Lipid-free apoA-I Research Articles

High density lipoproteins improve insulin sensitivity in high-fat diet-fed mice by suppressing hepatic inflammation

Obesity-induced liver inflammation can drive insulin resistance. HDL has anti-inflammatory properties, so we hypothesized that low levels of HDL would perpetuate inflammatory responses in the liver and that HDL treatment would suppress liver inflammation and insulin resistance. The aim of this study was to investigate the effects of lipid-free apoAI on hepatic inflammation and insulin resistance in mice. We also investigated apoAI as a component of reconstituted HDLs (rHDLs) in hepatocytes to confirm results we observed in vivo. To test our hypothesis, C57BL/6 mice were fed a high-fat diet (HFD) for 16 weeks and administered either saline or lipid-free apoAI. Injections of lipid-free apoAI twice a week for 2 or 4 weeks with lipid-free apoAI resulted in: i) improved insulin sensitivity associated with decreased systemic and hepatic inflammation; ii) suppression of hepatic mRNA expression for key transcriptional regulators of lipogenic gene expression; and iii) suppression of nuclear factor κB (NF-κB) activation. Human hepatoma HuH-7 cells exposed to rHDLs showed suppressed TNFα-induced NF-κB activation, correlating with decreased NF-κB target gene expression. We conclude that apoAI suppresses liver inflammation in HFD mice and improves insulin resistance via a mechanism that involves a downregulation of NF-κB activation.

The Conformation of Lipid-Free Human Apolipoprotein A-I in Solution

Apolipoprotein AI (apoA-I) is the principal acceptor of lipids from ATP-binding cassette transporter A1, a process that yields nascent high density lipoproteins. Analysis of lipidated apoA-I conformation yields a belt or twisted belt in which two strands of apoA-I lie antiparallel to one another. In contrast, biophysical studies have suggested that a part of lipid-free apoA-I was arranged in a four-helix bundle. To understand how lipid-free apoA-I opens from a bundle to a belt while accepting lipid it was necessary to have a more refined model for the conformation of lipid-free apoA-I. This study reports the conformation of lipid-free human apoA-I using lysine-to-lysine chemical cross-linking in conjunction with disulfide cross-linking achieved using selective cysteine mutations. After proteolysis, cross-linked peptides were verified by sequencing using tandem mass spectrometry. The resulting structure is compact with roughly four helical regions, amino acids 44-186, bundled together. C- and N-terminal ends, amino acids 1-43 and 187-243, respectively, are folded such that they lie close to one another. An unusual feature of the molecule is the high degree of connectivity of lysine40 with six other lysines, lysines that are close, for example, lysine59, to distant lysines, for example, lysine239, that are at the opposite end of the primary sequence. These results are compared and contrasted with other reported conformations for lipid-free human apoA-I and an NMR study of mouse apoA-I.

Influence of apolipoprotein A-I and apolipoprotein A-II availability on nascent HDL heterogeneity

It is important to understand HDL heterogeneity because various subspecies possess different functionalities. To understand the origins of HDL heterogeneity arising from the existence of particles containing only apoA-I (LpA-I) and particles containing both apoA-I and apoA-II (LpA-I+A-II), we compared the abilities of both proteins to promote ABCA1-mediated efflux of cholesterol from HepG2 cells and form nascent HDL particles. When added separately, exogenous apoA-I and apoA-II were equally effective in promoting cholesterol efflux, although the resultant LpA-I and LpA-II particles had different sizes. When apoA-I and apoA-II were mixed together at initial molar ratios ranging from 1:1 to 16:1 to generate nascent LpA-I+A-II HDL particles, the particle size distribution altered, and the two proteins were incorporated into the nascent HDL in proportion to their initial ratio. Both proteins formed nascent HDL particles with equal efficiency, and the relative amounts of apoA-I and apoA-II incorporation were driven by mass action. The ratio of lipid-free apoA-I and apoA-II available at the surface of ABCA1-expressing cells is a major factor in determining the contents of these proteins in nascent HDL. Manipulation of this ratio provides a means of altering the relative distribution of LpA-I and LpA-I+A-II HDL particles.

Hormonal modulators of glial ABCA1 and apoE levels

Apolipoprotein E (apoE) is the major lipid carrier in the central nervous system. As apoE plays a major role in the pathogenesis of Alzheimer disease (AD) and also mediates repair pathways after several forms of acute brain injury, modulating the expression, secretion, or function of apoE may provide potential therapeutic approaches for several neurological disorders. Here we show that progesterone and a synthetic progestin, lynestrenol, significantly induce apoE secretion from human CCF-STTG1 astrocytoma cells, whereas estrogens and the progesterone metabolite allopregnanolone have negligible effects. Intriguingly, lynestrenol also increases expression of the cholesterol transporter ABCA1 in CCF-STTG1 astrocytoma cells, primary murine glia, and immortalized murine astrocytes that express human apoE3. The progesterone receptor inhibitor RU486 attenuates the effect of progestins on apoE expression in CCF-STTG1 astrocytoma cells but has no effect on ABCA1 expression in all glial cell models tested, suggesting that the progesterone receptor (PR) may participate in apoE but does not affect ABCA1 regulation. These results suggest that selective reproductive steroid hormones have the potential to influence glial lipid homeostasis through liver X receptor-dependent and progesterone receptor-dependent pathways.

Recent advances in physiological lipoprotein metabolism

Research into lipoprotein metabolism has developed because understanding lipoprotein metabolism has important clinical indications. Lipoproteins are risk factors for cardiovascular disease. Recent advances include the identification of factors in the synthesis and secretion of triglyceride rich lipoproteins, chylomicrons (CM) and very low density lipoproteins (VLDL). These included the identification of microsomal transfer protein, the cotranslational targeting of apoproteinB (apoB) for degradation regulated by the availability of lipids, and the characterization of transport vesicles transporting primordial apoB containing particles to the Golgi. The lipase maturation factor 1, glycosylphosphatidylinositol-anchored high density lipoprotein binding protein 1 and an angiopoietin-like protein play a role in lipoprotein lipase (LPL)-mediated hydrolysis of secreted CMs and VLDL so that the right amount of fatty acid is delivered to the right tissue at the right time. Expression of the low density lipoprotein (LDL) receptor is regulated at both transcriptional and post-transcriptional level. Proprotein convertase subtilisin/kexin type 9 (PCSK9) has a pivotal role in the degradation of LDL receptor. Plasma remnant lipoproteins bind to specific receptors in the liver, the LDL receptor, VLDL receptor and LDL receptor-like proteins prior to removal from the plasma. Reverse cholesterol transport occurs when lipid free apoAI recruits cholesterol and phospholipid to assemble high density lipoprotein (HDL) particles. The discovery of ABC transporters (ABCA1 and ABCG1) and scavenger receptor class B type I (SR-BI) provided further information on the biogenesis of HDL. In humans HDL-cholesterol can be returned to the liver either by direct uptake by SR-BI or through cholesteryl ester transfer protein exchange of cholesteryl ester for triglycerides in apoB lipoproteins, followed by hepatic uptake of apoB containing particles. Cholesterol content in cells is regulated by several transcription factors, including the liver X receptor and sterol regulatory element binding protein. This review summarizes recent advances in knowledge of the molecular mechanisms regulating lipoprotein metabolism.

Abstract 155: Mutations In ABCA8 Result In HDL Deficiency And Cholesterol Efflux Defects

The molecular basis of extreme HDL-C levels is incompletely understood. To identify mutations in novel putative HDL genes, we sequenced select genes in 80 unrelated individuals with extreme low HDLc (HDL %ile≤10th) and as controls, 120 individuals with high HDL (HDL %ile≥90th). We identified two variants in ABCA8 in subjects with low HDL-C levels. The E18-2T>C mutation is located in a highly conserved region and gives rise to a disruption of a splice site. The G>C variant occurs at a site conserved in vertebrates, at position 64425884 (build HG18), and results in Pro609Arg substitution. No ABCA8 mutations were identified in HHDL individuals. These represent the first mutations identified in ABCA8, which belongs to the family of ATP binding cassette transporters. Family expansion followed by genotyping identified additional mutation carriers and first degree relative controls in whom segregation analyses were performed. Compared with controls, mutation carriers showed a significant 32.6% decrease in plasma HDLc levels and decreased HDL percentiles (HDLc: controls = 1.23±0.26, mutation carriers = 0.83±0.37, mmol/L, p=0.0007; HDL%ile: controls = 41.8±21.2, mutation carriers = 17.3±19.2, p=0.004, n=23 controls and 12 carriers). No changes in LDLc, triglycerides or BMI were observed. Since mutations in ABCA8 reduce plasma HDLc levels, we assessed the role of ABCA8 in lipid efflux. Cholesterol efflux to lipid-free APOA-I was significantly reduced by 62.1% in fibroblasts from the proband with the E18-2 T>C mutation. In comparison, fibroblasts from heterozygous ABCA1 mutation carriers showed a 30-60% decrease in efflux to APOA-I depending on mutation severity. Wild type and P609R ABCA8 cDNA clones were generated from human liver RNA. A significant ~200% increase in cholesterol efflux to lipid free APOA-I was observed with wild type ABCA8, further suggesting that ABCA8 is a lipid transporter. A significant 75% decrease in the efflux of cholesterol to lipid-free APOA-I (p=0.02) was observed with the mutant P609R ABCA8 protein compared to wild-type. We show here that ABCA8 is a cholesterol transporter, and that mutations in ABCA8 are a novel cause for reduced plasma HDLc levels in humans.

Polyunsaturated fatty acid relatively decreases cholesterol content in THP-1 macrophage-derived foam cell: partly correlates with expression profile of CIDE and PAT members

BackgroundPolyunsaturated fatty acids (PUFAs) have positive effect on the regulation of plasma lipids. But the mechanism for them to modulate lipid homeostasis in macrophage is still unclear. In this study, we employed PUFA to pretreat macrophages and evaluated the variations of lipid droplet (LD) content, lipid composition, and expressions of LD-associated genes in macrophage-derived foam cells.MethodTHP-1-derived macrophages or human peripheral blood monocyte-derived macrophages were pre-treated with four non-esterified fatty acids (NEFAs) separately: saturated fatty acid (SFA)-palmitic acid (PA), monounsaturated fatty acids (MUFAs)-oleic acid (OA), PUFAs-linoleic acid (LA) and eicosapentaenoic acid (EPA). Intracellular lipid content and cholesterol efflux were analyzed in THP-1 macrophage-derived foam cells. Related gene expressions were detected by quantitative real-time PCR.ResultsPUFA pre-treatment reduced cholesterol content in foam cells and increased cholesterol efflux to lipid-free apoAI in conditioned medium compared with PA or OA group. Cell death-inducing DFF45 like effector (CIDE) and Perilipin-Adipophilin-TIP47 (PAT) family members, as LD-associated proteins, showed specific gene expression profiles after PUFA pre-treatment. These results may help to explain the process of lipid metabolism within foam cells.ConclusionPUFA (LA or EPA) had a potential protective effect against cholesterol accumulation. The specific expressions of CIDE and PAT genes may provide clues to explore the protective mechanism of PUFA in foam cells.

Effect of apoA-I Mutations in the Capacity of Reconstituted HDL to Promote ABCG1-Mediated Cholesterol Efflux

ATP binding cassette transporter G1 (ABCG1) mediates the cholesterol transport from cells to high-density lipoprotein (HDL), but the role of apolipoprotein A-I (apoA-I), the main protein constituent of HDL, in this process is not clear. To address this, we measured cholesterol efflux from HEK293 cells or J774 mouse macrophages overexpressing ABCG1 using as acceptors reconstituted HDL (rHDL) containing wild-type or various mutant apoA-I forms. It was found that ABCG1-mediated cholesterol efflux was severely reduced (by 89%) when using rHDL containing the carboxyl-terminal deletion mutant apoA-I[Δ(185–243)]. ABCG1-mediated cholesterol efflux was not affected or moderately decreased by rHDL containing amino-terminal deletion mutants and several mid-region deletion or point apoA-I mutants, and was restored to 69–99% of control by double deletion mutants apoA-I[Δ(1–41)Δ(185–243)] and apoA-I[Δ(1–59)Δ(185–243)]. These findings suggest that the central helices alone of apoA-I associated to rHDL can promote ABCG1-mediated cholesterol efflux. Further analysis showed that rHDL containing the carboxyl-terminal deletion mutant apoA-I[Δ(185–243)] only slightly reduced (by 22%) the ABCG1-mediated efflux of 7-ketocholesterol, indicating that depending on the sterol type, structural changes in rHDL-associated apoA-I affect differently the ABCG1-mediated efflux of cholesterol and 7-ketocholesterol. Overall, our findings demonstrate that rHDL-associated apoA-I structural changes affect the capacity of rHDL to accept cellular cholesterol by an ABCG1-mediated process. The structure-function relationship seen here between rHDL-associated apoA-I mutants and ABCG1-mediated cholesterol efflux closely resembles that seen before in lipid-free apoA-I mutants and ABCA1-dependent cholesterol efflux, suggesting that both processes depend on the same structural determinants of apoA-I.

Mutation mapping of apolipoprotein A-I structure assisted with the putative cholesterol recognition regions

Twenty-nine from 52 missense mutations in apoA-I gene are predicted to be deleterious by both SIFT and PolyPhen-2 algorithms. Among those, eight mutations with a prominent change in structure stability as modeled by the SDM tool for both lipid-free (Mei and Atkinson (2011) PDB ID: 3R2P) and HDL-bound (Wu et al. (2009) PDB ID: 3K2S) apoA-I, are referred as structural. The remaining mutations with a preferential location in a long intrinsically disordered region, predicted by the SPINE-D and DNdisorder tools, may influence the functional sites. Among structural mutations, five amyloidosis-only-related mutations, significant in a lipid-free structure, are located in 1–90 region. Six amyloidosis- and hypoalphalipoproteinemia-associated mutations, differently significant in two chains of lipid-bound apoA-I, are distributed among the N-domain. Six cholesterol recognition putative motifs (5 CRAC/1 CCM) in apoA-I structure are suggested to interact with cholesterol. Among those, the K40-W50 partially conserved CCM sequence with a putative recognition feature, predicted by the MoRF tool, may underlie cholesterol binding to lipid-free apoA-I, the binding triggering the disorder-to-order transition within MoRF. Thus, the impairment of helix formation and accelerated protein aggregation may underlie the amyloidogenic effect of W50R substitution. Also, D102H substitution in conserved CRAC2 V97-K106 sequence may be harmful in reverse cholesterol transport. With PDBe Motifs and Sites algorithm, cholesterol is a ligand for L101, F104 and W108 residues in HDL-bound apoA-I. The influence of specific mutation on apoA-I structure and mutated apolipoprotein switch between different pathologies is suggested to depend on the surrounding phase properties.

Apolipoprotein A-I glycation by Glucose and Reactive Aldehydes Alters Phospholipid Affinity but Not Cholesterol Export from Lipid-Laden Macrophages

Increased protein glycation in people with diabetes may promote atherosclerosis. This study examined the effects of non-enzymatic glycation on the association of lipid-free apolipoproteinA-I (apoA-I) with phospholipid, and cholesterol efflux from lipid-loaded macrophages to lipid-free and lipid-associated apoA-I. Glycation of lipid-free apoA-I by methylglyoxal and glycolaldehyde resulted in Arg, Lys and Trp loss, advanced glycation end-product formation and protein cross-linking. The association of apoA-I glycated by glucose, methylglyoxal or glycolaldehyde with phospholipid multilamellar vesicles was impaired in a glycating agent dose-dependent manner, with exposure of apoA-I to both 30 mM glucose (42% decrease in kslow) and 3 mM glycolaldehyde (50% decrease in kfast, 60% decrease in kslow) resulting is significantly reduced affinity. Cholesterol efflux to control or glycated lipid-free apoA-I, or discoidal reconstituted HDL containing glycated apoA-I (drHDL), was examined using cholesterol-loaded murine (J774A.1) macrophages treated to increase expression of ATP binding cassette transporters A1 (ABCA1) or G1 (ABCG1). Cholesterol efflux from J774A.1 macrophages to glycated lipid-free apoA-I via ABCA1 or glycated drHDL via an ABCG1-dependent mechanism was unaltered, as was efflux to minimally modified apoA-I from people with Type 1 diabetes, or controls. Changes to protein structure and function were prevented by the reactive carbonyl scavenger aminoguanidine. Overall these studies demonstrate that glycation of lipid-free apoA-I, particularly late glycation, modifies its structure, its capacity to bind phospholipids and but not ABCA1- or ABCG1-dependent cholesterol efflux from macrophages.

Abstract 412: In Silico Structural and Metabolic Profiling of Non-Synonymous Single Nucleotide Polymorphisms in APOA-I Gene

Mature apoA-I contains 243 residues organized in two-domains as N-terminal four-helix bundle and C-terminal two helices. Cholesterol interaction with both lipid-free and lipid-bound apoA-I may be one of the key events in amyloidosis and reverse cholesterol transport (RCT). By the SIFT score, 20 non-synonymous missense mutations in APOA-I gene, located almost exclusively in N-domain, may impair apoA-I function. A majority (4 from 5) of putative cholesterol recognition amino acid consensus (CRAC: L/V-X(1-5)-Y-X(1-5)-R/K) and a single cholesterol consensus motif (CCM: R/K-X(2-6)-I/L/V-X(3)-W/Y) aligned within/between helices in the bundle. Importantly, CRAC2 in apoA-I co-aligned with CRAC2 in apoE and CRAC4 in apoA-IV. A short four amino acid sequence L44-L47 may present disorder-to-order transitioning binding region in apoA-I as predicted by molecular recognition feature (MoRF). Indeed, CCM corresponded to unstructured region with low stability in lipid-free apoA-I. Stabilizing effect due to the direct cholesterol-peptide interaction with the involvement of CRAC1 (L14-K23) and/or CCM (K40-W50) in amyloidogenic peptide 1-93 is suggested to underlie the known effect of cholesterol on the induction of helix formation in this peptide. Cholesterol may be speculated to act in helix formation as a chemical chaperone thus triggering disorder-to-order transition within MoRF; without cholesterol, this MoRF may be involved into protein-protein interaction. Indeed, the association of W50R substitution with the accelerated mutant aggregation rate and the involvement of L44-S55 segment into cross-beta-sheet formation were demonstrated by others. Also, D102H substitution in CRAC2 (V97-K106) may be harmful in RCT. With PDBe Motifs and Sites algorithm, cholesterol is a ligand for L101, F104 and W108 residues in CRAC2 motif of apoA-I in nascent HDL. Besides, the resistance of two-domain apoA-I structure to denaturation by GuHCl is sensitive to cholesterol level in discoidal HDL. Overall, different CRACs may contribute into amyloidosis-connected apoA-I misfolding and cholesterol efflux for lipid-free apoA-I. Cholesterol binding to lipid-bound apolipoprotein may change the structural plasticity of two domains and/or their relative dimensions.

Abstract 101: Reconstituted High-density Lipoprotein - More Than the Sum of Its Parts.

Rationale Reconstituted high-density lipoprotein (rHDL, CSL-111) particles have been shown to reduce multiple organ failure following hypovolemic shock, perturb irritable bowel syndrome and, more recently, to inhibit aneurysm formation in animal models. To identify new endothelial targets of the protective effect we examined the differential changes that occur in the proteome of activated primary endothelial cells following treatment with rHDLs. Methods Primary human endothelial cells were isolated from human umbilical cords, grown to confluence and activated with TNFα (10ng/ml), following a 4 hr incubation half the cells received rHDLs (1mg/ml, ApoAI) and the remaining cells received carrier only (10% sucrose). Protein lysates were prepared following 20 hr incubation and differential protein expression compared by scanning DiGE-2dimentional PAGE. Protein identities were confirmed using LC/MS of tryptic digests of proteins recovered from preparatory gels. Western blotting confirmed differential expression and established whether the response required the whole particle or solely the protein component of rHDLs. Results Of the proteins identified by LC/MS which were differentially expressed in response to rHDLs, Perilipin 3 (PLPN3, 1.3 fold, p<0.003); Growth factor receptor-bound protein 2 (GRB2, 2.5 fold, p<0.003) and Vimentin (1.6 fold, p<0.007) were induced, whilst sinexin 6 (SNX6, 1.2 fold p< 0.007); Glutathione S Transferase-1 (GST-1, 1.6 fold, p<0.02) and Crystallin-zeta like (CrystZL, 1.2 fold, p<0.004) were reduced. We found that the entire lipoprotein particle was necessary for the induction of PLN3 and reduction of SNX6. Concentration of rHDL < 100μg/ml were able to modulate the maximal effect observed. However, both rHDL and lipid free ApoAI were able to induce cytokine-inhibited expression of GRB2 (rHDL>ApoAI). Conclusion The PC component of rHDL is necessary for an acute effect on proteins involved in endosomal trafficking. Further studies are needed to understand the role this could play in the protective effect of HDL therapy.

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.

Discoidal HDL and apoA-I-derived peptides improve glucose uptake in skeletal muscle

Lipid-free apoA-I and mature spherical HDL have been shown to induce glucose uptake in skeletal muscle. To exploit apoA-I and HDL states for diabetes therapy, further understanding of interaction between muscle and apoA-I is required. This study has examined whether nascent discoidal HDL, in which apoA-I attains a different conformation from mature HDL and lipid-free states, could induce muscle glucose uptake and whether a specific domain of apoA-I can mediate this effect. Using L6 myotubes stimulated with synthetic reconstituted discoidal HDL (rHDL), we show a glucose uptake effect comparable to insulin. Increased plasma membrane GLUT4 levels in ex vivo rHDL-stimulated myofibers from HA-GLUT4-GFP transgenic mice support this observation. rHDL increased phosphorylation of AMP kinase (AMPK) and acetyl-coA carboxylase (ACC) but not Akt. A survey of domain-specific peptides of apoA-I showed that the lipid-free C-terminal 190–243 fragment increases plasma membrane GLUT4, promotes glucose uptake, and activates AMPK signaling but not Akt. This may be explained by changes in α-helical content of 190–243 fragment versus full-length lipid-free apoA-I as assessed by circular dichroism spectroscopy. Discoidal HDL and the 190–243 peptide of apoA-I are potent agonists of glucose uptake in skeletal muscle, and the C-terminal α-helical content of apoA-I may be an important determinant of this effect.

Abstract 426: Selective Proteolysis of Apolipoprotein A-I by Secretory Phospholipase A2: New Insights Into sPLA2-dependent Catabolism of HDL

Introduction Secretory phospholipase A2 (sPLA2) is a pro-inflammatory enzyme that promotes atherogenesis via several mechanisms. One of these mechanisms probably involves high-density lipoproteins (HDL). Previous in vitro studies of HDL modified by sPLA2 showed increased exposure of certain domains of the major HDL protein, apoA-I, without protein fragmentation. Over-expression of sPLA2 in transgenic mice reduced plasma levels of HDL cholesterol due to rapid HDL catabolism. The origin of this enhanced HDL catabolism is unknown. Hypothesis In addition to hydrolysis of HDL phospholipids, sPLA2 can also hydrolyze apoA-I, leading to enhanced catabolism of sPLA2 modified HDL. Methods Human plasma HDL and lipid-free apoA-I were incubated with sPLA2 at 37 oC. The HDL lipids were removed, and the protein integrity was analyzed by gel electrophoresis and mass spectrometry. Results Consistent with previous studies, HDL hydrolysis by sPLA2 slightly reduced the particle size without release of protein. SDS PAGE of hydrolyzed HDL showed no significant changes in the protein composition. Interestingly, delipidation of sPLA2-hydrolyzed HDL showed that nearly 10% of apoA-I formed well-defined fragments of 6-15 kDa. Incubation of sPLA2 with lipid-free apoA-I produces very similar fragments, indicating direct action of sPLA2 on apoA-I. Similar incubation of sPLA2 with other HDL proteins, apoA-II and apoC-I, did not lead to proteolysis, indicating enzymatic specificity of sPLA2 for apoA-I. ApoA-I hydrolysis by sPLA2 was Ca-independent and hence, occurred via a different mechanism than the phospholipid hydrolysis by sPLA2 that is Ca-dependent. Mass spectrometry analysis of isolated proteolytic fragments indicated several N-and C-terminal fragments of apoA-I, suggesting at least two cleavage sites in the central domain of apoA-I. Conclusion We have identified a previously unknown proteolytic activity of sPLA2 which is specific to apoA-I and is independent of the apoA-I lipidation. This novel enzymatic activity of sPLA2 helps explain enhanced catabolism of HDL in vivo, and is expected to contribute to the pro-atherogenic action of this pro-inflammatory enzyme.

Abstract 3: Apolipoprotein A-I Inhibits Streptococcal Cell Wall-Induced Arthritis in the Rat in an ABCA1-dependent Manner

Introduction. Arthritis is a chronic inflammatory disease characterized by joint inflammation and destruction, reduced high-density lipoprotein (HDL) levels, and increased cardiovascular risk. Objective To determine if apolipoprotein (apo) A-I, the main HDL apolipoprotein, prevents joint inflammation in arthritis. Methods and Results In vivo: Arthritis was induced in female Lewis rats with a single 15 mg/kg intraperitoneal streptococcal cell wall peptidoglycan-polysaccharide (PG-PS) injection and quantified as a combined forepaw and hindpaw inflammation score. Arthritis progressed from an initial, acute phase of joint inflammation during the first 4 days post-PG-PS administration to remission by day 8, followed by chronic joint inflammation up to sacrifice at day 21. Two intravenous infusions of lipid-free apoA-I (8 mg/kg) 24 h pre- and 24 h post-PG-PS injection reduced the acute and chronic joint inflammation by 63±9% at day 3 and by 61±8% at day 21. Infusion of apoA-I at days 7, 9 and 11 post-PG-PS injection reduced the chronic response by 43±11% at day 21. ApoA-I infusions at 24 h prior to and at days 1, 7, 9, 11 post-PG-PS injection reduced joint inflammation by 61±5% at day 3 and by 90±5% at day 21 (p<0.05 for all vs saline infusion). These beneficial effects of apoA-I were accompanied by a reduced inflammatory white blood cell count, reduced pro-inflammatory cytokine levels in synovial fluid, and reduced macrophage accumulation, toll-like receptor 2 (TLR2) and inflammatory cytokine expression in synovial tissue. In vitro: Human monocyte-derived macrophages (HMDMs) were pre-incubated with lipid-free apoA-I, then stimulated with PG-PS (20 μg/mL). Pre-incubation with apoA-I inhibited PG-PS-induced TLR2 and MyD88, a TLR2 adapter protein, expression. Nuclear factor-κB activation and pro-inflammatory cytokine production were also attenuated. These anti-inflammatory effects of apoA-I were abolished in HMDMs transfected with ATP-binding cassette transporter 1 (ABCA1) siRNA. Conclusions These findings establish that apoA-I attenuates PG-PS induced arthritis in the rat. These effects may involve ABCA-1 and inhibition of TLR2 expression and activation.

Factors controlling nascent high‐density lipoprotein particle heterogeneity: ATP‐binding cassette transporter A1 activity and cell lipid and apolipoprotein AI availability

Nascent high-density lipoprotein (HDL) particles arise in different sizes. We have sought to uncover factors that control this size heterogeneity. Gel filtration, native PAGE, and protein cross-linking were used to analyze the size heterogeneity of nascent HDL produced by BHK-ABCA1, RAW 264.7, J774, and HepG2 cells under different levels of two factors considered as a ratio, the availability of apolipoprotein AI (apoAI) -accessible cell lipid, and concentration of extracellular lipid-free apoAI. Increases in the available cell lipid:apoAI ratio due to either elevated ATP-binding cassette transporter A1 (ABCA1) expression and activity or raised cell density (i.e., increasing numerator) shifted the production of nascent HDL from smaller particles with fewer apoAI molecules per particle and fewer molecules of choline-phospholipid and cholesterol per apoAI molecule to larger particles that contained more apoAI and more lipid per molecule of apoAI. A further shift to larger particles was observed in BHK-ABCA1 cells when the available cell lipid:apoAI ratio was raised still higher by decreasing the apoAI concentration (i.e., the denominator). These changes in nascent HDL biogenesis were reminiscent of the transition that occurs in the size composition of reconstituted HDL in response to an increasing initial lipid:apoAI molar ratio. Thus, the ratio of available cell lipid:apoAI is a fundamental cause of nascent HDL size heterogeneity, and rHDL formation is a good model of nascent HDL biogenesis.—Lyssenko, N. N., Nickel, M., Tang, C., Phillips, M. C. Factors controlling nascent high-density lipoprotein particle heterogeneity: ATP-binding cassette transporter A1 activity and cell lipid and apolipoprotein AI availability.

Effects of Low-Density Lipoproteins on Endothelium-Generation from Human Peripheral Blood Mononuclear Cells

Human peripheral blood mononuclear cells (PBMCs) are capable of rapidly transforming into endothelial-like cells after adhering to preexisting endothelium. This represents a potential source of endothelium repair. We have previously shown that Apolipoprotein (apo) A-I enhances PBMC-based endothelium-generation. The current study investigated the effect of low-density lipoproteins (LDL) on this process. PBMCs obtained from healthy HLA-A2+ donors were co-cultured with HLA-A2− human umbilical vein endothelial cells, thus allowing the PBMC-derived cells to be tracked and quantified by their HLA-A2 expression. The co-cultures were exposed to LDL at a protein concentration of 0.5 mg/ml. These were compared with co-cultures exposed to lipid-free apoA-I (0.5 mg/ml) and controls that received equal volume of buffered saline. LDL was isolated from human serum within seven days of the experiments. The cell layers were analysed by dual-color flow cytometry up to day three of co-culture. Endothelium transformation of the adherent PBMCs was evident by the up-regulation of the endothelial antigen CD105 and the down-regulation of the monocyte antigen CD11b. This was not affected by exposure to LDL or apoA-I. Exposure of the co-culture to LDL decreased the proportion of PBMC-derived endothelial cells from 10.6 ± 1% to 4.4 ± 0.2% (P < 0.01). In contrast, exposure of apoA-I increased the proportion of PBMC-derived endothelial cells from 11.5 ± 3.9% to 25 ± 4/6% (P < 0.01). In conclusion, LDL substantially impaired PBMC-based generation of endothelium whereas apoA-I has the opposite effect. This PBMC-based endothelium-generating mechanism has important implications in terms of the role of HDL in protecting against and the role of LDL in promoting the development of atherosclerosis.

Myeloperoxidase-derived oxidants modify apolipoprotein A-I and generate dysfunctional high-density lipoproteins: comparison of hypothiocyanous acid (HOSCN) with hypochlorous acid (HOCl)

Oxidative modification of HDLs (high-density lipoproteins) by MPO (myeloperoxidase) compromises its anti-atherogenic properties, which may contribute to the development of atherosclerosis. Although it has been established that HOCl (hypochlorous acid) produced by MPO targets apoA-I (apolipoprotein A-I), the major apolipoprotein of HDLs, the role of the other major oxidant generated by MPO, HOSCN (hypothiocyanous acid), in the generation of dysfunctional HDLs has not been examined. In the present study, we characterize the structural and functional modifications of lipid-free apoA-I and rHDL (reconstituted discoidal HDL) containing apoA-I complexed with phospholipid, induced by HOSCN and its decomposition product, OCN- (cyanate). Treatment of apoA-I with HOSCN resulted in the oxidation of tryptophan residues, whereas OCN- induced carbamylation of lysine residues to yield homocitrulline. Tryptophan residues were more readily oxidized on apoA-I contained in rHDLs. Exposure of lipid-free apoA-I to HOSCN and OCN- significantly reduced the extent of cholesterol efflux from cholesterol-loaded macrophages when compared with unmodified apoA-I. In contrast, HOSCN did not affect the anti-inflammatory properties of rHDL. The ability of HOSCN to impair apoA-I-mediated cholesterol efflux may contribute to the development of atherosclerosis, particularly in smokers who have high plasma levels of SCN- (thiocyanate).

Identification of sites in apolipoprotein A-I susceptible to chymase and carboxypeptidase A digestion

MCs (mast cells) adversely affect atherosclerosis by promoting the progression of lesions and plaque destabilization. MC chymase cleaves apoA-I (apolipoprotein A-I), the main protein component of HDL (high-density lipoprotein). We previously showed that C-terminally truncated apoA-I (cleaved at the carboxyl side of Phe225) is present in normal human serum using a newly developed specific mAb (monoclonal antibody). In the present study, we aimed to identify chymase-induced cleavage sites in both lipid-free and lipid-bound (HDL3) forms of apoA-I. Lipid-free apoA-I was preferentially digested by chymase, at the C-terminus rather than the N-terminus. Phe229 and Tyr192 residues were the main cleavage sites. Interestingly, the Phe225 residue was a minor cleavage site. In contrast, the same concentration of chymase failed to digest apoA-I in HDL3; however, a 100-fold higher concentration of chymase modestly digested apoA-I in HDL3 at only the N-terminus, especially at Phe33. CPA (carboxypeptidase A) is another MC protease, co-localized with chymase in severe atherosclerotic lesions. CPA, in vitro, further cleaved C-terminal Phe225 and Phe229 residues newly exposed by chymase, but did not cleave Tyr192. These results indicate that several forms of C-terminally and N-terminally truncated apoA-I could exist in the circulation. They may be useful as new biomarkers to assess the risk of CVD (cardiovascular disease).