High Density Lipoprotein Structure Research Articles

Structural investigation of reconstituted high density lipoproteins by scanning tunnelling microscopy

Being able to participate in the reverse cholesterol transport (RCT), high density lipoproteins (HDL) are known to be anti-atherogenic. In order to understand such a process, it is thus essential to have a detailed knowledge of the structure and molecular organisation of HDL. Reconstituted nascent high density lipoproteins (r-HDL), consisting of synthetic phospholipids together with different apolipoproteins (apo A-I, A-IV and E), were thus analysed by scanning tunnelling microscopy (STM). Both shape and dimensions of the discoidal HDL particles measured by this technique were found in good agreement with the data available from the literature. The accuracy of the STM pictures presented in this paper enables for the first time the visualisation of the molecular organisation of such macromolecules. The arrangement of the protein as antiparallel helical segments, is consistent with the general mode of organisation of apolipoprotein/phospholipid discoidal particles previously reported.

Endothelial lipase is a major genetic determinant for high-density lipoprotein concentration, structure, and metabolism.

High-density lipoprotein (HDL) protects against atherosclerosis. Endothelial lipase (EL) has been postulated to be involved in lipoprotein, and possibly HDL, metabolism, yet the evidence has been scarce and conflicting. We have inactivated EL in mice by gene targeting. EL(-/-) mice have elevated plasma and HDL cholesterol, and increased apolipoproteins A-I and E. NMR analysis reveals an abundance of large HDL particles. There is down-regulation of the transcripts for phospholipid transfer protein, but up-regulation of those for hepatic lipase and lipoprotein lipase. Plasma lecithin:cholesterol acyltransferase is unchanged despite an increase in hepatic mRNA; lecithin:cholesterol acyltransferase activity toward endogenous EL(-/-) substrate is, however, reduced by 50%. HDL clearance is decreased in EL(-/-) mice; both the structure of HDL and the presence of EL are factors that determine the rate of clearance. To determine EL's role in humans, we find a significant association between a single-nucleotide polymorphism 584C/T in the EL (LIPG) gene and HDL cholesterol in a well characterized population of 372 individuals. We conclude that EL is a major determinant of HDL concentration, structure, and metabolism in mice, and a major determinant of HDL concentration in humans.

Significant impact of the highly informative (CA)n repeat polymorphism of the APOA-II gene on the plasma APOA-II concentrations and HDL subfractions: The ECTIM study.

High density lipoproteins (HDL) are heterogeneous in their apolipoprotein composition and the role of apolipoprotein A-II (APOA-II) in HDL structure and metabolism is poorly understood. Yet, studies of naturally occurring variations of APOA-II in mice and experiments in transgenic mice overexpressing the APOA-II gene (APOA-II) have shown that APOA-II expression influences APOA-II plasma levels and HDL size and composition. In humans, two RFLPs (BstNI and MspI) have been described in the APOA-II gene. These RFLPs, however, have been inconstantly associated with variations in APOA-II plasma levels. In particular, the large multicentric ECTIM Study did not show any significant effect of the two RFLPs. Other polymorphisms consisting of repetitive sequences have been proposed as more informative markers than RFLPs. Thus, data from the ECTIM Study were reconsidered by integrating the additional information obtained from a highly informative multiallelic (CA)(n)-repeat polymorphism located in the second intron of the gene. The population study was composed of 763 non-treated male controls and 594 cases of myocardial infarction. In controls, the (CA)(19) allele was associated with significantly decreased APOA-II (P < 0.0009) and LpA-II:A-I (P < 0.02) plasma levels. Although the APOA-I plasma levels were not affected by the polymorphism, the (CA)(19) allele was associated with an increased LpA-I/LpA-II:A-I ratio (P < 0.004). No effect, however, could be detected on myocardial infarction. Study of the linkage disequilibrium and the estimation of haplotype frequencies indicated that the impact of the APOA-II locus could hardly be detected by using the BstNI and MspI RFLPs. These data revive interest in evaluating the role of the APOA-II locus in the control of APOA-II plasma levels and HDL composition.

Cyclosporine A transfer between high‐ and low‐density lipoproteins: Independent from lipid transfer protein I‐facilitated transfer of lipoprotein‐coated phospholipids because of high affinity of cyclosporine a for the protein component of lipoproteins

The objectives of this study were to determine if lipid transfer protein I (LTP I)‐facilitated phospholipid (PC) transfer activity regulates the plasma lipoprotein distribution of cyclosporine (CSA) and if the association of CSA with high‐density lipoproteins (HDL) is due to the high protein and/or alterations in coat lipid content of HDL. To assess if LTP I‐facilitated PC transfer activity regulates the plasma lipoprotein distribution of CSA, 14C‐PC‐ or 3H‐CSA‐enriched HDL or low‐density lipoproteins (LDL) were incubated in T150 buffer [pH 7.4, containing a 14C‐PC‐ or 3H‐CSA‐free lipoprotein counterpart ± exogenous LTP I (1.0 μg protein/mL)] or in delipidated human plasma that contained 1.0 μg protein/mL of endogenous LTP I in the presence or absence of a monoclonal antibody TP1 (30 μg protein/mL) directed against LTP I for 90min at 37°C. To assess the influence of HDL subfraction lipid composition and structure on the plasma distribution of CSA, CSA at 1000ng of drug/mL of plasma was incubated in human plasma pretreated for 24hwith a lecithin:cholesterol acyltransferase (LCAT) inhibitor, dithionitrobenzoate (DTNB; 3mM). To assess the binding of CSA to apolipoproteins AI, AII, and B, increasing concentrations of CSA were added to a constant concentration of either apolipoprotein AI, AII, or B. Equilibrium dialysis was used to determine free and bound fractions and Scatchard plot analysis was used to determine binding coefficients. To assess the influence of hydrophobic core lipid volume on the plasma distribution of CSA, CSA was incubated in plasma from patients with well‐characterized dyslipidemias. The hydrophobic core lipid volume (CE+TG) within each lipoprotein subfraction was correlated to the amount of CSA recovered in each plasma sample from the different human subjects. The percent transfer of PC from LDL to HDL was different than the percent transfer of CSA in T150 buffer or human plasma source. In the presence of TP1, only PC transfer from LDL to HDL decreased. For plasma incubated with CSA and separated into HDL2 and HDL3, 35–50% of drug originally incubated was recovered in the HDL3 fraction, with the remaining drug being found within the other fractions. When CSA was incubated in plasma pretreated with DTNB, the percentage of CSA recovered in the HDL3 and HDL2 fractions was not significantly different compared with that in the HDL3 and HDL2 fractions from untreated control plasma. CSA distribution into HDL inversely correlated with the hydrophobic core lipid volume of HDL, whereas distribution into LDL and triglyceride‐rich lipoproteins directly correlated with their respective hydrophobic core lipid volumes. We further observed that CSA has high binding affinity and multiple binding sites with apolipoproteins AI (kd = 188.9 nM; n = 2), AII (kd = 184.7 nM; n = 2), and B (kd = 191 nM; n = 3). These findings suggest that the transfer of CSA between different lipoprotein particles is not influenced by LTP I‐facilitated PC transfer activity probably because of the high affinity of CSA for the protein components of HDL and LDL. © 2001 Wiley‐Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 90:1308–1317, 2001

In vivo interactions of apoA-II, apoA-I, and hepatic lipase contributing to HDL structure and antiatherogenic functions

Studies with mice have revealed that increased expression of apolipoprotein A-II (apoA-II) results in elevations in high density lipoprotein (HDL), the formation of larger HDL, and the development of early atherosclerosis. We now show that the increased size of HDL results in part from an inhibition of the ability of hepatic lipase (HL) to hydrolyze phospholipids and triglycerides in the HDL and that the ratio of apoA-I to apoA-II determines HDL functional and antiatherogenic properties. HDL from apoA-II transgenic mice was relatively resistant to the action of HL in vitro. To test whether HL and apoA-II influence HDL size independently, combined apoA-II transgenic/HL knockout (HLko) mice were examined. These mice had HDL similar in size to apoA-II transgenic mice and HLko mice, suggesting that they do not increase HDL side by independent mechanisms. Overexpression of apoA-I from a transgene reversed many of the effects of apoA-II overexpression, including the ability of HDL to serve as a substrate for HL. Combined apoA-I/apoA-II transgenic mice exhibited significantly less atherosclerotic lesion formation than did apoA-II transgenic mice. These results were paralleled by the effects of the transgenes on the ability of HDL to protect against the proinflammatory effects of oxidized low density lipoprotein (LDL). Whereas nontransgenic HDL protected against oxidized LDL induction of adhesion molecules in endothelial cells, HDL from apoA-II transgenic mice was proinflammatory. HDL from combined apoA-I/apoA-II transgenic mice was equally as protective as HDL from nontransgenic mice. Our data suggest that as the ratio of apoA-II to apoA-I is increased, the HDL become larger because of inhibition of HL, and lose their antiatherogenic properties.

Role of SR-BI in regulating lipoprotein structure and metabolism.

The scavenger receptor class B type I (SR-BI) is a cell surface lipoprotein receptor that mediates physiologically relevant selective cholesteryl ester uptake from high-density lipoprotein (HDL). SR-BI appears to be required for the maintenance of reverse cholesterol transport, normal HDL levels and HDL structure by means of hepatic-selective HDL-cholesterol and phospholipid uptake in mice. SR-BI can also promote the selective uptake of cholesteryl ester from apoB-containing lipoproteins; however, unlike the effect of SR-BI on HDL-cholesterol, the effect on low-density lipoprotein is limited in vivo. Furthermore, SR-BI plays a protective role against atherosclerotic development. A pharmacological agent that increased reverse cholesterol transport by targeting SR-BI could become a powerful tool for prevention of or therapeutic intervention in atherosclerotic cardiovascular diseases.

A Key Point Mutation (V156E) Affects the Structure and Functions of Human Apolipoprotein A-I

A Key Point Mutation (V156E) Affects the Structure and Functions of Human Apolipoprotein A-I

Influence of the high density lipoprotein receptor SR-BI on reproductive and cardiovascular pathophysiology.

The high density lipoprotein (HDL) receptor SR-BI (scavenger receptor class B type I) mediates the selective uptake of plasma HDL cholesterol by the liver and steroidogenic tissues. As a consequence, SR-BI can influence plasma HDL cholesterol levels, HDL structure, biliary cholesterol concentrations, and the uptake, storage, and utilization of cholesterol by steroid hormone-producing cells. Here we used homozygous null SR-BI knockout mice to show that SR-BI is required for maintaining normal biliary cholesterol levels, oocyte development, and female fertility. We also used SR-BI/apolipoprotein E double homozygous knockout mice to show that SR-BI can protect against early-onset atherosclerosis. Although the mechanisms underlying the effects of SR-BI loss on reproduction and atherosclerosis have not been established, potential causes include changes in (i) plasma lipoprotein levels and/or structure, (ii) cholesterol flux into or out of peripheral tissues (ovary, aortic wall), and (iii) reverse cholesterol transport, as indicated by the significant reduction of gallbladder bile cholesterol levels in SR-BI and SR-BI/apolipoprotein E double knockout mice relative to controls. If SR-BI has similar activities in humans, it may become an attractive target for therapeutic intervention in a variety of diseases.

In vivo regulation of plasma platelet-activating factor acetylhydrolase during the acute phase response.

Plasma platelet-activating factor acetylhydrolase (PAF-AH) hydrolyzes PAF and oxidized phospholipids and is associated with lipoproteins in the circulation. Endotoxin [lipopolysaccharide (LPS)], a potent inducer of the acute phase response (APR), produces marked changes in several proteins that play important roles in lipoprotein metabolism. We now demonstrate that LPS produces a 2.5- to 3-fold increase in plasma PAF-AH activity in Syrian hamsters. The plasma PAF-AH activity is found in the high-density lipoprotein (HDL) fraction and is increased threefold with LPS treatment despite a decrease in plasma HDL levels, indicating that plasma PAF-AH activity is increased per HDL particle. LPS markedly increased PAF-AH mRNA levels in liver, spleen, lung, and small intestine. The maximal increase in plasma PAF-AH activity and mRNA expression in liver and spleen is seen 24 h after LPS treatment. Both tumor necrosis factor and interleukin-1 modestly increased plasma PAF-AH activity and mRNA levels in liver and spleen, suggesting that they may partly mediate the effect of LPS on PAF-AH. Surgical removal of spleen had no effect on basal or LPS-induced plasma PAF-AH activity, suggesting that spleen per se may not contribute to plasma PAF-AH activity. Finally, LPS, turpentine and zymosan increased plasma PAF-AH activity in mice and/or rats, indicating that multiple APR inducers upregulate plasma PAF-AH and this effect is consistent across different rodent species. Taken together, our results indicate that plasma PAF-AH activity and mRNA expression is markedly upregulated during the host response to infection and inflammation. An increase in plasma PAF-AH may enhance the degradation of PAF as well as alter the structure and function of HDL during infection and inflammation.

Modifications in High-Density Lipoprotein Lipid Composition and Structure Alter the Plasma Distribution of Free and Liposomal Annamycin

Recent studies have shown that changes in lipoprotein cholesterol and triglyceride concentration alters the plasma distribution of free (Ann.) and liposomal annamycin (LAnn) and that the majority of Ann. is associated with high-density lipoproteins (HDL) following the incubation in plasma of LAnn. To demonstrate that alterations in HDL lipid composition and HDL structure may influence the plasma distribution of Ann. and LAnn, Ann. and LAnn (20 ,μg/mL) were incubated in plasma pretreated with dithionitrobenzoate (DTNB, a compound which inhibits the conversion of free cholesterol to esterified cholesterol) 18h prior to the experiment or in untreated plasma for 60min at 37 °C. In addition, Ann. and LAnn were co-incubated with DTNB in plasma for 6min at 37 °C. Following incubation the plasma was separated into its HDL, low- density lipoprotein (LDL), very-low-density lipoprotein (VLDL), and lipoprotein-deficient plasma (LPDP) fractions by ultracentrifugation and assayed for Ann. by fluorimetry. The HDL plasma cholesterol:triglyceride concentration ratio was significantly decreased following 18h of DTNB pretreatment compared to untreated plasma controls. No significant differences in LDL/VlDL plasma cholesterol:triglyceride concentration ratio following 18h of DTNB pretreatment was observed. An increased number of discoidal HDL particles were observed following 18h of DTNB pretreatment. When Ann. was incubated in plasma pretreated with DTNB for 18h the percentage of Ann. recovered in the HDL, LDL, and VLDL fractions significantly increased. However, the percentage of Ann. recovered within the LPDP fraction was significantly decreased. When LAnn was incubated in plasma pretreated with DTNB for 18h the percentage of Ann. recovered in the HDL fraction significantly decreased. The percentage of Ann. recovered in the LPDP fraction significantly increased when LAnn was incubated in plasma pretreated with DTNB for 18 h. No significant differences in Ann. lipoprotein distribution were observed when Ann. and LAnn were co-incubated with DTNB in plasma for 1 h. These findings suggest that the cholesterol:triglyceride concentration ratio and physical structure of HDL maybe important in defining the capacity of hDl to sequester Ann.

Disruption of the murine lecithin:cholesterol acyltransferase gene causes impairment of adrenal lipid delivery and up-regulation of scavenger receptor class B type I.

Lecithin:cholesterol acyltransferase (LCAT) is the major determinant of the cholesteryl ester (CE) content of high density lipoprotein (HDL) in plasma. The selective uptake of HDL-CE is postulated to participate in delivery of tissue-derived cholesterol both to the liver and steroidogenic tissues. Recent studies comparing mice with similarly low levels of HDL, due to the absence of either of the two major HDL-associated apolipoproteins apoA-I and apoA-II, suggest that apoA-I is crucial in modulating this process, possibly through interaction with scavenger receptor class B type I (SR-BI). Because of the central role of LCAT in determining the size, lipid composition, and plasma concentration of HDL, we have created LCAT-deficient mice by gene targeting to examine the effect of LCAT deficiency on HDL structure and composition and adrenal cholesterol delivery. The HDL in the LCAT-deficient mice was reduced in its plasma concentration (92%) and CE content (96%). The HDL particles were heterogeneous in size and morphology and included numerous discoidal particles, mimicking those observed in LCAT-deficient humans. The adrenals of the male Lcat (-/-) mice were severely depleted of lipid stores, which was associated with a 2-fold up-regulation of the adrenal SR-BI mRNA. These studies demonstrate that LCAT deficiency, similar to apoA-I deficiency, is associated with a marked decrease in adrenal cholesterol delivery and supports the hypothesis that adrenal SR-BI expression is regulated by the adrenal cholesterol.

Fish-eye disease: Structural and in vivo metabolic abnormalities of high-density lipoproteins

Fish-eye disease (FED) in humans is characterized by corneal opacities and markedly decreased plasma concentrations of high-density lipoprotein (HDL) cholesterol, apolipoprotein (apo) AI, and apo AII, but no tendency to precocious atherosclerosis is present. To elucidate this paradox, the structure of HDL, the potential of serum to promote cholesterol efflux from cultured cells, and the in vivo metabolism of HDL were examined in a 53-year-old woman with a FED syndrome in association with a markedly decreased lecithin:cholesterol acyltransferase (LCAT) activity in HDL due to a mutation of the LCAT gene (Arg 158 → Cys). HDLs isolated by ultracentrifugation were small and enriched in unesterified cholesterol and phospholipids at the expense of cholesteryl esters and proteins. The apolipoprotein content showed an enrichment in apo E and apo AIV, whereas apo AI and apo AII were dramatically reduced. Sodium dodecyl sulfate—polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting using specific antibodies showed that the apo E was free or covalently bound to apo AII. These particles analyzed by electron microscopy were small and round lipoproteins with a size similar to the smallest fraction of normal HDL 3. The potential capacity of the serum to promote efflux from the cells was approximately 40% of control serum levels, but FED HDLs were as efficient as control HDLs in promoting cholesterol efflux from cells. To assess the metabolism of HDL apolipoproteins, in vivo apolipoprotein kinetic studies were performed using endogenous labeling techniques in the patient with FED and three control subjects. All subjects were administered D 3-labeled leucine by primed constant infusion for up to 10 hours. The fractional synthetic rates (FSRs) of apo AI and apo AII in the patient were 0.674 and 0.594 per day, clearly higher than in controls, 0.210 ± 0.053 and 0.148 ± 0.014 per day for apo AI and apo AII, respectively. Apo AI and apo AII production rates in the patient with FED were normal, 11.32 and 2.62 mg/kg · d, respectively, as compared with those in normal subjects, 11.45 ± 1.23 and 2.68 ± 0.17 mg/kg · d. These data established that hypoalphalipoproteinemia in FED was caused by marked hypercatabolism of apo AI and apo AII. This hypercatabolism could be the consequence of structural abnormalities due to the selective LCAT deficiency. In conclusion, two steps of reverse cholesterol transport, cholesterol efflux and apo-HDL metabolism, appeared particularly efficient. This efficiency could participate in the absence of premature atherosclerosis in FED patients as regards the low HDL level.

Bile salt stimulated cholesterol esterase increases uptake of high density lipoprotein-associated cholesteryl esters by HepG2 cells.

Bile salt stimulated cholesterol esterase is predominantly synthesized in the pancreas. However, this enzyme is also synthesized by the liver and was found to be present in plasma. The physiologic role of the systemic cholesterol esterase has not been clearly defined. In the current study, the human hepatoma cell line HepG2 was used as a model to determine the role of cholesterol esterase on hepatic uptake of high density lipoprotein (HDL)-associated cholesteryl esters. The results showed that hepatic uptake of the cholesteryl esters analog [3H]cholesteryl ether on reconstituted HDL was inhibited by anti-cholesterol esterase antibodies. The HDL-associated cholesteryl ester transported to HepG2 cells was also increased 2-fold in the presence of taurocholate, an activator of the cholesterol esterase. These results suggest that liver-derived cholesterol esterase may play an important role in cellular uptake of cholesteryl esters from HDL. This hypothesis was supported by demonstrating the ability of exogenously added cholesterol esterase to further enhance hepatic uptake of HDL-associated cholesteryl esters. The results of the current study also showed that cholesterol esterase increased free-to-esterified cholesterol ratio in the lipoprotein. Thus, alteration of HDL structure and composition contributes to the cholesterol esterase-induced cellular uptake of HDL-associated cholesteryl esters. On the basis of these observations, we propose that liver-derived cholesterol esterase may play an important role in lipoprotein metabolism.

Modification of LCAT activity and HDL structure. New links between cigarette smoke and coronary heart disease risk.

The mechanism(s) through which smoking influences the progression of atherosclerosis is poorly understood. Recent evidence suggests that oxidants present in the gas phase of cigarette smoke are involved. We exposed human plasma to the filtered gas phase of cigarette smoke to assess its effects on plasma components involved in the antiatherogenic reverse cholesterol transport pathway. In our model, freshly isolated plasma (24 mL) was exposed to filtered air or gas-phase cigarette smoke for up to 6 hours at 37 degrees C. Lecithin-cholesterol acyltransferase (LCAT) activity was dramatically inhibited by cigarette smoke. A single 15-minute exposure to the smoke from an eighth of a cigarette was sufficient to reduce LCAT activity by 7%; additional exposures resulted in further decreases in activity. At 6 hours, only 22% of control LCAT activity remained in plasma exposed to smoke. Compared with control, gas-phase cigarette smoke-exposed plasma possessed high-density lipoprotein (HDL) with increased (16%) negative charge and with cross-linked apolipoproteins AI and AII. These data demonstrate that gas-phase cigarette smoke can inhibit a key enzyme (LCAT) and modify an integral lipid transport particle (HDL) that are essential components for the normal function of the reverse cholesterol transport pathway. Gas-phase cigarette smoke-induced modification of the reverse cholesterol transport pathway may provide a new mechanistic link between cigarette smoke and coronary heart disease risk.

Effects of dietary manganese deficiency on high density lipoprotein composition and metabolism in Sprague-Dawley rats

Manganese (Mn) has been implicated in the development of atherosclorosis but its in vivo role in lipid and lipoprotein metabolism has been inconclusive to date. The objectives of this experiment were to study the effect of dietary Mn deificiency on high density lipoprotein (HDL) composition and structure. Weanling male Sprague-Dawley (SD) rats were fed either a Mn-deficient (MnD, 1.00 ppm) or a Mn-supplemented (MnS, 82.00 ppm) diet for 8 weeks. Very low density lipoproteins, low density lipoproteins and HDL, prestained with Sudan black, were separated by a discontinuous density gradient ultracentrifugation. High density lipoproteins were delipidated, apolipoproteins were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis (5–20% gradient) and quantified by laser densitometry. Fluorescence spectroscopy was used to study changes in HDL structure with dietary Mn deficiency. Body weights, liver weights, liver Mn levels, total plasma protein, HDL cholesterol and HDL protein were significantly lower (P≤0.05) in the MnD rats. The molecular weight of apolipoprotein C-I was significantly less (P<-0.01), and the relative peak area of apolipoprotein E3 was less (P≤0.1) in the MnD rats. Fluorescence studies of apo-HDL showed 71% of tryptophan residues accessible to quenching by Mn +2 with a quenching constant of 1.03×10 5 M −1 for the MnD rats vs. 58% and 1.29×10 5 M −1 respectively, for the MnS rats. The data indicate that Mn is involved in HDL metabolism. Changes in HDL composition and surface charge may significantly affect cholesterol transport and metabolism in the SD rat.

Simvastatin-induced decrease in the transfer of cholesterol esters from high density lipoproteins to very low and low density lipoproteins in normolipidemic subjects

Hyperlipidemic patients often have an accelerated esterified cholesterol transfer (ECT) from high density lipoproteins (HDL) to very low (VLDL) and low density lipoproteins (LDL). We investigated the effect of simvastatin on ECT in twelve normolipidemic subjects. After 6 weeks of simvastatin administration, ECT was decreased by 23%. To determine the mechanism of action of simvastatin, we measured ECT in different recombination experiments, using an isotopic assay in which the transfer of labelled EC from HDL to VLDL/LDL was determined. When HDL of the treated subjects were incubated with VLDL/LDL and CETP fractions isolated from control plasma, no effect of simvastatin was observed, indicating that the drug did not alter the HDL-dependent ECT. This might be expected since simvastatin induced only minor modifications of HDL structure. When HDL and VLDL/LDL of control plasma were incubated with CETP fractions of the treated subjects, a clear reduction of ECT occurred after simvastatin administration. The decrease of plasma transfer activity was correlated to that of CETP concentration and accounted for the simvastatin-induced lowering of ECT. The diminution of plasma CETP was correlated to that of the apo B-containing lipoproteins concentration. This finding confirms previous reports suggesting a relationship between LDL level and CETP activity. In conclusion, our work shows that simvastatin administration results in a decrease of ECT and that this effect occurs through a lowering of plasma CETP activity.

Effect of LCAT on HDL-mediated cholesterol efflux from loaded EA.hy 926 cells

1. 1. Human endothelial cells (EA.hy 926 line) were loaded with cholesterol, using cationized LDL, and the effect of lecithin:cholesterol acyltransferase (LCAT) on cellular cholesterol efflux mediated by high density lipoproteins (HDL) was measured subsequently. 2. 2. In plasma, lecithin:cholesterol acyltransferase (LCAT) converts unesterified HDL cholesterol into cholesteryl esters, thereby maintaining the low UC/PL ratio of HDL. It was tested if further decrease in UC/PL ratio of HDL by LCAT influences cellular cholesterol efflux in vitro. 3. 3. Efflux was measured as the decrease of cellular cholesterol after 24 hr of incubation with various concentrations of HDL in the presence and absence of LCAT. LCAT from human plasma (about 3000-fold purified) was added to the cell culture, resulting in activity levels in the culture media of 60–70% of human serum. 4. 4. Although LCAT had a profound effect on HDL structure (UC/TC and UC/PL ratio's decreased), the enzyme did not enhance efflux of cellular cholesterol, using a wide range of HDL concentrations (0.05–2.00 mg HDL protein/ml). 5. 5. The data indicate that the extremely low unesterified cholesterol content of HDL, induced by LCAT, does not enhance efflux of cholesterol from loaded EA.hy 926 cells. It is concluded that the HDL composition (as isolated from plasma by ultracentrifugation) is optimal for uptake of cellular cholesterol.

A rapid method for the synthesis of protein-lipid complexes using adsorption chromatography.

A novel and rapid method for the detergent-mediated synthesis of protein-lipid complexes has been developed and has several advantages over detergent dialysis methods. This new method involves co-incubation of human apolipoprotein A-I (apoA-I), the major protein component of high density lipoproteins (HDL), and dipalmitoylphosphatidylcholine for 1 hr in the presence of cholate, after which removal of greater than 99.7% of the detergent is achieved by a 2-hr batch adsorptive chromatography procedure. Complexes prepared by this method had a density of 1.10 g/ml, similar to plasma HDL. Chemical cross-linking of these products demonstrated that there was complete conversion of apoA-I to a protein-lipid complex that contained two molecules of apoA-I. One major band was resolved by gradient gel electrophoresis in the region of the gel expected for newly synthesized HDL. Results are described which show the application of this method to the study of lipid variation on the structure of model HDL, including the alteration of lipid-protein molar ratios and the addition of cholesterol.

Structural modifications of rat serum high density lipoprotein by pancreatic phospholipase A2.

An important and unusual aspect of the high density lipoprotein (HDL) in the rat is its tendency to undergo marked alterations in structure in response to physiological perturbations. In this study, the role of the surface lipids for maintenance of HDL integrity were investigated. Hydrolysis by pancreatic phospholipase A2 of the phospholipids of rat HDL in the presence of the d greater than 1.21 g/ml fraction of rat serum results in an increase in the particle diameter and an uptake of apo-E and apo A-IV from the lipoprotein-free fraction; augmentation of the albumin concentration in the incubation mixture intensified the observed changes, probably due to enhancement of the compositional changes brought about by phospholipase treatment. Phospholipase A2, treatment of the d less than 1.21 g/ml fraction of rat serum produces only minor changes in the properties of the isolated HDL. These data suggest that changes in apoprotein content reflect an uptake of A-IV and E by the rat HDL, rather than a net loss of apo A-I. Likewise, titration of the action of pancreatic phospholipase A2 on HDL apoprotein composition showed that initially a modest increase in apo A-IV content occurred, but with more extensive phospholipolysis there was a considerably greater increase in the apo-E content of the particle. The data suggest that hydrolysis of phospholipids such as occurs physiologically through the action of lecithin:cholesterol acyl transferase and hepatic lipase may alter the HDL structure independently from changes effected in the neutral lipid core.

Enzymatically induced alterations in the structure of rat serum lipoproteins

Incubation of freshly isolated rat serum induces a large number of changes in the properties of the serum lipoproteins, especially the high density lipoproteins (HDL). The particle diameter of the HDL increases from about 10.4 nm to 12.3 nm and the protein content appears to increase by about 60,000 Daltons. Reactions catalyzed by lecithin:cholesterol acyltransferase (LCAT) lead to a marked decrease in cholesterol and phospholipid content, and an even greater increase in cholesteryl ester content. Especially noteworthy are the marked increases in apoE and apoA-IV which are found associated with HDL as a result of this process. Data indicate that the affinity of apoE and apoA-IV for the HDL particles may be influenced by the proportion of surface to core lipid and by the presence of products of the LCAT reaction. Changes in the apoprotein content of very low density lipoproteins are also observed, with A-I and A-IV appearing in this density interval. All of the above changes can be prevented by the inclusion of 5,5'dithiobis(2-nitrobenzoate) or p-chloromercuriphenylsulfonate during the incubation, or by heat treatment of serum at 56 degrees C for 30 min; these treatments are known to inhibit LCAT activity. It is concluded that LCAT action is the major cause of the various changes in HDL structure that are observed and that alterations in apoprotein content occur to correct the resultant imbalance between core lipid and coverage of this core by amphiphilic components. Increased apoE association with cholesteryl ester-rich HDL may provide an efficient means for receptor-mediated removal of cholesterol from the circulation.