High Density Lipoprotein Apoprotein Research Articles

Cross-linking modifications of HDL apoproteins by oxidized phospholipids: structural characterization, in vivo detection, and functional implications

Apolipoprotein A-I (apoA-I) is cross-linked and dysfunctional in human atheroma. Although multiple mechanisms of apoA-I cross-linking have been demonstrated in vitro, the in vivo mechanisms of cross-linking are not well-established. We have recently demonstrated the highly selective and efficient modification of high-density lipoprotein (HDL) apoproteins by endogenous oxidized phospholipids (oxPLs), including γ-ketoalkenal phospholipids. In the current study, we report that γ-ketoalkenal phospholipids effectively cross-link apoproteins in HDL. We further demonstrate that cross-linking impairs the cholesterol efflux mediated by apoA-I or HDL3 in vitro and in vivo Using LC-MS/MS analysis, we analyzed the pattern of apoprotein cross-linking in isolated human HDL either by synthetic γ-ketoalkenal phospholipids or by oxPLs generated during HDL oxidation in plasma by the physiologically relevant MPO-H2O2-NO2- system. We found that five histidine residues in helices 5-8 of apoA-I are preferably cross-linked by oxPLs, forming stable pyrrole adducts with lysine residues in the helices 3-4 of another apoA-I or in the central domain of apoA-II. We also identified cross-links of apoA-I and apoA-II with two minor HDL apoproteins, apoA-IV and apoE. We detected a similar pattern of apoprotein cross-linking in oxidized murine HDL. We further detected oxPL cross-link adducts of HDL apoproteins in plasma and aorta of hyperlipidemic LDLR-/- mice, including cross-link adducts of apoA-I His-165-apoA-I Lys-93, apoA-I His-154-apoA-I Lys-105, apoA-I His-154-apoA-IV Lys-149, and apoA-II Lys-30-apoE His-227. These findings suggest an important mechanism that contributes to the loss of HDL's atheroprotective function in vivo.

Characterization of covalent modifications of HDL apoproteins by endogenous oxidized phospholipids.

High density lipoprotein (HDL) is cardioprotective, unless it is pathologically modified under oxidative stress. Covalent modifications of lipid-free apoA-I, the most abundant apoprotein in HDL, compromise its atheroprotective functions. HDL is enriched in oxidized phospholipids (oxPL) in vivo in oxidative stress. Furthermore, oxidized phospholipids can covalently modify HDL apoproteins. We have now carried out a systematic analysis of modifications of HDL apoproteins by endogenous oxPL. Human HDL or plasma were oxidized using a physiologically relevant MPO-H2O2-NO2- system or AIPH, or were exposed to synthetic oxPL. Protein adduction by oxPL was assessed using LC-MS/MS and MALDI-TOF MS. The pattern of HDL apoprotein modification by oxPL was independent of the oxidation systems used. ApoA-I and apoA-II were the major modification targets. OxPL with a γ-hydroxy (or oxo)-alkenal were mostly responsible for modifications, and the Michael adduct was the most abundant adduct. Histidines and lysines in helices 5-8 of apoA-I were highly susceptible to oxPL modifications, while lysines in helices 1, 2, 4 and 10 were resistant to modification by oxPL. In plasma exposed to oxidation or synthetic oxPL, oxPL modification was highly selective, and four histidines (H155, H162, H193 and H199) in helices 6-8 of apoA-I were the main modification target. H710 and H3613 in apoB-100 of LDL and K190 of human serum albumin were also modified by oxPL but to a lesser extent. Comparison of oxPL with short chain aldehyde HNE using MALDI-TOF MS demonstrated high selectivity and efficiency of oxPL in the modification of HDL apoproteins. These findings provide a novel insight into a potential mechanism of the loss of atheroprotective function of HDL in conditions of oxidative stress.

The Relationship Among the Level of Serum Amyloid A, High-Density Lipoprotein and Microalbuminuria in Patients With Familial Mediterranean Fever.

Serum amyloid A (SAA), which is produced in the liver, acts as an apoprotein of high-density lipoprotein (HDL) accumulation in extracellular matrix of tissues and organs. SAA elevations play a significant role in the development of amyloidosis. Microalbuminuria (MAU) is the early period of amyloidosis in patients with familial Mediterranean fever (FMF). We assessed the association between SAA as an important factor for the development of amyloidosis in patients with FMF and cytokines, HDL, and MAU. A total of 40 FMF patients diagnosed with Tel-Hashomer criteria and making regular follow-up visits at the tertiary referral center from 2012 to 2013 were included in this study, besides 40 age- and sex-matched individuals as controls. Compared with controls, FMF patients had higher SAA (25.20 ± 45.78 vs. 1.68 ± 0.63 ng/ml; P = 0.002). Also, FMF patients had higher MAU than controls (23.20 ± 39.86 vs. 9.40 ± 5.32 mg/day; P = 0.036). HDL was significantly lower in the patient group than in controls (39.35 ± 10.45 vs. 47.82 ± 15.31 mg/dl; P = 0.023). Interleukin-1 beta (IL-1), IL-6, and tumor necrosis factor alpha (TNF-α) levels were higher in the FMF group than in controls (P < 0.0001, P = 0.009, P = 0.003, respectively). Our results suggest that IL-1, IL-6, TNF-α, SAA, and HDL may serve as markers of subclinical inflammation in FMF patients. Due to increased plasma HDL levels, antiinflammatory and antioxidant effects may elevate in FMF patients.

ATP-Binding Cassette Transporter A1 in Lipoprotein Metabolism and Atherosclerosis

ATP-binding cassette transporter A1 (ABCA1) is a key protein determining high-density lipoprotein (HDL) function. In 1999, it was discovered by 3 independent groups that mutations in the gene for ABCA1 underlie the molecular defect in the HDL deficiency syndrome Tangier disease. Subsequent studies with genetically engineered mice lacking or overexpressing ABCA1 provided evidence that ABCA1 modulates atherosclerosis susceptibility on either end of the reverse cholesterol transport pathway. In the liver (and to a lesser extent in intestine), it determines the biogenesis of nascent HDL particles, whereas in macrophages it is essential for the prevention of the excess cholesterol accumulation by facilitating the transport of cellular cholesterol and phospholipid onto lipid-poor apo AI, the major apoprotein of HDL. See accompanying article on page 2288 Mice lacking ABCA1 in all tissues display a virtual absence of HDL cholesterol (HDL-C) in the circulation.1 This is primarily the consequence of the absence of hepatic ABCA1 as targeted inactivation of ABCA1 in liver resulted in a 83% reduction in the total amount of HDL-C in the circulation.2 Surprisingly, the first studies by Aiello et al3 investigating the effects of ABCA1 deletion in all endogenous tissues failed to show any affect on atherosclerosis susceptibility, neither in the apolipoprotein E knockout (apoE KO) background nor in the low-density lipoprotein receptor knockout (LDLr KO) background. Both models, however, did show marked decreases in proatherogenic apoB lipoproteins on deletion of ABCA1. One could thus also argue that total body ABCA1 KO mice still developed substantial atherosclerosis despite the markedly decreased levels of proatherogenic lipoproteins. Total body KO mice also lack ABCA1 in bone marrow–derived cells, including macrophages that accumulate in the arterial wall during atherosclerotic lesion development. Several bone marrow transplantation studies were performed in LDLr KO and apoE KO mice to investigate the effects of …

Mathematical Analysis of Hepatic High Density Lipoprotein Transport Based on Quantitative Imaging Data

Hepatocytes internalize high density lipoprotein (HDL) at the basolateral membrane. Most HDL is recycled while some is shuttled to the canalicular membrane by transcytosis. Here, transport of HDL was analyzed by mathematical modeling based on measurements in polarized hepatic HepG2 cells. Recycling of HDL from basolateral sorting endosomes was modeled by applying the rapid equilibrium approach. Analytical expressions were derived, which describe in one model the transport of HDL to the subapical compartment/apical recycling compartment, the biliary canaliculus (BC), and to late endosomes and lysosomes (LE/LYS). Apical endocytosis of HDL predicted by the model was confirmed for rhodamine-dextran and fluorescent asialoorosomucoid, markers for LE/LYS in living HepG2 cells. Budding of endocytic vesicles from the BC was directly observed by time lapse imaging of a fluorescent lipid probe. Based on fitted kinetic parameters and their covariance matrix a Monte Carlo simulation of HDL transport in hepatocytes was performed. The model was used to quantitatively assess release of HDL-associated free cholesterol by scavenger receptor BI. It is shown that only 6% of HDL-associated sterol reaches the BC as a constituent of the HDL particles, whereas the remaining sterol is rapidly released from HDL and shuttled to the BC by non-vesicular transport.

Biology: risk factor modification by OCs and HRT lipids and lipoproteins

The many effects that the oestrogens and progestagens used in oral contraceptive (OC) and postmenopausal hormone replacement therapies (HRTs) have on lipoprotein metabolism are of importance because of the involvement of lipoproteins in endothelial damage and arterial occlusion. Lipoproteins promoting endothelial damage include: low density lipoprotein (LDL), particularly the small dense subfraction (sdLDL), remnants of very low density lipoprotein (VLDL) and chylomicron metabolism, and lipoprotein (a). High density lipoprotein (HDL) has pleiotropic effects that prevent or alleviate endothelial damage. Orally administered oestrogens increase hepatic triglyceride synthesis and VLDL secretion and increase the proportion of sdLDL. Oestrogens increase the rates of elimination of LDL, VLDL remnants and chylomicrons, suppress the synthesis of key enzymes of lipoprotein metabolism, hepatic and lipoprotein lipase, and increase synthesis of the principal apoprotein of HDL, apoAI. In general, progestagens oppose these effects according to type and dose. In OC and HRT users, this leads to a range of different lipoprotein profiles, which may differ from those evaluated with respect to vascular disease risk in population studies. Accumulating evidence suggests that the clinical implications of steroid induced changes in the lipoprotein profile will need to be evaluated independently of population-based evidence.

Effect of Aluminium on Lipid Peroxidation of Human High Density Lipoproteins

We investigated the effect of aluminium (Al 3+ ) on lipid peroxidation and physico-chemical properties of high density lipoproteins (HDL) isolated from human plasma. Our results demonstrated that Al 3+ enhances lipid peroxidation of human HDL as shown by the significant increase in lipid hydroperoxides in Al-treated HDL with respect to control HDL. The oxidative effect was higher at acid pH (pH 5.5) with respect to pH 7.4. Moreover, a stimulating effect of Al 3+ on iron-induced lipid peroxidation of HDL was demonstrated. The study of the effect of Al 3+ on the physico-chemical properties of HDL, using the fluorescence polarization (Pf) of the probes TMA-DPH (1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene iodide) and DPH (1,6-diphenyl-1,3,5-hexatriene), showed a significant decrease of Pf in Al-treated HDL with respect to control. These results suggest that Al 3+ induces a decrease of molecular order at the lipoprotein surface. Moreover, the study of tryptophan (Trp) fluorescence demonstrated that aluminium induces structural modifications of HDL apoproteins and on HDL physico-chemical properties. The effect of Al 3+ on lipid peroxidation of HDL was observed at aluminium concentrations similar to those observed in the brain of patients affected by neurological diseases. Aluminium-induced oxidative damage of HDL could be involved in the development of neurological diseases.

Targeted mutation of plasma phospholipid transfer protein gene markedly reduces high-density lipoprotein levels.

It has been proposed that the plasma phospholipid transfer protein (PLTP) facilitates the transfer of phospholipids and cholesterol from triglyceride-rich lipoproteins (TRL) into high-density lipoproteins (HDL). To evaluate the in vivo role of PLTP in lipoprotein metabolism, we used homologous recombination in embryonic stem cells and produced mice with no PLTP gene expression. Analysis of plasma of F2 homozygous PLTP-/- mice showed complete loss of phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, sphingomyelin, and partial loss of free cholesterol transfer activities. Moreover, the in vivo transfer of [3H]phosphatidylcholine ether from very-low-density proteins (VLDL) to HDL was abolished in PLTP-/- mice. On a chow diet, PLTP-/- mice showed marked decreases in HDL phospholipid (60%), cholesterol (65%), and apo AI (85%), but no significant change in non-HDL lipid or apo B levels, compared with wild-type littermates. On a high-fat diet, HDL levels were similarly decreased, but there was also an increase in VLDL and LDL phospholipids (210%), free cholesterol (60%), and cholesteryl ester (40%) without change in apo B levels, suggesting accumulation of surface components of TRL. Vesicular lipoproteins were shown by negative-stain electron microscopy of the free cholesterol- and phospholipid-enriched IDL/LDL fraction. Thus, PLTP is the major factor facilitating transfer of VLDL phospholipid into HDL. Reduced plasma PLTP activity causes markedly decreased HDL lipid and apoprotein, demonstrating the importance of transfer of surface components of TRL in the maintenance of HDL levels. Vesicular lipoproteins accumulating in PLTP-/- mice on a high-fat diet could influence the development of atherosclerosis.

Increased High Density Lipoprotein (HDL), Defective Hepatic Catabolism of ApoA-I and ApoA-II, and Decreased ApoA-I mRNA inob/ob Mice: POSSIBLE ROLE OF LEPTIN IN STIMULATION OF HDL TURNOVER

Abnormalities of plasma high density lipoprotein (HDL) levels commonly reflect altered metabolism of the major HDL apolipoproteins, apoA-I and apoA-II, but the regulation of apolipoprotein metabolism is poorly understood. Two mouse models of obesity, ob/ob and db/db, have markedly increased plasma HDL cholesterol levels. The purpose of this study was to evaluate mechanisms responsible for increased HDL in ob/ob mice and to assess potential reversibility by leptin administration. ob/ob mice were found to have increased HDL cholesterol (2-fold), apoA-I (1.3-fold), and apoA-II (4-fold). ApoA-I mRNA was markedly decreased (to 25% of wild-type) and apoA-II mRNA was unchanged, suggesting a defect in HDL catabolism. HDL apoprotein turnover studies using nondegradable radiolabels confirmed a decrease in catabolism of apoA-I and apoA-II and a 4-fold decrease in hepatic uptake in ob/ob mice compared with wild-type, but similar renal uptake. Low dose leptin treatment markedly lowered HDL cholesterol and apoA-II levels in both ob/ob mice and in lean wild-type mice, and it restored apoA-I mRNA to normal levels in ob/ob mice. These changes occurred without significant alteration in body weight. Moreover, ob/ob neuropeptide Y-/- mice, despite marked attenuation of diabetes and obesity phenotypes, showed no change in HDL cholesterol levels relative to ob/ob mice. Thus, increased HDL levels in ob/ob mice reflect a marked hepatic catabolic defect for apoA-I and apoA-II. In the case of apoA-I, this is offset by decreased apoA-I mRNA, resulting in apoA-II-rich HDL particles. The studies reveal a specific HDL particle catabolic pathway that is down-regulated in ob/ob mice and suggest that HDL apolipoprotein turnover may be regulated by obesity and/or leptin signaling.

Embryogenesis-promoting factors in rat serum

Regarding whole rat embryo cultures in vitro, rat serum as a culture medium is known to support the normal growth of rat embryos in the organogenesis phase. The purpose of the present study was to isolate the embryogenesis-promoting factors from rat serum as a first step in the development of a defined serum-free medium for a whole embryo culture system. Pooled rat serum after heat inactivation was fractionated into three major peaks (frA, containing a region of void volume, frB, and frC) by gel filtration. The 9.5-day rat embryos that were cultivated for 48 hr in essential salt medium containing frB (with a molecular size range of 100-500 kDa) revealed normal growth. Three proteins (27 kDa, 76 kDa, and 190 kDa) that had the embryogenesis-promoting effects were isolated from 3-hr delayed centrifuged rat serum by the ion exchange chromatography. The 76-kDa protein was found to be rat transferrin by immunoblotting. The 27-kDa protein was identified as apo-AI (the major apoprotein of high-density lipoprotein) by immunoblotting. High-density lipoprotein obtained from pooled rat serum by a NaBr density gradient ultracentrifugation was found to have a positive effect on embryogenesis. The 10-kDa protein was also identified as alpha 1-inhibitor 3 by immunoblotting. In addition, the embryogenesis-promoting effect of the fraction containing 27-kDa and 190-kDa proteins declined within a short period of storage at -20 degrees C. This decrease was countered by supplementing its fraction (D-2) with albumin isolated from rat serum. These results in the present study suggest that transferrin, high-density lipoprotein, and alpha 1-inhibitor 3 in rat serum may be embryogenesis-promoting factors, and that albumin appeared to play a role in the embryogenesis of rat embryos in whole embryo cultures.

Hepatic lipase affects both HDL and ApoB-containing lipoprotein levels in the mouse

Transgenic mice were created overproducing a range of human HL (hHL) activities (4–23-fold increase) to further examine the role of hepatic lipase (HL) in lipoprotein metabolism. A 5-fold increase in heparin releasable HL activity was accompanied by moderate (approx. 20%) decreases in plasma total and high density lipoprotein (HDL) cholesterol and phospholipid (PL) but no significant change in triglyceride (TG). A 23-fold increase in HL activity caused a more significant decrease in plasma total and HDL cholesterol, PL and TG (77%, 64%, 60%, and 24% respectively), and a substantial decrease in lipoprotein lipids amongst IDL, LDL and HDL fractions. High levels of HL activity diminished the plasma concentration of apoA-I, A-II and apoE (76%, 48% and 75%, respectively). In contrast, the levels of apoA-IV-containing lipoproteins appear relatively resistant to increased titers of hHL activity. Increased hHL activity was associated with a progressive decrease in the levels and an increase in the density of LpAI and LpB48 particles. The increased rate of disappearance of 125I-labeled human HDL from the plasma of hHL transgenic mice suggests increased clearance of HDL apoproteins in the transgenic mice. The effect of increased HL activity on apoB100-containing lipoproteins was more complex. HL-deficient mice have substantially decreased apoB100-containing low density lipoproteins (LDL) compared to controls. Increased HL activity is associated with a transformation of the lipoprotein density profile from predominantly buoyant (VLDL/IDL) lipoproteins to more dense (LDL) fractions. Increased HL activity from moderate (4-fold) to higher (5-fold) levels decreased the levels of apoB100-containing particles. Thus, at normal to moderately high levels in the mouse, HL promotes the metabolism of both HDL and apoB-containing lipoproteins and thereby acts as a key determinant of plasma levels of both HDL and LDL.

The insulin-like growth factor axis and plasma lipid levels in the elderly.

The activity of the hypothalamic-GH-insulin-like growth factor (IGF) network declines with age. It has recently been shown that increased cardiovascular mortality occurs in adults with GH deficiency. As hypercholesterolemia is common in GH-deficient adults, and because there is experimental evidence that GH may play a role in regulating plasma cholesterol, we decided to investigate the activity of the GH-IGF axis in an elderly population by measuring serum IGF-I, IGF-II, and IGF-binding protein-3 (IGFBP-3) levels and to study their relationship with blood lipid levels. One hundred and thirty-two elderly subjects, 52 men and 80 women, were studied (age range, 60-91 yr). Men had significantly lower levels of IGFBP-3, high density lipoprotein cholesterol (HDL-C) and apoprotein A1 (ApoA1) compared to the women, whereas IGF-I and IGF-II were only slightly lower. Using linear regression analysis, we observed an inverse relationship of age with IGF-I (r = -0.35; P < 0.001), IGF-II (r = 0.40; P < 0.001), IGFBP-3 (r = 0.52; P < 0.001), body mass index, and lipid levels. Univariate regression analysis showed a strong and positive correlation of both IGF-I and IGFBP-3 with HDL-C and ApoA1. Partial correlation analysis, after adjustment for age and body mass index, showed that IGFBP-3 and IGF-II were still significantly and positively related to HDL-C and ApoA1. Furthermore, a strong association was documented among IGF-I, IGF-II, and IGFBP-3. These data demonstrate that even in an elderly population, further aging is accompanied by a progressive decline in circulating IGF-I, IGF-II, and IGFBP-3, suggesting a continuing diminution of the GH-IGF axis throughout aging. Moreover, the strong correlation between HDL-C and an index of GH secretion, such as IGFBP-3, suggests that GH might play an important role in lipid metabolism in healthy elderly subjects.

Segregation analysis of two-locus models regulating apolipoprotein-A1 levels.

For quantitative traits associated with risk to complex diseases, such as heart disease, single major locus models are likely to be too simplistic. Currently, researchers have begun to use oligogenic models of inheritance, but the resolving power of these models remains to be determined. As the major apoprotein of high density lipoprotein (HDL), apolipoprotein A1 (apo-A1) is generally accepted as a protective factor for coronary artery disease. Although familial aggregation of apo-A1 levels has been reported, the mode of inheritance of apo-A1 remains ill defined. In the present study, we conducted a segregation analysis comparing a series of one-locus and two-locus univariate models for apo-A1 levels in a sample of 137 families ascertained through probands undergoing elective, diagnostic coronary angiography. A two-locus Mendelian model fit these data significantly better than any one-locus model. The incorporation of the second major locus into the model of inheritance leads to a significant improvement in the fit, and a significant decrease of the residual heritability. The best-fitting model included two loci with a reciprocal pattern of epistasis generating 4 distinct genotypic distributions. Taken together, these two major loci account for 58% of the variance of adjusted apo-A1 levels. This demonstration of a second major locus controlling apo-A1 levels may explain the equivocal results obtained from previous studies. This two-locus model may be more powerful for linkage analysis to map one or both of these quantitative trait loci.

Lung, ileum and heart are predilection sites for AApoAII amyloid deposition in CD-1 Swiss mice used for toxicity studies. Pulmonary amyloid indicates AApoAII

Amyloid deposits represent frequent histological findings in SPF strains of mice mainly used for toxicological studies. Usually, these are deposits of reactive amyloid (AA-amyloid) derived from the acute phase protein serum amyloid A (SAA). The SAA is an apoprotein of high density lipoprotein (apoSAA). Senescence-accelerated amyloid (ASsam) occurs in a special strain of mice. This type of amyloid is derived from apolipoprotein-AII and, therefore, is called AApoAII. Recently, C57B1/Ka control mice not treated for long duration with immunosuppressive agents, were found to have developed AApoAII-amyloidosis with a predilection for the deposits in the ileum (HogenEsch et al. 1993). In the present study, SPF CD-1 Swiss outbred mice, used for chronic toxicity experiments, were investigated. Amyloidosis was diagnosed by haematoxylin and eosin staining. The tissue localization of amyloid was recorded and confirmed by Congo red staining. The chemical type of amyloid was investigated by peroxidase antiperoxidase (PAP)-immunostaining using anti-murine AA and anti-murine AApoAII antibodies. Those animals which died during the study and the mice killed at end of the experiment, aged 18 months, from treated as well as non-treated control groups, showed AApoAII-amyloid deposits with similar prevalence. The AApoAII amyloid had organ predilection for gut, heart and lung tissue. A group of animals was euthanazed intercurrently at a young age, since they suffered from spontaneous dermatitis associated with Staphylococcus aureus infection. Sixty-eight percent had reactive amyloid deposits found primarily in spleen, liver, kidney and gut. From these findings and literature data on various other mouse strains, it is concluded that in mice used for toxicity studies, AA and AApoAII types of amyloidosis may be expected. The deposition patterns of these types of amyloid are slightly different. AA-amyloid has a predilection for spleen, liver, gut and kidney, and is often associated with inflammatory lesions of the skin, whereas masses of amyloid in lung, heart and ileum suggest AApoAII. Pulmonary amyloid appears to represent the most reliable deposition criterion for discriminating between both types of amyloidosis.

HDL3‐mediated cholesterol efflux from cultured enterocytes: The role of apoproteins A‐I and A‐II

High density lipoproteins (HDL) were recently demonstrated in an enterocyte model (CaCo-2 cells) to mediate reverse cholesterol transport by retroendocytosis. The present study was carried out to define the role of the major HDL apoproteins (apo) A-I and apo A-II in this pathway. HDL3 was fractionated by heparin affinity chromatography into the two main fractions containing either apo A-I only (fraction A) or both apo A-I and apo A-II (fraction B). In addition, liposomes were reconstituted from purified apo A-I or apo A-II and dimyristoyl phosphatidylcholine. The cell binding properties and cholesterol efflux potential were studied in the lipoprotein fractions and the liposomes. Both fractions exhibited similar maximal binding capacities of 4427 (A) and 5041 (B) ng/mg cell protein, but their dissociation constants differed (40.5 and 167.7 micrograms/mL, respectively). Fraction A induced cholesterol efflux and stimulated cholesterol synthesis more than did fraction B. Fraction A mobilized both cellular free and esterified cholesterol, whereas fraction B preferentially mobilized cholesteryl esters. Liposomes, containing either apo A-I or apo A-II, showed specific binding, endocytosis and endosomal transport, and were released as intact particles. Apo A-I liposomes also mediated cholesterol efflux. In conclusion, there is evidence that the HDL3 subfractions A and B, as well as reconstituted liposomes containing either apo A-I or apo A-II, were specifically bound and entered a retroendocytosis pathway which was directly linked to cholesterol efflux. Quantitatively, the apo A-I subfraction appeared to play the dominant role in normal enterocytes. The apo A-II content of fraction B was related to the mobilization of cholesteryl esters.

Influence of apolipoprotein composition of high density lipoprotein particles on cholesteryl ester transfer protein activity. Particles containing various proportions of apolipoproteins AI and AII.

The effect of apolipoprotein (apo) composition of high density lipoproteins (HDL) on cholesteryl ester transfer protein (CETP) activity was studied by measuring the rate of radiolabeled cholesteryl esters transferred between low density lipoproteins (LDL) and HDL3 which contained various proportions of apoAI and apoAII. Ultracentrifugally isolated HDL3, which contained virtually only apoAI and apoAII in their protein moiety, were progressively enriched with apoAII upon the incubation with increasing amounts of delipidated HDL apolipoproteins. The substitution of apoAII for apoAI in HDL3 did not induce marked alteration of the lipid composition of the lipoprotein particles. The rates of cholesteryl ester exchanges with LDL in the presence of purified human CETP were significantly reduced with apoAII-enriched HDL3 as compared with non-enriched homologous particles. Consistent results were obtained by determining the rate of cholesteryl esters transferred either from LDL toward HDL3, or in the opposite direction, from HDL3 to LDL. The effect of the apoAI and apoAII content of HDL particles on CETP activity was also investigated by measuring the rate of cholesteryl esters transferred from LDL to plasma HDL3 particles which contained either only apoAI, HDL3-AI, or both apoAI and apoAII, HDL3-AIAII. HDL3-AI and HDL3-AIAII particles were isolated from human plasma by a sequential procedure which combined ultracentrifugation and anti-apoAII immunoaffinity chromatography. As observed with HDL3 artificially enriched with apoAII, cholesteryl ester transfer rates were significantly lower with plasma HDL3-AIAII than with plasma HDL3-AI particles. Kinetic analysis of the interaction of CETP with apoAII-enriched HDL3 revealed that apoAII could act as an uncompetitive inhibitor of the cholesteryl ester transfer reaction. Since the plasma levels of HDL-AI, HDL-AIAII, and HDL-AII may undergo significant physiological fluctuation, the present study suggests that HDL apoproteins may be important factors in modulating cholesteryl ester transfer rates in vivo.

In vivo protection against endotoxin by plasma high density lipoprotein.

Overwhelming bacterial infection is accompanied by fever, hypotension, disseminated intravascular coagulation, and multiple organ failure leading to death in 30-80% of cases. These classical symptoms of septic shock are caused by potent cytokines that are produced in response to endotoxin released from Gram-negative bacteria. Treatments with antibodies and receptor antagonists to block endotoxin or cytokine mediators have given mixed results in clinical trials. High density lipoprotein (HDL) is a natural component of plasma that is known to neutralize endotoxin in vitro. We report here that raising the plasma HDL concentration protects mice against endotoxin in vivo. Transgenic mice with 2-fold-elevated plasma HDL levels had more endotoxin bound to HDL, lower plasma cytokine levels, and improved survival rates compared with low-HDL mice. Intravenous infusion of HDL also protected mice, but only when given as reconstituted HDL prepared from phospholipid and either HDL apoprotein or an 18-amino acid peptide synthesized to mimic the structure of apolipoprotein A-I of HDL. Intact plasma HDL was mildly toxic, and HDL apoprotein was ineffective. The effectiveness of the reconstituted peptide renders very unlikely any significant contribution to protection by trace proteins in apo-HDL. These data suggest a simple leaflet insertion model for binding and neutralization of lipopolysaccharide by phospholipid on the surface of HDL. Plasma HDL may normally act to protect against endotoxin; this protection may be augmented by administration of reconstituted HDL or reconstituted peptides.

Comparison of the intravascular metabolism of cholesteryl esters and apoproteins of plasma low- and high-density lipoproteins in the rat ( Rattus norvegicus), an animal species without plasma cholesteryl ester transfer activity

1. 1. The intravascular metabolism of the cholesteryl esters (CE) and apoproteins of low density lipoproteins (LDL) and high density lipoproteins (HDL) was compared in the rat, an animal species without plasma cholesteryl ester transfer activity (CETA). 2. 2. The apoproteins and the CE of LDL had identical catabolic rates, and there was no transfer of LDL CE to other lipoprotein classes. 3. 3. The CE of the HDL, however, had higher catabolic rates than the apoproteins, and there was transfer of HDL CE to LDL but not to very low density lipoproteins.

Plasma lipid, lipoprotein cholesterol, and apoprotein distributions in selected US communities. The Atherosclerosis Risk in Communities (ARIC) Study.

The distributions of plasma lipids, lipoproteins, and apoproteins of 14,524 female and male black and white participants 45 to 64 years old in the Atherosclerosis Risk in Communities (ARIC) Study are presented. All specimens were analyzed at a central laboratory. Mean total cholesterol levels increased with increasing age across all ages from 204 to 229 mg/dL (12%) in women and from 208 to 213 mg/dL (2%) in men. Triglyceride levels increased with age in women, remained stable in men, and were higher in whites than blacks. High-density lipoprotein (HDL) cholesterol levels were higher in black and white women (range, 57 to 59 mg/dL) compared with black men (49 to 52 mg/dL) or white men (42 to 43 mg/dL). Cholesterol associated with HDL was distributed in a relatively constant proportion between HDL3 (70% to 76%) and HDL2 (24% to 30%) for all race/sex groups. Low-density lipoprotein (LDL) cholesterol levels increased with age in black (14.7%) and white (17.1%) women and in black (4.4%) and white (3.7%) men; more than 50% of all participants had LDL cholesterol levels > 130 mg/dL. Apoprotein A-I and B levels followed the same trends as HDL cholesterol and LDL cholesterol levels, respectively. Lipoprotein(a) [Lp(a)] levels were twice as high in blacks as in whites, and women's Lp(a) levels were higher than men's Lp(a) levels for each race. Menopause was associated with elevated total cholesterol, LDL cholesterol, apoprotein B, and Lp(a) levels, and hormone replacement medication use in postmenopausal subjects was associated with higher HDL cholesterol, triglyceride, and apoprotein A-I levels and lower LDL cholesterol, apoprotein B, and Lp(a) levels.

Uptake of high density lipoprotein cholesterol ester by HepG2 cells involves apolipoprotein E localized on the cell surface.

High density lipoprotein (HDL) cholesterol ester (CE) is taken up by many cells without simultaneous uptake of HDL apoprotein. The studies described herein demonstrate that the selective uptake of cholesterol ester by HepG2 cells is reduced by antibody directed against the receptor-binding domain of apoE (monoclonal antibody (mAb) 1D7) but not by antibody directed against the NH2-terminal portion of the molecule. The reduction, by 1D7, of HDL cholesteryl ester uptake is not due to apoE acquisition by the labeled HDL preparation or by the transfer of [3H]CE of HDL to apoE-containing lipoproteins and uptake by the apoB/E or apoE receptors. Rather, it appears that mAb 1D7 recognizes apoE localized at the cell surface of HepG2 cells. This conclusion is supported by the fact that: 1) reduction of HDL-CE uptake by HepG2 cells is observed within 15 min after the addition of the antibody-ligand mixture; 2) 1D7 is similarly effective in reducing the selective uptake of HDL-CE when added to the ligand or to the cells; 3) three different anti-apoE mAbs (1D7, 3B7, and 3H1) bind specifically to the surface of the cells. We have also demonstrated that heparin (5 mg/ml) does not reduce the amount of apoE-immunoreactive material bound at the cell surface when added before or after the binding period. 1D7, but not 3B7 or 3H1, binds less in the presence of heparin. The observations are consistent with a localization of apoE on the cell membrane rather than on lipoproteins bound to apoB/E or apoE receptors.