Pre-beta High-density Lipoprotein Research Articles

Innovation in the Treatment of Uremia: Proceedings from the Cleveland Clinic Workshop: Causes of Dysregulation of Lipid Metabolism in Chronic Renal Failure

End-stage renal disease (ESRD) is associated with accelerated atherosclerosis and premature death from cardiovascular disease. These events are driven by oxidative stress inflammation and lipid disorders. ESRD-induced lipid abnormalities primarily stem from dysregulation of high-density lipoprotein (HDL), triglyceride-rich lipoprotein metabolism, and oxidative modification of lipoproteins. In this context, production and plasma concentration of Apo-I and Apo-II are reduced, HDL maturation is impaired, HDL composition is altered, HDL antioxidant and anti-inflammatory functions are depressed, clearance of triglyceride-rich lipoproteins and their atherogenic remnants is impaired, their composition is altered, and their plasma concentration is elevated in ESRD. The associated defect in HDL maturation is largely caused by acquired lecithin-cholesterol acyltransferase deficiency while its triglyceride enrichment is due to hepatic lipase deficiency. Hypertriglyceridemia, abnormal composition, and impaired clearance of triglyceride-rich lipoproteins and their remnants are mediated by down-regulation of lipoprotein lipase, hepatic lipase, very low-density lipoprotein (VLDL) receptor, and LDL receptor-related protein, relative reduction in ApoC-II/ApoC-III ratio, up-regulation of acyl-CoA cholesterol acyltransferase, and elevated plasma level of cholesterol ester-poor prebeta HDL. Impaired clearance and accumulation of oxidation-prone VLDL and chylomicron remnants and abnormal LDL composition in the face of oxidative stress and inflammation favors their uptake by macrophages and resident cells in the artery wall. The effect of heightened influx of lipids is compounded by impaired HDL-mediated reverse cholesterol transport leading to foam cell formation which is the central event in atherosclerosis plaque formation and subsequent plaque rupture, thrombosis, and tissue damage.

Lipid and Lipoprotein Metabolism in Chronic Kidney Disease

The risk of cardiovascular events and mortality increases as renal function declines although the relative risk of mortality contributed by the standard Framingham risk factors are altered or replaced. Low-density lipoprotein (LDL) cholesterol does not predict mortality but low high-density lipoprotein (HDL) cholesterol and triglycerides remain risk factors. The lipoproteins within each class are shifted to smaller, more dense isoforms. The accumulation of apolipoprotein B-containing lipoproteins, including lipoprotein(a) results primarily from decreased clearance rather than from increased synthesis. Lipoprotein(a) levels are also associated with cardiovascular outcome among dialysis patients. Decreased clearance of very low-density lipoprotein and intermediate-density lipoprotein is a result of decreased lipoprotein lipase, structural alterations in the lipoproteins rendering them poorer substrates, and a decrease in receptor number for these proteins. HDL levels are decreased as a result of an increased fractional catabolic rate both among obese patients with normal renal function and among dialysis patients, but the mechanisms responsible for increased HDL fractional catabolic rate may differ. In patients with advanced kidney disease, HDL fails to mature normally as a result of decreased lecithin cholesterol ester transfer protein, leaving cholesterol ester-poor, triglyceride-rich HDL(3) and pre-beta HDL. HDL in patients with chronic kidney disease is a less effective antioxidative agent than is HDL from normal subjects because of a decrease in paroxonase activity, allowing the accumulation of oxidized LDL.

ApoA-I cleaved by transthyretin has reduced ability to promote cholesterol efflux and increased amyloidogenicity

A fraction of plasma transthyretin (TTR) circulates in HDL through binding to apolipoprotein A-I (apoA-I). Moreover, TTR is able to cleave the C terminus of lipid-free apoA-I. In this study, we addressed the relevance of apoA-I cleavage by TTR in lipoprotein metabolism and in the formation of apoA-I amyloid fibrils. We determined that TTR may also cleave lipidated apoA-I, with cleavage being more effective in the lipid-poor prebeta-HDL subpopulation. Upon TTR cleavage, discoidal HDL particles displayed a reduced capacity to promote cholesterol efflux from cholesterol-loaded THP-1 macrophages. In similar assays, TTR-containing HDL from mice expressing human TTR in a TTR knockout background had a decreased ability to perform reverse cholesterol transport compared with similar particles from TTR knockout mice, reinforcing the notion that cleavage by TTR reduces the ability of apoA-I to promote cholesterol efflux. As amyloid deposits composed of N-terminal apoA-I fragments are common in the atherosclerotic intima, we assessed the impact of TTR cleavage on apoA-I aggregation and fibrillar growth. We determined that TTR-cleaved apoA-I has a high propensity to form aggregated particles and that it formed fibrils faster than full-length apoA-I, as assessed by electron microscopy. Our results show that apoA-I cleavage by TTR may affect HDL biology and the development of atherosclerosis by reducing cholesterol efflux and increasing the apoA-I amyloidogenic potential.

Functional LCAT deficiency in human apolipoprotein A-I transgenic, SR-BI knockout mice

Reduction of plasma LCAT activity has been observed in several conditions in which the size of HDL particles is increased; however, the mechanism of this reduction remains elusive. We investigated the plasma activity, mass, and in vivo catabolism of LCAT and its association with HDL particles in human apolipoprotein A-I transgenic, scavenger receptor class B type I knockout (hA-ITg SR-BI-/-) mice. Compared with hA-ITg mice, hA-ITg SR-BI-/- mice had a 4-fold higher total plasma cholesterol concentration, which occurred predominantly in 13-18 nm diameter HDL particles, a significant reduction in plasma esterified cholesterol-total cholesterol (EC/TC) ratio, and significantly lower plasma LCAT activity, suggesting a decrease in LCAT protein. However, LCAT protein in plasma, hepatic mRNA for LCAT, and in vivo turnover of 35S-radiolabeled LCAT were similar in both genotypes of mice. HDL from hA-ITg SR-BI-/- mice was enriched in sphingomyelin (SM), relative to phosphatidylcholine, and had less associated [35S]LCAT radiolabel and endogenous LCAT activity compared with HDL from hA-ITg mice. We conclude that the decreased EC/TC ratio in the plasma of hA-ITg SR-BI-/- mice is attributed to a reduction in LCAT reactivity with SM-enriched HDL particles.

Apolipoprotein M – a novel player in high-density lipoprotein metabolism and atherosclerosis

Apolipoprotein M is a recently described apolipoprotein predominantly associated with high-density lipoprotein, but also found in chylomicrons, very low-density lipoproteins, and low-density lipoprotein. The purpose is to review recent information on the unusual structural properties of apolipoprotein M and its possible role in formation of pre-beta high-density lipoprotein and reverse cholesterol metabolism. Apolipoprotein M is a lipocalin having a coffee filter-like structure with a hydrophobic ligand-binding pocket. Mature apolipoprotein M retains its signal peptide, which serves as a hydrophobic anchor. In mice, silencing of expression in the liver with siRNA led to disappearance of pre-beta high-density lipoprotein and appearance of unusually large high-density lipoproteins. This suggests that apolipoprotein M is important for the formation of pre-beta high-density lipoprotein and reverse cholesterol transport. In accordance with this idea, hepatic overexpression of apolipoprotein M with an adenovirus in low-density lipoprotein-receptor deficient mice led to an approximately 70% reduction of atherosclerosis. In addition to the liver, apolipoprotein M is also expressed in the kidney. Kidney-derived apolipoprotein M binds to megalin, a member of the low-density lipoprotein-receptor family, which interacts with many lipocalins in renal tubuli. Apolipoprotein M is excreted in the urine of mice with a kidney-specific megalin deficiency but not in the urine of normal mice, suggesting megalin-mediated uptake of apolipoprotein M in the tubular epithelium of normal mice. Apolipoprotein M is a novel apolipoprotein with unusual structural features that appears to play important roles in high-density lipoprotein metabolism and prevention of atherosclerosis.

Mass kinetics of apolipoprotein A-I in interstitial fluid after administration of intravenous apolipoprotein A-I/lecithin discs in humans

Apolipoprotein kinetics are customarily determined by modeling time curves of specific radioactivity or isotopic enrichment in plasma after intravenous infusion of radiolabeled lipoproteins or stable isotope-enriched amino acids. However, this provides no information on the fractional rate of transfer of the apolipoprotein from plasma to interstitial fluid (k(p-if)) or its mean residence time in interstitial fluid (MRT(if)). To determine these parameters for a pharmacologic dose of exogenous apolipoprotein A-I (apoA-I) given intravenously as apoA-I/lecithin discs, we measured apoA-I in plasma and prenodal leg lymph in five healthy men before, during, and after a 4 h infusion at 10 mg/kg/h. ApoA-I concentrations in plasma and lymph were modeled by linear compartmental models (SAAM II version 1.1), using lymph albumin to adjust for the effects of variations in lymph flow rate. k(p-if) averaged 0.75%/h (range, 0.33-1.32), and MRT(if) averaged 29.1 h (14.1-40.0). Neither parameter was correlated with the distribution volume (57-105 ml/kg) or the fractional elimination rate (1.44-2.91%/h) of apoA-I, determined by modeling plasma apoA-I concentration alone. Although used here to study the mass kinetics of apoA-I, if combined with infusion of a tracer, analysis of lymph could also expand the modeling of endogenous apolipoprotein kinetics.

Purification and ATPase Activity of Human ABCA1

ATP-binding cassette protein A1 (ABCA1) plays a major role in cholesterol homeostasis and high density lipoprotein metabolism. Apolipoprotein A-I binds to ABCA1 and cellular cholesterol and phospholipids, mainly phosphatidylcholine, are loaded onto apoA-I to form pre-beta high density lipoprotein (HDL). It is proposed that ABCA1 translocates phospholipids and cholesterol directly or indirectly to form pre-beta HDL. To explore the mechanism of ABCA1-mediated pre-beta HDL formation, we expressed human ABCA1 in insect Sf9 cells and purified it. Trypsin limited-digestion of purified ABCA1 in the detergent-soluble form suggested that it retained conformation similar to ABCA1 expressed in the membranes of human fibroblast WI-38 cells. Purified ABCA1 showed robust ATPase activity when reconstituted in liposomes made of synthetic phosphatidylcholine. ABCA1 showed lower ATPase activity when reconstituted in liposomes containing phosphatidylserine, phosphatidylethanolamine, or phosphatidylglycerol and also showed weak specificity in acyl chain species. ATPase activity was reduced by the addition of cholesterol and decreased by 25% in the presence of 20% cholesterol. Beta-sitosterol and campesterol showed similar inhibitory effects but stigmasterol did not, suggesting structure-specific interaction between ABCA1 and sterols. Glibenclamide suppressed ABCA1 ATPase, suggesting that it inhibits apoA-I-dependent cellular cholesterol efflux by suppressing ABCA1 ATPase activity. These results suggest that the ATPase activity of ABCA1 is stimulated preferentially by phospholipids with choline head groups, phosphatidylcholine and sphingomyelin. This study with purified human ABCA1 provides the first biochemical basis of the mechanism for HDL formation mediated by ABCA1.

Mechanism of Prebeta-HDL Formation and Activation

The mechanism of formation of functional high-density lipoprotein (HDL) from secreted lipid-free apolipoprotein A1 (apo A1) was determined using human liver-derived (HepG2) cells, human intestine-derived (CaCO2) cells, and CHO cells stably expressing full-length human apo A1 (CHO-A1 cells). In each cell line, a significant proportion of secreted apo A1 had a Stokes radius of 2.6 nm and was inactive in binding phospholipids (PL) or free cholesterol (FC). Extracellularly, in a reaction dependent on membrane transporter ABCA1, prealpha-migrating 2.6 nm apo A1 was converted to a prebeta-migrating product that was able to bind PL. Both forms were reactive with mAb55201, a monoclonal antibody specific for native plasma lipid-poor (prebeta1) HDL [Nakamura, Y., et al. (2004) Biochemistry 43, 14311-14318]. The physical properties of precursor and product apo A1 suggested that both are monomers, with Stokes radii of 2.6 and 3.6 nm, respectively, consistent with the absence of intermolecular cross-linking of apo A1 in lipid-poor HDL, reported previously. Product but not precursor apo A1 promoted reverse cholesterol transport (RCT) from human aortic smooth muscle cells. These studies suggest an important contribution of secreted lipid-free apo A1 to HDL formation.

Kinetics of preβ1 HDL and αHDL in type II diabetic patients

The aim of this study was to analyze the recycling of high density lipoprotein (HDL) in six type II diabetic patients compared with six control subjects by endogenous labelling of apolipoprotein A-I (Apo A-I) with stable isotope Apo A. The -I-HDL kinetics were performed by infusion of (5.5.5-(2)H3)-leucine for 14 h. The prebeta1 and alphaHDL were separated by gel filtration fast protein liquid chromatrography system (FPLC). Kinetics of isotopic enrichment of Apo A-I were analyzed with a multi-compartmental model software (SAAM II, SAAM Institute, Seattle, WA). Plasma Apo A-I concentration was decreased in patients with type II diabetes as a result of a decrease in Apo A-I-alphaHDL (P < 0.05). Diabetic patients were also characterized by an increased relative contribution of Apo A-I in prebeta1 HDL (18.3 +/- 2.8% vs 11.9 +/- 3.7%, P < 0.01). The synthetic rate of prebeta1 HDL was slightly increased in diabetic patients compared with control (NS) and an increase of recycling rate of alpha to prebeta1 HDL was observed (11.67 +/- 3.14 d(-1) vs 7.09 +/- 4.51 d(-1), P < 0.05). The clearance rate of Apo A-I was higher in diabetic patients (P < 0.05 for Apo A-I-prebeta1 HDL and P < 0.005 for Apo A-I-alphaHDL). This study suggests that the usual increase in prebeta1 HDL in type II diabetic patients is mainly related to an increased conversion rate of alpha to prebeta1 HDL.

HDLs in apoA-I transgenic Abca1 knockout mice are remodeled normally in plasma but are hypercatabolized by the kidney

Patients homozygous for Tangier disease have a near absence of plasma HDL as a result of mutations in ABCA1 and hypercatabolize normal HDL particles. To determine the relationship between ABCA1 expression and HDL catabolism, we investigated intravascular remodeling, plasma clearance, and organ-specific uptake of HDL in mice expressing the human apolipoprotein A-I (apoA-I) transgene in the Abca1 knockout background. Small HDL particles (7.5 nm), radiolabeled with (125)I-tyramine cellobiose, were injected into recipient mice to quantify plasma turnover and the organ uptake of tracer. Small HDL tracer was remodeled to 8.2 nm diameter particles within 5 min in human apolipoprotein A-I transgenic (hA-I(Tg)) mice (control) and knockout mice. Decay of tracer from plasma was 1.6-fold more rapid in knockout mice (P < 0.05) and kidney uptake was twice that of controls, with no difference in liver uptake. We also observed 2-fold greater hepatic expression of ABCA1 protein in hA-I(Tg) mice compared with nontransgenic mice, suggesting that overexpression of human apoA-I stabilized hepatic ABCA1 protein in vivo. We conclude that ABCA1 is not required for in vivo remodeling of small HDLs to larger HDL subfractions and that the hypercatabolism of normal HDL particles in knockout mice is attributable to a selective catabolism of HDL apoA-I by the kidney.

Cellular cholesterol efflux to plasma from proteinuric patients is elevated and remains unaffected by antiproteinuric treatment

Lipid derangements are assumed to contribute to the elevated cardiovascular risk in proteinuric patients. The impact of proteinuria on reverse cholesterol transport (RCT) is unknown. The first step in RCT, cellular cholesterol efflux to plasma, may be altered in proteinuria, consequent to changes in pre-beta high-density lipoprotein (HDL) formation and plasma phospholipid transfer protein (PLTP) activity. In six non-diabetic male patients with nephrotic-range proteinuria and 12 matched healthy men, plasma (apo)lipoproteins, pre-beta HDL formation, PLTP activity as well as the ability of plasma to promote cholesterol efflux out of cultured human skin fibroblasts were determined. These variables were also measured in response to antiproteinuric treatment, consisting of single and dual RAAS blockade by losartan and lisinopril. Plasma total cholesterol (P<0.05), triglycerides (P<0.05), apolipoprotein (apo) A-I (P<0.001), apo B (P<0.001), PLTP activity (P<0.005) and pre-beta HDL formation (P<0.001) were higher in proteinuric patients. Cellular cholesterol efflux to plasma from proteinuric patients was 41% higher than to plasma from healthy subjects (P<0.001). Reduction of proteinuria from 5.0 to 1.4 g/day by dual RAAS blockade was associated with a 23% reduction in plasma apo B levels (P<0.05). Pre-beta HDL formation and plasma PLTP activity did not change significantly. Combined antiproteinuric treatment did not reduce the elevated cellular cholesterol efflux. Cellular cholesterol efflux to plasma from patients with nephrotic-range proteinuria is enhanced, in conjunction with elevated pre-beta HDL formation and plasma PLTP activity. These changes may attenuate the cardiovascular risk associated with proteinuria-associated hyperlipidaemia. Antiproteinuric therapy lowers plasma apo B, but does not affect cell-derived cholesterol efflux, suggesting that this therapy beneficially affects cardiovascular risk in proteinuric patients.

Serum phospholipid transfer protein mass as a possible protective factor for coronary heart diseases.

Phospholipid transfer protein (PLTP) can generate pre-beta high-density lipoprotein (HDL), an efficient acceptor of peripheral cholesterol, by mediating a process called HDL conversion. The transfer of phospholipids to immature HDL is also essential in maintaining reverse cholesterol transport. The phospholipid transfer activity of PLTP has been associated with various patho-physiological conditions; however, little information is available concerning the relationship between PLTP mass and disease. Using a sandwich enzyme-linked immunosorbent assay, PLTP concentration was measured and related to the risk of developing cardiovascular disease in a worksite-based cohort of Japanese men (n=2,567). Multiple linear regression analysis showed significant associations between PLTP and HDL cholesterol, triglycerides, low-density lipoprotein cholesterol, and body mass index (standardized beta=0.395, -0.191, -0.064, and -0.064, respectively; R(2)=0.31). During the follow-up period, there were 10 cases of coronary heart disease (CHD) and 7 of stroke. The multivariate adjusted relative risk of CHD was 0.46 (95% confidence interval, 0.20-1.07) for an increase of 1 standard deviation in the PLTP value (p=0.071). PLTP concentration was not related to the risk of stroke. The results of this prospective study indicate that the serum PLTP concentration would serve as a predictor of CHD, independent of HDL cholesterol, triglycerides and other established risk factors.

The effects of physical exercise on plasma prebeta-1 high-density lipoprotein

The impact of physical exercise on high-density lipoprotein (HDL) metabolism is recognized as a major mechanism of coronary artery disease (CAD) risk reduction. Prebeta-1 HDL subparticle species play a pivotal role in initiating reverse cholesterol transport (RCT). We examined the effect of acute physical exercise on plasma prebeta-1 HDL levels. Nineteen nonsmoking, healthy men (n = 11) and women (n = 8) not receiving lipid-altering medications completed dietary surveys, and had percent body fat determinations, and fasting blood drawn for measurements of plasma lipids, lipoproteins, apolipoprotein A-I (Apo A-I), and absolute and percent prebeta-1 HDL. Each subject completed cardiopulmonary exercise stress testing to Vo2max followed by a 4-km course of run-jogging. Laboratory measurements were repeated from blood drawn immediately after exercise. Mean ± SD values were determined for age, percent body fat, dietary calories, dietary cholesterol, dietary fat, and plasma lipids, lipoproteins, Apo A-I, and absolute and percent prebeta-1 HDL using 1-way analysis of variance (ANOVA). One-way ANOVA comparisons were made for measurements of plasma lipids, lipoproteins, Apo A-I, and absolute and percent prebeta HDL measurements taken before and after exercise for all subjects combined. Entry characteristics showed the following (mean ±SD): age, 24 ± 5.8 years; body mass index (BMI), 22.4 ± 2.6; percent body fat, 13 ± 5.7; and Vo2max, 49.1 ± 7.9 mL O2/kg/min. Exercise significantly increased absolute plasma prebeta HDL (0.10 ± 0.05 to 0.130 ± 0.07 μg/mL, P =.039) and decreased plasma HDL-triglycerides (23.3 ± 10.8 to 12.5 ± 5.6 mg/dL, P =.012). Our findings indicate that prebeta-1 HDL and HDL-triglyceride metabolism are significant components of the effect of acute exercise on RCT. These findings have important relevance for studies pertaining to exercise-related effects on HDL metabolism as pertains to CAD risk reduction. Copyright 2003 Elsevier, Inc. All rights reserved.

Down-regulation of hepatic lecithin:cholesterol acyltransferase gene expression in chronic renal failure

Chronic renal failure (CRF) is associated with premature arteriosclerosis, impaired high-density lipoprotein (HDL) maturation, increased pre-beta HDL (a lipid-poor HDL species), reduced HDL/total cholesterol ratio, hypertriglyceridemia, and depressed lipolytic activity. The latter has been, in part, attributed to elevated pre-beta HDL, which is a potent inhibitor of lipoprotein lipase (LPL). Accumulation of cholesterol in the arterial wall is a critical step in atherogenesis, and HDL-mediated cholesterol removal from peripheral tissues mitigates atherosclerosis. Lecithin:cholesterol acyltransferase (LCAT) is essential for maturation of HDL and cholesterol removal by HDL from peripheral tissues. Earlier studies have revealed depressed plasma LCAT enzymatic activity in patients with CRF. This study was conducted to determine whether impaired LCAT activity can be confirmed in CRF animals and if so whether it is due to down-regulation of hepatic LCAT expression. Hepatic tissue LCAT mRNA and plasma LCAT enzymatic activity were measured in male Sprague-Dawley rats six weeks after excisional 5/6 nephrectomy or sham operation. Compared with the controls, the CRF group exhibited a significant reduction of hepatic tissue LCAT mRNA abundance. The reduction in hepatic LCAT mRNA was accompanied by a marked reduction of plasma LCAT activity and elevation of serum-free cholesterol in the CRF animals. LCAT activity correlated positively with the HDL/total cholesterol ratio and inversely with free cholesterol and triglyceride concentrations. CRF leads to a marked down-regulation of hepatic LCAT mRNA expression and plasma LCAT activity. This abnormality can impair HDL-mediated cholesterol uptake from the vascular tissue and contribute to cardiovascular disease. In addition, LCAT deficiency can, in part, account for elevated serum-free cholesterol, reduced HDL/total cholesterol, and elevated pre-beta HDL in CRF. The latter can, in turn, depress lipolytic activity and hinder triglyceride-rich lipoprotein clearance in CRF.

Role of phospholipid transfer protein and preß-high density lipoproteins in maintaining cholesterol efflux from Fu5AH cells to plasma from insulin-resistant subjects

Plasma phospholipid transfer protein (PLTP) enhances the generation of prebeta-high density lipoproteins (HDL) that may act as initial acceptors of cellular cholesterol, and are likely to play an important role in the antiatherogenic process of reverse cholesterol transport. We examined the interrelationships between insulin resistance, the ability of plasma to stimulate cellular cholesterol efflux, HDL cholesterol, plasma PLTP activity and prebeta-HDL in 12 non-diabetic, non-smoking, normotriglyceridaemic men. Cholesterol efflux from Fu5AH cells to plasma, plasma lipoproteins, PLTP activity and prebeta-HDL formation as measured in incubated plasma were determined after a 12-h fast. Insulin sensitivity was assessed by a euglycaemic, hyperinsulinaemic clamp (M-value). HDL cholesterol was positively correlated with the M-value (r=0.65, p< 0.05), whereas plasma PLTP activity (r= -0.59, p <0.05) and prebeta-HDL in incubated plasma (r= -0.66, p<0.05) were negatively correlated with the M-value. Thus, the lower the insulin sensitivity, the lower was HDL cholesterol and the higher were plasma PLTP activity and prebeta-HDL. Cellular cholesterol efflux tended to be correlated with HDL cholesterol (r=0.55, p < 0.10) as well as with plasma PLTP activity (r=0.56, p<0.10) and was positively correlated with prebeta-HDL in incubated plasma (r=0.74, p<0.01). No positive correlation between the M-value and cellular cholesterol efflux was found (r= -0.34, ns). These preliminary results support the hypothesis that, despite a lower HDL cholesterol, the ability of plasma from insulin-resistant subjects to promote cellular cholesterol efflux is not impaired, as a consequence of a higher plasma PLTP activity and enhanced prebeta-HDL formation.

The antiatherogenic role of high-density lipoprotein cholesterol

Landmark clinical studies in the past 5 years that demonstrated diminished mortality and first coronary events following lowering of low-density lipoprotein (LDL) cholesterol stimulated considerable interest in the medical community. Yet, high-density lipoprotein (HDL) cholesterol, which transports circulating cholesterol to the liver for clearance, clearly also exerts antiatherogenic effects. The Framingham Heart Study produced compelling epidemiologic evidence indicating that a low level of HDL cholesterol was an independent predictor of coronary artery disease (CAD). Emerging experimental and clinical findings are, collectively, now furnishing a solid scientific foundation for this relation. First, the reverse cholesterol transport pathway—including the roles of nascent (pre-beta) HDL, apolipoprotein A-I, lecithin–cholesterol acyltransferase (LCAT), cholesteryl ester transport protein, and hepatic uptake of cholesteryl ester from HDL by liver—is better understood. For example, the identification of a hepatic HDL receptor, SR-BI, suggests a mechanism of delivery of cholesteryl ester to liver that differs from the receptor-mediated uptake of LDL. Second, apolipoprotein A-I, the major protein component of HDL, and 2 enzymes on HDL, paraoxonase and platelet-activating factor acetylhydrolase appear to diminish the formation of the highly atherogenic oxidized LDL. Third, lower levels of HDL cholesterol are associated in a dose–response fashion with the severity and number of angiographically documented atherosclerotic coronary arteries. Fourth, low HDL cholesterol predicts total mortality in patients with CAD and desirable total cholesterol levels (<200 mg/dL). Fifth, low HDL cholesterol concentrations appear to be associated with increased rates of restenosis after percutaneous transluminal coronary angioplasty. In terms of elevating HDL cholesterol, cessation of cigarette smoking, reduction to ideal body weight, and regular aerobic exercise all appear important. Most medications used to treat dyslipidemias will raise HDL cholesterol levels modestly; however, niacin appears to have the greatest potential to do so, and can increase HDL cholesterol up to 30%. Recognizing these data, the most recent report of the National Cholesterol Education Program identified low HDL cholesterol as a CAD risk factor and recommended that all healthy adults be screened for both total cholesterol and HDL cholesterol levels.

Selective down-regulation by protein kinase C inhibitors of apolipoprotein-mediated cellular cholesterol efflux in macrophages.

Extracellular apolipoprotein A-I removed cholesterol and phospholipid from cholesterol-loaded mouse peritoneal macrophage and thereby generated a prebeta high-density lipoprotein (HDL) particle having a weight ratio of cholesterol to phosphatidylcholine of approximately 1:1. Treatment of the cells with phorbol myristate slightly increased cholesterol efflux by this mechanism without influencing the nonspecific cholesterol efflux to the lipid microemulsion. When the cells were treated by protein kinase C (PKC) inhibitors, H7 and staurosporine, apolipoprotein-mediated cellular cholesterol efflux was substantially reduced without a significant change in phosphatidylcholine efflux, resulting in generation of cholesterol-poor prebeta-HDL particles having a weight ratio of cholesterol to phosphatidylcholine as low as 1:10. In spite of this change, specific binding of apoA-I to the cellular surface was unaffected. Cellular cholesterol available for acylCoA:cholesterol acyltransferase (ACAT) was rapidly depleted by adding apoA-I to the medium, and the PKC inhibitor treatment reversed this effect. In contrast, nonspecific cellular cholesterol efflux to the lipid microemulsion did not influence the ACAT-available cellular cholesterol pool, and it was not influenced by the PKC inhibitors. Thus, we concluded that apolipoprotein-mediated cellular cholesterol efflux is linked to mobilization of cholesterol from an intracellular pool used by ACAT to a specific pool for apolipoprotein-mediated prebeta-HDL generation, in response to apolipoprotein-cell interaction and subsequent intracellular signaling. Binding of apolipoprotein to the cell surface is required for assembly of the prebeta-HDL particle with cellular phospholipid, and the intracellular cholesterol mobilization is needed for enrichment with cholesterol of the prebeta-HDL. These reactions are largely independent of diffusion-mediated nonspecific cell cholesterol efflux.

Inhibitory effects of HepG2 cell-derived apolipoprotein A-I-containing lipoproteins on cholesteryl ester accumulation in macrophages.

We investigated the mechanisms of inhibitory effects on foam cell formation of apolipoprotein A-I-containing lipoproteins secreted by HepG2 cells (HepG2-HDL) using mouse peritoneal macrophages. When macrophages were incubated with acetylated low-density lipoprotein (acetyl-LDL) in the presence of HepG2-HDL, cholesterol ester (CE) accumulation in cells was reduced by 63%. This inhibitory capacity was almost similar to that of plasma high-density lipoprotein (HDL). When macrophages were converted to foam cells with acetyl-LDL and then reacted with HepG2-HDL or plasma HDL, the HDL-induced CE reduction was 2.2-fold greater than HepG2-HDL. Similar results were obtained using apo E-free HepG2-HDL. Since the inhibitory effect of HDL on acetyl-LDL-induced CE accumulation in macrophages is due largely to its cholesterol efflux capacity, these results suggest the presence of an additional mechanism for the inhibition of CE accumulation by HepG2-HDL. To investigate the mechanism, acetyl-LDL was reisolated from HepG2-HDL by Sephacryl S-300 gel filtration after incubation in a cell-free system. Reisolated acetyl-LDL showed a significant reduction in electrophoretic mobility. The extent of CE accumulation by reisolated acetyl-LDL was reduced by 20% compared with control acetyl-LDL. Moreover, its endocytic degradation by macrophages was reduced by 28%. HepG2-HDL also inhibited macrophage degradation of acetyl-LDL as well as oxidized LDL, a likely atherogenic lipoprotein. This inhibitory effect was ascribed to the HepG2-HDL subfraction containing pre-beta HDL. Our results indicated that apo A-I-containing lipoproteins as a physiological model of nascent HDL may inhibit foam cell formation by reducing ligand activity of atherogenic lipoproteins. These data possibly suggest inhibitory function of nascent HDL for the formation of foam cells in vivo.

Increased prebeta-high density lipoprotein, apolipoprotein AI, and phospholipid in mice expressing the human phospholipid transfer protein and human apolipoprotein AI transgenes.

Human plasma phospholipid transfer protein (PLTP) circulates bound to high density lipoprotein (HDL) and mediates both net transfer and exchange of phospholipids between different lipoproteins. However, its overall function in lipoprotein metabolism is unknown. To assess the effects of increased plasma levels of PLTP, human PLTP transgenic mice were established using the human PLTP gene driven by its natural promoter. One line of PLTP transgenic mice with moderate expression of PLTP mRNA and protein was obtained. The order of human PLTP mRNA expression in tissues was: liver, kidney, brain, small intestine > lung > spleen > heart, adipose tissue. Western blotting using a human PLTP monoclonal antibody revealed authentic human PLTP (Mr 80 kD) in plasma. Plasma PLTP activity was increased by 29% in PLTP transgenic mice. However, plasma lipoprotein analysis, comparing PLTP transgenic mice to control littermates, revealed no significant changes in the plasma lipoprotein lipids or apolipoproteins. Since previous studies have shown that human cholesteryl ester transfer protein and lecithin:cholesterol acyltransferase only function optimally in human apoAI transgenic mice, the human PLTP transgenic mice were cross-bred with human apoAI transgenic mice. In the human apoAI transgenic background, PLTP expression resulted in increased PLTP activity (47%), HDL phospholipid (26%), cholesteryl ester (24%), free cholesterol (37%), and apoAI (22%). There was a major increase of apoAI in prebeta-HDL (56%) and a small increase in alpha-HDL (14%). The size distribution of HDL particles within alpha- and prebeta-migrating species was not changed. The results suggest that PLTP increases the influx of phospholipid and secondarily cholesterol into HDL, leading to an increase in potentially antiatherogenic prebeta-HDL particles.

Lecithin:cholesterol acyltransferase reaction on cellular lipid released by free apolipoprotein-mediated efflux.

Lecithin:cholesterol acyltransferase (LCAT) reaction was studied in free apolipoprotein-mediated cellular lipid efflux from mouse peritoneal macrophages and human skin fibroblasts. When the cells were incubated with lipid-free human apolipoproteins (apo) A-I or A-II, pre-beta high density lipoprotein (HDL) particles were generated by removing cellular cholesterol and phospholipid. Cholesterol was esterified by LCAT in such particles generated with human apoA-I, but not in those with apoA-II. The reactivity of the apoA-I-pre-beta-HDL particles with LCAT was in the same order as that in human plasma HDL and in phosphatidylcholine/cholesterol unilamellar vesicles activated by apoA-I when compared on the rate of percent cholesterol esterification. However, cholesterol efflux mediated by apoA-I was not enhanced by active cholesterol esterification in the medium from either type of cells. Thus, it is unlikely the LCAT reaction on newly generated pre-beta-HDL directly causes further cellular cholesterol efflux. In control experiments, LCAT esterified cholesterol on human plasma HDL in the cell medium regardless of its origin, either HDL or cells. Cholesterol esterification on HDL was unable to enhance cellular cholesterol efflux significantly but reduced the influx of cholesterol from HDL to cell, resulting in the increase of net efflux of cellular cholesterol, in agreement with the results previously demonstrated.