Discoidal HDL Research Articles

Abstract 2052: Macrophage-specific Ripk1 Knockdown Reduces Macrophage Inflammation And Atherosclerosis Progression In Ldlr -/- Mice.

Background: Chronic activation of the innate immune system drives inflammation and contributes to atherosclerosis. Pro-inflammatory agents (oxidised LDL, IL-1β) drive lesional inflammation and development, whereas anti-inflammatory agents (IL-10, HDL, Apolipoprotein-AI), promote an anti-inflammatory milieu and resolution. RIPK1 (Receptor interacting protein kinase 1) is a master regulatory gene that decides whether a cell undergoes inflammation, apoptosis and/or necroptosis, depending on its phosphorylation or ubiquitination status. We have previously observed that RIPK1 gene expression is increased in both endothelial cells and macrophages in early atherosclerotic lesions (Karunakaran et al, Circulation 2022). Here, we seek to determine whether macrophage-specific RIPK1 contributes to atherosclerosis. Methods: Given that Ripk1 -/- mice are lethal, we performed bone marrow (BM) transplants of Ripk1 +/- and wild-type (wt) littermate BM into female and male Ldlr -/- mice (n=8-12/grp) and mice were fed a western diet for 12-16 wks. In vitro experiments were performed in BM derived macrophages from wt and Ripk1 +/- mice. Results: Ldlr -/- mice recipient of Ripk1 +/- BM and western diet feeding for 12 or 16 wk had a marked reduction in aortic sinus lesion area (68.9% and 49.7% respectively, p<0.05) compared to their wt controls. Preliminary analysis show that there is a reduction in lesional macrophage area in Ripk1 +/- BM recipient Ldlr -/- mice relative to control (0.02±0.01 vs 0.01±0.006mm, p<0.01), suggesting that macrophage infiltration was reduced. Interestingly, anti-inflammatory agents, including recombinant IL10, IL13 (both 100ng/ml), Apolipoprotein-AI and discoidal HDL (both 500ug/mL) markedly reduced macrophage Ripk1 expression in vitro (by ~50%, p<0.05), likely reducing Ripk1-dependent inflammation and promoting an anti-inflammatory milieu. Current mechanistic studies are investigating the transcriptional regulator, STAT3, as a key regulator of Ripk1 expression. Conclusion: We identified macrophage-RIPK1 as a central driver of atherosclerosis. These timely studies highlight the therapeutic potential of dampening Ripk1 overexpression, as opposed to the detrimental abolishment of Ripk1, in treating atherosclerosis.

Human apoA-I[Lys107del] mutation affects lipid surface behavior of apoA-I and its ability to form large nascent HDL

Population studies have found that a natural human apoA-I variant, apoA-I[K107del], is strongly associated with low HDL-C but normal plasma apoA-I levels. We aimed to reveal properties of this variant that contribute to its unusual phenotype associated with atherosclerosis. Our oil-drop tensiometry studies revealed that compared to WT, recombinant apoA-I[K107del] adsorbed to surfaces of POPC-coated triolein drops at faster rates, remodeled the surfaces to a greater extent, and was ejected from the surfaces at higher surface pressures on compression of the lipid drops. These properties may drive increased binding of apoA-I[K107del] to and its better retention on large triglyceride-rich lipoproteins, thereby increasing the variant’s content on these lipoproteins. While K107del did not affect apoA-I capacity to promote ABCA1-mediated cholesterol efflux from J774 cells, it impaired the biogenesis of large nascent HDL particles resulting in the formation of predominantly smaller nascent HDL. Size-exclusion chromatography of spontaneously reconstituted 1,2-dimyristoylphosphatidylcholine-apoA-I complexes showed that apoA-I[K107del] had a hampered ability to form larger complexes but formed efficiently smaller-sized complexes. CD analysis revealed a reduced ability of apoA-I[K107del] to increase α-helical structure on binding to 1,2-dimyristoylphosphatidylcholine or in the presence of trifluoroethanol. This property may hinder the formation of large apoA-I[K107del]-containing discoidal and spherical HDL but not smaller HDL. Both factors, the increased content of apoA-I[K107del] on triglyceride-rich lipoproteins and the impaired ability of the variant to stabilize large HDL particles resulting in reduced lipid:protein ratios in HDL, may contribute to normal plasma apoA-I levels along with low HDL-C and increased risk for CVD.

Abstract 193: Apolipoprotein A-I Rotamers Determine The Size And Cholesterol Efflux Capacity Of High-Density Lipoprotein

Cardiovascular disease (CVD) risk is strongly and inversely associated with plasma levels of high-density lipoprotein cholesterol (HDL-C). Recent clinical studies suggest that the association between HDL-C and CVD risk is indirect and that HDL’s cardioprotective function may be attributed to its ability to promote cholesterol efflux from macrophages. Apolipoprotein A-I (APOA1), the major protein in HDL, serves as a structural scaffold to facilitate protein binding and enzyme activation. Our previous work demonstrated that two or three molecules of APOA1 form antiparallel dimer structures (rotamers) that wrap around discoidal and spherical HDL. Human HDL contains both LL5/5 and LL5/4 rotamers. Because little is known about the impact of different rotamers on HDL function, we generated human APOA1 mutants with single cysteine mutations that produced reconstituted HDL (rHDL) locked in specific rotamer (K133C for LL5/5 and L122C for LL5/4). The LL5/5 rotamer of rHDL demonstrated four-fold higher LCAT activation efficiency than the LL5/4 rotamer. To investigate the roles of APOA1 rotamers in vivo , we used a liver-targeted adeno-associated virus (AAV) to express WT hAPOA1 and the two cysteine mutants in APOA1-deficient ( Apoa1 -/- ) mice. HDL particle concentration (HDL-P) in WT and LL5/5 mice were similar but were 250% higher (n=8; P<0.0001) than those of LL5/4 mice. Importantly, mice expressing different APOA1 rotamers had markedly different HDL size patterns: HDL in LL5/4 hAPOA1 mice consisted mainly of extra-small HDL (7.8 nm in diameter), consistent with our finding that the LL5/4 rotamer had a low efficiency in activating LCAT. HDL in WT and LL5/5 mice had the same size distribution with one major peak at 10 nm. LC-MS/MS analysis identified 48 HDL proteins. One-third of the proteins (including APOA2, APOC1, APOC3 and LCAT) differed significantly in relative abundance in the different rotamers. Importantly, on a per particle basis, HDL from LL5/4 mice demonstrated three times higher ABCA1 cholesterol efflux capacity than HDL from WT and LL5/5 mice. Our observations strongly suggest that APOA1 rotamers play distinct roles in HDL metabolism, raising the possibility that differences in APOA1 rotamer distribution alter the cardioprotective functions of HDL.

Effect of thyroid function on pre-β1 high-density lipoprotein levels in patients with Graves' disease undergoing radioiodine treatment.

The metabolism of HDL is altered in thyroid dysfunctions. Preβ-1 HDL is a very small discoidal precursor HDL and promotes cholesterol efflux via ABCA1. The effects of thyroid dysfunctions on pre-β1 HDL are unknown. Thyroid hormone regulates ANGPTL3 expression, which may participate in HDL metabolism in thyroid dysfunctions. To determine the variation of HDL subfractions, especially preβ-1 HDL in thyroid dysfunctions, and whether ANGPTL3 mediates the effect of thyroid function on HDL metabolism. We recruited 26 patients with Graves' disease undergoing radioiodine treatment. They were evaluated at three time points: at baseline, when they were hypothyroid after radioiodine treatment, and when they were on stable levothyroxine replacement and euthyroid. The concentrations of smaller HDL particles Preβ-1 HDL and HDL3 were highest at the hyperthyroid state, and lowest at the hypothyroid state. While the larger HDL particles HDL2 and HDL1 changed just the opposite. Preβ1-HDL and HDL3 were positively correlated to fT3 and fT4, while were negatively correlated to TSH. In contrast, HDL1 was negatively associated with fT3 and fT4, while was positively associated with TSH. The correlations between thyroid hormones and HDL subfractions remained significant after adjusting for ANGPTL3. There is a shift form smaller HDL particles pre-β1 HDL and HDL3 to larger HDL particles HDL2 and HDL1 in hypothyroidism, while the change is just the opposite in hyperthyroidism. In future, cholesterol efflux capacity should be measured to determine if the function of HDL particles also changes with the shifting of HDL subfractions.

ApoE and ApoE Nascent-Like HDL Particles at Model Cellular Membranes: Effect of Protein Isoform and Membrane Composition.

Apolipoprotein E (ApoE), an important mediator of lipid transportation in plasma and the nervous system, plays a large role in diseases such as atherosclerosis and Alzheimer's. The major allele variants ApoE3 and ApoE4 differ only by one amino acid. However, this difference has major consequences for the physiological behaviour of each variant. In this paper, we follow (i) the initial interaction of lipid-free ApoE variants with model membranes as a function of lipid saturation, (ii) the formation of reconstituted High-Density Lipoprotein-like particles (rHDL) and their structural characterisation, and (iii) the rHDL ability to exchange lipids with model membranes made of saturated lipids in the presence and absence of cholesterol [1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) with and without 20 mol% cholesterol]. Our neutron reflection results demonstrate that the protein variants interact differently with the model membranes, adopting different protein conformations. Moreover, the ApoE3 structure at the model membrane is sensitive to the level of lipid unsaturation. Small-angle neutron scattering shows that the ApoE containing lipid particles form elliptical disc-like structures, similar in shape but larger than nascent or discoidal HDL based on Apolipoprotein A1 (ApoA1). Neutron reflection shows that ApoE-rHDL do not remove cholesterol but rather exchange saturated lipids, as occurs in the brain. In contrast, ApoA1-containing particles remove and exchange lipids to a greater extent as occurs elsewhere in the body.

Analysis of pyrene-labelled apolipoprotein A-I oligomerization in solution: Spectra deconvolution and changes in P-value and excimer formation

ApoA-I is the main protein of HDL which has anti-atherogenic properties attributed to reverse cholesterol transport. It shares with other exchangeable apolipoproteins a high level of structural plasticity. In the lipid-free state, the apolipoprotein amphipathic α-helices interact intra- and inter-molecularly, providing structural stabilization by a complex self-association mechanism. In this study, we employed a multi-parametric fluorescent probe to study the self-association of apoA-I. We constructed six single cysteine mutants spanning positions along three helices: F104C, K107C (H4), K133C, L137C (H5), F225C and K226C (H10); and labelled them with N-Maleimide Pyrene. Taking advantage of its spectral properties, namely formation of an excited dimer (excimer) and polarity-dependent changes in its fluorescence fine structure (P-value), we monitored the apoA-I self-association in its lipid-free form as a function of its concentration. Interactions in helices H5 (K133C) and H10 (F225C and K226C) were highlighted by excimer emission; while polarity changes were reported in helix H4 (K107C), as well as in helices H5 and H10. Mathematical models were developed to enrich data analysis and estimate association constants (KA) and oligomeric species distribution. Furthermore, we briefly discuss the usefulness of the multi-parametric fluorescent probe to monitor different equilibria, even at a single labelling position. Results suggest that apoA-I self-association must be considered to fully understand its physiological roles. Particularly, some contacts that stabilize discoidal HDL particles seem to be already present in the lipid-free apoA-I oligomers.

Arginine 123 of apolipoprotein A-I is essential for lecithin:cholesterol acyltransferase activity

ApoA-I activates LCAT that converts lipoprotein cholesterol to cholesteryl ester (CE). Molecular dynamic simulations suggested earlier that helices 5 of two antiparallel apoA-I molecules on discoidal HDL form an amphipathic tunnel for migration of acyl chains and unesterified cholesterol to the active sites of LCAT. Our recent crystal structure of Δ(185-243)apoA-I showed the tunnel formed by helices 5/5, with two positively charged residues arginine 123 positioned at the edge of the hydrophobic tunnel. We hypothesized that these uniquely positioned residues Arg123 are poised for interaction with fatty acids produced by LCAT hydrolysis of the sn-2 chains of phosphatidylcholine, thus positioning the fatty acids for esterification to cholesterol. To test the importance of Arg123 for LCAT phospholipid hydrolysis and CE formation, we generated apoA-I[R123A] and apoA-I[R123E] mutants and made discoidal HDL with the mutants and WT apoA-I. Neither mutation of Arg123 changed the particle composition or size, or the protein conformation or stability. However, both mutations of Arg123 significantly reduced LCAT catalytic efficiency and the apparent Vmax for CE formation without affecting LCAT phospholipid hydrolysis. A control mutation, apoA-I[R131A], did not affect LCAT phospholipid hydrolysis or CE formation. These data suggest that Arg123 of apoA-I on discoidal HDL participates in LCAT-mediated cholesterol esterification.

Is ABCA1 a lipid transfer protein?

ABCA1 functions as a lipid transporter because it mediates the transfer of cellular phospholipid (PL) and free (unesterified) cholesterol (FC) to apoA-I and related proteins present in the extracellular medium. ABCA1 is a membrane PL translocase and its enzymatic activity leads to transfer of PL molecules from the cytoplasmic leaflet to the exofacial leaflet of a cell plasma membrane (PM). The presence of active ABCA1 in the PM promotes binding of apoA-I to the cell surface. About 10% of this bound apoA-I interacts directly with ABCA1 and stabilizes the transporter. Most of the pool of cell surface-associated apoA-I is bound to lipid domains in the PM that are created by the activity of ABCA1. The amphipathic α-helices in apoA-I confer detergent-like properties on the protein enabling it to solubilize PL and FC in these membrane domains to create a heterogeneous population of discoidal nascent HDL particles. This review focuses on current understanding of the structure-function relationships of human ABCA1 and the molecular mechanisms underlying HDL particle production.

A thumbwheel mechanism for APOA1 activation of LCAT activity in HDL[S

APOA1 is the most abundant protein in HDL. It modulates interactions that affect HDL's cardioprotective functions, in part via its activation of the enzyme, LCAT. On nascent discoidal HDL, APOA1 comprises 10 α-helical repeats arranged in an anti-parallel stacked-ring structure that encapsulates a lipid bilayer. Previous chemical cross-linking studies suggested that these APOA1 rings can adopt at least two different orientations, or registries, with respect to each other; however, the functional impact of these structural changes is unknown. Here, we placed cysteine residues at locations predicted to form disulfide bonds in each orientation and then measured APOA1's ability to adopt the two registries during HDL particle formation. We found that most APOA1 oriented with the fifth helix of one molecule across from fifth helix of the other (5/5 helical registry), but a fraction adopted a 5/2 registry. Engineered HDLs that were locked in 5/5 or 5/2 registries by disulfide bonds equally promoted cholesterol efflux from macrophages, indicating functional particles. However, unlike the 5/5 registry or the WT, the 5/2 registry impaired LCAT cholesteryl esterification activity (P < 0.001), despite LCAT binding equally to all particles. Chemical cross-linking studies suggest that full LCAT activity requires a hybrid epitope composed of helices 5-7 on one APOA1 molecule and helices 3-4 on the other. Thus, APOA1 may use a reciprocating thumbwheel-like mechanism to activate HDL-remodeling proteins.

Abstract 564: Structure-Function Studies of ApoA-I Mimetic Peptides for ABCA1-Dependent Cholesterol Efflux and HDL Formation

To examine the mechanism of action of ApoA-I mimetics, we designed 4 peptides with a variable number of E, L, K and A residues and determined their ability to form HDL-like particle and promote ABCA1-dependent cholesterol efflux. They were named based on a unique physical property: N=ELK-neutral (EKLKELLEKLLEKLKELL), H=ELK-hydrophobic (EKLLELLKKLLELLKELL), P=ELK-positive (EKLKALLEKLKAKLKELL), Neg=ELK-negative (EELKEKLEELKEKLEEKL). CD-spectroscopy showed that H and P were mostly helical in an aqueous buffer (52% and 22%, respectively), and in a TFE-containing solvent, mimicking a lipid environment, all peptides showed greater than 40% helicity. In DMPC vesicle solubilization assay, we observed following order: P&gt;N&gt;H&gt;&gt;Neg. By non-denaturing gel electrophoresis, N, H, P formed approximately 8, 8, and 18 nm size lipid particles, respectively when combined with DMPC. However, Neg did not form any detectable particle. A mixture of natural lipids (PC:SM:LysoPC:PE:PS:PI; mol% 55:12:5:10:8:10), intended to simulate ABCA1 lipid membrane microdomain, yielded similar results . Using BHK-ABCA1 transfected cells, cholesterol efflux studies showed following results: H&gt;N&gt;&gt;P, whereas Neg peptide was inactive. All-atom MD simulation of N carried out on a 12–nm disc containing POPC:cholest (200:20), 26 peptides, and initially placed in belt-like configuration as head-to-tail dimers, the starting structure was maintained for at least 3 μ-sec, indicated a strong preference for this configuration. However, when N was initially oriented in a picket-fence configuration, it distorted to a belt-like pattern within 1 μ-sec. For Neg, both starting configurations were much less stable with dimers losing their connectivity and monomers migrating to the top and bottom of the disc, leaving large hydrophobic patches of acyl chains exposed. Cross-linking studies were consistent with the ability of H and N to form dimers to stabilize the HDL structure, and thereby, were also consistent with simulation. Together, we show that a net neutral charge, a relatively large hydrophobic moment (0.78), and a broad hydrophobic face (180 degrees) are optimum features for an apoA-I mimetic peptide to promote cholesterol efflux and to stabilize a nascent discoidal HDL structure.

High-density lipoprotein subpopulation profiles in lipoprotein lipase and hepatic lipase deficiency

Background and aimsOur aim was to gain insight into the role that lipoprotein lipase (LPL) and hepatic lipase (HL) plays in HDL metabolism and to better understand LPL- and HL-deficiency states. MethodsWe examined the apolipoprotein (apo) A-I-, A-II-, A-IV-, C-I-, C-III-, and E-containing HDL subpopulation profiles, assessed by native 2-dimensional gel-electrophoresis and immunoblotting, in 6 homozygous and 11 heterozygous LPL-deficient, 6 homozygous and 4 heterozygous HL-deficient, and 50 control subjects. ResultsLPL-deficient homozygotes had marked hypertriglyceridemia and significant decreases in LDL-C, HDL-C, and apoA-I. Their apoA-I-containing HDL subpopulation profile was shifted toward small HDL particles compared to controls. HL-deficient homozygotes had moderate hypertriglyceridemia, modest increases in LDL-C and HDL-C level, but normal apoA-I concentration. HL-deficient homozygotes had a unique distribution of apoA-I-containing HDL particles. The normally apoA-I:A-II, intermediate-size (α-2 and α-3) particles were significantly decreased, while the normally apoA-I only (very large α-1, small α-4, and very small preβ-1) particles were significantly elevated. In contrast to control subjects, the very large α-1 particles of HL-deficient homozygotes were enriched in apoA-II. Homozygous LPL- and HL-deficient subjects also had abnormal distributions of apo C-I, C-III, and E in HDL particles. Values for all measured parameters in LPL- and HL-deficient heterozygotes were closer to values measured in controls than in homozygotes. ConclusionsOur data are consistent with the concept that LPL is important for the maturation of small discoidal HDL particles into large spherical HDL particles, while HL is important for HDL remodeling of very large HDL particles into intermediate-size HDL particles.

Lack of LCAT reduces the LPS-neutralizing capacity of HDL and enhances LPS-induced inflammation in mice

HDL has important immunomodulatory properties, including the attenuation of lipopolysaccharide (LPS)-induced inflammatory response. As lecithin–cholesterol acyltransferase (LCAT) is a critical enzyme in the maturation of HDL we investigated whether LCAT-deficient (Lcat−/−) mice present an increased LPS-induced inflammatory response. LPS (100μg/kg body weight)-induced cytokine response in Lcat−/− mice was markedly enhanced and prolonged compared to wild-type mice. Importantly, reintroducing LCAT expression using adenovirus-mediated gene transfer reverted their phenotype to that of wild-type mice. Ex vivo stimulation of whole blood with LPS (1–100ng/mL) showed a similar enhanced pro-inflammatory phenotype. Further characterization in RAW 264.7 macrophages in vitro showed that serum and HDL, but not chylomicrons, VLDL or the lipid-free protein fraction of Lcat−/− mice, had a reduced capacity to attenuate the LPS-induced TNFα response. Analysis of apolipoprotein composition revealed that LCAT-deficient HDL lacks significant amounts of ApoA-I and ApoA-II and is primarily composed of ApoE, while HDL from Apoa1−/− mice is highly enriched in ApoE and ApoA-II. ApoA-I-deficiency did not affect the capacity of HDL to neutralize LPS, though Apoa1−/− mice showed a pronounced LPS-induced cytokine response. Additional immunophenotyping showed that Lcat−/− , but not Apoa1−/− mice, have markedly increased circulating monocyte numbers as a result of increased Cd11b+Ly6Cmed monocytes, whereas ‘pro-inflammatory’ Cd11b+Ly6Chi monocytes were reduced. In line with this observation, peritoneal macrophages of Lcat−/− mice showed a markedly dampened LPS-induced TNFα response. We conclude that LCAT-deficiency increases LPS-induced inflammation in mice due to reduced LPS-neutralizing capacity of immature discoidal HDL and increased monocyte number.

Abstract 113: Phospholipid Physical State Determines the Efficiency of Cholesteryl Ester Transfer by Cholesteryl Ester Transfer Protein between Discoidal High Density Lipoproteins

The transfer of cholesteryl ester by recombinant cholesteryl ester transfer protein (CETP) between reconstituted discoidal HDL was studied. Particles contained apolipoprotein A-I, unsaturated palmitoyllinoleoylphosphatidylcholine (PLPC), palmitoyloleoylphosphatidylcholine (POPC) or saturated dipalmitoylphosphatidylcholine (DPPC) and cholesteryl ester up to 3 mol% as cholesteryl 1-pyrenedecanoate (CPD) or cholesteryl laurate (CL) in donor and acceptor rHDL, respectively. The concentration dependencies of excimerization upward deviated from a linear dependence, being more curvilinear for DPPC. Separately for DPPC complexes, monomer fluorescence compared to excimer was more efficiently quenched by acrylamide. The nonradiative energy transfer between apoA-I tryptophan residues and CPD molecules was more efficient for PLPC compared to DPPC complexes. Data fulfilled the quenching sphere-of-action model, the sphere with adjacent probe molecules being larger and more distant from apoA-I tryptophan residues in DPPC complexes. CETP-catalyzed cholesteryl ester exchange between donor and acceptor HDL followed by probe excimerization was characterized by a heterogeneous kinetics; the fast exchanging CPD pool was much higher in a case of POPC compared to DPPC complexes. Probe fraction accessible to CETP increased with temperature, suggesting a more homogeneous probe distribution. The noncompetitive inhibition of probe transfer by acceptor particles seems to satisfy the zero off-rate compatible with probe tunneling. The values of V max (0.063 μM·min -1 ) and k cat (0.42 s -1 ) together with a similarity of K m (0.9 μM CPD) and K I (2.8 μM CL) values for POPC-containing rHDL suggest the efficient cholesteryl ester transfer between nascent HDL with unsaturated phosphatidylcholine in vivo opposite to much less efficiency with HDL with saturated phosphatidylcholine. The physical state of phospholipid matrix may underlie CETP activity with discoidal HDL through self-association and location of cholesteryl ester in the bilayer, the availability of cholesteryl ester to cholesterol-binding site in apoA-I structure and the binding of apoA-I-interacting cholesteryl ester to CETP. We are grateful to Dr. A. Tall for providing the recombinant CETP.

Direct detection of ABCA1-dependent HDL formation based on lipidation-induced hydrophobicity change in apoA-I

ABCA1 mediates the efflux of cholesterol and phospholipids into apoA-I to form HDL, which is important in the prevention of atherosclerosis. To develop a novel method for the evaluation of HDL formation, we prepared an apoA-I-POLARIC by labeling the specific residue of an apoA-I variant with a hydrophobicity-sensitive fluorescence probe that detects the environmental change around apoA-I during HDL formation. apoA-I-POLARIC possesses the intact ABCA1-dependent HDL formation activity and shows 4.0-fold higher fluorescence intensity in HDL particles than in the lipid-free state. Incubation of apoA-I-POLARIC with ABCA1-expressing cells, but not ABCA1-non-expressing cells, caused a 1.7-fold increase in fluorescence intensity. Gel filtration analysis demonstrated that the increase in fluorescence intensity of apoA-I-POLARIC represents the amount of apoA-I incorporated into the discoidal HDL particles rather than the amount of secreted cholesterol. THP-1 macrophage-mediated HDL formation and inhibition of HDL formation by cyclosporine A could also be measured using apoA-I-POLARIC. Furthermore, HDL formation-independent lipid release induced by microparticle formation or cell death was not detected by apoA-I-POLARIC. These results demonstrate that HDL formation by ABCA1-expressing cells can be specifically detected by sensing hydrophobicity change in apoA-I, thus providing a novel method for assessing HDL formation and screening of the HDL formation modulator.

Cholesteryl ester diffusion, location and self-association constraints determine CETP activity with discoidal HDL: Excimer probe study

The transfer of cholesteryl ester by recombinant cholesteryl ester transfer protein (CETP) between reconstituted discoidal high-density lipoprotein (rHDL) was studied. Particles contained apolipoprotein A-I, unsaturated POPC or saturated DPPC and cholesteryl ester as cholesteryl 1-pyrenedecanoate (CPD) or cholesteryl laurate (CL) in donor and acceptor rHDL, respectively. Probe dynamics fulfilled the quenching sphere-of-action model. The cholesteryl ester exchange between donor and acceptor particles was characterized by a heterogeneous kinetics; the fast exchanging CPD pool was much higher in a case of POPC compared to DPPC complexes. Probe fraction accessible to CETP increased with temperature, suggesting a more homogeneous probe distribution. Noncompetitive inhibition of probe transfer by acceptor particles was observed. The values of Vmax (0.063μMmin−1) and catalytic rate constant kcat (0.42s−1) together with a similarity of Km (0.9μM CPD) and KI (2.8μM CL) values for POPC-containing rHDL suggest the efficient cholesteryl ester transfer between nascent HDL with unsaturated phosphatidylcholine in vivo. The phospholipid matrix in discoidal HDL may underlie CETP activity through the self-association, diffusivity and location of cholesteryl ester in the bilayer, the accessibility of cholesteryl ester to cholesterol-binding site in apoA-I structure and the binding of cholesteryl ester, positionable by apoA-I, to CETP.

ApoE3[K146N/R147W] acts as a dominant negative apoE form that prevents remnant clearance and inhibits the biogenesis of HDL

The K146N/R147W substitutions in apoE3 were described in patients with a dominant form of type III hyperlipoproteinemia. The effects of these mutations on the in vivo functions of apoE were studied by adenovirus-mediated gene transfer in different mouse models. Expression of the apoE3[K146N/R147W] mutant in apoE-deficient (apoE(-/-)) or apoA-I-deficient (apoA-I(-/-))×apoE(-/-) mice exacerbated the hypercholesterolemia and increased plasma apoE and triglyceride levels. In apoE(-/-) mice, the apoE3[K146N/R147W] mutant displaced apoA-I from the VLDL/LDL/HDL region and caused the accumulation of discoidal apoE-containing HDL. The WT apoE3 cleared the cholesterol of apoE(-/-) mice without induction of hypertriglyceridemia and promoted formation of spherical HDL. A unique property of the truncated apoE3[K146N/R147W]202 mutant, compared with similarly truncated apoE forms, is that it did not correct the hypercholesterolemia. The contribution of LPL and LCAT in the induction of the dyslipidemia was studied. Treatment of apoE(-/-) mice with apoE3[K146N/R147W] and LPL corrected the hypertriglyceridemia, but did not prevent the formation of discoidal HDL. Treatment with LCAT corrected hypertriglyceridemia and generated spherical HDL. The combined data indicate that the K146N/R147W substitutions convert the full-length and the truncated apoE3[K146N/R147W] mutant into a dominant negative ligand that prevents receptor-mediated remnant clearance, exacerbates the dyslipidemia, and inhibits the biogenesis of HDL.

High-density lipoprotein metabolism, composition, function, and deficiency.

To examine the recent advances in our knowledge of HDL metabolism, composition, function, and coronary heart disease (CHD), as well as marked HDL deficiency states because of mutations in the apolipoprotein (apo) A-I, ATP-binding cassette transfer protein A1 and lecithin cholesterol acyltransferase (LCAT) gene loci. It has been documented that apoA-I, myeloperoxidase and paraoxonase 1 (PON1) form a complex in HDL that is critical for HDL binding and function. Myeloperoxidase has a negative impact on HDL function, whereas PON1 has a beneficial effect. Patients who lack apoA-I develop markedly premature CHD. Patients who lack ATP-binding cassette transfer protein A1 transporter function have only very small discoidal preβ-1 HDL, and develop hepatosplenomegaly, intermittent neuropathy and premature CHD, although significant heterogeneity for these disorders has been reported. Patients with LCAT deficiency have abnormal small discoidal LDLs and HDL particles, and develop kidney failure. Enzyme replacement therapy is being developed for the latter disorder. Recent data indicates that proteins other than apoA-I and apoA-II such as MPO and PON1 have important effects on HDL function. There has been considerable recent progress made in our understanding of HDL protein content and function.

Abstract 584: ApoE3[K146N/R147W] Acts as a Dominant Negative ApoE Form that Prevents Remnant Clearance and Inhibits the Biogenesis of HDL

Introduction: The K146N/R147W substitutions in human apolipoproteinE3 (apoE3) have been associated with a dominant form of type III hyperlipoproteinemia which is expressed at an early age. Methods: The effects of the K146N,R147W substitutions on the lipid and lipoprotein profiles and the HDL phenotypes were studied by adenovirus mediated gene transfer of the full length and a truncated apoE3[K146N/R147W]-202 mutant using different mouse models. Results: A low dose of adenovirus expressing the apoE3[K146N/R147W] mutant in apoE deficient or in apoA-I x apoE double deficient mice exacerbated the hypercholesterolemia and increased greatly plasma apoE and triglycerides levels. In apoE deficient mice, the apoE3[K146N/R147W] mutant displaced apoA-I from the VLDL/LDL/HDL region and resulted in the accumulation of discoidal apoE containing HDL particles in plasma. Similar doses of WT apoE3 cleared the cholesterol of apoE deficient mice without induction of hypertriglyceridemia and promoted formation of spherical HDL particles. A unique feature of the truncated apoE3[K146N/R147W]-202 mutant is that it prevented the induction of hypertriglyceridemia but did not correct the hypercholesterolemia. Other apoE-202 truncated mutants tested did not induce hypertriglyceridemia but corrected hypercholesterolemia. Infection of apoE deficient mice with adenoviruses expressing the apoE3[K146N/R147W] and lipoprotein lipase corrected the hypertriglyceridemia but did not prevent the formation of discoidal HDL particles. Infection of apoE deficient mice with adenoviruses expressing the apoE3[K146N/R147W] and lecithin:cholesterol (LCAT) acyltransferase corrected hypertriglyceridemia, normalized the plasma cholesteryl ester to total cholesterol ratio and generated spherical HDL particles. The combined data indicate that the K146N/R147W substitutions in the full length and the truncated apoE3[K146N/R147W] mutant prevent receptor mediated remnant clearance. Conclusion: The lipid/lipoprotein and HDL abnormalities observed in the different mouse models suggest that the apoE3[K146N/R147W] mutant acts a dominant negative ligand that exacerbates the dyslipidemia and also affects the activity of LCAT and thus inhibits the maturation of HDL.

Evidence for the presence of lipid-free monomolecular apolipoprotein A-1 in plasma

The first step in reverse cholesterol transport is a process by which lipid-free or lipid-poor apoA-1 removes cholesterol from cells through the action of ATP binding cassette transporter A1 at the plasma membrane. However the structure and composition of lipid-free or -poor apoA-1 in plasma remains obscure. We previously obtained a monoclonal antibody (MAb) that specifically recognizes apoA-1 in preβ1-HDL, the smallest apoA-1-containing particle in plasma, which we used to establish a preβ1-HDL ELISA. Here, we purified preβ1-HDL from fresh normal plasma using said antibody, and analyzed the composition and structure. ApoA-1 was detected, but neither phospholipid nor cholesterol were detected in the purified preβ1-HDL. Only globular, not discoidal, particles were observed by electron microscopy. In nondenaturing PAGE, no difference in the mobility was observed between the purified preβ1-HDL and original plasma preβ1-HDL, or between the preβ1-HDL and lipid-free apoA-1 prepared by delipidating HDL. In sandwich ELISA using two anti-preβ1-HDL MAbs, reactivity with intact plasma preβ1-HDL was observed in ELISA using two MAbs with distinct epitopes but no reactivity was observed in ELISA using a single MAb, and the same phenomenon was observed with monomolecular lipid-free apoA-1. These results suggest that plasma preβ1-HDL is lipid-free monomolecular apoA-1.

MD simulations suggest important surface differences between reconstituted and circulating spherical HDL

Since spheroidal HDL particles (sHDL) are highly dynamic, molecular dynamics (MD) simulations are useful for obtaining structural models. Here we use MD to simulate sHDL with stoichiometries of reconstituted and circulating particles. The hydrophobic effect during simulations rapidly remodels discoidal HDL containing mixed lipids to sHDL containing a cholesteryl ester/triglyceride (CE/TG) core. We compare the results of simulations of previously characterized reconstituted sHDL particles containing two or three apoA-I created in the absence of phospholipid transfer protein (PLTP) with simulations of circulating human HDL containing two or three apoA-I without apoA-II. We find that circulating sHDL compared with reconstituted sHDL with the same number of apoA-I per particle contain approximately equal volumes of core lipid but significantly less surface lipid monolayers. We conclude that in vitro reconstituted sHDL particles contain kinetically trapped excess phospholipid and are less than ideal models for circulating sHDL particles. In the circulation, phospholipid transfer via PLTP decreases the ratio of phospholipid to apolipoprotein for all sHDL particles. Further, sHDL containing two or three apoA-I adapt to changes in surface area by condensation of common conformational motifs. These results represent an important step toward resolving the complicated issue of the protein and lipid stoichiometry of circulating HDL.