximately 5%. HDL from CEA patients differ in its ability to promote cholesterol efflux. The range of CEC observed was: min 14.7%, max 34.0% mean 22.4±0.6%; (IQR 18.9-27.3% median 22.0). We found a weak correlation between HDL-C and CEC in patients CEA (r= 0.20, P=0.012); a significant correlation was found between apo A-I and CEC (r=0.23, P=0.005). Subjects with similar HDL-C or apoA-I differed in their ability to promote cellular cholesterol efflux, suggesting that the HDL-C mass does not reflect functionality. We found similar CEC in patients with CAD and ACS recovery. Here, we report that patients with severe carotid atherosclerosis have low HDL-C and a CEC similar to that observed in CAD patients. The function of HDL to promote cellular cholesterol efflux, as determined by CEC, varied over a wide range and was only weakly correlated with HDL-C. The significant correlation between apo AI and CEC suggests that HDL particle size -and composition may also be an important determinant of the ability to promote cellular cholesterol efflux.

High-capacity selective uptake of cholesteryl ester from native LDL during macrophage foam cell formation

Macrophage foam cells are a defining pathologic feature of atherosclerotic lesions. Recent studies have demonstrated that at high concentrations associated with hypercholesterolemia, native LDL induces macrophage lipid accumulation. LDL particles are taken up by macrophages as part of bulk fluid pinocytosis. However, the uptake and metabolism of cholesterol from native LDL during foam cell formation has not been clearly defined. Previous reports have suggested that selective cholesteryl ester (CE) uptake might contribute to cholesterol uptake from LDL independently of particle endocytosis. In this study we demonstrate that the majority of macrophage LDL-derived cholesterol is acquired by selective CE uptake in excess of LDL pinocytosis and degradation. Macrophage selective CE uptake does not saturate at high LDL concentrations and is not down-regulated during cholesterol accumulation. In contrast to CE uptake, macrophages exhibit little selective uptake of free cholesterol (FC) from LDL. Following selective uptake from LDL, CE is rapidly hydrolyzed by a novel chloroquine-sensitive pathway. FC released from LDL-derived CE hydrolysis is largely effluxed from cells but also is subject to ACAT-mediated reesterification. These results indicate that selective CE uptake plays a major role in macrophage metabolism of LDL.

Scavenger Receptor Class B Type I Is a Plasma Membrane Cholesterol Sensor

Signal initiation by the high-density lipoprotein (HDL) receptor scavenger receptor class B, type I (SR-BI), which is important to actions of HDL on endothelium and other processes, requires cholesterol efflux and the C-terminal transmembrane domain. The C-terminal transmembrane domain uniquely interacts with plasma membrane (PM) cholesterol. The molecular basis and functional significance of SR-BI interaction with PM cholesterol are unknown. We tested the hypotheses that the interaction is required for SR-BI signaling, and that it enables SR-BI to serve as a PM cholesterol sensor. In studies performed in COS-M6 cells, mutation of a highly conserved C-terminal transmembrane domain glutamine to alanine (SR-BI-Q445A) decreased PM cholesterol interaction with the receptor by 71% without altering HDL binding or cholesterol uptake or efflux, and it yielded a receptor incapable of HDL-induced signaling. Signaling prompted by cholesterol efflux to methyl-β-cyclodextrin also was prevented, indicating that PM cholesterol interaction with the receptor enables it to serve as a PM cholesterol sensor. Using SR-BI-Q445A, we further demonstrated that PM cholesterol sensing by SR-BI does not influence SR-BI-mediated reverse cholesterol transport to the liver in mice. However, the PM cholesterol sensing does underlie apolipoprotein B intracellular trafficking in response to postprandial micelles or methyl-β-cyclodextrin in cultured enterocytes, and it is required for HDL activation of endothelial NO synthase and migration in cultured endothelial cells and HDL-induced angiogenesis in vivo. Through interaction with PM cholesterol, SR-BI serves as a PM cholesterol sensor, and the resulting intracellular signaling governs processes in both enterocytes and endothelial cells.

Lipid-Sensing High-Throughput ApoA-I Assays

Apolipoprotein A-I (ApoA-I), a primary protein component of high-density lipoprotein (HDL), plays an important role in cholesterol metabolism mediating the formation of HDL and the efflux of cellular cholesterol from macrophage foam cells in arterial walls. Lipidation of ApoA-I is mediated by adenosine triphosphate (ATP) binding cassette A1 (ABCA1). Insufficient ABCA1 activity may lead to increased risk of atherosclerosis due to reduced HDL formation and cholesterol efflux. The standard radioactive assay for measuring cholesterol transport to ApoA-I has low throughput and poor dynamic range, and it fails to measure phospholipid transfer. We describe the development of two sensitive, nonradioactive high-throughput assays that report on the lipidation of ApoA-I: a homogeneous assay based on time-resolved fluorescence resonance energy transfer (TR-FRET) and a discontinuous assay that uses the label-free Epic platform. The TR-FRET assay employs HiLyte Fluor 647–labeled ApoA-I with N-terminal biotin bound to streptavidin-terbium. When fluorescent ApoA-I was incorporated into HDL, TR-FRET decreased proportionally to the increase in the ratio of lipids to ApoA-I, demonstrating that the assay was sensitive to the amount of lipid bound to ApoA-I. In the Epic assay, biotinylated ApoA-I was captured on a streptavidin-coated biosensor. Measured resonant wavelength shift was proportional to the amount of lipids associated with ApoA-I, indicating that the assay senses ApoA-I lipidation.

IMB2026791, a Xanthone, Stimulates Cholesterol Efflux by Increasing the Binding of Apolipoprotein A-I to ATP-Binding Cassette Transporter A1

It is known that the ATP-binding cassette transporter A1 (ABCA1) plays a major role in cholesterol homeostasis and high density lipoprotein (HDL) metabolism. Several laboratories have demonstrated that ABCA1 binding to lipid-poor apolipoprotein A-I (apoA-I) will mediate the assembly of nascent HDL and cellular cholesterol efflux, which suggests a possible receptor-ligand interaction between ABCA1 and apoA-I. In this study, a cell-based-ELISA-like high-throughput screening (HTS) method was developed to identify the synthetic and natural compounds that can regulate binding activity of ABCA1 to apoA-I. The cell-based-ELISA-like high-throughput screen was conducted in a 96-well format using Chinese hamster ovary (CHO) cells stably transfected with ABCA1 pIRE2-EGFP (Enhanced Green Fluorecence Protein) expression vector and the known ABCA1 inhibitor glibenclamide as the antagonist control. From 2,600 compounds, a xanthone compound (IMB 2026791) was selected using this HTS assay, and it was proved as an apoA-I binding agonist to ABCA1 by a flow cytometry assay and western blot analysis. The 3H cholesterol efflux assay of IMB2026791 treated ABCA1-CHO cells and PMA induced THP-1 macrophages (human acute monocytic leukemia cell) further confirmed the compound as an accelerator of cholesterol efflux in a dose-dependent manner with an EC50 of 25.23 μM.

Impact of Mifepristone, a Glucocorticoid/Progesterone Antagonist, on HDL Cholesterol, HDL Particle Concentration, and HDL Function

Mifepristone is a glucocorticoid and progestin antagonist under investigation for the treatment of Cushing's syndrome. Mifepristone decreases high-density lipoprotein (HDL) cholesterol (HDL-C) levels in treated patients, but the clinical significance of this is unclear because recent studies suggest that functional properties of HDL predict cardiovascular disease status better than does HDL-C concentration. The aim of the study was to characterize the impact of mifepristone administration on HDL particle concentration and function. We conducted a double-blind, randomized, placebo-controlled trial at a single-site, clinical research center. Thirty healthy postmenopausal female volunteers participated in the study. Individuals were randomized to receive daily oral mifepristone (600 mg) or placebo for 6 wk. We measured HDL-C, serum HDL particle concentration, and HDL-mediated cholesterol efflux by treatment group. As expected, ACTH, cortisol, estradiol, and testosterone levels increased in the mifepristone group. Mifepristone treatment decreased HDL-C and HDL particle concentration by 26 and 25%, respectively, but did not alter pre-β HDL concentration. In contrast, the serum HDL-mediated cholesterol efflux decreased with mifepristone treatment by only 12%, resulting in an effective increase of the efflux capacity per HDL particle. No changes were observed in cholesterol ester transfer protein or lecithin:cholesterol acyltransferase activity. Treatment with mifepristone reduced HDL-C, HDL particle concentration, and serum HDL cholesterol efflux in postmenopausal women. However, on a per particle basis, the efflux capacity of serum HDL increased. These observations support the concept that a decrease in HDL-C may not represent proportional impairment of HDL function.

High-Density Lipoprotein Cholesterol Efflux, Nitration of Apolipoprotein A-I, and Endothelial Function in Obese Women

Subjects at risk of atherosclerosis might have dysfunctional high-density lipoprotein (HDL) despite normal cholesterol content in the plasma. We considered whether the efflux of excess cellular cholesterol to HDL from obese subjects is associated with impaired arterial endothelial function, a biomarker of cardiovascular risk. A total of 54 overweight (body mass index [BMI] 25 to 29.9 kg/m(2)) or obese (BMI ≥30 kg/m(2)) women, aged 46 ± 11 years, were enrolled in a worksite wellness program. The HDL cholesterol averaged 57 ± 17 mg/dl and was inversely associated with the BMI (r = -0.419, p = 0.002). Endothelial function was assessed using brachial artery flow-mediated dilation. Cholesterol efflux from (3)H-cholesterol-labeled baby hamster kidney cells transfected with the adenosine triphosphate-binding cassette transporter 1 showed 8.2% to 22.5% cholesterol efflux within 18 hours when incubated with 1% serum and was positively correlated with brachial artery flow-mediated dilation (p <0.05), especially in the 34 subjects with BMI ≥30 kg/m(2) (r = 0.482, p = 0.004). This relation was independent of age, HDL or low-density lipoprotein cholesterol concentrations in plasma, blood pressure, or insulin resistance on stepwise multiple regression analysis (β = 0.31, R(2) = 0.21, p = 0.007). Nitration of apolipoprotein A-I tyrosine residues (using sandwich enzyme-linked immunosorbent assay) was significantly greater in women with a BMI ≥30 kg/m(2) and the lowest cholesterol efflux than in women with a BMI of 25 to 29.9 kg/m(2) and the greatest cholesterol efflux (p = 0.01). In conclusion, we have shown that decreased cholesterol efflux by way of the adenosine triphosphate-binding cassette transporter 1 is associated with increased nitration of apolipoprotein A-I in HDL and is an independent predictor of impaired endothelial function in women with a BMI of ≥30 kg/m(2). This finding suggests that the functional measures of HDL might be better markers for cardiovascular risk than the HDL cholesterol levels in this population.

Relationships between cholesterol efflux and high-density lipoprotein particles in patients with type 2 diabetes mellitus

High-density lipoprotein (HDL) encompasses a heterogeneous population of lipoproteins with differences in functionality. The impact of HDL heterogeneity on its ability to support HDL-mediated cholesterol efflux has not been previously studied in patients with type 2 diabetes mellitus (T2DM). To examine the relationships between various HDL subtypes and cholesterol efflux from macrophages in patients with T2DM. Lipoprotein molecular profiles of 44 patients were studied by NMR spectroscopy. Cholesterol efflux was expressed as percentage efflux of radioactivity from lipid-laden THP-1 macrophages preincubated with (3)H-cholesterol and then incubated with serum depleted of apolipoprotein Bto provide an HDL-enriched acceptor medium. There was a predominance of small HDL particles (59%) and small putatively atherogenic low-density lipoprotein particles (56%). Neither HDL-C nor ApoA-I concentrations showed statistically significant correlations with percentage cholesterol efflux, but a significant positive relationship was found with the total HDL particle concentration (r= 0.41, P= .005) contributed to largely by medium HDL particles (r= 0.41, P= .006). The correlation between medium-sized HDL particle concentration remained significantly associated with cholesterol efflux when assessed with the use of a linear regression model that included all the HDL lipoprotein subclass concentrations as well as apolipoprotein A-I. Importantly, no statistically significant association was observed between the number of small HDL particles and cholesterol efflux. Hemoglobin A1c showed a significant inverse correlation with cholesterol efflux (r= -0.31, P= .04). In patients with moderately controlled type 2 diabetes mellitus, cholesterol efflux from macrophages incubated with apolipoprotein B-depleted plasmas correlated significantly and positively with the concentration of total and medium-sized HDL and not with that of the smallest particles.

ATP-Binding Cassette Transporters A1 and G1, HDL Metabolism, Cholesterol Efflux, and Inflammation: Important Targets for the Treatment of Atherosclerosis

Atherosclerosis has been characterized as a chronic inflammatory response to cholesterol deposition in arteries. Plasma high density lipoprotein (HDL) levels bear a strong independent inverse relationship with atherosclerotic cardiovascular disease. One central antiatherogenic role of HDL is believed to be its ability to remove excessive peripheral cholesterol back to the liver for subsequent catabolism and excretion, a physiologic process termed reverse cholesterol transport (RCT). Cholesterol efflux from macrophage foam cells, the initial step of RCT is the most relevant step with respect to atherosclerosis. The ATP-binding cassette (ABC) transporters ABCA1 and ABCG1 play crucial roles in the efflux of cellular cholesterol to HDL and its apolipoproteins. Moreover, ABCA1 and ABCG1 affect cellular inflammatory cytokine secretion by modulating cholesterol content in the plasma membrane and within intracellular compartments. In humans, ABCA1 mutations can cause a severe HDL-deficiency syndrome characterized by cholesterol deposition in tissue macrophages and prevalent atherosclerosis. Disrupting Abca1 or Abcg1 in mice promotes accumulation of excessive cholesterol in macrophages, and physiological manipulation of ABCA1 expression affects atherogenesis. Here we review recent advances in the role of ABCA1 and ABCG1 in HDL metabolism, macrophage cholesterol efflux, inflammation, and atherogenesis. Next, we summarize the structure, expression, and regulation of ABCA1 and ABCG1. Finally, we give an update on the progress and pitfalls of therapeutic approaches that target ABCA1 and ABCG1 to stimulate the flux of lipids through the RCT pathway.

Cholesterol efflux pathways and other potential mechanisms involved in the athero‐protective effect of high density lipoproteins

Plasma high density lipoprotein (HDL) levels bear a strong independent inverse relationship with atherosclerotic cardiovascular disease. Although HDL has anti-oxidant, anti-inflammatory, vasodilating and anti-thrombotic properties, the central anti-atherogenic activity of HDL is likely to be its ability to remove cholesterol and oxysterols from macrophage foam cells, smooth muscle cells and endothelial cells in the arterial wall. To some extent, the pleotropic athero-protective properties of HDL may be related to its ability to promote sterol and oxysterol efflux from arterial wall cells, as well as to detoxify oxidized phospholipids. In cholesterol-loaded macrophages, activation of liver X receptors (LXRs) leads to increased expression of adenosine triphosphate (ATP) binding cassetter transporter (ABCA1), ATP binding cassetter transporter gene (ABCG1) and apoE and promotes cholesterol efflux. ABCA1 stimulates cholesterol efflux to lipid-poor apolipoproteins, whilst ABCG1 promotes efflux of cholesterol and oxysterols to HDL. Despite some recent setbacks in the clinical arena, there is still intense interest in therapeutically targeting HDL and macrophage cholesterol efflux pathways, via treatments with niacin, cholesterol ester transfer protein inhibitors, LXR activators and infusions of apoA-1, phospholipids and peptides.

The Scavenger Receptor Class B Type I Adaptor Protein PDZK1 Maintains Endothelial Monolayer Integrity

Circulating levels of high-density lipoprotein (HDL) cholesterol are inversely related to the risk of cardiovascular disease, and HDL and the HDL receptor scavenger receptor class B type I (SR-BI) initiate signaling in endothelium through src that promotes endothelial NO synthase activity and cell migration. Such signaling requires the C-terminal PDZ-interacting domain of SR-BI. Here we show that the PDZ domain-containing protein PDZK1 is expressed in endothelium and required for HDL activation of endothelial NO synthase and cell migration; in contrast, endothelial cell responses to other stimuli, including vascular endothelial growth factor, are PDZK1-independent. Coimmunoprecipitation experiments reveal that Src interacts with SR-BI, and this process is PDZK1-independent. PDZK1 also does not regulate SR-BI abundance or plasma membrane localization in endothelium or HDL binding or cholesterol efflux. Alternatively, PDZK1 is required for HDL/SR-BI to induce Src phosphorylation. Paralleling the in vitro findings, carotid artery reendothelialization following perivascular electric injury is absent in PDZK1-/- mice, and this phenotype persists in PDZK1-/- mice with genetic reconstitution of PDZK1 expression in liver, where PDZK1 modifies SR-BI abundance. Thus, PDZK1 is uniquely required for HDL/SR-BI signaling in endothelium, and through these mechanisms, it is critically involved in the maintenance of endothelial monolayer integrity.

Detergent-Mediated Phospholipidation of Plasma Lipoproteins Increases HDL Cholesterophilicity and Cholesterol Efflux via SR-BI

Cellular cholesterol efflux is an early, obligatory step in reverse cholesterol transport, the putative antiatherogenic mechanism by which human plasma high-density lipoproteins (HDL) transport cholesterol from peripheral tissue to the liver for recycling or disposal. HDL-phospholipid content is the essential cholesterol-binding component of lipoproteins and therefore a major determinant of cholesterol efflux. Thus, increased phospholipidation of lipoproteins, particularly HDL, is one strategy for increasing cholesterol efflux. This study validates a simple, new detergent perturbation method for the phospholipidation of plasma lipoproteins; we have quantified the cholesterophilicity of human plasma lipoproteins and the effects of lipoprotein phospholipidation on cholesterophilicity and cellular cholesterol efflux mediated by the class B type I scavenger receptor (SR-BI). We determined that low-density lipoproteins (LDL) are more cholesterophilic than HDL and that LDL has a higher affinity for phospholipids than HDL whereas HDL has a higher phospholipid capacity than LDL. Phospholipidation of total human plasma lipoproteins enhances cholesterol efflux, an effect that occurs largely through the preferential phospholipidation of HDL. We conclude that increasing HDL phospholipid increases its cholesterophilicity, thereby making it a better acceptor of cellular cholesterol efflux. Phospholipidation of lipoproteins by detergent perturbation is a simple way to increase HDL cholesterophilicity and cholesterol efflux in a way that may be clinically useful.

SR-BI-mediated High Density Lipoprotein (HDL) Endocytosis Leads to HDL Resecretion Facilitating Cholesterol Efflux

The high density lipoprotein (HDL) receptor, scavenger receptor class B, type I (SR-BI), mediates selective cholesteryl ester uptake from lipoproteins into liver and steroidogenic tissues but also cholesterol efflux from macrophages to HDL. Recently, we demonstrated the uptake of HDL particles in SR-BI overexpressing Chinese hamster ovarian cells (ldlA7-SRBI) using ultrasensitive microscopy. In this study we show that this uptake of entire HDL particles is followed by resecretion. After uptake, HDL is localized in endocytic vesicles and organelles en route to the perinuclear area; many HDL-positive compartments were classified as multivesiculated and multilamellated organelles by electron microscopy. By using 125I-labeled HDL, we found that approximately 0.8% of the HDL added to the media is taken up by the ldlA7-SRBI cells within 1 h, and almost all HDL is finally resecreted. 125I-Labeled low density lipoprotein showed a very similar association, uptake, and resecretion pattern in ldlA7-SRBI cells that do not express any low density lipoprotein receptor. Moreover, we demonstrate that the process of HDL cell association, uptake, and resecretion occurs in three physiologically relevant cell systems, the liver cell line HepG2, the adrenal cell line Y1BS1, and phorbol myristate acetate-differentiated THP-1 cells as a model for macrophages. Finally, we present evidence that HDL retroendocytosis represents one of the pathways for cholesterol efflux.

Differential abilities of mouse liver parenchymal and nonparenchymal cells in HDL and LDL (native and oxidized) association and cholesterol efflux

The aim of this study was to quantify the abilities of mouse liver parenchymal and nonparenchymal cells with respect to (i) cholesteryl ester (CE) selective uptake from low-density lipoproteins (LDL), oxidized LDL (OxLDL), and high-density lipoprotein (HDL); and (ii) their free cholesterol efflux to HDL. The preparations of cells were incubated with lipoproteins labelled either in protein with iodine-125 or in CE with 3H-cholesterol oleate, and lipoprotein-protein and lipoprotein-CE associations were measured. The associations of LDL-protein and LDL-CE with nonparenchymal cells were 5- and 2-fold greater, respectively, than with parenchymal cells. However, in terms of CE-selective uptake (CE association minus protein association) both types of cell were equivalent. Similar results were obtained with OxLDL, but both types of cell showed higher abilities in OxLDL-CE than in LDL-CE selective uptake (on average by 3.4-fold). The association of HDL-protein with nonparenchymal cells was 3x that with parenchymal cells; however, nonparenchymal cells associated 45% less HDL-CE. Contrary to parenchymal cells, nonparenchymal cells did not show HDL-CE selective uptake activity. Thus parenchymal cells selectively take CE from the 3 types of lipoproteins, whereas nonparenchymal cells exert this function only on LDL and OxLDL. Efflux was 3.5-fold more important in nonparenchymal than in parenchymal cells.

Glycine 420 Near the C-terminal Transmembrane Domain of SR-BI Is Critical for Proper Delivery and Metabolism of High Density Lipoprotein Cholesteryl Ester

Scavenger receptor BI, SR-BI, is a physiologically relevant receptor for high density lipoprotein (HDL) that mediates the uptake of cholesteryl esters and delivers them to a metabolically active membrane pool where they are subsequently hydrolyzed. A previously characterized SR-BI mutant, A-VI, with an epitope tag inserted into the extracellular domain near the C-terminal transmembrane segment, revealed a separation-of-function between SR-BI-mediated HDL cholesteryl ester uptake and cholesterol efflux to HDL, on one hand, and cholesterol release to small unilamellar phospholipid vesicle acceptors and an increased cholesterol oxidase-sensitive pool of membrane free cholesterol on the other. To further elucidate amino acid residues responsible for this separation-of-function phenotype, we engineered alanine substitutions and point mutations in and around the site of epitope tag insertion, and tested these for various cholesterol transport functions. We found that changing amino acid 420 from glycine to histidine had a profound effect on SR-BI function. Despite the ability to mediate selective HDL cholesteryl ester uptake, the G420H receptor had a greatly reduced ability to: 1) enlarge the cholesterol oxidase-sensitive pool of membrane free cholesterol, 2) mediate cholesterol efflux to HDL, even at low concentrations of HDL acceptor where binding-dependent cholesterol efflux predominates, and 3) accumulate cholesterol mass within the cell. Most importantly, the G420H mutant was unable to deliver the HDL cholesteryl ester to a metabolically active membrane compartment for efficient hydrolysis. These observations have important implications regarding SR-BI function as related to its structure near the C-terminal transmembrane domain.