High Density Lipoprotein Remodeling Research Articles

Differences in HDL Remodeling during Healthy Pregnancy and Pregnancy with Cardiometabolic Complications.

This study investigated the longitudinal trajectory of changes in antioxidative and anti-inflammatory high-density lipoprotein (HDL) components during healthy pregnancy and pregnancy with cardiometabolic complications. We recruited and longitudinally followed 84 women with healthy pregnancies and 46 pregnant women who developed cardiometabolic pregnancy complications (gestational diabetes mellitus and hypertensive disorders of pregnancy). Their general lipid profiles, oxidative stress status, inflammatory status, and antioxidative and anti-inflammatory HDL components were analyzed. The results of our study confirmed the expected trajectory for the routine lipid parameters. Our study results indicate more intensive oxidative stress and a higher level of inflammation in the group with complications compared with the control group. Sphingosine-1-phosphate (S1P) was significantly lower in the first trimester in the group with complications compared with the control group (p < 0.05). We did not find significant differences in the apolipoprotein A1 (Apo A1) concentrations in the first trimester between the control group and the group with complications, but in the second and third trimesters, the group with complications had significantly higher concentrations (p < 0.001, p < 0.05, respectively). The S1P, paraoxonase 1 (PON1), and serum amyloid A (SAA) concentrations were significantly lower in the group with complications in the first trimester. During the second trimester, only the SAA concentrations were identified as significantly lower in the group with complications compared with the control group, while in the third trimester, the PON1, apolipoprotein M (Apo M), and SAA concentrations were all significantly lower in the group with complications. Through a multivariate binary logistic regression analysis, the S1P concentration in the first trimester was distinguished as an HDL-associated marker independently associated with cardiometabolic pregnancy complications. In conclusion, our study results showed that HDL remodeling differs between healthy pregnancies and pregnancies with maternal cardiometabolic complications, with changed HDL composition and functionality consequently impacting its biological functionality in the latter case.

Abstract 3040: Quantitative Proteomic Analysis Of HDL Shows Apolipoprotein M (ApoM) As A Possible Marker For COVID-19 Mortality

Introduction: High-density lipoprotein (HDL) comprises a heterogeneous group of particles that differ in many aspects, such as density, size, composition and function. The use of mass spectrometry-based proteomics to investigate HDL protein composition has brought some new insights into the field. The qualitative and quantitative HDL proteome is remodeled in different disease states, such as diabetes mellitus, hypercholesterolemia and kidney dysfunction. Beyond its established role in inflammatory and cardiovascular diseases, HDL's involvement in infectious diseases, such as COVID-19, has recently come to light. Here, we investigate the remodeling of HDL proteome in hospitalized and non-hospitalized COVID-19 patients. Hypothesis: We hypothesize HDL proteome is remodeled in infectious diseases and may be a marker and perhaps a predictor of a poor prognosis. Methods: We implemented a robust, global label-free quantification proteomic approach to unravel relative changes in HDL protein composition. We analyzed the proteome of COVID-19 subjects from Brazil categorized in two groups, hospitalized (n=30) and non-hospitalized (n=11). Results: Comparative analysis between hospitalized and non-hospitalized patients revealed a more than 50% increase in relative levels of certain proteins in hospitalized patients, including the inflammatory proteins serum amyloid A1 and A2, as well as pulmonary surfactant-associated protein B. We also observed reductions in the lipid-metabolism related apolipoprotein A2 and phospholipid transfer protein. These findings, independent of sex, triglyceride and HDL-cholesterol, suggest an inflammatory remodeling of the HDL particles. Further analysis of surviving (n=35) and deceased patients (n=6) indicated a negative association between ApoM levels and odds of death due to COVID-19 complications (odds ratio per 1-SD increase in ApoM was 0.27 [0.07 to 0.72], P=0.007). Conclusions: Our results point out HDL remodeling is a marker of COVID-19 severity and ApoM-carried sphingosine-1-phosphate signaling pathway may play a pivotal role in the disease outcome.

High-density lipoprotein remodeling affects the osmotic properties of plasma in goldfish under critical salinity.

To investigate the stress response and physiological adaptations of goldfish (Carassius auratus) to critical salinity (CS) waters, we analyzed high-density lipoprotein (HDL) stoichiometry, stress markers (cortisol, glucose), and plasma osmotic properties (Na+ , osmolality, water content) using ichthyology, biochemistry, and proteomics approaches. After 21 days of exposure to CS, plasma concentrations of cortisol, glucose, and Na+ increased, indicating stress. Total plasma osmolality (Osmtotal ) and osmolality generated by inorganic (Osminorg ) and organic osmolytes (Osmorg ) also increased, the latter by ~2%. We associated the increase of Osmorg with (1) increased metabolite concentration (glucose), (2) dissociation of HDL particles resulting in increased HDL number per unit plasma volume (~1.5-2-fold) and (3) increased HDL osmotic activity. HDL remodeling may be the reason for the redistribution of bound and free water in plasma, which may contribute to water retention in plasma and, at the same time, to hemodynamic disturbances under CS conditions. The study's findings suggest that HDL remodeling is an important mechanism for maintaining osmotic homeostasis in fish, which is consistent with current capillary exchange models in vertebrates.

A Current Update on the Role of HDL-Based Nanomedicine in Targeting Macrophages in Cardiovascular Disease.

High-density lipoproteins (HDL) are complex endogenous nanoparticles involved in important functions such as reverse cholesterol transport and immunomodulatory activities, ensuring metabolic homeostasis and vascular health. The ability of HDL to interact with a plethora of immune cells and structural cells places it in the center of numerous disease pathophysiologies. However, inflammatory dysregulation can lead to pathogenic remodeling and post-translational modification of HDL, rendering HDL dysfunctional or even pro-inflammatory. Monocytes and macrophages play a critical role in mediating vascular inflammation, such as in coronary artery disease (CAD). The fact that HDL nanoparticles have potent anti-inflammatory effects on mononuclear phagocytes has opened new avenues for the development of nanotherapeutics to restore vascular integrity. HDL infusion therapies are being developed to improve the physiological functions of HDL and to quantitatively restore or increase the native HDL pool. The components and design of HDL-based nanoparticles have evolved significantly since their initial introduction with highly anticipated results in an ongoing phase III clinical trial in subjects with acute coronary syndrome. The understanding of mechanisms involved in HDL-based synthetic nanotherapeutics is critical to their design, therapeutic potential and effectiveness. In this review, we provide a current update on HDL-ApoA-I mimetic nanotherapeutics, highlighting the scope of treating vascular diseases by targeting monocytes and macrophages.

Plasma Lecithin-Cholesterol Acyltransferase Activity in Pregnant Women Enrolled in the iLiNS-DYAD Trial in Ghana

ObjectivesThe International Lipid-Based Nutrient Supplement (iLiNS) Project developed small-quantity lipid-based nutrient supplements (SQ-LNS) for mothers and their children with the goal of reducing malnutrition. The aim of this secondary outcome analysis is to investigate the effects of SQ-LNS on the activity of plasma lecithin-cholesterol acyltransferase (LCAT), a key enzyme involved in the remodeling of high-density lipoprotein (HDL) particles, which increases their cholesterol carrying capacity.MethodsPlasma samples from a subset of 197 out of 1320 women at 36-wk gestation enrolled in the iLiNS-DYAD trial in Ghana were analyzed. In the main cohort, women were enrolled during the second trimester (≤ 20 wk gestation) and randomly assigned to receive SQ-LNS, iron and folic acid (IFA), or a multiple micronutrient supplement. Women in this subset received either SQ-LNS or IFA. Plasma LCAT activity was measured using a commercial fluorometric kit and the non-transformed data was compared by Wilcoxon test (mean ± SD). LCAT activity is expressed as a ratio (390 nm/470 nm), where greater ratio indicates higher enzyme activity. We also explored whether LCAT activity differed by enrollment season (Dry, November–April; Wet, May-October) or was associated with biomarkers of inflammation.ResultsThere was no significant difference in LCAT activity between the SQ-LNS and IFA groups (1.44 ± 0.17 vs. 1.41 ± 0.14, P = 0.24). Women enrolled during the wet season had higher LCAT activity than women enrolled during the dry season (1.47 ± 0.15 vs. 1.37 ± 0.14, P < 0.001), with no differences between intervention groups stratified by season (P > 0.05). LCAT activity was negatively correlated with inflammatory biomarkers α1-acid glycoprotein (R = −0.19, P = 0.007) and C-reactive protein (R = −0.28, P < 0.001) measured at 36-wk gestation.ConclusionsPlasma LCAT activity, as a measure of HDL maturation and cholesterol carrying capacity, is more sensitive to factors mediated by seasonal variation (e.g., infection, inflammation) than by nutrient supplementation in this cohort of women in Ghana. These findings confirm a previously established link between HDL and inflammation.Funding SourcesThis project was supported by Bill & Melinda Gates Foundation grant (OPP124589) to the University of California, Davis.

High-density lipoprotein remodeling by phospholipid nanoparticles improves cholesterol efflux capacity and protects from atherosclerosis

The efficiency of cholesterol efflux from cells promoted by high-density lipoproteins (HDLs) depends on HDL concentration and functional properties. The term “dysfunctional HDL” describes HDLs with impaired protective properties. Cholesterol efflux capacity (CEC) of HDL is reduced in patients with atherosclerosis, but the exact mechanisms underlying this impairment are not well characterized. Enriching HDLs with phospholipids (PLs) improves CEC. Herein, we assessed the potential of PL nanoparticles in improving HDL functionality. We lipidated HDL subfractions by incubating with PL nanoparticles containing soybean polyunsaturated phosphatidylcholine. Incubating blood plasma with PL nanoparticles resulted in the dose-dependent lipidation of all HDL subfractions. Changes in apolipoprotein A1 (apoA-1) and PL concentrations were the most prominent in the HDL2 fraction. Concentrations of PL in the HDL3 fraction and the fraction with a density > 1.21 g/mL increased by 30–50%, whereas apoA-1 levels decreased. We hypothesized that PL nanoparticles may cause HDL remodeling that can improve their functions. The CECs of lipidated HDLs were analyzed by incubating apolipoprotein B (apoB)-depleted plasma with 3H-cholesterol-labeled THP-1 macrophages. The findings revealed a two-fold increase in cholesterol efflux compared with native apoB-depleted plasma. Moreover, intravenous administration of PL nanoparticles restored lipid profiles and effectively protected blood vessels from atherosclerosis progression in cholesterol-fed rabbits compared with that of fenofibrate and atorvastatin. PL nanoparticles also protected against atherosclerosis and decreased the atherogenic index. Altogether, these results indicate that PL nanoparticles can be used to correct the lipid composition and CEC of HDLs. Data AvailabilityAdditional data can be provided upon reasonable request from the date of publication of this article within 5 years. The request should be sent to the author-correspondent at the address cd95@mail.ru.

High-sensitivity CRP may be a marker of HDL dysfunction and remodeling in patients with acute coronary syndrome

In patients with coronary artery disease (CAD), further increasing the level of high-density lipoprotein (HDL) cholesterol (HDL-C) as an add-on to statins cannot reduce cardiovascular risk. And it has been reported that HDL functional metric—cholesterol efflux capacity (CEC) may be a better predictor of CAD risk than HDL-C. CEC measurement is time-consuming and not applicable in clinical settings. Thus, it is meaningful to explore an easily acquired index for evaluating CEC. Thirty-six CAD patients and sixty-one non-CAD controls were enrolled in this cross-sectional study. All CAD patients had acute coronary syndrome (ACS). CEC was measured using a [3H] cholesterol loading Raw 264.7 cell model with apolipoprotein B-depleted plasma (a surrogate for HDL). Proton nuclear magnetic resonance (NMR) spectroscopy was used to assess HDL components and subclass distribution. CEC was significantly impaired in CAD patients (11.9 ± 2.3%) compared to controls (13.0 ± 2.2%, p = 0.022). In control group, CEC was positively correlated with enzymatically measured HDL-C levels (r = 0.358, p = 0.006) or with NMR-determined HDL-C levels (NMR-HDL-C, r = 0.416, p = 0.001). However, in CAD group, there was no significant correlation between CEC and HDL-C (r = 0.216, p = 0.206) or NMR-HDL-C (r = 0.065, p = 0.708). Instead, we found that the level of high-sensitivity C-reactive protein (hsCRP) was inversely associated with CEC (r = − 0.351, p = 0.036). Multiple regression analysis showed that the hsCRP level was associated with CEC after adjusting other cardiovascular risk factors and HDL-C, although the association would not reach significance if adjusting for multiple testing. NMR spectroscopy showed that HDL particles shifted to larger ones in patients with high hsCRP levels, and this phenomenon was accompanied by decreased CEC. In patients with CAD, the level of HDL-C cannot reflect HDL function. The impaired correlation between HDL-C and CEC is possibly due to an inflammation-induced HDL subclass remodeling. These hypothesis-generating data suggest that hsCRP levels, a marker of acute inflammation, may associate with HDL dysfunction in ACS subjects. Due to the design limited to be correlative in nature, not permitting causal inference and a larger, strictly designed study is still needed.

The HDL from septic-ARDS patients with composition changes exacerbates pulmonary endothelial dysfunction and acute lung injury induced by cecal ligation and puncture (CLP) in mice

BackgroundSeptic-acute respiratory distress syndrome (ARDS), characterized by the acute lung injury (ALI) secondary to aberrant systemic inflammatory response, has high morbidity and mortality. Despite increased understanding of ALI pathogenesis, the therapies to prevent lung dysfunction underlying systemic inflammatory disorder remain elusive. The high density lipoprotein (HDL) has critical protective effects in sepsis and its dysfunction has a manifested contribution to septic organ failure. However, the adverse changes in HDL composition and function in septic-ARDS patients are large unknown.MethodsTo investigate HDL remodeling in septic-ARDS, we analyzed the changes of HDL composition from 40 patients with septic-ARDS (A-HDL) and 40 matched normal controls (N-HDL). To determine the deleterious functional remodeling of HDL, A-HDL or N-HDL was administrated to C57BL/6 and apoA-I knock-out (KO) mice after cecal ligation and puncture (CLP) procedure. Mouse lung microvascular endothelial cells (MLECs) were further treated by these HDLs to investigate whether the adverse effects of A-HDL were associated with endothelial dysfunction.ResultsSeptic-ARDS patients showed significant changes of HDL composition, accompanied with significantly decreased HDL-C. We further indicated that A-HDL treatment aggravated CLP induced ALI. Intriguingly, these deleterious effects of A-HDL were associated with pulmonary endothelial dysfunction, rather than the increased plasma lipopolysaccharide (LPS). Further in vitro results demonstrated the direct effects of A-HDL on MLECs, including increased endothelial permeability, enhanced expressions of adhesion proteins and pro-inflammatory cytokines via activating NF-κB signaling and decreased junction protein expression.ConclusionsOur results depicted the remodeling of HDL composition in sepsis, which predisposes lung to ARDS via inducing ECs dysfunction. These results also demonstrated the importance of circulating HDL in regulating alveolar homeostasis.

Early Pro-Inflammatory Remodeling of HDL Proteome in a Model of Diet-Induced Obesity: 2H2O-Metabolic Labeling-Based Kinetic Approach.

Mice fed a high-fat diet for 12 weeks or longer develop hyperglycemia, insulin resistance, dyslipidemia, and fatty liver. Additionally, a high-fat diet induces inflammation that remodels and affects the anti-inflammatory and antiatherogenic property of the high-density lipoprotein (HDL). However, the precise time course of metabolic disease progression and HDL remodeling remains unclear. Short-term (four weeks) high-fat feeding (60% fat calories) was performed in wild-type male C57BL/6J mice to gain insights into the early metabolic disease processes in conjunction with a HDL proteome dynamics analysis using a heavy water metabolic labeling approach. The high-fat diet-fed mice developed hyperglycemia, impaired glucose tolerance, hypercholesterolemia without hypertriglyceridemia or hepatic steatosis. A plasma HDL proteome dynamics analysis revealed increased turnover rates (and reduced half-lives) of several acute-phase response proteins involved in innate immunity, including complement C3 (12.77 ± 0.81 vs. 9.98 ± 1.20 h, p < 0.005), complement factor B (12.71 ± 1.01 vs. 10.85 ± 1.04 h, p < 0.05), complement Factor H (19.60 ± 1.84 vs. 16.80 ± 1.58 h, p < 0.05), and complement factor I (25.25 ± 1.29 vs. 19.88 ± 1.50 h, p < 0.005). Our findings suggest that an early immune response-induced inflammatory remodeling of the plasma HDL proteome precedes the diet-induced steatosis and dyslipidemia.

HDL (High-Density Lipoprotein) Remodeling and Magnetic Resonance Imaging-Assessed Atherosclerotic Plaque Burden: Study in a Preclinical Experimental Model.

HDL (high-density lipoprotein) role in atherosclerosis is controversial. Clinical trials with CETP (cholesterylester transfer protein)-inhibitors have not provided benefit. We have shown that HDL remodeling in hypercholesterolemia reduces HDL cardioprotective potential. We aimed to assess whether hypercholesterolemia affects HDL-induced atherosclerotic plaque regression. Approach and Results: Atherosclerosis was induced in New Zealand White rabbits for 3-months by combining a high-fat-diet and double-balloon aortic denudation. Then, animals underwent magnetic resonance imaging (basal plaque) and randomized to receive 4 IV infusions (1 infusion/wk) of HDL isolated from normocholesterolemic (NC-HDL; 75 mg/kg; n=10), hypercholesterolemic (HC-HDL; 75 mg/Kg; n=10), or vehicle (n=10) rabbits. Then, animals underwent a second magnetic resonance imaging (end plaque). Blood, aorta, and liver samples were obtained for analyses. Follow-up magnetic resonance imaging revealed that NC-HDL administration regressed atherosclerotic lesions by 4.3%, whereas, conversely, the administration of HC-HDLs induced a further 6.5% progression (P<0.05 versus basal). Plaque characterization showed that HC-HDL administered animals had a 2-fold higher lipid and cholesterol content versus those infused NC-HDL and vehicle (P<0.05). No differences were observed among groups in CD31 levels, nor in infiltrated macrophages or smooth muscle cells. Plaques from HC-HDL administered animals exhibited higher Casp3 (caspase 3) content (P<0.05 versus vehicle and NC-HDL) whereas plaques from NC-HDL infused animals showed lower expression of Casp3, Cox1 (cyclooxygenase 1), inducible nitric oxide synthase, and MMP (metalloproteinase) activity (P<0.05 versus HC-HDL and vehicle). HDLs isolated from animals administered HC-HDL displayed lower antioxidant potential and cholesterol efflux capacity as compared with HDLs isolated from NC-HDL-infused animal and vehicle or donor HDL (P<0.05). There were no differences in HDL-ApoA1 content, ABCA1 (ATP-binding cassette transporter A1) vascular expression, and SRB1 (scavenger receptor B1) and ABCA1 liver expression. HDL particles isolated from a hypercholesterolemic milieu lose their ability to regress and stabilize atherosclerotic lesions. Our data suggest that HDL remodeling in patients with co-morbidities may lead to the loss of HDL atheroprotective functions.

Advances in HDL: Much More than Lipid Transporters.

High Density Lipoprotein (HDL) particles, beyond serving as lipid transporters and playing a key role in reverse cholesterol transport, carry a highly variable number of proteins, micro-RNAs, vitamins, and hormones, which endow them with the ability to mediate a plethora of cellular and molecular mechanisms that promote cardiovascular health. It is becoming increasingly evident, however, that the presence of cardiovascular risk factors and co-morbidities alters HDLs cargo and protective functions. This concept has led to the notion that metrics other than HDL-cholesterol levels, such as HDL functionality and composition, may better capture HDL cardiovascular protection. On the other hand, the potential of HDL as natural delivery carriers has also fostered the design of engineered HDL-mimetics aiming to improve HDL efficacy or as drug-delivery agents with therapeutic potential. In this paper, we first provide an overview of the molecules known to be transported by HDL particles and mainly discuss their functions in the cardiovascular system. Second, we describe the impact of cardiovascular risk factors and co-morbidities on HDL remodeling. Finally, we review the currently developed HDL-based approaches.

HDL flux is higher in patients with nonalcoholic fatty liver disease.

Altered lipid metabolism and inflammation are involved in the pathogenesis of both nonalcoholic fatty liver disease (NAFLD) and cardiovascular disease (CVD). Even though high-density lipoprotein (HDL), a CVD protective marker, is decreased, whether HDL metabolism and function are perturbed in NAFLD are currently unknown. We examined the effect of NAFLD and disease severity on HDL metabolism and function in patients with biopsy-proven simple steatosis (SS), nonalcoholic steatohepatitis (NASH), and healthy controls. HDL turnover and HDL protein dynamics in SS (n = 7), NASH (n = 8), and healthy controls (n = 9) were studied in vivo. HDL maturation and remodeling, antioxidant, cholesterol efflux properties, and activities of lecithin-cholesterol ester acyltransferase and cholesterol ester transfer protein (CETP) were quantified using in vitro assays. All patients with NAFLD had increased turnover of both HDL cholesterol (HDLc; 0.16 ± 0.09 vs. 0.34 ± 0.18 days, P < 0.05) and apolipoprotein A1 (ApoAI) (0.26 ± 0.04 vs. 0.34 ± 0.06 days, P < 0.005) compared with healthy controls. The fractional catabolic rates of other HDL proteins, including ApoAII (and ApoAIV) were higher (P < 0.05) in patients with NAFLD who also had higher CETP activity, ApoAI/HDLc ratio (P < 0.05). NAFLD-induced alterations were associated with lower antioxidant (114.2 ± 46.6 vs. 220.5 ± 48.2 nmol·mL-1·min-1) but higher total efflux properties of HDL (23.4 ± 1.3% vs. 25.5 ± 2.3%) (both P < 0.05), which was more pronounced in individuals with NASH. However, no differences were observed in either HDL turnover, antioxidant, and cholesterol efflux functions of HDL or HDL proteins' turnover between subjects with SS and subjects with NASH. Thus, HDL metabolism and function are altered in NAFLD without any significant differences between SS and NASH.

Attenuation of atherogenic apo B-48-dependent hyperlipidemia and high density lipoprotein remodeling induced by vitamin C and E combination and their beneficial effect on lethal ischemic heart disease in mice

Background and aimsAtherosclerotic cardiovascular disease is highly prevalent and its underlying pathogenesis involves dyslipidemia including pro-atherogenic high density lipoprotein (HDL) remodeling. Vitamins C and E have been proposed as atheroprotective agents for cardiovascular disease management. However, their effects and benefits on high density lipoprotein function and remodeling are unknown. In this study, we evaluated the role of vitamin C and E on non HDL lipoproteins as well as HDL function and remodeling, along with their effects on inflammation/oxidation biomarkers and atherosclerosis in atherogenic diet-fed SR-B1 KO/ApoER61h/h mice.Methods and resultsMice were pre-treated for 5 weeks before and during atherogenic diet feeding with vitamin C and E added to water and diet, respectively. Compared to a control group, combined vitamin C and E administration reduced serum total cholesterol and triglyceride levels by decreasing apo B-48-containing lipoproteins, remodeled HDL particles by reducing phospholipid as well as increasing PON1 and apo D content, and diminished PLTP activity and levels. Vitamin supplementation improved HDL antioxidant function and lowered serum TNF-α levels. Vitamin C and E combination attenuated atherogenesis and increased lifespan in atherogenic diet-fed SR-B1 KO/ApoER61h/h mice.ConclusionsVitamin C and E administration showed significant lipid metabolism regulating effects, including HDL remodeling and decreased levels of apoB-containing lipoproteins, in mice. In addition, this vitamin supplementation generated a cardioprotective effect in a murine model of severe and lethal atherosclerotic ischemic heart disease.

Abstract 026: Apolipoprotein A-I Helical Registry Modulates Lecithin:cholesterol Acyl Transferase Activity

The structure of apolipoprotein (APOA1), the major protein of high density lipoproteins (HDL), can modulate protein interactions affecting the cardioprotective functions of HDL particles. APOA1 on nascent discoidal HDL is composed of ten, tandem, 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 may adopt two different orientations, or registries, with respect to each other. These orientations include one where the bulk of APOA1 orients with the 5 th helix of one molecule opposing the 5 th helix of the other (5/5 helical registry), and another orientation adopting a 5/2 helical registry. Engineered HDL particles locked in both 5/5 and 5/2 registries, or rotamers, equally promoted cholesterol efflux from macrophages, indicating functional particles. However, the ability of lecithin:cholesterol acyl transferase (LCAT) to esterify cholesterol was increased over WT in the 5/5 rotamer, and dramatically impaired in the 5/2 rotamer ( p&lt;0.001 ), despite surface plasmon resonance studies showing that LCAT bound equally to both. Cross-linking experiments indicated that LCAT activity requires a hybrid epitope composed of helices 5-7 on one APOA1 molecule and 3-4 on the other. Thus, the reciprocating nature of APOA1’s structure may represent a thumbwheel mechanism that influences the activity of HDL remodeling factors in plasma.

Detrimental Effect of Hypercholesterolemia on High-Density Lipoprotein Particle Remodeling in Pigs

BackgroundBeneficial effects of high-density lipoproteins (HDL) seem altered in patients with symptomatic cardiovascular disease. We recently demonstrated in a swine model of ischemia-reperfusion (IR) that hypercholesterolemia abolishes HDL-related cardioprotection. ObjectivesThis study sought to investigate, using the same animal model, whether the reported impairment of HDL cardioprotective function was associated with alterations in HDL remodeling and functionality. MethodsPigs were fed a normocholesterolemic (NC) or hypercholesterolemic (HL) diet for 10 days, reaching non-HDL cholesterol concentrations of 38.2 ± 3.5 mg/dl and 218.6 ± 27.6 mg/dl, respectively (p < 0.0001). HDLs were isolated, and lipidomics and differential proteomics tests were performed to determine HDL molecular changes. HDL functionality and particle size were determined. ResultsUsing principal component analysis, we identified 255 molecular lipid species differentially clustered in NC-HDL and HL-HDL. Ninety lipid metabolites were differentially expressed, and 50 showed at least 1.5-fold variation (false discovery rate adjustment q value <0.05). HL-HDLs presented a core enriched in cholesteryl esters and a surface depleted of phosphatidylcholine species containing polyunsaturated and long-chain fatty acids, indicating the presence of mature HDL particles with low surface fluidity. Hypercholesterolemia induced an important change in HDL-transported proteins (576 spots in HL-HDL vs. 621 spots in NC-HDL). HL-HDLs showed a reduced content of lipocalin retinol binding protein 4 and apolipoprotein M and in the retinoic acid-transporter cellular retinoic acid binding protein 1 (p < 0.05 vs. NC-HDL). No changes were observed in apolipoprotein A-I content and profile. Functionally, HL-HDL showed lower antioxidant activity (−35%) and a reduced capacity to efflux cholesterol (−60%) compared to NC-HDL (p < 0.05). Hypercholesterolemia induced larger HDL particles. ConclusionsWe demonstrate that hypercholesterolemia induces HDL lipidomic changes, losing phosphatidylcholine-lipid species and gaining cholesteryl esters, and proteomic changes, with losses in cardioprotective proteins. These remodeling changes shifted HDL particles toward a dysfunctional state.

Self-Assembling Cyclic d,l-α-Peptides as Modulators of Plasma HDL Function. A Supramolecular Approach toward Antiatherosclerotic Agents.

There is great interest in developing new modes of therapy for atherosclerosis to treat coronary heart disease and stroke, particularly ones that involve modulation of high-density lipoproteins (HDLs). Here, we describe a new supramolecular chemotype for altering HDL morphology and function. Guided by rational design and SAR-driven peptide sequence enumerations, we have synthesized and determined the HDL remodeling activities of over 80 cyclic d,l-α-peptides. We have identified a few distinct sequence motifs that are effective in vitro in remodeling human and mouse plasma HDLs to increase the concentration of lipid-poor pre-beta HDLs, which are key initial acceptors of cholesterol in the reverse cholesterol transport (RCT) process, and concomitantly promote cholesterol efflux from macrophage cells. Functional assays with various control peptides, such as scrambled sequences, linear and enantiomeric cyclic peptide variants, and backbone-modified structures that limit peptide self-assembly, provide strong support for the supramolecular mode of action. Importantly, when the lead cyclic peptide c[wLwReQeR] was administered to mice (ip), it also promoted the formation of small, lipid-poor HDLs in vivo, displayed good plasma half-life (∼6 h), did not appear to have adverse side effects, and exerted potent anti-inflammatory effects in an acute in vivo inflammation assay. Given that previously reported HDL remodeling peptides have been based on α-helical apoA-I mimetic architectures, the present study, involving a new structural class, represents a promising step toward new potential therapeutics to combat atherosclerosis.

Immunochemical Approach for Monitoring of Structural Transition of ApoA-I upon HDL Formation Using Novel Monoclonal Antibodies

Apolipoprotein A-I (apoA-I) undergoes a large conformational reorganization during remodeling of high-density lipoprotein (HDL) particles. To detect structural transition of apoA-I upon HDL formation, we developed novel monoclonal antibodies (mAbs). Splenocytes from BALB/c mice immunized with a recombinant human apoA-I, with or without conjugation with keyhole limpet hemocyanin, were fused with P3/NS1/1-Ag4-1 myeloma cells. After the HAT-selection and cloning, we established nine hybridoma clones secreting anti-apoA-I mAbs in which four mAbs recognize epitopes on the N-terminal half of apoA-I while the other five mAbs recognize the central region. ELISA and bio-layer interferometry measurements demonstrated that mAbs whose epitopes are within residues 1–43 or 44–65 obviously discriminate discoidal and spherical reconstituted HDL particles despite their great reactivities to lipid-free apoA-I and plasma HDL, suggesting the possibility of these mAbs to detect structural transition of apoA-I on HDL. Importantly, a helix-disrupting mutation of W50R into residues 44–65 restored the immunoreactivity of mAbs whose epitope being within residues 44–65 against reconstituted HDL particles, indicating that these mAbs specifically recognize the epitope region in a random coil state. These results encourage us to develop mAbs targeting epitopes in the N-terminal residues of apoA-I as useful probes for monitoring formation and remodeling of HDL particles.

Abstract 387: HDL-apolipoprotein A-I Exchange Occurs in the Absence of HDL Particle Remodeling

Objective: Apolipoprotein A-I (apoA-I) spontaneously exchanges between high-density lipoprotein (HDL)-bound and lipid-free states. This is a key event in reverse cholesterol transport, necessary for de novo HDL biogenesis. However, the mechanism of HDL-apoA-I exchange (HAE) is poorly understood. In this study, we test whether HDL remodeling is obligatory to HAE or an independent process. Method and Results: Recombinant human apoA-I modified with single cysteine mutations were expressed in bacteria and labeled with Alexa 488 and Alexa 647 fluorophores. ApoA-I Alexa647 was used to synthesize rHDL particles of defined sizes with phosphatidyl choline (POPC) and cholesterol. Reconstituted HDL of 17.0, 12.2, 9.6, 8.4 and 7.8 nm were obtained. These particles were incubated with excess lipid-free apoA-I Alexa488 to initiate HAE, which was quantified using non-denaturing gradient gel electrophoresis to separate HDL particles from lipid-free apoA-I. Gels were imaged using FITC and Cy5 filters. The extent of apoA-I Alexa488 binding and apoA-I Alexa647 released from HDL was determined by densitometry. HAE occurred without significant changes in rHDL particle size for all subclasses of rHDL. HAE in the forward (lipid-free to bound) direction did not exhibit size dependence, but HAE in the reverse (lipid-bound to free) direction appeared to be size-dependent, with the 8.4 nm rHDL displaying 2-fold higher apoA-I Alexa647 dissociation compared to the 17.0 nm particle. Conclusions: The bidirectional rate of HAE can be measured simultaneously using fluorescent apoA-I probes. Reconstituted HDL particles predominantly retained their size, indicating that apoA-I exchange occurs without remodeling of HDL particles. This result has significant implications as to the possible molecular processes driving apoA-I exchange with HDL.

Abstract 13218: Remodeling of High Density Lipoprotein Induced by CSL112: Structure and Function of the Remodeled Lipoproteins

Introduction: CSL112 is a novel formulation of human apolipoprotein A-I (apoA-I) suitable for intravenous infusion, currently in development for reducing the high rate of early recurrent events after acute myocardial infarction. Infusion of CSL112 into healthy volunteers causes HDL remodeling, with an immediate rise in lipid-poor apoA-I and a strong and immediate increase in the ABCA1-dependent cholesterol efflux capacity of plasma. Interaction of CSL112 with whole plasma or purified HDL gives rise to three new remodeled HDL species: large (L-HDL rem ) and small (S-HDL rem ) species, and lipid-poor apoA-I. Here we characterized the composition and morphology of remodeled HDL species and compared their ability to efflux cholesterol via different transporters. Methods and Results: CSL112 and HDL3, fluorescently labeled on protein or lipid, were incubated together for varying periods of time at 37°C. Resultant HDL particle size distribution was assessed by native gel electrophoresis followed by fluorescence imaging. Morphology of the individual purified products of HDL remodeling was examined by negative stain electron microscopy. We show that L-HDL rem is a large, spherical species composed of apoA-I and phospholipid from both native HDL and CSL112. S-HDL rem is a small, disc-shaped species composed of apoA-I from CSL112 that is smaller due to the loss of phospholipids. The smallest species, lipid-poor apoA-I, is composed of apoA-I from HDL and CSL112. Lipid-poor apoA-I and S-HDL rem were found to have the greatest ability to efflux cholesterol via ABCA1. While S-HDL rem and L-HDL rem showed comparable ability to efflux cholesterol via ABCG1, L-HDL rem mediated additional efflux via SR-B1 and by an aqueous diffusion process. Conclusion: The generation of these remodeled HDL species can account for the strong and immediate increase in cholesterol efflux capacity observed upon infusion of CSL112, and make CSL112 a promising approach for rapidly removing cholesterol from atherosclerotic plaque.

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.