High-density Lipoprotein Subpopulations Research Articles

Relationships between HDL subpopulation proteome and HDL function in overweight/obese people with and without coronary heart disease

Background and aimsThe structure-function relationships of high-density lipoprotein (HDL) subpopulations are not well understood. Our aim was to examine the interrelationships between HDL particle proteome and HDL functionality in subjects with and without coronary heart disease (CHD). MethodsWe isolated 5 different HDL subpopulations based on charge, size, and apolipoprotein A1 (APOA1) content from the plasma of 33 overweight/obese CHD patients and 33 age-and body mass index (BMI)-matched CHD-free subjects. We measured the relative molar concentration of HDL-associated proteins by liquid chromatography tandem mass spectrometry (LC-MS/MS) and assessed particle functionality. ResultsWe quantified 110 proteins associated with the 5 APOA1-containing HDL subpopulations. The relative molar concentration of these proteins spanned five orders of magnitude. Only 10 proteins were present in >1% while 73 were present in <0.1% concentration. Only 6 of the 10 most abundant proteins were apolipoproteins. Interestingly, the largest (α-1) and the smallest (preβ-1) HDL particles contained the most diverse proteomes. The protein composition of each HDL subpopulation was altered in CHD cases as compared to controls with the most prominent differences in preβ-1 and α-1 particles. APOA2 concentration was positively correlated with preβ-1 particle functionality (ABCA1-CEC/mg APOA1 in preβ-1) (R2 = 0.42, p = 0.005), while APOE concentration was inversely correlated with large-HDL particle functionality (SRBI-CEC/mg APOA1 in α-1+α-2) (R2 = 0.18, p = 0.01). ConclusionsThe protein composition of the different HDL subpopulations was altered differentially in CHD patients. The functionality of the small and large HDL particles correlated with the protein content of APOA2 and APOE, respectively. Our data indicate that distinct particle subspecies and specific particle associated proteins provide new information about the role of HDL in CHD.

High-Density Lipoprotein Particle Concentration and Size Predict Incident Coronary Artery Disease Events in a Cohort With Type 1 Diabetes.

The cholesterol efflux capacity of high density lipoprotein (HDL) is negatively associated with cardiovascular risk. Small HDL particles account almost quantitatively for cholesterol efflux capacity, perhaps mediated through efflux of cholesterol and outer leaflet plasma membrane phospholipids by ABCA1 (ATP binding cassette subfamily A member 1). People with type 1 diabetes are at increased coronary artery disease (CAD) risk despite normal HDL-cholesterol concentrations. We therefore tested the hypothesis that small HDL particles (HDL-P)-rather than HDL-cholesterol-predict incident CAD in type 1 diabetes. Incident CAD (CAD death, myocardial infarction, or coronary revascularization) was determined in 550 individuals with childhood-onset type 1 diabetes. HDL-P was quantified by calibrated ion mobility analysis and cholesterol efflux capacity was quantified with validated assays. During a median follow-up of 26 years, 36.5% of the participants developed incident CAD, for an incidence density of 181.3 per 10 000 person-years. In multivariable Cox models, neither HDL-cholesterol nor apolipoprotein A1 concentration was significantly associated with CAD risk. In contrast, higher extra-small HDL-P concentrations were significantly associated with decreased CAD risk (hazard ratio [HR], 0.26 [95% CI, 0.14-0.50]). Weaker associations were observed for total HDL-P (HR, 0.88 [95% CI, 0.83-0.93]), small HDL (HR, 0.83 [95% CI, 0.68-1.02]), medium HDL (HR, 0.79 [95% CI, 0.71-0.89]), and large HDL (HR, 0.72 [95% CI, 0.59-0.89]). Although cholesterol efflux capacity was negatively associated with incident CAD, this association was no longer significant after adjustment for total HDL-P. Lower concentrations of total HDL-P and HDL subpopulations were positively associated with incident CAD independently of HDL-cholesterol, apolipoprotein A1, and other common CVD risk factors. Extra-small HDL was a much stronger predictor of risk than the other HDLs. Our data are consistent with the proposal that extra-small HDL plays a critical role in cardioprotection in type 1 diabetes, mediated by macrophage cholesterol efflux by the ABCA1 pathway.

An atomistic characterization of high-density lipoproteins and the conserved “LN” region of apoA-I

The physicochemical characteristics of the various subpopulations of high-density lipoproteins (HDLs) and, in particular, their surface properties determine their ability to scavenge lipids and interact with specific receptors and peptides. Five representative spheroidal HDL subpopulation models were mapped from a previously reported equilibrated coarse-grained (CG) description to an atomistic representation for subsequent molecular dynamics simulation. For each HDL model a range of finer-level analyses was undertaken, including the component-wise characterization of HDL surfaces, the average size and composition of hydrophobic surface patches, dynamic protein secondary structure monitoring, and the proclivity for solvent exposure of the proposed β-amyloid (Aβ) binding region of apolipoprotein A-I (apoA-I), “LN.” This study reveals that previously characterized ellipsoidal HDL3a and HDL2a models revert to a more spherical geometry in an atomistic representation due to the enhanced conformational flexibility afforded to the apoA-I protein secondary structure, allowing for enhanced surface lipid packing and lower overall surface hydrophobicity. Indeed, the proportional surface hydrophobicity and apoA-I exposure reduced with increasing HDL size, consistent with previous characterizations. Furthermore, solvent exposure of the “LN” region of apoA-I was exclusively limited to the smallest HDL3c model within the timescale of the simulations, and typically corresponded to a distinct loss in secondary structure across the “LN” region to form part of a significant contiguous hydrophobic patch on the HDL surface. Taken together, these findings provide preliminary evidence for a subpopulation-specific interaction between HDL3c particles and circulating hydrophobic species such as Aβ via the exposed “LN” region of apoA-I.

Acute myocardial infarction preferentially alters low-abundant, long-chain unsaturated phospholipid and sphingolipid species in plasma high-density lipoprotein subpopulations

AimHigh-density lipoprotein (HDL) particles in ST-segment elevation myocardial infarction (STEMI) are deficient in their anti-atherogenic function. Molecular determinants of such deficiency remain obscure. MethodsFive major HDL subpopulations were isolated using density-gradient ultracentrifugation from STEMI patients (n = 12) and healthy age- and sex-matched controls (n = 12), and 160 species of phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, sphingomyelin and ceramide were quantified by LC-MS/MS. ResultsMultiple minor species of proinflammatory phosphatidic acid and lysophosphatidylcholine were enriched by 1.7–27.2-fold throughout the majority of HDL subpopulations in STEMI. In contrast, minor phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, sphingomyelin and ceramide species were typically depleted up to 3-fold in STEMI vs. control HDLs, while abundances of their major species did not differ between the groups. Intermediate-to-long-chain phosphatidylcholine, phosphatidylinositol and phosphatidylglycerol species were more affected by STEMI than their short-chain counterparts, resulting in positive correlations between their fold decrease and the carbon chain length. Additionally, fold decreases in the abundances of multiple lipid species were positively correlated with the double bond number in their carbon chains. Finally, abundances of several phospholipid and ceramide species were positively correlated with cholesterol efflux capacity and antioxidative activity of HDL subpopulations, both reduced in STEMI vs controls. KEGG pathway analysis tied these species to altered glycerophospholipid and linoleic acid metabolism. ConclusionsMinor unsaturated intermediate-to-long-chain phospholipid and sphingolipid species in HDL subpopulations are most affected by STEMI, reflecting alterations in glycerophospholipid and linoleic acid metabolism with the accumulation of proinflammatory lysolipids and maintenance of homeostasis of major phospholipid species.

Unravelling the influence of surface lipids on the structure, dynamics and interactome of high-density lipoproteins

Surface lipids influence the biological activities of high-density lipoproteins (HDLs) but their species-specific effects on HDL structure, dynamics, and surface interactome has remained unclear. Building upon the five-lipid species HDL models developed and characterised in previous work, representative models of the major HDL subpopulations found in human plasma containing apolipoprotein A-I (apoA-I) have been studied using molecular dynamics simulation to describe their varying degrees of surface lipidome complexity. Specifically, two additional sets of representative HDL subpopulation particles were developed, one with sphingomyelin (SM) and the other with SM, phosphatidylethanolamine, phosphatidylinositol, and ceramide in quantities reflecting average levels characterised for HDL subpopulations derived from normolipidemic patients. These lipid species were assessed in terms of HDL size, morphology, dynamics, and overall interactome. The findings reveal that the presence of a representative SM fraction marginally enhanced HDL interfacial curvature and surface monolayer rigidity, manifesting in tighter phospholipid packing and slower surface lipid dynamics relative to SM-deficient HDL models. Furthermore, the presence of SM resulted in a reduction in the solvent exposure of core lipids and cholesterol molecules, whilst also enhancing apolipoprotein conformational flexibility and its overall twisting across the HDL surface. The hydrophobicity of apoA-I-bound lipid patches and the proportion of apoA-I hydrophobic surface area is enhanced by the overall lipidation of apoA-I irrespective of lipid composition. These findings offer new insights into how the surface lipid composition of different HDL subpopulations can significantly impact the overall interactome of HDL particles, potentially influencing subpopulation-specific biological functions like lipid scavenging and receptor interactions.

CSL112 Infusion Rapidly Increases APOA1 Exchange Rate via Specific Serum Amyloid-Poor HDL Subpopulations When Administered to Patients Post-Myocardial Infarction.

To characterize the effects of CSL112 (human APOA1 [apolipoprotein A1]) on the APOA1 exchange rate (AER) and the relationships with specific HDL (high-density lipoprotein) subpopulations when administered in the 90-day high-risk period post-acute myocardial infarction. A subset of patients (n=50) from the AEGIS-I (ApoA-I Event Reducing in Ischemic Syndromes I) study received either placebo or CSL112 post-acute myocardial infarction. AER was measured in AEGIS-I plasma samples incubated with lipid-sensitive fluorescent APOA1 reporter. HDL particle size distribution was assessed by native gel electrophoresis followed by fluorescent imaging and detection of APOA1 and SAA (serum amyloid A) by immunoblotting. CSL112 infusion increased AER peaking at 2 hours and returning to baseline 24 hours post-infusion. AER correlated with cholesterol efflux capacity (r=0.49), HDL-cholesterol (r=0.30), APOA1 (r=0.48), and phospholipids (r=0.48; all P<0.001) over all time points. Mechanistically, changes in cholesterol efflux capacity and AER induced by CSL112 reflected HDL particle remodeling resulting in increased small HDL species that are highly active in mediating ABCA1 (ATP-binding cassette transporter 1)-dependent efflux, and large HDL species with high capacity for APOA1 exchange. The lipid-sensitive APOA1 reporter predominantly exchanged into SAA-poor HDL particles and weakly incorporated into SAA-enriched HDL species. Infusion of CSL112 enhances metrics of HDL functionality in patients with acute myocardial infarction. This study demonstrates that in post-acute myocardial infarction patients, HDL-APOA1 exchange involves specific SAA-poor HDL populations. Our data suggest that progressive enrichment of HDL with SAA may generate dysfunctional particles with impaired HDL-APOA1 exchange capacity, and that infusion of CSL112 improves the functional status of HDL with respect to HDL-APOA1 exchange. URL: https://www. gov; Unique identifier: NCT02108262.

The Differential Effects of HDL Subpopulations on Lipoprotein Lipase (LPL)-Mediated VLDL Catabolism.

High-density lipoprotein (HDL) subpopulations functional assessment is more relevant for HDL anti-atherogenic activity than cholesterol level. The aim of the study was to assess the impact of HDL-2 and HDL-3 on lipoprotein lipase (LPL)-mediated very-low-density lipoprotein (VLDL) catabolism related to hypertriglyceridemia development. VLDL and HDLs were isolated from serum by ultracentrifugation. VLDL was incubated with LPL in the absence and presence of total HDL or HDL subpopulations. Next, VLDL remnants were separated, and their composition and electrophoretic mobility was assessed. Both HDL subpopulations increased the efficiency of triglyceride lipolysis and apolipoprotein CII and CIII removal from VLDL up to ~90%. HDL-3 exerted significantly greater impact than HDL-2 on apolipoprotein E (43% vs. 18%, p < 0.001), free cholesterol (26% vs. 18%, p < 0.05) and phospholipids (53% vs. 43%, p < 0.05) removal from VLDL and VLDL remnant electrophoretic mobility (0.18 vs. 0.20, p < 0.01). A greater release of these components was also observed in the presence of total HDL with a low HDL-2/HDL-3 cholesterol ratio. Both HDL subpopulations affect VLDL composition during lipolysis, but HDL-3 exhibited a greater effect on this process. Altered composition of HDL related to significant changes in the distribution between HDL-2 and HDL-3 can influence the VLDL remnant features, affecting atherosclerosis progression.

Abstract 10220: CSL112 (Apolipoprotein A-I (human)) Infusion Rapidly Increases ApoA-I Exchange Rate via Specific Serum Amyloid-Poor HDL Sub-Populations When Administered to Patients Post Myocardial Infarction

Background: Apolipoprotein A-I (apoA-I) exchange rate (AER) into plasma high density lipoprotein (HDL) may represent a cell-free measure of HDL function, and predict recurrent major adverse cardiovascular events post-acute myocardial infarction (AMI). Purpose: To characterise the effects of CSL112 (apoA-I (human)) on AER and the relationships with specific HDL subpopulations when administered in the 90-day high-risk period post AMI. Methods: Patients from the AEGIS-I (ApoA-I Event Reducing in Ischemic Syndromes I) study received either placebo, 2g or 6g CSL112 post AMI. The rate of apoA-I exchange into HDL was measured in AEGIS-I plasma samples incubated with fluorescent apoA-I-NBD (7-nitrobenz-2-oxa-1,3-diazole) lipid-sensitive reporter for 30 min at 37°C. HDL particle size distribution was assessed by native gel electrophoresis followed by fluorescent imaging and detection of apoA-I and serum amyloid A (SAA1/SAA2) by western blotting. Results: CSL112 infusion increased AER peaking 2 h post infusion and returning to baseline by 24 h. AER was correlated with cholesterol efflux capacity, HDL-C, apoA-I and phosphatidylcholine (all p&lt;0.001). CSL112 infusion induced transient changes in HDL particle size distribution. Small remodelled HDL (S-HDL rem ) derived from CSL112 were detected in plasma 2-4 h after infusion. Large remodeled HDL species (L-HDL rem ) accumulated 2-12 h post infusion. The apoA-1-NBD reporter was confined to distinct HDL subpopulations that contained little SAA. In plasma samples characterized by the baseline pattern of HDL particle size distribution (0 h and 24-48 h post infusion), the apoA-I-NBD reporter predominantly exchanged into the HDL3 subpopulation, but not into large HDL2. Conclusion: Infusion of CSL112 increased AER via exchange into SAA-poor HDL particles. AER may represent a clinically amenable biomarker of HDL functionality. Figure: HDL particle remodelling and AER in a representative subject infused with 6g CSL112 .

Subpopulations of High-Density Lipoprotein: Friends or Foes in Cardiovascular Disease Risk in Chronic Kidney Disease?

Dyslipidemia is a major traditional risk factor for cardiovascular disease (CVD) in chronic kidney disease (CKD) patients, although the altered lipid profile does not explain the number and severity of CVD events. High-density lipoprotein (HDL) is a heterogeneous (size, composition, and functionality) population of particles with different atherogenic or atheroprotective properties. HDL-cholesterol concentrations per se may not entirely reflect a beneficial or a risk profile for CVD. Large HDL in CKD patients may have a unique proteome and lipid composition, impairing their cholesterol efflux capacity. This lack of HDL functionality may contribute to the paradoxical coexistence of increased large HDL and enhanced risk for CVD events. Moreover, CKD is associated with inflammation, oxidative stress, diabetes, and/or hypertension that are able to interfere with the anti-inflammatory, antioxidative, and antithrombotic properties of HDL subpopulations. How these changes interfere with HDL functions in CKD is still poorly understood. Further studies are warranted to fully clarify if different HDL subpopulations present different functionalities and/or atheroprotective effects. To achieve this goal, the standardization of techniques would be valuable.

Structure and intermolecular interactions in spheroidal high-density lipoprotein subpopulations

Human serum high-density lipoproteins (HDLs) are a population of small, dense protein-lipid aggregates that are crucial for intravascular lipid trafficking and are protective against cardiovascular disease. The spheroidal HDL subfraction can be separated by size and density into five major subpopulations with distinct molecular compositions and unique biological functionalities: HDL3c, HDL3b, HDL3a, HDL2a and HDL2b. Representative molecular models of these five subpopulations were developed and characterised for the first time in the presence of multiple copies of its primary protein component apolipoprotein A-I (apoA-I) using coarse-grained molecular dynamics simulations. Each HDL model exhibited size, morphological and compositional profiles consistent with experimental observables. With increasing particle size the separation of core and surface molecules became progressively more defined, resulting in enhanced core lipid mixing, reduced core lipid exposure at the surface, and the formation of an interstitial region between core and surface molecules in HDL2b. Cholesterol molecules tended to localise around the central helix-5 of apoA-I, whilst triglyceride molecules predominantly interacted with aromatic, hydrophobic residues located within the terminal helix-10 across all subpopulation models. The three intermediate HDL models exhibited similar surface profiles despite having distinct molecular compositions. ApoA-I in trefoil, quatrefoil and pentafoil arrangements across the surface of HDL particles exhibited significant warping and twisting, but largely retained intermolecular contacts between adjacent apoA-I chains. Representative HDL subpopulations differed in particle size, morphology, intermolecular interaction profiles and lipid and protein dynamics. These findings reveal how different HDL subpopulations might exhibit distinct functional associations depending on particle size, form and composition.

Effects of fatty liver on the size and composition of high-density lipoprotein cholesterol subpopulations in adolescents with type 2 diabetes mellitus.

Type 2 diabetes mellitus (T2DM) is an emerging disease in the pediatric population. The association between T2DM and non-alcoholic fatty liver disease (NAFLD) has been described. Recent evidence suggests that sizes and composition of high-density lipoprotein (HDL) may be more important that HDL-C levels in predicting coronary heart disease. There is not data regarding the HDL subclasses distribution and composition in T2DM youths with NAFLD. This cross-sectional study included 47 adolescents with T2DM and 23 non-diabetic controls of both sexes aged 10 to 18 years. The presence of NAFLD was determined estimated proton density fat fraction (PDFF) by magnetic resonance by spectroscopy. We compared the HDL subclasses distribution (HDL2b, HDL2a, HDL3a HDL3b and HDL3c) and the HDL chemical composition (total protein, triglyceride, phospholipid, cholesteryl esters, and free cholesterol) between the groups of adolescents with T2DM and the control group. Patients with T2DM and NAFLD had a significantly lower proportion HDL2b (P = .040) and a higher proportion of HDL3c (P = .035); higher proportion of TG (P = .032) and a lower CE (P = .002) and FC (P < .001). A negative association was observed between PDFF and the percentages of HDL2b (r2 = -0.341, P = .004) and the average particle size (r2 = -0.327, P = .05), and a positive association with HDL3c subpopulations (r2 = 0.327, P = .015); about composition inside HDL particle, a positive association with PDFF and the TG (r2 = 0.299, P = .013) and negative with CE (r2 = -0.265, P = .030). In adolescents diagnosed with T2DM, the presence of NAFLD is associated with abnormalities in the distribution of HDL subpopulations and the lipid composition of HDL particles.

Small HDL, diabetes, and proinflammatory effects in macrophages

Atherosclerosis, an inflammatory disease of the arterial wall, is the commonest cause of cardiovascular disease (CVD) events. Elevated levels of high‐density lipoprotein (HDL) cholesterol are generally believed to protect against CVD and to reduce inflammatory activation of macrophages, a key cell type in lesions of atherosclerosis. However, HDL can also exert proinflammatory effects in macrophages by inducing cholesterol depletion. Because small HDL (7–8.5 nm diameter) is most effective in removing cholesterol from cells by the ABCA1 pathway, we hypothesized that small HDL is the most proinflammatory HDL population. Small‐, medium‐ and large HDL populations were isolated from a human apolipoprotein A‐I transgenic LDL receptor‐deficient mouse model (APOA1Tg;Ldlr−/−). Depletion of cholesterol by HDL increased the ability of toll‐like receptor (TLR) ligands to induce proinflammatory mediators in macrophages. This effect was prevented by cholesterol loading of the macrophages. Small HDL had a significantly stronger effect on Tnfa (3‐fold over LPS alone) and Il1b (2.5‐fold over LPS alone) gene expression than medium or large HDL particles. Inhibition or deletion of the sheddase ADAM17 in macrophages prevented the proinflammatory effects of HDL. Patients with type 1 diabetes mellitus (T1DM) exhibit an increased risk of CVD despite having normal or high HDL‐cholesterol. To investigate the effect of diabetes on HDL subpopulations, we crossed a virally‐induced mouse model of T1DM (Ldlr−/−;GpTg) with the human APOA1Tg;Ldlr−/− mouse. The mice (APOA1Tg;Ldlr−/−;GpTg, n=8–10) were injected with a liver‐specific adeno‐associated virus driving expression of human cholesteryl ester transfer protein (hCETP) to mimic human physiology. Diabetic mice had an increased HDL particle concentration (~2‐fold) associated with a significant increase in serum HDL cholesterol efflux capacity through both ABCA1 and ABCG1, compared with non‐diabetic mice. The largest increase was in the small HDL particle concentration (15 μM vs 6 μM in non‐diabetic mice). Moreover, resident peritoneal macrophages from diabetic mice exhibited increased cytokine gene expression, as compared with non‐diabetic littermates. Our findings demonstrate that higher levels of small HDL, as observed in a mouse model of diabetes, can exacerbate inflammatory activation in non‐lipid loaded macrophages in an ADAM17‐dependent manner. We have found similar changes in HDL concentration and function in humans with T1DM, suggesting that our mouse model might provide novel insights into CVD risk in T1DM patients.Support or Funding Informationfunded in part by the National Institutes of Health, grants DP3DK108209, P01HL092969, P30 DK017047, and P01HL128203This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Distinct phospholipid and sphingolipid species are linked to altered HDL function in apolipoprotein A-I deficiency

Familial apolipoprotein A-I (apoA-I) deficiency (FAID) involving low levels of both apoA-I and high-density lipoprotein (HDL) cholesterol is associated with accelerated atherosclerosis. The objective of this study was to define distinctive patterns in the lipidome of HDL subpopulations in FAID in relationship to antiatherogenic activities. Five HDL subfractions were isolated by ultracentrifugation from plasma of FAID Caucasian patients (n=5) and age-matched healthy normolipidemic Caucasian controls (n=8), and the HDL lipidome (160 molecular species of 9 classes of phospholipids and sphingolipids) was quantitatively evaluated. Increased concentrations of numerous molecular species of lysophosphatidylcholine (up to 12-fold), ceramides (up to 3-fold), phosphatidylserine (up to 34-fold), phosphatidic acid (up to 71-fold), and phosphatidylglycerol (up to 20-fold) were detected throughout all five HDL subpopulations as compared with their counterparts from controls, whereas concentrations of phosphatidylethanolamine species were decreased (up to 5-fold). Moderately to highly abundant, within their lipid class, species of phosphatidylcholine, sphingomyelin, phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine, and ceramide featuring multiple unsaturations were primarily affected by apoA-I deficiency; their HDL content, particularly that of phosphatidylcholine (34:2), was strongly correlated with HDL function, impaired in FAID. Metabolic pathway analysis revealed that sphingolipid, glycerophospholipid, and linoleic acid metabolism was significantly affected by FAID. These data reveal that altered content of specific phospholipid and sphingolipid species is linked to deficient antiatherogenic properties of HDL in FAID.

Poor glycemic control in type 2 diabetes enhances functional and compositional alterations of small, dense HDL3c

High-density lipoprotein (HDL) possesses multiple biological activities; small, dense HDL3c particles displaying distinct lipidomic composition exert potent antiatherogenic activities which can be compromised in dyslipidemic, hyperglycemic insulin-resistant states. However, it remains indeterminate (i) whether such functional HDL deficiency is related to altered HDL composition, and (ii) whether it originates from atherogenic dyslipidemia, dysglycemia, or both.In the present work we analyzed compositional characteristics of HDL subpopulations and functional activity of small, dense HDL3c particles in treatment-naïve patients with well-controlled (n=10) and poorly-controlled (n=8) type 2 diabetes (T2D) and in normolipidemic age- and sex-matched controls (n=11).Our data reveal that patients with both well- and poorly-controlled T2D displayed dyslipidemia and low-grade inflammation associated with altered HDL composition. Such compositional alterations in small, dense HDL subfractions were specifically correlated with plasma HbA1c levels. Further analysis using a lipidomic approach revealed that small, dense HDL3c particles from T2D patients with poor glycemic control displayed additional modifications of their chemical composition. In parallel, antioxidative activity of HDL3c towards oxidation of low-density lipoprotein was diminished.These findings indicate that defective functionality of small, dense HDL particles in patients with T2D is not only affected by the presence of atherogenic dyslipidemia, but also by the level of glycemic control, reflecting compositional alterations of HDL.

Functionality of High-Density Lipoprotein as Antiatherosclerotic Therapeutic Target.

A great majority of the morbidity and mortality worldwide can still be attributed to cardiovascular diseases, such as ischemic (coronary) heart disease, angina pectoris, and myocardial and cerebral infarction. Atherosclerosis, narrowing of the arteries because of arterial cholesterol deposition in macrophage foam cells, is the driving force behind the cardiovascular disease pathology. Water-soluble protein/lipid complexes called lipoproteins mediate the transport of cholesterol and other lipoid substances through the blood compartment. Relatively high levels of cholesterol associated with apolipoprotein B–containing low-density lipoprotein (LDL) particles predispose human subjects to the development of atherosclerosis and, thereby, increase the risk for cardiovascular disease.1,2 Apolipoprotein B–containing lipoproteins are, therefore, generally regarded as being proatherogenic factors. Cholesterol ester–rich high-density lipoprotein (HDL) particles use apolipoprotein A1 (apoA1) as their primary protein component. In sharp contrast to LDL, HDL is considered a potent anti-atherogenic agent. This notion is based on the fact that, in the general population, a strong inverse correlation exists between plasma levels of HDL cholesterol and the risk of cardiovascular disease.1 Of note, this inverse association seems to be independent of the level of cholesterol associated with proatherogenic LDL particles. As such, increasing plasma levels of HDL cholesterol has long been regarded a promising alternative therapy to supplement classical statin–based LDL cholesterol–lowering strategies that are able to reduce cardiovascular disease by only ≈30%.3 However, over the last decade, the enthusiasm for HDL as an interesting therapeutic target has been challenged by the HDL hypothesis critics because genetic association studies have excluded HDL cholesterol levels as determinants for cardiovascular disease risk.2,4 Furthermore, several therapeutic HDL-targeting approaches have proven insufficient to secure benefit for cardiovascular disease patients. Niacin is the most effective drug available in the clinic to raise plasma HDL cholesterol levels. Despite the fact that niacin is …

Impact of genetic deletion of platform apolipoproteins on the size distribution of the murine lipoproteome

Given their association with cardiovascular disease protection, there has been intense interest in understanding the biology of high density lipoproteins (HDL). HDL is actually a family of diverse particle types, each made up of discrete - but as yet undetermined – combinations of proteins drawn from up to 95 lipophilic plasma proteins. The abundant apolipoproteins (apo) of the A class (apoA-I, apoA-II and apoA-IV) have been proposed to act as organizing platforms for auxiliary proteins, but this concept has not been systematically evaluated. We assessed the impact of genetic knock down of each platform protein on the particle size distribution of auxiliary HDL proteins. Loss of apoA-I or apoA-II massively reduced HDL lipids and changed the plasma size pattern and/or abundance of several plasma proteins. Surprisingly though, many HDL proteins were not affected, suggesting they assemble on lipid particles in the absence of apoA-I or apoA-II. In contrast, apoA-IV ablation had minor effects on plasma lipids and proteins, suggesting that it forms particles that largely exclude other apolipoproteins. Overall, the data indicate that distinct HDL subpopulations exist that do not contain, nor depend on, apoA-I, apoA-II or apoA-IV and these contribute substantially to the proteomic diversity of HDL. Biological significancePlasma levels of high density lipoproteins (HDL) are inversely correlated with cardiovascular disease. These particles are becoming known as highly heterogeneous entities that have diverse compositions and functions that may impact disease. Unfortunately, we know little about the forces that maintain the composition of each particle in plasma. It has been suggested that certain ‘scaffold’ proteins, such as apolipoprotein (apo) A-I, apoA-II and apoA-IV, may act as organizing centers for the docking of myriad accessory proteins. To test this hypothesis, we took advantage of the genetic tractability of the mouse model and ablated these three proteins individually. We then tracked the abundance and size profile of the remaining HDL proteins by gel filtration chromatography combined with mass spectrometry. The results clearly show that certain cohorts of proteins depend on each scaffold molecule to assemble normal sized HDL particles under wild-type conditions. This work forms the basis for more detailed studies that will define the specific compositions of HDL subspecies with the possibility of connecting them to specific functions or roles in disease.

Abstract 631: Proteomic Characterization of Apolipoprotein A-II Defined Subfractions of Human High Density Lipoproteins

High-density lipoproteins (HDL) play a critical role in lipid transport and vascular lipid homeostasis. HDL is heterogeneous, with particles varying in size, composition, and functionality. Proteomic studies have shown that HDL can host as many as 94 different proteins which can segregate into distinct subclasses. The two major apolipoproteins (apo)A-I and apoA-II account for 70% and 15-20%, respectively of HDL total protein. We hypothesized that the presence or absence of apoA-II can affect the binding of other proteins thus influencing HDL function. Immunoaffinity chromatography was used to isolate particles containing both apoA-I and apoA-II (LpA-I/LpA-II) and those with apoA-I with no apoA-II (LpA-I). These were isolated from both i ) total HDL isolated by ultracentrifugation (UC) and ii ) directly from plasma. The proteomic fingerprint of each population was determined by mass spectrometry. Within both populations, 54 total proteins were identified in UC isolated HDL compared to 98 proteins in particles isolated directly from plasma. In the UC isolated HDL, LpA-I/A-II particles contained 39 proteins compared to LpA-I particles which contained about 30. Interestingly, the opposite was true in particles isolated from plasma where upwards of 77 proteins on LpA-I/A-II particles were identified compared to LpA-I particles where 98 proteins were identified. Limited tryptic digestion experiments showed that LpA-I/A-II were more prone to proteolysis compared to LpA-I. Additionally, we found that LpA-I/A-II exhibited an increased capacity to efflux cholesterol from cultured macrophages (normalized by phospholipid content) suggesting a protein mediated difference in HDL functionality. Taken together, these results suggest the proteomic fingerprint of HDL subpopulations is heavily influenced by the method of isolation and that the proteomic pattern on HDL influences the physiological function of the particle.

HDL particle number and size as predictors of cardiovascular disease.

Previous studies indicate that reduced concentrations of circulating high-density lipoprotein (HDL) particles can be superior to HDL-cholesterol (HDL-C) levels as a predictor of cardiovascular disease. Measurements of HDL particle numbers, therefore, bear a potential for the improved assessment of cardiovascular risk. Furthermore, such measurement can be relevant for the evaluation of novel therapeutic approaches targeting HDL. Modern in-depth analyses of HDL particle profile may further improve evaluation of cardiovascular risk. Although clinical relevance of circulating concentrations of HDL subpopulations to cardiovascular disease remains controversial, the negative relationship between the number of large HDL particles and cardiovascular disease suggests that assessment of HDL particle profile can be clinically useful. Reduced mean HDL size is equally associated with cardiovascular disease in large-scale clinical studies. Since HDL-C is primarily carried in the circulation by large, lipid-rich HDL particles, the inverse relationship between HDL size and cardiovascular risk can be secondary to those established for plasma levels of HDL particles, HDL-C, and large HDL. The epidemiological data thereby suggest that HDL particle number may represent a more relevant therapeutic target as compared to HDL-C.

A complex phenotype in a child with familial HDL deficiency due to a novel frameshift mutation in APOA1 gene (apoA-IGuastalla)

We describe a kindred with high-density lipoprotein (HDL) deficiency due to APOA1 gene mutation in which comorbidities affected the phenotypic expression of the disorder. An overweight boy with hypertriglyceridemia (HTG) and HDL deficiency (HDL cholesterol 0.39mmol/L, apoA-I 40mg/dL) was investigated. We sequenced the candidate genes for HTG (LPL, APOC2, APOA5, GPIHBP1, LMF1) and HDL deficiency (LCAT, ABCA1 and APOA1), analyzed HDL subpopulations, measured cholesterol efflux capacity (CEC) of sera and constructed a model of the mutant apoA-I. No mutations in HTG-related genes, ABCA1 and LCAT were found. APOA1 sequence showed that the proband, his mother and maternal grandfather were heterozygous of a novel frameshift mutation (c.546_547delGC), which generated a truncated protein (p.[L159Afs*20]) containing 177 amino acids with an abnormal C-terminal tail of 19 amino acids. Trace amounts of this protein were detectable in plasma. Mutation carriers had reduced levels of LpA-I, preβ-HDL and large HDL and no detectable HDL-2 in their plasma; their sera had a reduced CEC specifically the ABCA1-mediated CEC. Metabolic syndrome in the proband explains the extremely low HDL cholesterol level (0.31mmol/L), which was half of that found in the other carriers. The proband's mother and grandfather, both presenting low plasma low-density lipoprotein cholesterol, were carriers of the β-thalassemic trait, a condition known to be associated with a reduced low-density lipoprotein cholesterol and a reduced prevalence of cardiovascular disease. This trait might have delayed the development of atherosclerosis related to HDL deficiency. In these heterozygotes for apoA-I truncation, the metabolic syndrome has deleterious effect on HDL system, whereas β-thalassemia trait may delay the onset of cardiovascular disease.

ApoE Containing HDL Subspecies: Their Ability to Inhibit Key Steps in the Atherosclerotic Process

Cardiovascular disease is the leading cause of death in people with type-2 diabetes. Cardiovascular disease develops when low-density lipoproteins (LDL) bind to proteoglycans in arterial walls and form atherosclerotic plaques. High-density lipoproteins (HDL) are thought to protect against the development of cardiovascular disease by inhibiting this process. Previous work has shown that HDL exists as numerous subpopulations and that adolescents with type-2 diabetes have decreased apolipoprotein E (apoE) rich large HDL subpopulations compared to lean youth. The goal of this project was to show that decreased amounts of the apoE containing HDL subpopulation is linked to increased risk for cardiovascular disease in type-2 diabetics. Ultracentrifugation and immunoaffinity chromatography were used to isolate the apoE containing large HDL subpopulation from lean and type-2 diabetic adolescents. The composition of these samples was identified using mass spectrometry. Fluorescent proteoglycan binding assays were used to establish if the apoE containing particles from healthy individuals inhibited LDL from binding to a surrogate proteoglycan. Our data support the hypothesis that apoE-containing large HDL subpopulations protect against atherosclerosis. Our findings suggest the possibility of preventing cardiovascular disease in diabetic patients by manipulating the apoE containing particles in adolescents to resemble the composition of a healthy individual.