HDL Biogenesis Research Articles

Phospholipid Transfer Protein Is Expressed in Cerebrovascular Endothelial Cells and Involved in High Density Lipoprotein Biogenesis and Remodeling at the Blood-Brain Barrier

Phospholipid transfer protein (PLTP) is a key protein involved in biogenesis and remodeling of plasma HDL. Several neuroprotective properties have been ascribed to HDL. We reported earlier that liver X receptor (LXR) activation promotes cellular cholesterol efflux and formation of HDL-like particles in an established in vitro model of the blood-brain barrier (BBB) consisting of primary porcine brain capillary endothelial cells (pBCEC). Here, we report PLTP synthesis, regulation, and its key role in HDL metabolism at the BBB. We demonstrate that PLTP is highly expressed and secreted by pBCEC. In a polarized in vitro model mimicking the BBB, pBCEC secreted phospholipid-transfer active PLTP preferentially to the basolateral ("brain parenchymal") compartment. PLTP expression levels and phospholipid transfer activity were enhanced (up to 2.5-fold) by LXR activation using 24(S)-hydroxycholesterol (a cerebral cholesterol metabolite) or TO901317 (a synthetic LXR agonist). TO901317 administration elevated PLTP activity in BCEC from C57/BL6 mice. Preincubation of HDL3 with human plasma-derived active PLTP resulted in the formation of smaller and larger HDL particles and enhanced the capacity of the generated HDL particles to remove cholesterol from pBCEC by up to 3-fold. Pre-β-HDL, detected by two-dimensional crossed immunoelectrophoresis, was generated from HDL3 in pBCEC-derived supernatants, and their generation was markedly enhanced (1.9-fold) upon LXR activation. Furthermore, RNA interference-mediated PLTP silencing (up to 75%) reduced both apoA-I-dependent (67%) and HDL3-dependent (30%) cholesterol efflux from pBCEC. Based on these findings, we propose that PLTP is actively involved in lipid transfer, cholesterol efflux, HDL genesis, and remodeling at the BBB.

A novel gene regulator, pyrrole–imidazole polyamide targeting ABCA1 gene increases cholesterol efflux from macrophages and plasma HDL concentration

Pyrrole-imidazole (PI) polyamides are nuclease-resistant novel compounds that inhibit transcription factors by binding to the minor groove of DNA. A PI polyamide that targets mouse ABCA1 and increases ABCA1 gene expression was designed and evaluated as an agent to increase plasma HDL concentration. A PI polyamide was designed to bind the activator protein-2 binding site of the mouse ABCA1 promoter. The effect of this PI polyamide on ABCA1 expression was evaluated by real-time RT-PCR and Western blotting using RAW264 cells. In vivo effects of this polyamide on ABCA1 gene expression and plasma HDL level were examined in C57B6 mice. One milligram per kilogram of body weight of PI polyamide was injected via the tail veins every 2 days for 1 week, and plasma lipid profiles were evaluated. PI polyamide showed a specific binding to the target DNA in gel mobility shift assay. Treatment of RAW264 cells with 1.0 μM PI polyamide significantly increased ABCA1 mRNA expression. PI polyamide also significantly increased apolipoprotein AI-mediated HDL biogenesis in RAW264 cells. Cellular cholesterol efflux mediated by apolipoprotein AI was significantly increased by the PI polyamide treatment. PI polyamide significantly increased expression of ABCA1 mRNA in the liver of C57B6 mice. Plasma HDL concentration was increased by PI polyamide administration. All of the HDL sub-fractions showed a tendency to increase after PI polyamide administration. The designed PI polyamide that targeted ABCA1 successfully increased ABCA1 expression and HDL biogenesis. This novel gene-regulating agent is promising as a useful compound to increase plasma HDL concentration. A novel pyrrole-imidazole (PI) polyamide binds to ABCA1. PI polyamide interfered with binding of AP-2ɑ protein to the ABCA1 gene promoter. PI polyamide inhibited the AP-2ɑ-mediated reduction of ABCA1 gene and protein expression. PI polyamide increased ABCA1 protein and apolipoprotein AI mediated HDL biogenesis. PI polyamide is a new gene regulator for the prevention of atherosclerotic diseases.

Endoplasmic reticulum stress impairs cholesterol efflux and synthesis in hepatic cells

Metabolic disorders such as type 2 diabetes cause hepatic endoplasmic reticulum (ER) stress, which affects neutral lipid metabolism. However, the role of ER stress in cholesterol metabolism is incompletely understood. Here, we show that induction of acute ER stress in human hepatic HepG2 cells reduced ABCA1 expression and caused ABCA1 redistribution to tubular perinuclear compartments. Consequently, cholesterol efflux to apoA-I, a key step in nascent HDL formation, was diminished by 80%. Besides ABCA1, endogenous apoA-I expression was reduced upon ER stress induction, which contributed to reduced cholesterol efflux. Liver X receptor, a key regulator of ABCA1 in peripheral cells, was not involved in this process. Despite reduced cholesterol efflux, cellular cholesterol levels remained unchanged during ER stress. This was due to impaired de novo cholesterol synthesis by reduction of HMG-CoA reductase activity by 70%, although sterol response element-binding protein-2 activity was induced. In mice, ER stress induction led to a marked reduction of hepatic ABCA1 expression. However, HDL cholesterol levels were unaltered, presumably because of scavenger receptor class B, type I downregulation under ER stress. Taken together, our data suggest that ER stress in metabolic disorders reduces HDL biogenesis due to impaired hepatic ABCA1 function.

Significance of the hydrophobic residues 225–230 of apoA-I for the biogenesis of HDL

We studied the significance of four hydrophobic residues within the 225-230 region of apoA-I on its structure and functions and their contribution to the biogenesis of HDL. Adenovirus-mediated gene transfer of an apoA-I[F225A/V227A/F229A/L230A] mutant in apoA-I⁻/⁻ mice decreased plasma cholesterol, HDL cholesterol, and apoA-I levels. When expressed in apoA-I⁻/⁻ × apoE⁻/⁻ mice, approximately 40% of the mutant apoA-I as well as mouse apoA-IV and apoB-48 appeared in the VLDL/IDL/LDL. In both mouse models, the apoA-I mutant generated small spherical particles of pre-β- and α4-HDL mobility. Coexpression of the apoA-I mutant and LCAT increased and shifted the-HDL cholesterol peak toward lower densities, created normal αHDL subpopulations, and generated spherical-HDL particles. Biophysical analyses suggested that the apoA-I[225-230] mutations led to a more compact folding that may limit the conformational flexibility of the protein. The mutations also reduced the ability of apoA-I to promote ABCA1-mediated cholesterol efflux and to activate LCAT to 31% and 66%, respectively, of the WT control. Overall, the apoA-I[225-230] mutations inhibited the biogenesis of-HDL and led to the accumulation of immature pre-β- and α4-HDL particles, a phenotype that could be corrected by administration of LCAT.

Role of the hydrophobic and charged residues in the 218–226 region of apoA-I in the biogenesis of HDL

We investigated the significance of hydrophobic and charged residues 218-226 on the structure and functions of apoA-I and their contribution to the biogenesis of HDL. Adenovirus-mediated gene transfer of apoA-I[L218A/L219A/V221A/L222A] in apoA-I⁻/⁻ mice decreased plasma cholesterol and apoA-I levels to 15% of wild-type (WT) control mice and generated pre-β- and α4-HDL particles. In apoA-I⁻/⁻ × apoE⁻/⁻ mice, the same mutant formed few discoidal and pre-β-HDL particles that could not be converted to mature α-HDL particles by excess LCAT. Expression of the apoA-I[E223A/K226A] mutant in apoA-I⁻/⁻ mice caused lesser but discrete alterations in the HDL phenotype. The apoA-I[218-222] and apoA-I[E223A/K226A] mutants had 20% and normal capacity, respectively, to promote ABCA1-mediated cholesterol efflux. Both mutants had ∼65% of normal capacity to activate LCAT in vitro. Biophysical analyses suggested that both mutants affected in a distinct manner the structural integrity and plasticity of apoA-I that is necessary for normal functions. We conclude that the alteration of the hydrophobic 218-222 residues of apoA-I disrupts apoA-I/ABCA1 interactions and promotes the generation of defective pre-β particles that fail to mature into α-HDL subpopulations, thus resulting in low plasma apoA-I and HDL. Alterations of the charged 223, 226 residues caused milder but discrete changes in HDL phenotype.

In This Issue

HomeCirculation ResearchVol. 112, No. 12In This Issue Free AccessResearch ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessResearch ArticlePDF/EPUBIn This Issue Originally published7 Jun 2013https://doi.org/10.1161/RES.0b013e31829c6689Circulation Research. 2013;112:1521Cezanne regulates NF-κB activation by hypoxia (p 1583)Luong et al discover how the enzyme Cezanne reduces ischemia-reperfusion injury.Download figureDownload PowerPointIschemia-reperfusion, which occurs in a number of clinical settings—such as during coronary angioplasty or organ transplant—can induce inflammation causing damage to the reperfused tissue. This inflammation is in part controlled by the activation of the transcription factor NF-κB, which in turn activates inflammatory genes. The protein Cezanne is a deubiquitinating enzyme and a known negative regulator NF-κB. Luong et al now report that a substrate of Cezanne is a protein called TRAF6, the polyubiquitination of which activates NF-κB and the inflammatory signaling. The team found that the silencing of Cezanne in cultured cells led to greater TRAF6 ubiquitination, while the deletion of Cezanne in mice increased injury and inflammation following ischemia-reperfusion. Interestingly, the team also found that the mRNA and protein levels of Cezanne were increased following hypoxia and reoxygenation in both cultured cells and animals, suggesting that Cezanne is automatically activated to dampen the effects of NF-κB and prevent over-inflammation. Artificially boosting Cezanne activity could further reduce inflammation and thus serve as an effective clinical strategy to prevent reperfusion injury, suggest the authors.miR-144 Regulates Cholesterol Metabolism (p 1592)Ramirez et al identify a microRNA that reduces cholesterol export from macrophages as well as the body.Download figureDownload PowerPointThe transporter protein ABCA1 is essential for removing cholesterol from macrophages and it helps in preventing these cells from developing into foam cells that form atherosclerotic lesions. In the liver, ABCA1 controls the biogenesis of HDL, which promotes cholesterol removal from the blood for excretion in bile. A number of microRNAs (miRs) have been shown to regulate ABCA1, and now Ramirez and colleagues have identified miR-144 as a new ABCA1 regulator through an unbiased screen for miRs upregulated in macrophages that had been stimulated to undergo cholesterol efflux. They found that ABCA1 mRNA has seven potential binding sites for miR-144, and that over-expressing miR-144 in macrophages and in mice suppressed ABCA1 expression, confirming that miR-144 and ABCA1 mRNA do indeed interact. Importantly, the inhibition of miR-144 by an antisense oligonucletide not only increased ABCA1 levels and the efflux of cholesterol in macrophages, but also increased HDL levels in mice, thereby potentially boosting cholesterol export in two ways. Thus, miR-144 inhibition could be a novel therapeutic strategy for lowering cholesterol.miR-144 Regulates ABCA1/HDL After FXR Activation (p 1602)de Aguiar Vallim et al independently identify microRNA-144 as a regulator of ABCA1.Download figureDownload PowerPointAt the same time when Ramirez and colleagues were screening macrophages for microRNAs (miRs) related to cholesterol efflux, de Aguiar Vallim and his team were searching for miRs that might be involved in lipid and lipoprotein metabolism in the liver. And both teams independently identified miR-144. Initially, de Aguiar Vallim’s team identified two miRs—miR-144 and miR-451. Both were highly conserved and also co-transcribed in response to hepatocyte stimulation with FXR—a transcription factor that regulates lipoprotein metabolism, among other things. But in a search for putative targets of miRs-, only miR-144 yielded strong, highly conserved candidates. And at the top of the list was ABCA1. The team over-expressed miR-144 in human and mouse hepatocytes and found that, ABCA1 protein levels were suppressed in both these cells. Similarly, the over-expression of miR-144 in mice decreased ABCA1 in the liver and HDL in the blood. Silencing of miR-144 with antisense oligonucleotides, on the other hand, increased ABCA1 and HDL levels, just as Ramirez and colleagues observed. Although FXR induced miR-144, the transcription factor has also been reported to reduce atherosclerosis in mice, suggesting FXR’s particular role in cholesterol efflux is complex and requires additional study. Previous Back to top Next FiguresReferencesRelatedDetails June 7, 2013Vol 112, Issue 12 Advertisement Article InformationMetrics © 2013 American Heart Association, Inc.https://doi.org/10.1161/RES.0b013e31829c6689 Originally publishedJune 7, 2013 PDF download Advertisement SubjectsCell Signaling/Signal TransductionGeneticsVascular Biology

Recent advances in niacin and lipid metabolism

This review focuses on the current understanding of the physiological mechanisms of action of niacin on lipid metabolism and atherosclerosis. Emerging findings indicate that niacin decreases hepatic triglyceride synthesis and subsequent VLDL/LDL secretion by directly and noncompetitively inhibiting hepatocyte diacylglycerol acyltransferase 2. Recent studies in mice lacking niacin receptor GPR109A and human clinical trials with GPR109A agonists disproved the long believed hypothesis of adipocyte triglyceride lipolysis as the mechanism for niacin's effect on serum lipids. Niacin, through inhibiting hepatocyte surface expression of β-chain ATP synthase, inhibits the removal of HDL-apolipoprotein (apo) AI resulting in increased apoAI-containing HDL particles. Additional recent findings suggest that niacin by increasing hepatic ATP-binding cassette transporter A1-mediated apoAI lipidation increases HDL biogenesis, thus stabilizing circulation of newly secreted apoAI. New concepts have also emerged on lipid-independent actions of niacin on vascular endothelial oxidative and inflammatory events, myeloperoxidase release from neutrophils and its impact on HDL function, and GPR109A-mediated macrophage inflammatory events involved in atherosclerosis. Recent advances have provided physiological mechanisms of action of niacin on lipid metabolism and atherosclerosis. Better understanding of niacin's actions on multiple tissues and targets may be helpful in designing combination therapy and new treatment strategies for atherosclerosis.

Single nucleotide polymorphisms in CETP, SLC46A1, SLC19A1, CD36, BCMO1, APOA5, and ABCA1 are significant predictors of plasma HDL in healthy adults

BackgroundIn a marker-trait association study we estimated the statistical significance of 65 single nucleotide polymorphisms (SNP) in 23 candidate genes on HDL levels of two independent Caucasian populations. Each population consisted of men and women and their HDL levels were adjusted for gender and body weight. We used a linear regression model. Selected genes corresponded to folate metabolism, vitamins B-12, A, and E, and cholesterol pathways or lipid metabolism.MethodsExtracted DNA from both the Sacramento and Beltsville populations was analyzed using an allele discrimination assay with a MALDI-TOF mass spectrometry platform. The adjusted phenotype, y, was HDL levels adjusted for gender and body weight only statistical analyses were performed using the genotype association and regression modules from the SNP Variation Suite v7.ResultsStatistically significant SNP (where P values were adjusted for false discovery rate) included: CETP (rs7499892 and rs5882); SLC46A1 (rs37514694; rs739439); SLC19A1 (rs3788199); CD36 (rs3211956); BCMO1 (rs6564851), APOA5 (rs662799), and ABCA1 (rs4149267). Many prior association trends of the SNP with HDL were replicated in our cross-validation study. Significantly, the association of SNP in folate transporters (SLC46A1 rs37514694 and rs739439; SLC19A1 rs3788199) with HDL was identified in our study.ConclusionsGiven recent literature on the role of niacin in the biogenesis of HDL, focus on status and metabolism of B-vitamins and metabolites of eccentric cleavage of β-carotene with lipid metabolism is exciting for future study.

Abstract 430: PCPE2 a Novel Determinant in HDL Biogenesis and Metabolism

Recent attempts to reduce cardiovascular events by pharmacologically raising plasma HDL concentration have not proven efficacious. This has lead to investigations on how to increase functional versus dysfunctional plasma HDL. Our work shows that a functional subfraction from ABCA1 mediated biogenesis of nascent HDL (nHDL) consists of 3 apoA-I, 105 phosphotidylcholine, 27 sphingomyelin and 108 free cholesterol molecules, compositionally reminiscent of lipid rafts. Our studies are now focused on procollagen C-proteinase enhancer 2 (PCPE2), first described by Francone, OL, et al. (2011), to play a significant role in the ABCA1 mediated efflux of cholesterol to apoA-I. Based on previous studies, we hypothesize that PCPE2 binds lipid-free apoA-I thereby aligning specific helices among 3 molecules of apoA-I for lipidation. To test this, we created loss-of-function apoA-I mutant proteins to identify specific helical repeats that participated in nHDL formation. The various mutations span the central helices as well as the N- and C- termini of apoA-I. Opposing cysteines were placed within 3-5 å using a composite 3-D model of lipid-free apoA-I conformation. The mutant apoA-I disulfide “locked” (oxidized) and “unlocked” (reduced) status was monitored using mass spectrometric sequencing. Using both locked and unlocked versions of each of the mutant proteins, secondary structure as well as the extent of lipidation was investigated. In addition, studies, utilizing LDL receptor, PCPE2 double knockout mice (LDLr -/- , PCPE2 -/- ) fed an atherogenic diet have been carried out. Despite having a higher HDL concentration LDLr -/- , PCPE2 -/- mice had significantly more aortic lipid deposition compared to LDLr -/- mice. The absence of PCPE2 substantially reduced the ability to efflux cholesterol suggesting that the HDL was dysfunctional. These data strongly suggest that PCPE2 participates in lipidation by helping apoA-I to either open or target a site on ABCA1 that promotes apoA-I opening.

A big role for small RNAs in HDL homeostasis

High-density lipoproteins play a central role in systemic cholesterol homeostasis by stimulating the efflux of excess cellular cholesterol and transporting it to the liver for biliary excretion. HDL has long been touted as the "good cholesterol" because of the strong inverse correlation of plasma HDL cholesterol levels with coronary heart disease. However, the disappointing outcomes of recent clinical trials involving therapeutic elevations of HDL cholesterol have called this moniker into question and revealed our lack of understanding of this complex lipoprotein. At the same time, the discovery of microRNAs (miRNAs) that regulate HDL biogenesis and function have led to a surge in our understanding of the posttranscriptional mechanisms regulating plasma levels of HDL. Furthermore, HDL has recently been shown to selectively transport miRNAs and thereby facilitate cellular communication by shuttling these potent gene regulators to distal tissues. Finally, that miRNA cargo carried by HDL may be altered during disease states further broadened our perspective of how this lipoprotein can have complex effects on target cells and tissues. The unraveling of how these tiny RNAs govern HDL metabolism and contribute to its actions promises to reveal new therapeutic strategies to optimize cardiovascular health.

Abstract 402: HDL Mimetic Peptides ATI-5261 and CS-6253 Induce ABCA1 Oligomerization, Cellular Cholesterol Efflux and Generate Nascent HDL-Like Lipoproteins

Novel approaches that target HDL biogenesis may increase nascent HDL-like particle formation that can be used for therapeutic purposes. Here, we evaluated two 26-amino acid apoA-I mimetic peptides designed from c-terminus of apoE, ATI-5261 and CS-6253 in their ability to promote cellular cholesterol efflux via the ABCA1 transporter. Using mifepristone-inducible ABCA1 BHK cells and cAMP-stimulated J774 macrophages, we examined ABCA1 oligomerization with dithiobis[succinimidyl propionate] (DSP) as a cross linker. Like native apoA-I, ATI-5261 and CS-6253 induced ABCA1 oligomerization at equimolar concentrations, and appear to interact with native oligomers of ABCA1, similar to apoA-I, as judged by crosslinking revealed by SDS-PAGE. By sucrose density gradient fractionation, we show that ABCA1 oligomerization occurs in non-raft domains. ATI-5261 and CS-6253 promoted cellular cholesterol efflux in a dose-dependent saturable manner. In BHK cells, the efficiency of cholesterol efflux (Km) on an equimolar basis was: apoA-I (0.16±0.02), ATI-5261 (0.45±0.08) and CS-6253 (0.68±0.15) and the efflux capacity (Vmax) (% in 24h) was: apoA-I (14.54±0.61), ATI-5261 (11.98±0.51) and CS-6253 (10.87±0.66), indicating a higher efflux efficiency for the apoA-I mimetics but with less capacity than native apoA-I. A similar trend was observed in J774 cells. Desorption of cellular cholesterol at 45 min occurred in both non-raft and raft-like membrane domains. We then examined by HPLC and 2-dimentional PAGGE the size and types of HDL-like particles formed. In 3[H]-cholesterol loaded BHK-ABCA1 cells, incubation with ATI-5261 and CS-6253 for 45 min and 12 hours yielded α-migrating particles with an apparent size ranging between 9 and 17 nm, as revealed by specific antibodies directed against ATI-5261 and CS-6253. Taken together, these observations suggest that ATI-5261 and CS-6253 induce cellular cholesterol efflux via ABCA1 oligomerization at molar concentrations similar to apoA-I and with higher efficiency. The particles generated by ATI-5261 and CS-6253 have α-migrating mobility with a size ranging between 9 and 17 nm and contain cholesterol, similar to nascent HDL particles.

ProAlgaZyme and its subfractions increase plasma HDL cholesterol via upregulation of ApoA1, ABCA1, and SRB1, and inhibition of CETP in hypercholesterolemic hamsters

ProAlgaZyme and its subfractions increase plasma HDL cholesterol via upregulation of ApoA1, ABCA1, and SRB1, and inhibition of CETP in hypercholesterolemic hamsters Andreea Geamanu, Nadia Saadat, Arvind Goja, Monika Wadehra, Xiangming Ji, Smiti V GuptaNutrition and Food Science, Wayne State University, Detroit, MI, USABackground: Plasma HDL cholesterol levels are inversely related to cardiovascular disease, which is the leading cause of death worldwide. This study investigated the effect of an algae infusion, ProAlgaZyme (PAZ), and its subfractions (P1, P2, P3, P4) on plasma HDL in a hamster model.Methods: Sixty male golden Syrian hamsters (8 weeks old) were randomized into controls (W) or PAZ (P), P1, P2, P3, and P4 (n = 10 per group). An infusion of either 5% (P1, P2, P3) or 20% (P, P4) concentration (v/v) was administered via the drinking water for 4 weeks, while the hamsters were being fed a high-fat diet (30% of calories from fat). Serum lipids were assayed and liver samples subjected to reverse transcription polymerase chain reaction to determine the relative transcription levels of genes involved in HDL/reverse cholesterol transport metabolism, ie, ApoA1, ABCA1, CETP, and SRB1.Results: Non-HDL cholesterol was significantly reduced in the P (P < 0.05), P3 and P4 (P < 0.001) groups as compared with the W group, while HDL cholesterol showed a significant increase in the P, P3, and P4 groups (P < 0.001). Moreover, the total cholesterol/HDL ratio was significantly improved in the P, P1, and P2 (P < 0.05), and P3 and P4 (P < 0.001) groups. The shift in cholesterol towards the higher density fractions was validated by density gradient ultracentrifugation. Real-time quantitative polymerase chain reaction showed a significant increase in hepatic ApoA1 (P, P4) and ABCA1 (P3, P4) expression, consistent with an increase in HDL production, biogenesis, and maturation. A two-fold increase in SRB1 expression indicates that P4 further augments the reverse cholesterol transport mechanism. Reduction of CETP expression (P4) is consistent with a decrease in the transfer of cholesteryl ester to LDL, further increasing the amount of cholesterol held as HDL particles.Conclusion: ProAlgaZyme and its subfractions significantly improved the plasma cholesterol profile by lowering non-HDL and increasing HDL, possibly via the reverse cholesterol transport mechanism.Keywords: ProAlgaZyme, ApoA1, ABCA1, SRB1, CETP, reverse cholesterol transport

Niacin increases HDL biogenesis by enhancing DR4-dependent transcription of ABCA1 and lipidation of apolipoprotein A-I in HepG2 cells

The lipidation of apoA-I in liver greatly influences HDL biogenesis and plasma HDL levels by stabilizing the secreted apoA-I. Niacin is the most effective lipid-regulating agent clinically available to raise HDL. This study was undertaken to identify regulatory mechanisms of niacin action in hepatic lipidation of apoA-I, a critical event involved in HDL biogenesis. In cultured human hepatocytes (HepG2), niacin increased: association of apoA-I with phospholipids and cholesterol by 46% and 23% respectively, formation of lipid-poor single apoA-I molecule-containing particles up to ~2.4-fold, and pre β 1 and α migrating HDL particles. Niacin dose-dependently stimulated the cell efflux of phospholipid and cholesterol and increased transcription of ABCA1 gene and ABCA1 protein. Mutated DR4, a binding site for nuclear factor liver X receptor alpha (LXR α ) in the ABCA1 promoter, abolished niacin stimulatory effect. Further, knocking down LXR α or ABCA1 by RNA interference eliminated niacin-stimulated apoA-I lipidation. Niacin treatment did not change apoA-I gene expression. The present data indicate that niacin increases apoA-I lipidation by enhancing lipid efflux through a DR4-dependent transcription of ABCA1 gene in HepG2 cells. A stimulatory role of niacin in early hepatic formation of HDL particles suggests a new mechanism that contributes to niacin action to increase the stability of newly synthesized circulating HDL.

Abstract 411: An Anion-Exchange Chromatography Isolated Subfraction of Mouse Apolipoprotein A-I Is Unable to Activate Cellular Cholesterol Release from Mouse Peritoneal Macrophage Foam Cells

Aim: Human and mouse plasma apoA-I separate on anion exchange chromatography (6M urea) in distinct subfractions. To investigate the nature of this diversity, we analyzed the protein subfractions by IEF and Urea-PAGE, and compared their ability to generate HDL in vitro. Methods: Human and mouse plasma HDLs were delipidated then applied to an DE52 ion-exchange column and eluted by 30-60mM Tris-HCl, pH7.80 containing 6M urea. Mouse, human, and recombinant apoA-Is were analyzed by IEF (Ettan IPGphor3) followed by CBB protein staining. Urea-PAGE analysis was performed with 14% acrylamide gel in 6M urea buffer. ApoA-Is abilities to efflux cholesterol from mouse peritoneal foam cells and to shift the size of the ACAT-accessible cholesterol pool were determined. Results: Human plasma and recombinant human apoA-I showed two apoA-I subtypes by IEF and Urea-PAGE analysis; whereas HDL from C57BL/6 and 129SV mice contained three apoA-Is at pI=5.37, 5.31, 5.27 (mP3-1, mP2, mP3-3), and pI=5.44, 5.37, 5.32, respectively. Subfractions mP2 and mP3-3 were isolated on a DE52 column and, despite their difference in pI, were able to mediate cellular cholesterol efflux at levels similar to human plasma apoA-Is. Subfraction mP3-1, with the highest pI, eluted between mP2 and mP3-3 on DE-52 column. Interestingly, for both mouse strains, mP3-1 showed very poor cholesterol efflux and the incorporation of 14C-oleic acid to cellular CE was significantly higher when compared to mP2 and mP3-3. Conclusion: We found that differences in the pI of anion-exchange chromatography apoA-I subfractions did not directly affect HDL generation except for mouse subtype mP3-1. mP3-1 binds strongly to the anionic DE52 resin, probably by hydrophobic interaction, and is unable to mediate cellular cholesterol efflux. Further investigation of the nature of the differences in these apoA-I subtypes will provide insights on factors that may be implicated in the biogenesis of HDL in vivo.

Abstract 167: PLTP Acts in Concert with SAA to Reduce Plasma HDL Levels in Mice and Modulates Nascent HDL Formation in Hepatocytes

Phospholipid transfer protein (PLTP), which binds phospholipids and facilitates their transfer between lipoproteins in plasma, plays a key role in lipoprotein remodeling. PLTP levels increase during acute inflammation and increased PLTP activity is associated with the inflammatory markers C-reactive protein (CRP) and serum amyloid A (SAA) as seen in patients with type 2 diabetes and cardiovascular disease. SAA, an HDL apolipoprotein, is highly induced during inflammation. In this study we investigated whether HDL remodeling by PLTP is affected by SAA, and the significance of PLTP on HDL biogenesis. Over-expression of PLTP in mice using an adenoviral vector reduced HDL-C and phospholipid (PL) in a dose dependent manner with the liberation of lipid-poor apoA-I. Co-expression of PLTP and SAA produced a significantly greater reduction in HDL-C and PL than expression of either PLTP or SAA alone. Studies were carried out to examine the lipidation of apoA-I and formation of nascent HDL particles by primary mouse hepatocytes. Over-expression of PLTP in hepatocytes markedly affected the levels as well as the species of nascent HDL particles produced by cells. Although the formation of smaller nascent HDLs was reduced by PLTP, the formation of the larger HDLs was increased. PLTP expression in hepatocytes did not change ABCA1 levels. Co-expression of SAA with PLTP exerted only a modest effect on the levels and types of HDL generated in the presence of PLTP. Our findings suggest that the remodeling of plasma HDL by PLTP and SAA contributes to the reduced HDL-C levels observed during inflammation. PLTP also significantly affects apoA-I lipidation and nascent HDL biogenesis in hepatocytes.

Abstract 434: A Novel 5-Step Model for the Mechanism of ABCA1-Mediated Nascent HDL Biogenesis

Objective— Lipid-poor apoA-I acts as an acceptor for cell cholesterol and phospholipids via the cell membrane protein ABCA1, generating nascent HDL. However, the mechanism of this process is not understood at the molecular level. Methods and Results— We propose a novel five-step model of nascent HDL biogenesis: ABCA1 remodeling of the plasma membrane lipids exposing phosphatidylserine and apoA-I binding to ABCA1 are the first two independent steps; third, ABCA1 facilitates apoA-I partial unfolding; forth, partially unfolded apoA-I inserts into the modified plasma membrane resulting in apoA-I lipidation; and fifth, nascent HDL is released from the cell. We created fluorescent apoA-I indicators that can monitor apoA-I unfolding and lipidation states. In cell free assays of reconstituted HDL (rHDL) generation from apoAI and DMPC liposomes, the fluorescent indicators demonstrated apoA-I unfolding and lipidation concurrent with rHDL formation. Next, HEK293 cells were stably transfected with different ABCA1 vectors encoding wild type (WT) and W590S and C1477R Tangier disease mutation isoforms. WT ABCA1 mediated cholesterol efflux to apoA-I (requires all steps) and sodium taurocholate (NaTC, requires only the membrane remodeling step,). Although neither mutant could efflux cholesterol efficiently to apoA-I, they were blocked at different steps. The W590S mutant bound apoAI but could not efflux cholesterol to NaTC, thus it was blocked at the membrane remodeling step. However, the C1477R mutant could not bind apoAI but could efflux cholesterol to NaTC, thus its activity was blocked at the apoAI binding step. When the lipidation indicator apoA-I was incubated with stably transfected HEK cells, we observed cell associated lipidated apoA-I in cells expressing WT ABCA1, but mostly unlipidated apoA-I was associated with the cells expressing W590S ABCA1. Conclusion— Our results support a novel five-step model for nascent HDL biogenesis: 1, 2) ABCA1 remodeling of the plasma membrane and apoA-I binding to ABCA1, which facilitate 3) apoA-I partial unfolding and 4) and lipidation by the remodeled membrane, followed by 5) the release of nascent HDL.

Genetics of Cholesterol Efflux

Plasma levels of high-density lipoprotein cholesterol (HDL-C) show an inverse association with coronary heart disease (CHD). As a biological trait, HDL-C is strongly genetically determined, with a heritability index ranging from 40 % to 60 %. HDL represents an appealing therapeutic target due to its beneficial pleiotropic effects in preventing CHD. This review focuses on the genetic basis of cellular cholesterol efflux, the rate-limiting step in HDL biogenesis. There are several monogenic disorders (e.g., Tangier disease, caused by mutations within ABCA1) affecting HDL biogenesis. Importantly, many disorders of cellular cholesterol homeostasis cause a reduced HDL-C. We integrate information from family studies and linkage analyses with that derived from genome-wide association studies (GWAS) and review the recent identification of micro-RNAs (miRNA) involved in cellular cholesterol metabolism. The identification of genomic pathways related to HDL may help pave the way for novel therapeutic approaches to promote cellular cholesterol efflux as a therapeutic modality to prevent atherosclerosis.

Calpain-mediated ABCA1 degradation: Post-translational regulation of ABCA1 for HDL biogenesis

Helical apolipoproteins remove cellular phospholipid and cholesterol to generate nascent HDL and this reaction is the major source of plasma HDL. ABCA1 is mandatory and rate-limiting for this reaction. Besides regulation of the gene expression by transcriptional factors including LXR, AP2 and SREBP, the ABCA1 activity is regulated post-translationally by calpain-mediated proteolytic degradation of ABCA1 protein that occurs in the early endosome after its endocytosis. When the HDL biogenesis reaction is ongoing as helical apolipoproteins interact with ABCA1, ABCA1 becomes resistant to calpain and is recycled to cell surface after endocytosis. Biogenesis of HDL is most likely to take place on cell surface. Clearance rate of ABCA1 by this mechanism is also retarded by various factors that interact with ABCA1, such as α1-syntrophin, LXRβ and calmodulin. Physiological relevance of the retardation by these factors is not entirely clear. Pharmacological inhibition of the calpain-mediated ABCA1 degradation results in the increase of the ABCA1 activity and HDL biogenesis in vitro and in vivo, and potentially suppresses atherogenesis. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945–2010).

Alteration of negatively charged residues in the 89 to 99 domain of apoA-I affects lipid homeostasis and maturation of HDL

In this study, we investigated the role of positively and negatively charged amino acids within the 89-99 region of apolipoprotein A-I (apoA-I), which are highly conserved in mammals, on plasma lipid homeostasis and the biogenesis of HDL. We previously showed that deletion of the 89-99 region of apoA-I increased plasma cholesterol and phospholipids, but it did not affect plasma triglycerides. Functional studies using adenovirus-mediated gene transfer of two apoA-I mutants in apoA-I-deficient mice showed that apoA-I[D89A/E91A/E92A] increased plasma cholesterol and caused severe hypertriglyceridemia. HDL levels were reduced, and approximately 40% of the apoA-I was distributed in VLDL/IDL. The HDL consisted of mostly spherical and a few discoidal particles and contained preβ1 and α4-HDL subpopulations. The lipid, lipoprotein, and HDL profiles generated by the apoA-I[K94A/K96A] mutant were similar to those of wild-type (WT) apoA-I. Coexpression of apoA-I[D89A/E91A/E92A] and human lipoprotein lipase abolished hypertriglyceridemia, restored in part the α1,2,3,4 HDL subpopulations, and redistributed apoA-I in the HDL2/HDL3 regions, but it did not prevent the formation of discoidal HDL particles. Physicochemical studies showed that the apoA-I[D89A/E91A/E92A] mutant had reduced α-helical content and effective enthalpy of thermal denaturation, increased exposure of hydrophobic surfaces, and increased affinity for triglyceride-rich emulsions. We conclude that residues D89, E91, and E92 of apoA-I are important for plasma cholesterol and triglyceride homeostasis as well as for the maturation of HDL.

Fenofibrate, homocysteine, cholesterol efflux and primum non nocere

The FIELD study investigated the effect of long-term treatment ith fenofibrate on cardiovascular events in diabetic patients [1]. he primary outcomes were somewhat disappointing, with no eduction in coronary events and less than the expected reducion in total cardiovascular events. However, the long duration and onsiderable size of the study, as well as thoroughness of analyis, has generated many exciting opportunities to investigate the nterrelationship between variousmetabolic parameters. One such pportunitywas explored in the study byMaranghi et al. published n this issue ofAtherosclerosis [2]. The authors took advantage of the nverse correlation between changes in plasma levels of homocyseine and HDL found in the FIELD study to investigate the possible echanismsof suchassociation. Thehypothesiswas thathomocyseinemay affect cholesterol efflux, and cholesterol efflux is amajor eterminant of plasma HDL concentration and, possibly, functionlity. The effect of homocysteine on cholesterol efflux could be irect or indirect; therefore several possibilities of indirect effect formationof pre -HDLandactivities of Phospholipid Transfer Proein (PLTP) and PON-1) were also investigated. If functionality of DL was impaired by fenofibrate, through homocysteine or somehing else, that may have negated the beneficial effects of higher DL concentration and in part explain the outcomes of the FIELD tudy. However, no effect of fenofibrate, or higher plasma homoysteine level, on cholesterol efflux was found. Cholesterol efflux is the first, and often the rate limiting, process n two pathways, HDL biogenesis and reverse cholesterol transort. The rate of cholesterol efflux depends on the ability of cells o release cholesterol and on the ability of plasma lipoproteins