Major Apolipoprotein Research Articles

Apolipoproteins in fish: From lipid transport to innate immunity

The fish has a strong innate immune system, and antimicrobial peptides play a major role in fish innate immunity, providing potential defence against broad spectrum of fish pathogens. Apolipoproteins,that are abundant proteins of plasma, playing important role in lipid transport and metabolism, also have potential antimicrobial activity. The resent review describes the classes, structural details and important biological functions of apolipoproteins reported in both mammals and fish with an emphasis on their roles in host defence. The role of fish apolipoprotein A-I, a major component of high-density lipoproteins (HDL), is described in great detail using different infection models along with its bactericidal and immunomodulatory activities in various fish species against wide range of fish pathogens.Further, role of some novel fish-specific apolipoproteins, including the mammalian ones, have also been defined with a special focus on the molecules described in Indian carp species. As the understanding on major apolipoproteins is limited in fish species, this review might serve as a foundation to explore further their functional diversity in Indian fish species.

In silico analyses of deleterious missense SNPs of human apolipoprotein E3

ApoE3 is the major chylomicron apolipoprotein, binding in a specific liver peripheral cell receptor, allowing transport and normal catabolism of triglyceride-rich lipoprotein constituents. Point mutations in ApoE3 have been associated with Alzheimer’s disease, type III hyperlipoproteinemia, atherosclerosis, telomere shortening and impaired cognitive function. Here, we evaluate the impact of missense SNPs in APOE retrieved from dbSNP through 16 computational prediction tools, and further evaluate the structural impact of convergent deleterious changes using 100 ns molecular dynamics simulations. We have found structural changes in four analyzed variants (Pro102Arg, Arg132Ser, Arg176Cys and Trp294Cys), two of them (Pro102Arg and Arg176Cys) being previously associated with human diseases. In all cases, except for Trp294Cys, there was a loss in the number of hydrogen bonds between CT and NT domains that could result in their detachment. In conclusion, data presented here could increase the knowledge of ApoE3 activity and be a starting point for the study of the impact of variations on APOE gene.

Abstract 189: Improved Triglyceride-Rich Lipoprotein Clearance In Human Apolipoprotein A-Ii Knockin Rabbits

Apolipoprotein A-II (apoA-II) is a second major apolipoprotein in HDL particles, which account for 15-20 % of total HDL protein in human. Compared with apolipoprotein A-I (apoA-I) which is a most major apolipoproteins in HDL, physiological role of apoA-II is not fully understood. We previously reported that overexpression of apoA-II in rabbits significantly suppressed cholesterol-rich diet induced atherosclerosis. Because rabbits are the animal of naturally deficient in apoA-II, so it becomes a good animal model to study the functions of apoA-II. To elucidate the role of apoA-II independent from apoA-I in HDL on lipid metabolism and atherogenesis, we generated a rabbit model in which the human apoA-II was knocked in to rabbit apoA-I locus by TALEN technology. The model was confirmed by genomic PCR, sequencing, RT-PCR and proteomics. With this model, we found that the triglyceride (TG) levels are significantly lower in homozygote than that in wild type control. The results of lipoprotein analysis using ultracentrifugation and FPLC also show that TG-rich lipoproteins were lower in homozygote than that of wild type. TGs were not increased in homozygote even after feeding of food. Small LDL and HDL2 particles were increased in homozygote rabbits. In addition, increased apoE was shown in HDL3 particle from homozygote. These results suggest that apoA-II knock-in has critical roles in the metabolism of TG-rich lipoproteins. Roles of apoA-II only HDLs from homozygote on cholesterol efflux and atherogenesis are under investigation.

Abstract 380: SAA Lipidation and Delipidation by Hepatocytes

Serum amyloid A (SAA) is one of the most striking acute phase reactants that can rapidly increase 1000-fold in plasma concentration in response to inflammatory cytokines. SAA in lipid-free form exhibits pro-inflammatory activities, but its putative physiological function(s) are poorly understood. SAA is produced and secreted largely by the liver and is present in plasma mainly as an HDL apolipoprotein. The pathways by which SAA is lipidated and incorporated into HDL are poorly understood. Plasma SAA is cleared more rapidly than the other major HDL apolipoproteins, but pathways involved in its delipidation and plasma clearance have also not been defined. In this study we examined how SAA is lipidated in primary hepatocytes and how such lipidation relates to the formation of nascent HDL particles. Endogenous hepatocyte SAA was lipidated and released from cells as large particles that were distinct from apoA-I-containing nascent HDL’s. Unlike apoA-I, formation of these SAA-containing particles was independent of ABCA-I. Similarly, when SAA was exogenously added to cells, SAA was lipidated to form nascent particles that were distinct from apoA-I-containing particles. We further studied the interaction of lipid-free and HDL-bound SAA with hepatocytes. Both in lipid-free form and as part of HDL, SAA exhibited significantly greater binding to cells than apoA-I or apoA-II. Binding studies were also carried out with normal and acute phase HDL’s isolated from control and SAA-deficient mice. Together, the results suggested that SAA, unlike apoA-I, is selectively removed from HDL by binding to hepatocytes. These findings may provide new insights into SAA metabolism and function.

Improved triglyceride‐rich lipoprotein clearance in human apolipoprotein A‐II knockin rabbits

Apolipoprotein A‐II is a second major apolipoprotein in HDL particles, which account for 15–20 % of total HDL protein in human. Compared with apolipoprotein A‐I which is a most major apolipoproteins in HDL, physiological role of apoA‐II is not fully understood. We previously reported that overexpression of apoA‐II in rabbits significantly suppressed cholesterol‐rich diet induced atherosclerosis. Because rabbits are the animal of naturally deficient in apoA‐II, so it becomes a good animal model to study the functions of apoA‐II. To elucidate the role of apoA‐II independent from apoA‐I in HDL on lipid metabolism and atherogenesis, we generated a rabbit model in which the human apoA‐II was knocked in to rabbit apoA‐I locus by TALEN technology. The model was confirmed by genomic PCR, sequencing, RT‐PCR and proteomics. With this model, we found that the triglyceride levels are significantly lower in homozygote than that in wild type control. The results of lipoprotein analysis using ultracentrifugation and FPLC also show that TG‐rich lipoproteins were lower in homozygote than that of wild type. TGs were not increased in homozygote even after feeding of food. Small LDL and HDL2 particles were increased in homozygote rabbits. In addition, increased apoE was shown in HDL3 particle from homozygote. These results suggest that apoA‐II knock‐in has critical roles in the metabolism of TG‐rich lipoproteins. Roles of apoA‐II only HDLs from homozygote on cholesterol efflux and atherogenesis are under investigation.

In situ AFM imaging of apolipoprotein A-I directly derived from plasma HDL

Background and aimsThe major apolipoproteins of plasma lipoproteins play vital roles in the structural integrity and physiological functions of lipoproteins. More than ten structural models of apolipoprotein A-I (apoA-I), the major apolipoprotein of high-density lipoprotein (HDL), have been developed successively. In these models, apoA-I was supposed to organize in a ring-shaped form. To date, however, there is no direct evidence under physiological condition. MethodsHere, atomic force microscopy (AFM) was used to in situ visualize the organization of apoA-I, which was exposed via depletion of the lipid component of plasma HDL pre-immobilized on functionalized mica sheets. ResultsFor the first time, the ring-shaped coarse structure and three detailed structures (crescent-shaped, gapped “O”-shaped, and parentheses-shaped structures, respectively) of apoA-I in plasma HDL, which have the ability of binding scavenger receptors, were directly observed and quantitatively measured by AFM. The three detailed structures probably represent the different extents to which the lipid component of HDL was depleted. Data on lipid depletion of HDL may provide clues to understand lipid insertion of HDL. ConclusionsThese data provide important information for the understanding of the structure/maturation of plasma HDL. Moreover, they suggest a powerful method for directly visualizing the major apolipoproteins of plasma lipoproteins or the protein component of lipoprotein-like lipid-protein complexes.

Methods for Monitoring ABCA1-Dependent Sterol Release.

Releasing sterols to the extracellular milieu is an important part of sterol homeostasis in cells and in the body. ATP-binding cassette transporter A1 (ABCA1) plays an essential role in cellular phospholipid and sterol release to lipid-free or lipid-poor apolipoprotein A-I (apoA-I), the major apolipoprotein in high-density lipoprotein (HDL), and constitutes the first step in the formation of nascent HDL. Loss-of-function mutations in the ABCA1 gene lead to a rare disease known as Tangier disease that causes severe deficiency in plasma HDL level. Mammalian cells receive exogenous cholesterol mainly from low-density lipoprotein. In addition, they synthesize cholesterol endogenously, as well as multiple precursor sterols that are sterol intermediates en route to be converted to cholesterol. HDL contains phospholipids, cholesterol, and precursor sterols, and ABCA1 has an ability to release phospholipids and various sterol molecules. Recent studies using model cell lines showed that ABCA1 prefers to use sterols newly synthesized endogenously as its preferred substrate, rather than cholesterol derived from LDL or cholesterol being recycled within the cells. Here, we describe several methods at the cell culture level to monitor ABCA1-dependent release of sterol molecules to apoA-I present at the cell exterior. Sterol release can be assessed by using a simple colorimetric enzymatic assay, and/or by monitoring the radioactivities of radiolabeled cholesterol incorporated into the cells, and/or of sterols biosynthesized from radioactive acetate, and/or by using gas chromatography-mass spectrometry analysis of various sterols present in medium and in cells. We also discuss the pros and cons of these methods. Together, these methods allow researchers to detect the release not only of cholesterol but also of other sterols present in minor quantities.

Potential Mechanisms for Enhanced Activity of Von Willebrand Factor in Patients with Sickle Cell Disease

Sickle cell disease (SCD) is a hemoglobinopathy characterized by vaso-occlusion and hemolysis. Even at disease baseline, von Willebrand factor (VWF) quantity and activity is markedly increased and correlates with the extent of hemolysis [Chen et al. Blood, 2011, 117:3680].VWF is an adhesive plasma protein capable of binding platelets, and secondarily, erythrocytes, and leukocytes. Its adhesive activity is regulated at several levels: being decreased by proteolysis by the metalloprotease ADAMTS13 [Dong et al. Blood, 2002, 100:4033] or by inhibition of self-association into more adhesive forms by high density lipoprotein (HDL) [Chung et al. Blood, 2016, 127:637], or enhanced by oxidative modification [Chen et al. Blood, 2010, 115:706; Fu et al. Blood, 2011, 118:5283]. Here, we explored several potential mechanisms accounting for the increased total active VWF in SCD patients: endothelial activation, enhanced VWF self-association, and defective cleavage of endogenous VWF because of ADAMTS13 dysfunction and/or VWF resistance to cleavage due to oxidation.Methods: We analyzed plasma collected from 12 adult SCD patients at disease baseline; for 3 of these we also have samples collected during vaso-occlusive crisis. We also analyzed multiple crisis samples from one additional patient with no baseline sample. To determine if SCD plasma could activate endothelial cells, we incubated plasma with human umbilical vein endothelial cells (HUVECs) cultured in parallel-plate flow chambers for 20 min at 37˚C then washed the plasma away before perfusing fixed platelets through the chamber. The number and length of platelet-decorated VWF strings were then quantified as a measure of endothelial activation. The plasma levels of VWF, apolipoprotein A-I (ApoA-I, the major apolipoprotein in HDL), myeloperoxidase (MPO), and ADAMTS13 were measured by sandwich ELISA. The activity of ADAMTS13 was evaluated by 1) cleavage of a small VWF A2 peptide substrate; 2) cleavage of multimeric VWF in patient plasma in 1.5 M urea and examining the VWF multimer pattern over time; 3) cleavage of preformed VWF-platelet strings on activated HUVECs; and 4) examining the extent of VWF cleavage in vivo using mass spectrometry (MS). The oxidation of specific methionine residues in VWF and ADAMTS13 from patient plasma was measured by MS as described previously [Chen et al. Blood, 2010, 115:706; Wang et al. JBC, 2015, 290:1422].Results: 1) HUVECs incubated with SCD plasma were activated and produced 2 to 25 fold more VWF strings than cells treated with pooled normal plasma (PNP). Activation was inhibited if the plasma was pre-incubated with hemopexin (0.5 mg/ml), implicating heme as an activator. However, purified heme alone even at high concentration did not activate HUVECs to secrete VWF strings, indicating that heme is required but not sufficient. 2) The ratio of VWF to ApoA-I was markedly elevated in SCD patients, especially during crisis (2.3 ± 0.7 fold of PNP), indicating predisposition to VWF self-association. 3) ADAMTS13 from SCD plasma was defective in cleaving VWF strings (11% to 57% of PNP) and multimeric VWF, but not the A2 peptide substrate. 4) Almost all of the SCD plasma had elevated MPO (3.8 ± 2.5 and 12.8 ± 7.6 fold of PNP in baseline and crisis samples, respectively). 5) Our previous studies showed that HOCl oxidation of VWF Met1606 at the ADAMTS13 cleavage site rendered the VWF noncleavable, and oxidation at other sites increased its platelet-binding activity. We also found that HOCl can inactivate ADAMTS13 by oxidizing Met249 within the metalloprotease domain. We therefore evaluated the extent of VWF Met1606 and ADAMTS13 Met249 oxidation, and VWF cleavage by ADAMTS13 in vivo in patient plasma using tandem MS. We found that ADAMTS13 in SCD plasma had elevated oxidation at Met249 (1.6 ± 0.2 fold of normal controls); VWF cleavage by ADAMTS13 in vivo was decreased by as much as 40% in several of these patients, and the extent of cleavage correlated negatively with the extent of VWF Met1606 oxidation.Summary: Multiple mechanisms contribute to enhanced VWF activity in SCD patients. These include increased secretion by activated endothelial cells, enhanced VWF self-association, and defective cleavage of VWF by endogenous ADAMTS13, partially due to oxidative modification of both VWF and ADAMTS13. DisclosuresNo relevant conflicts of interest to declare.

Significance of Lipid-Free and Lipid-Associated ApoA-I in Cellular Cho-lesterol Efflux.

The structure and stability of apolipoprotein (apo)A-I, the major apolipoprotein of human plasma high-density lipoproteins (HDL), determine the efficiency of the protein in the process of HDL generation and affect HDL properties in binding and exchanging its constituents, thus playing an essential role in reverse cholesterol transport. The equilibrium stability of an apoA-I molecule at the lipid interface (12.7 kcal/mol) predicted by a thermodynamic cycle for apolipoprotein folding-unfolding in water and at interface, largely exceeds apoA-I helix stability in HDL against chemical denaturation (3-5 kcal/mol). An ensemble of structures of lipid-bound apoA-I with different stabilities is assumed to exist. The conformational transitions between apoA-I conformers in water and lipid phases correspond to Lumry-Eyring model OL ⇔ CL ⇒ MW, where OL and CL are open and closed structures of HDLbound apoA-I, and MW is the molten globule in water. The model includes the reversible foldingunfolding transitions of N- and C-domains at HDL interface and apolipoprotein irreversible dissociation. We gathered published data on cholesterol efflux for apoA-I proteins with missense mutations in C-domain and calculated the stability of these mutants as a change of free energy relative to a wild type protein. Significant negative correlation was found between this stability and the efficiency of cAMP-stimulated cholesterol efflux. Thus, besides the known role of C-domain hydrophobicity, structure-destabilizing changes may significantly contribute to ABCA1-mediated cholesterol efflux by free apolipoprotein.

LPA Gene, Ethnicity, and Cardiovascular Events.

The relationship of LPA single nucleotide polymorphisms (SNPs), apolipoprotein(a) isoforms, and lipoprotein(a) [Lp(a)] levels with major adverse cardiovascular events (MACE) in different ethnic groups is not well known. LPA SNPs, apolipoprotein(a) isoforms, Lp(a), and oxidized phospholipids on apolipoprotein B-100 (OxPL-apoB) levels were measured in 1792 black, 1030 white, and 597 Hispanic subjects enrolled in the Dallas Heart Study. Their interdependent relationships and prospective association with MACE after median 9.5-year follow-up were determined. LPA SNP rs3798220 was most prevalent in Hispanics (42.38%), rs10455872 in whites (14.27%), and rs9457951 in blacks (32.92%). The correlation of each of these SNPs with the major apolipoprotein(a) isoform size was highly variable and in different directions among ethnic groups. In the entire cohort, Cox regression analysis with multivariable adjustment revealed that quartiles 4 of Lp(a) and OxPL-apoB were associated with hazard ratios (95% confidence interval) for time to MACE of 2.35 (1.50-3.69, P<0.001) and 1.89 (1.26-2.84, P=0.003), respectively, versus quartile 1. Addition of the major apolipoprotein(a) isoform and the 3 LPA SNPs to these models attenuated the risk, but significance was maintained for both Lp(a) and OxPL-apoB. Evaluating time to MACE in specific ethnic groups, Lp(a) was a positive predictor and the size of the major apolipoprotein(a) isoform was an inverse predictor in blacks, the size of the major apolipoprotein(a) isoform was an inverse predictor in whites, and OxPL-apoB was a positive predictor in Hispanics. The prevalence and association of LPA SNPs with size of apolipoprotein(a) isoforms, Lp(a), and OxPL-apoB levels are highly variable and ethnicity-specific. The relationship to MACE is best explained by elevated plasma Lp(a) or OxPL-apoB levels, despite significant ethnic differences in LPA genetic markers.

ANGKA BANDING APO B/APO A-I PADA GEJALA KORONER AKUT

Coronary Artery Disease has a high prevalence and is frequently occurred and associated with the high mortality and morbidity. Dyslipidemia is one of the risk factors of Coronary Heart Disease (CHD). ApoB contained in very low-density lipoproteins (VLDL), intermediate-density lipoproteins (IDL), LDL and small dense LDL (sd-LDL), with one molecule of apoB in each particle. Apo A-I is the major apolipoprotein in HDL particles. The ratio of apoB/apoA-I is a balance between apoB-containing particles and potentially atherogenic apoA-I that is antiaterogenik. This study is carried to know the determination whether there are differences between apoB/ apoA-I ratio in patients dyslipidemia with ACS and non ACS. The research used a cross-sectional study design with patients dyslipidemia subjects suffering Acute Coronary Syndrome (ACS) and non ACS who enter to the Laboratory of Pathology Clinic at Dr. Moewardi Hospital between July and November 2011. To determine the pattern of data distribution, the researchers used Kolmogorov Smirnov test. For the analysis of differences in mean apoB/apoA-I ratio in the two population groups is used the T test, using a computer program, with the significance level p&lt;0.05, 95% confidence interval. From 74 samples examined the mean age is 56.42 year old. This patients consisted of 33 males (44.6%) and 41 women (55.4%). All subjects are grouped into two groups, dyslipidemia ACS and non dyslipidemia ACS. The results showed apoB/apoA-I ratio significantly different in patients with dyslipidemia with ACS and non ACS. The mean apoB/apoA-I ratio of women and men subjects in both groups, including groups at high risk of myocardial myokard and higher than the cut-off ratio of apoB/ apoA-I (men 0.9 and women 0.8). It can be concluded that the apoB/apoA-I ratio of women and men subjects in both groups, included the high risk category for infarct myokard although lipid abnormalities are still not demonstrated to the risk of infarct myokard.

Hypercholesterolemia Tunes Hematopoietic Stem/Progenitor Cells for Inflammation and Atherosclerosis.

As the pathological basis of cardiovascular disease (CVD), atherosclerosis is featured as a chronic inflammation. Hypercholesterolemia is an independent risk factor for CVD. Accumulated studies have shown that hypercholesterolemia is associated with myeloid cell expansion, which stimulates innate and adaptive immune responses, strengthens inflammation, and accelerates atherosclerosis progression. Hematopoietic stem/progenitor cells (HSPC) in bone marrow (BM) expresses a panel of lipoprotein receptors to control cholesterol homeostasis. Deficiency of these receptors abrogates cellular cholesterol efflux, resulting in HSPC proliferation and differentiation in hypercholesterolemic mice. Reduction of the cholesterol level in the lipid rafts by infusion of reconstituted high-density lipoprotein (HDL) or its major apolipoprotein, apoA-I, reverses hypercholesterolemia-induced HSPC expansion. Apart from impaired cholesterol metabolism, inhibition of reactive oxygen species production suppresses HSPC activation and leukocytosis. These data indicate that the mechanisms underlying the effects of hypercholesterolemia on HSPC proliferation and differentiation could be multifaceted. Furthermore, dyslipidemia also regulates HSPC-neighboring cells, resulting in HSPC mobilization. In the article, we review how hypercholesterolemia evokes HSPC activation and mobilization directly or via its modification of BM microenvironment. We hope this review will bring light to finding key molecules to control HSPC expansion, inflammation, and atherosclerosis for the treatment of CVD.

ApoA-I induces S1P release from endothelial cells through ABCA1 and SR-BI in a positive feedback manner.

Sphingosine-1-phosphate (S1P), which has emerged as a pivotal signaling mediator that participates in the regulation of multiple cellular processes, is derived from various cells, including vascular endothelial cells. S1P accumulates in lipoproteins, especially HDL, and the majority of free plasma S1P is bound to HDL. We hypothesized that HDL-associated S1P is released through mechanisms associated with the HDL maturation process. ApoA-I, a major HDL apolipoprotein, is a critical factor for nascent HDL formation and lipid trafficking via ABCA1. Moreover, apoA-I is capable of promoting bidirectional lipid movement through SR-BI. In the present study, we confirmed that apoA-I can facilitate the production and release of S1P by HUVECs. Furthermore, we demonstrated that ERK1/2 and SphK activation induced by apoA-I is involved in the release of S1P from HUVECs. Inhibitor and siRNA experiments showed that ABCA1 and SR-BI are required for S1P release and ERK1/2 phosphorylation induced by apoA-I. However, the effects triggered by apoA-I were not suppressed by inhibiting ABCA1/JAK2 or the SR-BI/Src pathway. S1P released due to apoA-I activation can stimulate the (ERK1/2)/SphK1 pathway through S1PR (S1P receptor) 1/3. These results indicated that apoA-I not only promotes S1P release through ABCA1 and SR-BI but also indirectly activates the (ERK1/2)/SphK1 pathway by releasing S1P to trigger their receptors. In conclusion, we suggest that release of S1P induced by apoA-I from endothelial cells through ABCA1 and SR-BI is a self-positive-feedback process: apoA-I-(ABCA1 and SR-BI)-(S1P release)-S1PR-ERK1/2-SphK1-(S1P production)-(more S1P release induced by apoA-I).

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.

Apolipoprotein A-I Mimetic Peptide D-4F Reduces Cardiac Hypertrophy and Improves Apolipoprotein A-I-Mediated Reverse Cholesterol Transport From Cardiac Tissue in LDL Receptor-null Mice Fed a Western Diet.

Epidemiological studies have suggested that hypercholesterolemia is an independent determinant of increased left ventricular (LV) mass. Because high-density lipoprotein and its major protein apolipoprotein A-I (apoA-I) mediate reverse cholesterol transport (RCT) and have cardiac protective effects, we hypothesized that the apoA-I mimetic peptide D-4F could promote RCT in cardiac tissue and decrease cardiac hypertrophy induced by hypercholesterolemia. Low-density lipoprotein receptor-null mice were fed by a Western diet for 18 weeks and then randomized to receive water, or D-4F 0.3 mg/mL, or D-4F 0.5 mg/mL added to drinking water for 6 weeks. After D-4F administration, an increase in high-density lipoprotein cholesterol and a decrease in low-density lipoprotein cholesterol, total cholesterol, and triglyceride in a trend toward dose-responsivity were found in cardiac tissue. Ultrasound biomicroscopy revealed a reduction in LV posterior wall end-diastolic dimension, and an increase in mitral valve E/A ratio and LV ejection fraction. Hematoxylin-eosin staining showed reduced LV wall thickness and myocardial cell diameter. The protein levels of ABCA1 and LXRα were elevated in cardiac tissue of D-4F treated mice compared with the controls (P < 0.05). These results demonstrated that D-4F treatment reduced cardiac hypertrophy, and improved cardiac performance in low-density lipoprotein receptor-null mice fed a Western diet, presumably through the LXRα-ABCA1 pathway associated with enhanced myocardial RCT.

Multiplexed peptide analysis for kinetic measurements of major human apolipoproteins by LC/MS/MS

A multiplexed assay was developed by MS to analyze, in a single run, six major human Apos involved in lipoprotein metabolism: ApoA-I, ApoA-II, ApoB100, ApoC-II, ApoC-III, and ApoE. This method was validated in vivo in six subjects who received a 14 h constant infusion of [5,5,5-(2)H3]L-leucine at 10 μM/kg/h. Plasma lipoprotein fractions were isolated from collected blood samples and were digested with trypsin. Proteotypic peptides were subsequently analyzed by LC/MS/MS. Enrichment measurement data were compared with those obtained by the standard method using GC/MS. The required time to obtain the LC/MS/MS data was less than that needed for GC/MS. The enrichments from both methods were correlated for ApoA-I (r = 0.994; P < 0.0001) and ApoB100 (r = 0.999; P < 0.0001), and the Bland-Altman plot confirmed the similarity of the two methods. Intra- and inter-assay variability calculated for the six Apos of interest did not exceed 10.7 and 12.5%, respectively, and kinetic parameters were similar and/or in agreement with previously reported data. Therefore, LC/MS/MS can be considered as a useful tool for human Apo kinetic studies using stable isotopes.

High-density lipoprotein modulates thrombosis by preventing von Willebrand factor self-association and subsequent platelet adhesion

AbstractThe ability of von Willebrand factor (VWF) to initiate platelet adhesion depends on the number of monomers in individual VWF multimers and on the self-association of individual VWF multimers into larger structures. VWF self-association is accelerated by shear stress. We observed that VWF self-association occurs during adsorption of VWF onto surfaces, assembly of secreted VWF into hyperadhesive VWF strings on the endothelial surface, and incorporation of fluid-phase VWF into VWF fibers. VWF adsorption under static conditions increased with increased VWF purity and was prevented by a component of plasma. We identified that component as high-density lipoprotein (HDL) and its major apolipoprotein ApoA-I. HDL and ApoA-I also prevented VWF on the endothelium from self-associating into longer strands and inhibited the attachment of fluid-phase VWF onto vessel wall strands. Platelet adhesion to VWF fibers was reduced in proportion to the reduction in self-associated VWF. In a mouse model of thrombotic microangiopathy, HDL also largely prevented the thrombocytopenia induced by injection of high doses of human VWF. Finally, a potential role for ApoA-I in microvascular occlusion associated with thrombotic thrombocytopenic purpura and sepsis was revealed by the inverse relationship between the concentration of ApoA-I and that of hyperadhesive VWF. These results suggest that interference with VWF self-association would be a new approach to treating thrombotic disorders.

High yield of recombinant human Apolipoprotein A-I expressed in Pichia pastoris by using mixed-mode chromatography.

A vast majority of the cardioprotective properties exhibited by High-Density Lipoprotein (HDL) is mediated by its major protein component Apolipoprotein A-I (ApoA1). In order to develop a simplified bioprocess for producing recombinant human Apolipoprotein A-I (rhApoA1) in its near-native form, rhApoA1was expressed without the use of an affinity tag in view of its potential therapeutic applications. Expressed in Pichia pastoris at expression levels of 58.2 mg ApoA1 per litre of culture in a reproducible manner, the target protein was purified by mixed-mode chromatography using Capto™ MMC ligand with a purity and recovery of 84% and 68%, respectively. ApoA1 purification was scaled up to Mixed-mode Expanded Bed Adsorption chromatography to establish an 'on-line' process for the efficient capture of rhApoA1 directly from the P. pastoris expression broth. A polishing step using anion exchange chromatography enabled the recovery of ApoA1 up to 96% purity. Purified ApoA1 was identified and verified by RPLC-ESI-Q-TOF mass spectrometry. This two-step process would reduce processing times and therefore costs in comparison to the twelve-step procedure currently used for recovering rhApoA1 from P. pastoris.

A Novel Anti-Inflammatory Effect for High Density Lipoprotein.

High density lipoprotein has anti-inflammatory effects in addition to mediating reverse cholesterol transport. While many of the chronic anti-inflammatory effects of high density lipoprotein (HDL) are attributed to changes in cell adhesion molecules, little is known about acute signal transduction events elicited by HDL in endothelial cells. We now show that high density lipoprotein decreases endothelial cell exocytosis, the first step in leukocyte trafficking. ApoA-I, a major apolipoprotein of HDL, mediates inhibition of endothelial cell exocytosis by interacting with endothelial scavenger receptor-BI which triggers an intracellular protective signaling cascade involving protein kinase C (PKC). Other apolipoproteins within the HDL particle have only modest effects upon endothelial exocytosis. Using a human primary culture of endothelial cells and murine apo-AI knockout mice, we show that apo-AI prevents endothelial cell exocytosis which limits leukocyte recruitment. These data suggest that high density lipoprotein may inhibit diseases associated with vascular inflammation in part by blocking endothelial exocytosis.

Identification of Sequence Variation in the Apolipoprotein A2 Gene and Their Relationship with Serum High-Density Lipoprotein Cholesterol Levels.

Background:Apolipoprotein A2 (APOA2) is the second major apolipoprotein of the high-density lipoprotein cholesterol (HDL-C). The study aim was to identify APOA2 gene variation in individuals within two extreme tails of HDL-C levels and its relationship with HDL-C level. Methods:This cross-sectional survey was conducted on participants from Tehran Glucose and Lipid Study (TLGS) at Research Institute for Endocrine Sciences, Tehran, Iran from April 2012 to February 2013. In total, 79 individuals with extreme low HDL-C levels (≤5th percentile for age and gender) and 63 individuals with extreme high HDL-C levels (≥95th percentile for age and gender) were selected. Variants were identified using DNA amplification and direct sequencing. Results:Screen of all exons and the core promoter region of APOA2 gene identified nine single nucleotide substitutions and one microsatellite; five of which were known and four were new variants. Of these nine variants, two were common tag single nucleotide polymorphisms (SNPs) and seven were rare SNPs. Both exonic substitutions were missense mutations and caused an amino acid change. There was a significant association between the new missense mutation (variant Chr.1:16119226, Ala98Pro) and HDL-C level. Conclusion: None of two common tag SNPs of rs6413453 and rs5082 contributes to the HDL-C trait in Iranian population, but a new missense mutation in APOA2 in our population has a significant association with HDL-C.