High-density Lipoprotein Species Research Articles

Quantification of high-density lipoprotein particle number by proton nuclear magnetic resonance: don't believe the numbers.

Proton nuclear magnetic resonance (NMR) can rapidly assess lipoprotein concentrations and sizes in biological samples. It may be especially useful for quantifying high-density lipoprotein (HDL), which exhibits diverse particle sizes and concentrations. We provide a critical review of the strengths and limitations of NMR for quantifying HDL subclasses. Recent studies using NMR have shed light on HDL's role in various disorders, ranging from residual cardiovascular risk to host susceptibility to infection. However, accurately quantifying HDL particle number, size, and concentration (HDL-P) remains a challenge. Discrepancies exist between NMR and other methods such as gel electrophoresis, ion mobility analysis and size-exclusion chromatography in estimating the abundance of HDL species and the ratio of apolipoprotein A-I (APOA1) to HDL particles. NMR is a low-cost method for quantifying HDL-P that is readily applicable to clinical and translational studies. However, inconsistencies between the results of NMR quantification of HDL-P and other independent methods hinder the interpretation of NMR results. Because proton NMR apparently fails to accurately quantify the sizes and concentrations of HDL, the relevance of such studies to HDL biology poses challenges. This limits our understanding of pathophysiological implications of HDL-P as determined by NMR, particularly in determining cardiovascular disease (CVD) risk.

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.

CSL112 Increases Plasma Cholesterol Efflux Capacity and Reduces Erythrocyte Membrane Cholesterol Content in Blood Samples from Patients with Sickle Cell Disease

Background: Lipid homeostasis is abnormal in sickle cell disease (SCD) and contributes to elevated erythrocyte cholesterol content which has been implicated in sickling. The highly oxidative environment in blood from patients with SCD alters high-density lipoprotein (HDL) composition and impairs HDL particle remodeling. Specifically, it negatively impacts on the capacity of HDL particles to mediate apolipoprotein A-I (apoA-I) exchange and protect against inflammation. Low apoA-I levels and altered composition of HDL have been well-documented at steady state, associated with high risk features of SCD, including endothelial dysfunction and risk of pulmonary hypertension. The low levels and activity of apoA-I fall even further during vaso-occlusive events in SCD. This diminished pool of apoA-I activity may mediate reduced capacity of HDL to extract cholesterol from erythrocyte membranes and increase risk of vascular dysfunction. Purpose: In this study we aimed to determine whether plasma derived apoA-I (CSL112; apolipoprotein A-I (human)) could elevate the cholesterol efflux capacity (CEC) of plasma from SCD patients and thereby reduce erythrocyte membrane cholesterol content. Methods: All SCD subjects (n=14) had confirmed HbSS genotype except one who was with HbS/β0-thalassemia, healthy control subjects (n=13) were not race-matched. CSL112 was incubated with fresh blood derived from control subjects and SCD patients for 6 hours at 37°C. The cholesterol content of ghost membranes of isolated erythrocytes was measured. HDL particle size distribution was assessed by native gel electrophoresis followed by western blotting. The ex vivo capacity of apoB-depleted plasma to efflux cholesterol was determined using [3H]cholesterol-loaded RAW264.7 macrophages. Results: CEC of SCD plasma was attenuated (75.9 ± 7.4%, n=14) compared to controls (100%, n=13), with a predominant decrease of ATP binding cassette transporter A1 (ABCA1)-dependent CEC (77.7 ± 9.2% vs 100%, respectively). Consistently, SCD plasma showed a trend towards lower levels of pre-β1 HDL (78.5 ± 6.5%) as compared to controls (100 %). Incubation with CSL112 raised plasma levels of small HDL species and pre-β1 HDL, resulting in a more than 3.5 fold increase of CEC in both patients and controls. Treatment of SCD blood with CSL112 reduced erythrocyte cholesterol content to a greater extent compared to control blood (% reduction: 12.8% ± 3.1% in SCD, 6.3% ± 5.4% in controls). Conclusion: Our data suggest that reduced levels of lipid-poor pre-β1 HDL in SCD plasma reflect impaired HDL particle remodeling and account for a decreased CEC. CSL112 may acutely generate HDL species which optimally support ABCA1-dependent cholesterol efflux and thereby improve HDL function, normalize erythrocyte membrane cholesterol content and correct the imbalance in cellular cholesterol homeostasis in SCD patients. ApoA-I as a potential therapeutic could have implications in the pathobiologic understanding and future clinical management of vaso-occlusive crisis and of chronic vascualopathy such as pulmonary hypertension.

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 .

Supramolecular Assembly of High-Density Lipoprotein Mimetic Nanoparticles Using Lipid-Conjugated Core Scaffolds.

Synthetic high-density lipoprotein (HDL) mimics have emerged as promising therapeutic agents. However, approaches to date have been unable to reproduce key features of spherical HDLs, which are the most abundant human HDL species. Here, we report the synthesis and characterization of spherical HDL mimics using lipid-conjugated organic core scaffolds. The core design motif constrains and orients phospholipid geometry to facilitate the assembly of soft-core nanoparticles that are approximately 10 nm in diameter and resemble human HDLs in their size, shape, surface chemistry, composition, and protein secondary structure. These particles execute salient HDL functions, including efflux of cholesterol from macrophages, cholesterol delivery to hepatocytes, support lecithin:cholesterol acyltransferase activity, and suppress inflammation. These results represent a significant step toward a genuine functional mimic of human HDLs.

Sex Bias in Cytokines Transported by High‐Density Lipoproteins in Patients with Coronary Artery Disease

Inflammation plays an important role in the induction of Coronary Artery Disease (CAD) and the progression to deadly coronary syndromes. Cytokines are key mediators of inflammation, both in pro‐ and anti‐inflammatory capacities. We have recently discovered that cytokines are transported by high‐density lipoproteins (HDL), suggesting a novel mechanism contributing to the anti‐inflammatory effects of HDL. We examined the HDL‐associated cytokine profiles of male and female patients with CAD to better understand the differences in coronary disease presentation and therapeutic response between men and women.MethodsPatients with confirmed CAD, defined by history of myocardial infarction, coronary artery bypass surgery, or angiography, with controlled serum lipid levels were included in the study. Native HDL species were isolated from plasma samples by selected‐affinity immunosorption. Plasma and isolated HDL samples were assayed for cytokine content by a multiplex array detecting the quantity of 35 cytokines.ResultsThe majority of cytokines assayed were measurable in HDL samples. The distribution of HDL‐associated cytokines varied significantly between males and females. Female HDL samples indicate a much greater degree of remodeling of cytokine cargo than males. Cluster analysis of cytokine expression patterns suggests a difference in inflammatory pathway activation and response in females with CAD.ConclusionsThe cytokines transported by HDL in CAD patients differs greatly between men and women. The pattern of HDL‐associated cytokines provide new insights into the inflammatory processes contributing to CAD in a sex‐dependent manner. The amount of cytokine‐HDL remodeling is exhibited to a greater degree in females; the physiological relevance of this phenomenon is unknown. We aim to elucidate the mechanism of HDL transport of these cytokines and the clinical implications of sex differences in inflammatory response in CAD in future studies.Support or Funding InformationThis work is supported by the Read Family Foundation and the Campini Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Apolipoprotein E-containing high-density lipoprotein (HDL) modifies the impact of cholesterol-overloaded HDL on incident coronary heart disease risk: A community-based cohort study

Experimental studies have shown that cholesterol-overloaded high-density lipoprotein (HDL) can promote the formation of apolipoprotein E (APOE)-containing HDL, a process correcting the atherogenic function of cholesterol-overloaded HDL. The objective of the study was to explore whether APOE-containing HDL can attenuate the defective impact of cholesterol-overloaded HDL on the development of coronary heart disease (CHD) in humans. We measured APOE-HDL cholesterol (APOE-HDLC), HDL cholesterol (HDLC), and HDL particle number in 1112 participants aged 45 to 74years at baseline in a community-based cohort study. Cholesterol molecules per HDL particle (HDL-C/P ratio) were calculated as the ratio of HDLC to HDL particle number. The ratio of APOE-HDLC to total HDLC (APOE-HDLC/HDLC ratio) was calculated to assess the relative proportion of APOE-HDLC in total HDLC. The HDL-C/P ratio was strongly correlated with APOE-HDLC (partial-r: 0.615). Participants with cholesterol-overloaded HDL (indicated by the highest level of the HDL-C/P ratio) had a high APOE-HDLC/HDLC ratio. Baseline cholesterol-overloaded HDL significantly increased the 10-year risk of incident CHD (hazard ratio=2.42; 95% confidence interval=1.06-8.32), but this was attenuated by an increased APOE-HDLC/HDLC ratio. Participants with high HDL-C/P ratio and APOE-HDLC/HDLC ratio had a 42% lower risk, whereas those with a high HDL-C/P ratio and low APOE-HDLC/HDLC ratio had a 2.54-fold higher risk, than those with low HDL-C/P ratio and APOE-HDLC/HDLC ratio after multiple adjustments. Cholesterol-overloaded HDLs are related with increased APOE-containing HDL species. APOE-containing HDL was found to attenuate the impact of cholesterol-overloaded HDL on increased incident CHD risk, suggesting that APOE-containing HDL may correct the dysfunction of cholesterol-overloaded HDL.

Scavenger receptor B1 (SR-B1) profoundly excludes high density lipoprotein (HDL) apolipoprotein AII as it nibbles HDL-cholesteryl ester

Reverse cholesterol transport (transfer of macrophage-cholesterol in the subendothelial space of the arterial wall to the liver) is terminated by selective high density lipoprotein (HDL)-cholesteryl ester (CE) uptake, mediated by scavenger receptor class B, type 1 (SR-B1). We tested the validity of two models for this process: "gobbling," i.e. one-step transfer of all HDL-CE to the cell and "nibbling," multiple successive cycles of SR-B1-HDL association during which a few CEs transfer to the cell. Concurrently, we compared cellular uptake of apoAI with that of apoAII, which is more lipophilic than apoAI, using HDL-[3H]CE labeled with [125I]apoAI or [125I]apoAII. The studies were conducted in CHO-K1 and CHO-ldlA7 cells (LDLR-/-) with (CHO-SR-B1) and without SR-B1 overexpression and in human Huh7 hepatocytes. Relative to CE, both apoAI and apoAII were excluded from uptake by all cells. However, apoAII was more highly excluded from uptake (2-4×) than apoAI. To distinguish gobbling versus nibbling mechanisms, media from incubations of HDL with CHO-SR-B1 cells were analyzed by non-denaturing PAGE, size-exclusion chromatography, and the distribution of apoAI, apoAII, cholesterol, and phospholipid among HDL species as a function of incubation time. HDL size gradually decreased, i.e. nibbling, with the concurrent release of lipid-free apoAI; apoAII was retained in an HDL remnant. Our data support an SR-B1 nibbling mechanism that is similar to that of streptococcal serum opacity factor, which also selectively removes CE and releases apoAI, leaving an apoAII-rich remnant.

Nanoparticle‐based biosensors for the detection of lecithin:cholesterol acyltransferase activity

BackgroundLecithin:cholesterol acyltransferase (LCAT) is an enzyme that catalyzes the esterification of free cholesterol carried in high‐density lipoproteins (HDL). Esterification of cholesterol results in internalization of cholesteryl ester (CE) within the core of maturing HDLs. Of the HDL species, increased levels of CE‐rich HDLs are inversely correlated with the development of cardiovascular disease. Prior research has established that the activity of LCAT is influenced by numerous external physiological stimuli. Our lab has previously developed nanoparticles (HDL‐NP) that mimic natural HDLs with regard to size, shape, surface chemistry, and cholesterol binding function. Herein, we describe the application of HDL‐NP as specific activators of LCAT and biosensors for assaying LCAT activity.MethodsHDL‐NP were synthesized using 5 nm diameter gold nanoparticles (AuNP) as a scaffold for the assembly of a lipid bilayer containing a mixture of phosphotidylcholine (PC), phosphotidylethanolamine (PE), and apolipoportein A‐I (apoAI). ApoAI, found in natural HDL, serves as a cofactor for LCAT. 10% of total nanoparticle phospholipid content consisted of a PC lipid labeled with the fluorophore, TopFluor, thus forming TopFluor‐labeled HDL‐NP (TF‐NP). A subset of HDL‐NP was formulated without apoAI, having only the phospholipid bilayer (BL‐NP), to serve as a control to demonstrate specificity for LCAT. These nanoparticle groups were incubated for 24 hours in PBS with cholesterol, LCAT, or cholesterol and LCAT. In addition, the particles were incubated in dilute serum. An increase in the fluorescence intensity was used as a measure of LCAT activity, presumably, due to a reduced quenching effect of the core AuNP on the TopFluor‐labeled PC.ResultsTF‐NP and BL‐NP were incubated with cholesterol alone, LCAT alone, or a mixture of cholesterol and LCAT (Fig. A). A significant increase in fluorescent signal was observed for the TF‐NP incubated with cholesterol and LCAT over that of the TF‐NP alone (p=0.0000005) indicating activity of LCAT on TF‐NP. Interestingly, the presence of cholesterol alone with TF‐NP and BL‐NP results in a fluorescence response, independent of apoAI content. The importance of apoAI as a cofactor for promoting LCAT activity is apparent through comparing the fluorescent response of TF‐NP and BL‐NP where the apoAI‐containing TF‐NP exhibits significantly increased fluorescence over BL‐NP when incubated with both cholesterol and LCAT (p=0.00012). Next, TF‐NP and BL‐NP were incubated in samples of diluted serum for 24 hours (Fig. B), demonstrating a dose‐response whereby increased serum resulted in a stronger fluorescent response from the nanoparticles. Furthermore, this study further confirms the specificity of LCAT towards apoAI‐containing TF‐NP, where fluorescence intensity was significantly increased over BL‐NP groups for all serum dilutions.ConclusionThese results demonstrate the ability of fluorescent HDL‐NP to serve as biosensors for detecting LCAT activity, and that detection of LCAT activity directly from serum samples is possible using this technology. Future work will aim to better understand cholesterol and cholesteryl binding to HDL‐NP through the observed quenching and de‐quenching fluorescent response, and to apply this biosensor for the detection of LCAT activity changes in response to both acute and prolonged exercise, both previously demonstrated to induce changes in LCAT activity in human subjects.Support or Funding InformationAFRL FA8650‐15‐2‐5518

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 …

Enhanced HDL Functionality in Small HDL Species Produced Upon Remodeling of HDL by Reconstituted HDL, CSL112

Rationale: CSL112, human apolipoprotein A-I (apoA-I) reconstituted with phosphatidylcholine, is known to cause a dramatic rise in small high-density lipoprotein (HDL). Objective: To explore the mechanisms by which the formation of small HDL particles is induced by CSL112. Methods and Results: Infusion of CSL112 into humans caused elevation of 2 small diameter HDL fractions and 1 large diameter fraction. Ex vivo studies showed that this remodeling does not depend on lipid transfer proteins or lipases. Rather, interaction of CSL112 with purified HDL spontaneously gave rise to 3 HDL species: a large, spherical species composed of apoA-I from native HDL and CSL112; a small, disc-shaped species composed of apoA-I from CSL112, but smaller because of the loss of phospholipids; and the smallest species, lipid-poor apoA-I composed of apoA-I from HDL and CSL112. Time-course studies suggest that remodeling occurs by an initial fusion of CSL112 with HDL and subsequent fission leading to the smaller forms. Functional studies showed that ATP-binding cassette transporter 1–dependent cholesterol efflux and anti-inflammatory effects in whole blood were carried by the 2 small species with little activity in the large species. In contrast, the ability to inactivate lipid hydroperoxides in oxidized low-density lipoprotein was carried predominantly by the 2 largest species and was low in lipid-poor apoA-I. Conclusions: We have described a mechanism for the formation of small, highly functional HDL species involving spontaneous fusion of discoidal HDL with spherical HDL and subsequent fission. Similar remodeling is likely to occur during the life cycle of apoA-I in vivo.

Abstract 29: Alpha-1-antitrypsin Protects High Density Lipoprotein From Functional Inactivation by Elastase

High density lipoproteins (HDL) are complexes of lipid and protein with several known atheroprotective functions. These functions are driven by specific lipids and proteins contained on the HDL particle and include reverse cholesterol transport, suppression of inflammation, and modulation of endothelial function. These activities are most important within atherosclerotic plaque, a harsh environment where HDL interact with macrophage foam cells, activated neutrophils, and dysfunctional endothelial cells. Neutrophils and macrophages secrete proteases, such as elastase, which damage structural components and soluble proteins and propagate inflammatory signaling. It has been suggested that, in plaque, HDL become damaged and dysfunctional. We recently characterized a subspecies of HDL that carries the protein alpha-1-antitrypsin (A1AT), an abundant plasma serine protease inhibitor. In the current study, we tested the hypothesis that A1AT enriched HDL are protected from proteolytic damage and functional inactivation by elastase, the main protease inhibited by A1AT. Human HDL was isolated by ultracentrifugation and was enriched with A1AT by co-incubation and unbound A1AT was removed. Treatment of native HDL with elastase resulted in significant proteolytic degradation of both apoA-I and apoA-II, visualized by coomassie stained SDS-PAGE. Analysis of lipoprotein size by one dimensional native gel electrophoresis revealed that pre-beta HDL were completely degraded by elastase. Compared to native HDL, A1AT enriched HDL samples were protected from protein and pre-beta particle degradation by elastase. We next tested the effect of elastase treatment on HDL function. In native HDL, elastase had damaging effects on ABCA1 mediated cholesterol efflux (-32%; p&lt;0.0001) and the ability to esterify free cholesterol (-14%; p&lt;0.02). A1AT enriched HDL displayed no loss of functionality upon treatment with elastase. Both of these activities are required for HDL to perform what is thought to be its most important function, reverse cholesterol transport. In conclusion, the data presented indicate that HDL particles which contain A1AT may represent a functionally important species of HDL, which have an advantage in the protease-rich plaque environment.

Abstract 426: The Remodeling of HDL-SAA by Macrophage Surface Heparan Sulfate is Necessary for Efficient Cholesterol Efflux

Serum amyloid A (SAA) is an acute-phase protein that circulates associated with high density lipoprotein (HDL) and can replace apoA-I as the predominant apoprotein. SAA can increase HDL’s binding affinity for macrophages and we provide evidence that this targeting process is dependent on cell surface heparan sulfate (HS) and facilitated by at least one of two HS binding sites that we have been previously identified in SAA. Additional studies utilizing various assays including native-PAGE, turbidity, thioflavin T fluorescence and surface plasmon resonance have demonstrated that HDL-SAA binding to HS (or heparin) causes SAA to dissociate from the HDL particle, leaving behind novel HDL species with enhanced cholesterol carrying capacities. In the present study we explore this remodeling phenomenon further and assess its potential importance for cholesterol efflux from macrophages. The chemical disruption of HDL-SAA with chaotropic agents, temperatures below 37°C and lipid oxidation with copper all interfere with HDL-SAA/heparin remodeling. The oxidation of HDL-SAA also significantly reduces its cholesterol efflux potential and SAA’s ability to be converted into AA-amyloid fibrils in cell culture, a process that requires the dissociation of SAA from HDL. Finally, we demonstrate that the removal of cell surface HS chains, by either heparinase treatment or by preventing HS biosynthesis with sodium chlorate, impairs cholesterol efflux rates from macrophages to HDL-SAA. Our interpretation of these findings is that the reduction in HDL-lipid fluidity (low temperatures or conjugated dienes) significantly reduces the ability of HS:SAA interactions to dissociate SAA from HDL. We postulate that HS normally plays an important role in separating SAA from HDL but during chronic inflammatory diseases with elevated lipoprotein oxidation, HDL-SAA species are generated in which SAA is more tightly associated with HDL, thus preventing its dissociation from HDL and impairing atheroprotective functions.

Abstract 223: The Structural Organization of Apolipoprotein A-I in Reconstituted High-Density Lipoprotein Discs Using Stable Isotope-Assisted Cross-Linking

X-ray crystallography studies of lipid-free apolipoprotein A-I (apoA-I) and related apoA-IV have shown that the proteins form a reciprocating dimer which could be directly related to the structural organization of lipid-bound apoA-I. Previous work has demonstrated the success of chemical cross-linking combined with mass spectrometry to derive distance constraints for molecular modeling of apoA-I in reconstituted high density lipoprotein (HDL) particles. However, these studies have been limited by i) the relative paucity of distance constraint data, and ii) ambiguities in determining the molecular span of the cross-links. Here, we have addressed the first issue by using an affinity-tagged cross-linker to purify cross-linked peptides away from non-modified peptides prior to sampling with mass spectrometry. This essentially boosts the signal from cross-linked peptides of interest that would have otherwise been suppressed by detection of non-modified peptides. Using this method, we identified &gt;25 cross-linked peptides; the most reported to date for these particles. To address the issue of molecular span, we used a stable isotopic-labeling approach in which wild-type human recombinant apoA-I ( 14 N) was mixed 1:1 with an isotopically labeled ( 15 N) apoA-I. Analysis of the isotopic patterns of the resulting mass spectra allowed the unambiguous determination of the molecular span of the cross-linked peptides; i.e. whether the links were within the same molecule of apoA-I (intramolecular) or between the dimers of apoA-I (intermolecular). This information will be used to derive a detailed novel model of lipid-bound apoA-I in reconstituted HDL, an important step in our overall understanding of how native HDL species are organized and how they interact with plasma remodeling factors and cell surface proteins.

Abstract 631: Lipid Composition of Gold Nanoparticle-Templated High-Density Lipoprotein Analogs Dictates Cholesterol Efflux Function

Background: Reverse cholesterol transport, the process by which cholesterol is effluxed from cells to high-density lipoproteins (HDL) and is delivered to the liver for clearance, is a promising pathway to augment for treatment of atherosclerosis. Though structure-function relationships for nascent, discoidal HDL and cholesterol efflux have been well studied, how the lipid composition of spherical HDL species - which varies in pathophysiological conditions - impacts their ability to mediate cholesterol efflux has not been investigated. Methods and Results: Spherical gold nanoparticles (5 nm) were used to synthesize spherical HDL analogs (HDL-NP) by adding ApoAI protein, and various lipids. With this strategy a panel of HDL-NP varying in lipid content was generated. HDL-NP designs tested include: dipalmitylphosphatidylcholine (DPPC, saturated fatty acid), dioleoylphosphatidylcholine (DOPC, unsaturated fatty acid), sphingomyelin, lysophosphatidylcholine (LPC), and mixtures thereof. All of these species are found in natural HDL. After characterizing protein and lipid stoichiometry of the purified HDL-NP, these HDL-NP designs were tested in the cellular reverse cholesterol transport assay using J774 mouse macrophages. These studies demonstrate that all HDL-NP designs mediate more efflux than equimolar amounts of ApoAI protein control, and further demonstrate that HDL-NP designs incorporating unsaturated phospholipid (DOPC), sphingomyelin, and LPC - each of which can increase disorder in the lipid membrane and thus give rise to opportunity for cholesterol to intercalate and bind - enhance cholesterol efflux compared to saturated phospholipid (DPPC) design. Conclusion: In summary, these results demonstrate that lipid content of HDL-NP - analogs of spherical HDL - dictates cholesterol efflux function, a finding which sheds light on the functional importance of lipid content variation seen in mature, spherical HDL species.

Abstract 18311: Lipid Composition of Gold Nanoparticle-Templated High-Density Lipoprotein Analogs Dictates Cholesterol Efflux Function

Background: Reverse cholesterol transport, the process by which cholesterol is effluxed from cells to high-density lipoproteins (HDL) and is delivered to the liver for clearance, is a promising pathway to augment for treatment of atherosclerosis. Though structure-function relationships between nascent, discoidal HDL and cholesterol efflux have been well studied, how the lipid composition of spherical HDL species, which varies in pathophysiological conditions, impacts their ability to mediate cholesterol efflux has not been investigated. Methods and Results: Spherical gold nanoparticles (5 nm) were used to synthesize spherical HDL analogs (HDL-NP) by adding ApoAI protein and various lipids to the particle surface. With this strategy a panel of HDL-NP varying in lipid content was generated. HDL-NP designs tested include: dipalmitylphosphatidylcholine (DPPC, saturated fatty acid), dioleoylphosphatidylcholine (DOPC, unsaturated fatty acid), sphingomyelin, lysophosphatidylcholine (LPC), and mixtures thereof. All of these lipids are found in natural HDL. After characterizing the purified HDL-NP, these HDL-NP designs were tested in the cellular reverse cholesterol transport assay using J774 mouse macrophages. These studies demonstrate that all HDL-NP designs mediate more efflux than equimolar amounts of ApoAI protein control, and further demonstrate that HDL-NP designs incorporating unsaturated phospholipid (DOPC), sphingomyelin, and LPC--each of which can increase disorder in the lipid membrane and thus give rise to opportunity for cholesterol to intercalate and bind--enhance cholesterol efflux compared to the saturated phospholipid (DPPC) design. Conclusion: In summary, these results demonstrate that lipid content of HDL-NP,analogs of spherical HDL, dictates cholesterol efflux function, a finding which sheds light on the functional importance of lipid content variation seen in mature, spherical HDL species.

Relation of Increased Prebeta-1 High-Density Lipoprotein Levels to Risk of Coronary Heart Disease

Preβ-1 high-density lipoprotein (HDL) plays a key role in reverse cholesterol transport by promoting cholesterol efflux. Our aims were (1) to test previous associations between preβ-1 HDL and coronary heart disease (CHD) and (2) to investigate whether preβ-1 HDL levels also are associated with risk of myocardial infarction (MI). Plasma preβ-1 HDL was measured by an ultrafiltration-isotope dilution technique in 1,255 subjects recruited from the University of California-San Francisco Lipid and Cardiovascular Clinics and collaborating cardiologists. Preβ-1 HDL was significantly and positively associated with CHD and MI even after adjustment for established risk factors. Inclusion of preβ-1 HDL in a multivariable model for CHD led to a modest improvement in reclassification of subjects (net reclassification index 0.15, p = 0.01; integrated discrimination improvement 0.003, p = 0.2). In contrast, incorporation of preβ-1 HDL into a risk model of MI alone significantly improved reclassification of subjects (net reclassification index 0.21, p = 0.008; integrated discrimination improvement 0.01, p = 0.02), suggesting that preβ-1 HDL has more discriminatory power for MI than for CHD in our study population. In conclusion, these results confirm previous associations between preβ-1 HDL and CHD in a large well-characterized clinical cohort. Also, this is the first study in which preβ-1 HDL was identified as a novel and independent predictor of MI above and beyond traditional CHD risk factors.

Nanotechnology in molecular medicine

Nanotechnology in molecular medicine

High-density lipoproteins: Marker of cardiovascular risk and therapeutic target

The high-density lipoproteins (HDLs) are complex, polymolecular assemblies produced by the jejunum, liver, in serum, and on the surface of macrophages. HDL cholesterol (HDL-C) levels are an independent predictor of risk for cardiovascular events in both men and women. High serum levels of this lipoprotein are associated with reduced risk for atherosclerosis and its clinical sequelae, such as myocardial infarction, ischemic stroke, and death. The molecular basis for the apparent vasculoprotection afforded by elevated HDL-C is widely attributed to the ability of HDL particles to drive reverse cholesterol transport. The proteosome (protein cargo) of HDL also appears to endow this lipoprotein with a capacity to reduce oxidized lipids, promote endothelial cell nitric oxide production, reduce adhesion molecule expression, inhibit platelet activation, stimulate endothelial proliferation and inhibit apoptosis, and reduce inflammatory mediator expression, among other functions. Recent investigation suggests that among patients with coronary artery disease, HDL can also be dysfunctional, yielding a pro-inflammatory and pro-oxidative phenotype. Numerous drugs are currently in development in an effort to devise the means by which to optimally increase serum levels of functional, antiatherogenic HDL species. These drugs exploit a diverse range of mechanisms with potential for beneficially impacting the development and progression of atherosclerotic disease.

The Effects of Cholesterol Ester Transfer Protein Inhibition on Cholesterol Efflux

Cholesterol ester transfer protein (CETP) deficiency or inhibition results in dramatic elevations of high-density lipoprotein (HDL) levels, but there has been concern that HDL might be dysfunctional in its ability to promote efflux of cholesterol from macrophage foam cells or to mediate reverse cholesterol transport. Using cholesterol-loaded cultured macrophages, HDL that was isolated from subjects with homozygous CETP deficiency or who had been treated with high levels of CETP inhibitor (120 mg torcetrapib) had an increased cholesterol efflux potential when matched for unit mass of HDL in media. This correlated with the accumulation of HDL(2) species enriched in apolipoprotein E and lecithin-cholesterol acyltransferase. At lower levels of inhibition (60 mg torcetrapib), HDL had a similar ability to promote cholesterol efflux as pretreatment HDL but showed increased cholesterol efflux in parallel with the increase in plasma HDL concentration. Cholesterol efflux measurements appear to correlate with the finding that subjects who attained the highest levels of HDL on torcetrapib showed regression of coronary atheroma as determined by intravascular ultrasound. Although these in vitro measurements may not fully capture the in vivo complexities of HDL metabolism, they suggest that increased HDL attributable to CETP inhibition results in particles that have normal or enhanced ability to promote cholesterol efflux from macrophage foam cells.