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Introduction: Gene therapy using a helper-dependent adenovirus (HDAd) to express rabbit apolipoprotein (apo) AI in carotids of fat-fed rabbits reduces atherosclerosis. We hypothesize that transgenic apoAI promotes reverse cholesterol transport from the artery wall; however, this could not be tested because the transgenic rabbit apoAI is indistinguishable from endogenous rabbit apoAI. We therefore constructed an HDAd expressing GFP-tagged rabbit apoAI. Methods: We constructed HDAdGFPapoAI by fusing the GFP sequence to the N-terminus of the rabbit APOA1 gene with a short linker. Bovine aortic endothelial cells (BAEC) were transduced with HDAdGFPapoAI, washed, and incubated in serum-free medium for 24h. Conditioned medium (CM) was collected and fluorescence measured. Cell extracts and CM were analyzed by western blotting for protein size, expression level, and presence of GFP and apoAI antigens. CM cholesterol-efflux activity was measured using cholesterol-loaded BHK cells expressing ABCA1. Results: CM of HDAdGFPapoAI-transduced BAEC had green fluorescence and contained a protein of the size predicted for the GFP-apoAI fusion (~56 kDa), which was detected with antibodies to both GFP and apoAI. A smaller protein (~30 kDa) with GFP and apoAI antigens was also detected. Compared to a vector expressing WT rabbit apoAI, GFP-tagging decreased expression of apoAI protein by ~50% and the percentage of secreted apoAI from 55% to 13%. In combination, this reduced secreted GFP-apoAI to only 12% of levels of secreted WT apoAI. While CM containing WT rabbit apoAI strongly stimulated cholesterol efflux from BHK cells (46%), GFP-apoAI-containing CM did not promote cholesterol efflux compared to CM from untransduced BAEC (7.8% vs 7.4% efflux), even when present in CM at a concentration similar to that of WT apoAI. Conclusion: GFP-rabbit apoAI protein has reduced expression and secretion (likely due to misfolding/degradation) and does not promote cholesterol efflux (n-terminal GFP may disrupt the phospholipid/cholesterol organization needed for efflux via ABCA1 to this dynamic apoprotein). It will not be useful to investigate mechanisms of atheroprotection by vessel wall-expressed apoAI. Alternatives include use of a smaller tag or expression of human apoAI.

Atherosclerosis is a disease of large- and medium-sized arteries that is caused by cholesterol accumulation in arterial intimal cells, including macrophages and smooth muscle cells (SMC). Cholesterol accumulation in these cells can be prevented or reversed in preclinical models—and atherosclerosis reduced—by transgenesis that increases expression of molecules that control cholesterol efflux, including apolipoprotein AI (apoAI) and ATP-binding cassette subfamily A, member 1 (ABCA1). In a previous work, we showed that transduction of arterial endothelial cells (EC)—with a helper-dependent adenovirus (HDAd) expressing apoAI—enhanced EC cholesterol efflux in vitro and decreased atherosclerosis in vivo. Similarly, overexpression of ABCA1 in cultured EC increased cholesterol efflux and decreased inflammatory gene expression. These EC-targeted gene-therapy strategies might be improved by concurrent upregulation of cholesterol-efflux pathways in other intimal cell types. Here, we report modification of this strategy to enable delivery of therapeutic nucleic acids to cells of the sub-endothelium. We constructed an HDAd (HDAdXMoAntimiR33a5p) that expresses an antagomiR directed at miR-33a-5p (a microRNA that suppresses cholesterol efflux by silencing ABCA1). HDAdXMoAntimiR33a5p contains a sequence motif that enhances uptake of anti-miR-33a-5p into exosomes. Cultured EC release exosomes containing small RNA, including miR-33a-5p. After transduction with HDAdXMoAntimiR33a5p, EC-derived exosomes containing anti-miR-33a-5p accumulate in conditioned medium (CM). When this CM is added to macrophages or SMC, anti-miR-33a-5p is detected in these target cells. Exosome-mediated transfer of anti-miR-33a-5p reduces miR-33a-5p by ∼65–80%, increases ABCA1 protein by 1.6–2.2-fold, and increases apoAI-mediated cholesterol efflux by 1.4–1.6-fold (all p ≤ 0.01). These effects were absent in macrophages and SMC incubated in exosome-depleted CM. EC transduced with HDAdXMoAntimiR33a5p release exosomes that can transfer anti-miR-33a-5p to other intimal cell types, upregulating cholesterol efflux from these cells. This strategy provides a platform for genetic modification of intimal and medial cells, using a vector that transduces only EC.

Production of high density lipoprotein (HDL) requires ATP-binding cassette transporter A1 (ABCA1) to drive phospholipid (PL) from the plasma membrane into extracellular apolipoprotein A-I. Here, we use simulations to show that domains of ABCA1 within the plasma membrane remove PL from the membrane’s outer leaflet. In our simulations, after the lipid diffuses into the interior of ABCA1’s outward-open cavity, PL extracted by the gateway passes through a ring-shaped domain, the annulus orifice, which forms the base of an elongated hydrophobic tunnel in the transporter’s extracellular domain. Engineered mutations in the gateway and annulus strongly inhibit lipid export by ABCA1 without affecting cell-surface expression levels. Our finding that ABCA1 extracts lipid from the outer face of the plasma membrane and forces it through its gateway and annulus into an elongated hydrophobic tunnel contrasts with the alternating access model, which proposes that ABCA1 flops PL substrate from the inner leaflet to the outer leaflet of the membrane. Consistent with our model, ABCA1 lacks the charged amino acid residues in the transmembrane domain found in the floppase members of the ABC transporter family.

TheAPOEε4 allele is the strongest genetic risk factor for late-onset Alzheimer's disease (AD). ApoE protein aggregation plays a central role in AD pathology, including the accumulation of β-amyloid (Aβ). Lipid-poor ApoE4 protein is prone to aggregate and lipidating ApoE4 protects it from aggregation. The mechanisms regulating ApoE4 aggregationin vivoare surprisingly not known. ApoE lipidation is controlled by the activity of the ATP binding cassette A1 (ABCA1). ABCA1 recycling and degradation is regulated by ADP-ribosylation factor 6 (ARF6). We found that ApoE4 promoted greater expression of ARF6 compared with ApoE3, trapping ABCA1 in late-endosomes and impairing its recycling to the cell membrane. This was associated with lower ABCA1-mediated cholesterol efflux activity, a greater percentage of lipid-free ApoE particles, and lower Aβ degradation capacity. Human CSF fromAPOEε4/ε4 carriers showed a lower ability to induce ABCA1-mediated cholesterol efflux activity and greater percentage of aggregated ApoE protein compared with CSF fromAPOEε3/ε3 carriers. Enhancing ABCA1 activity rescued impaired Aβ degradation in ApoE4-treated cells and reduced both ApoE and ABCA1 aggregation in the hippocampus of male ApoE4-targeted replacement mice. Together, our data demonstrate that aggregated and lipid-poor ApoE4 increases ABCA1 aggregation and decreases ABCA1 cell membrane recycling. Enhancing ABCA1 activity to reduce ApoE and ABCA1 aggregation is a potential therapeutic strategy for the prevention of ApoE4 aggregation-driven pathology.SIGNIFICANCE STATEMENTApoE protein plays a key role in the formation of amyloid plaques, a hallmark of Alzheimer's disease (AD). ApoE4 is more aggregated and hypolipidated compared with ApoE3, but whether enhancing ApoE lipidationin vivocan reverse ApoE aggregation is not known. ApoE lipidation is controlled by the activity of the ATP binding cassette A1 (ABCA1). In this study, we demonstrated that the greater propensity of lipid-poor ApoE4 to aggregate decreased ABCA1 membrane recycling and its ability to lipidate ApoE. Importantly, enhancing ABCA1 activity to lipidate ApoE reduced ApoE and ABCA1 aggregation. This work provides critical insights into the interactions among ABCA1, ApoE lipidation and aggregation, and underscores the promise of stabilizing ABCA1 activity to prevent ApoE-driven aggregation pathology.

Objective: Expression of the extrahepatic acute-phase protein Saa3 (serum amyloid A3) increases in response to acute and chronic inflammatory stimuli and is elevated in adipose tissue and macrophages in obese mice. A recent report suggested that Saa3 is proatherogenic in male ApoE −/− mice. Because of our previous observation that female but not male Saa3-deficient mice are protected from obesity, adipose inflammation, and hyperlipidemia, we sought to determine whether Saa3 differentially modulates atherosclerosis in mice of both sexes. Approach and Results: To promote atherosclerosis, Saa3 +/+ and Saa3 −/− male and female mice were crossed with Ldlr −/− mice. All mice consumed a diet high in saturated fat and sucrose with 0.15% added cholesterol for 16 weeks. Plasma lipids and atherosclerosis levels were assessed. Female Saa3 −/− Ldlr −/− mice exhibited elevated cholesterol levels relative to Saa3 +/+ Ldlr −/− controls and exhibited increased atherosclerosis, while male Saa3 −/− Ldlr −/− mice were protected from atherosclerosis. Data from the hybrid mouse diversity panel revealed that Saa3 associates strongly with inflammatory, Trem2-associated, and tissue remodeling genes and pathways in males but not females, an effect confirmed in liver tissue, atherosclerotic lesions, and cultured macrophages. Macrophages isolated from male and female mice showed differential inflammatory effects of Saa3 deficiency, an effect linked with sex steroid signaling. Cholesterol efflux capacity was increased in Saa3 −/− males only. Conclusions: Saa3 is proatherogenic in male but atheroprotective in female mice, effects that may be related to sex-specific relationships between Saa3, cholesterol metabolism, inflammatory genes, and Trem2 macrophages.

Rationale: HDL (high-density lipoprotein) may be cardioprotective because it accepts cholesterol from macrophages via the cholesterol transport proteins ABCA1 (ATP-binding cassette transporter A1) and ABCG1 (ATP-binding cassette transporter G1). The ABCA1-specific cellular cholesterol efflux capacity (ABCA1 CEC) of HDL strongly and negatively associates with cardiovascular disease risk, but how diabetes mellitus impacts that step is unclear. Objective: To test the hypothesis that HDL’s cholesterol efflux capacity is impaired in subjects with type 2 diabetes mellitus. Methods and Results: We performed a case-control study with 19 subjects with type 2 diabetes mellitus and 20 control subjects. Three sizes of HDL particles, small HDL, medium HDL, and large HDL, were isolated by high-resolution size exclusion chromatography from study subjects. Then we assessed the ABCA1 CEC of equimolar concentrations of particles. Small HDL accounted for almost all of ABCA1 CEC activity of HDL. ABCA1 CEC―but not ABCG1 CEC―of small HDL was lower in the subjects with type 2 diabetes mellitus than the control subjects. Isotope dilution tandem mass spectrometry demonstrated that the concentration of SERPINA1 (serpin family A member 1) in small HDL was also lower in subjects with diabetes mellitus. Enriching small HDL with SERPINA1 enhanced ABCA1 CEC. Structural analysis of SERPINA1 identified 3 amphipathic α-helices clustered in the N-terminal domain of the protein; biochemical analyses demonstrated that SERPINA1 binds phospholipid vesicles. Conclusions: The ABCA1 CEC of small HDL is selectively impaired in type 2 diabetes mellitus, likely because of lower levels of SERPINA1. SERPINA1 contains a cluster of amphipathic α-helices that enable apolipoproteins to bind phospholipid and promote ABCA1 activity. Thus, impaired ABCA1 activity of small HDL particles deficient in SERPINA1 could increase cardiovascular disease risk in subjects with diabetes mellitus.

HDLs are nanoparticles with more than 80 associated proteins, phospholipids, cholesterol, and cholesteryl esters. The potential inverse relation of HDL to coronary artery disease (CAD) and the effects of HDL on myriad other inflammatory conditions warrant a better understanding of the genetic basis of the HDL proteome. We conducted a comprehensive genetic analysis of the regulation of the proteome of HDL isolated from a panel of 100 diverse inbred strains of mice (the hybrid mouse diversity panel) and examined protein composition and efflux capacity to identify novel factors that affect the HDL proteome. Genetic analysis revealed widely varied HDL protein levels across the strains. Some of this variation was explained by local cis-acting regulation, termed cis-protein quantitative trait loci (QTLs). Variations in apoA-II and apoC-3 affected the abundance of multiple HDL proteins, indicating a coordinated regulation. We identified modules of covarying proteins and defined a protein-protein interaction network that describes the protein composition of the naturally occurring subspecies of HDL in mice. Sterol efflux capacity varied up to 3-fold across the strains, and HDL proteins displayed distinct correlation patterns with macrophage and ABCA1-specific cholesterol efflux capacity and cholesterol exchange, suggesting that subspecies of HDL participate in discrete functions. The baseline and stimulated sterol efflux capacity phenotypes were associated with distinct QTLs with smaller effect size, suggesting a multigenetic regulation. Our results highlight the complexity of HDL particles by revealing the high degree of heterogeneity and intercorrelation, some of which is associated with functional variation, and support the concept that HDL-cholesterol alone is not an accurate measure of HDL's properties, such as protection against CAD.

Background: The microRNA miR-33a-5p impairs apoAI-mediated cholesterol efflux by silencing ABCA1 expression. Systemic injection of antagomirs to miR-33a-5p reduces atherosclerosis but can cause hepatic steatosis and hypertriglyceridemia. In contrast, local delivery of miR-33a-5p antagomirs to lipid-laden intimal cells [smooth muscle cells (SMC) and macrophages (Mϕ)] could potentially treat atherosclerosis without causing metabolic abnormalities. Helper-dependent adenoviral vectors (HDAd) provide durable transgene expression in endothelial cells (EC) in vivo; however, HDAd does not cross an intact large vessel EC layer and therefore cannot deliver antagomirs directly to SMC and Mϕ. We hypothesized that transducing EC with an HDAd expressing anti-miR-33a-5p (HDAdAnti) could result in packaging of anti-miR-33a-5p into EC-derived exosomes that, in turn, deliver anti-miR-33a-5p to neighboring SMC and Mϕ. Inhibition of miR-33a-5p in SMC and Mϕ would upregulate ABCA1, and enhance apoAI-mediated cholesterol efflux. Methods: We transduced cultured EC with either HDAdAnti or a control HDAd expressing a “scrambled” shRNA (HDAdScr), harvested conditioned medium (CM) from the EC, and treated SMC and Mϕ (some of which were cholesterol-loaded) with the CM. We measured levels of anti-miR-33a-5p and miR-33a-5p, ABCA1 protein, and apoAI-mediated cholesterol efflux in the treated SMC and Mϕ. Results: Exosomes containing anti-miR-33a-5p were present only in CM from HDAdAnti-transduced EC. Compared to Mϕ incubated with CM from EC transduced with HDAdScr, Mϕ incubated with CM from HDAdAnti-transduced EC had 80% lower levels of miR-33a-5p, a 2.2-fold increase in ABCA1 protein, and a 1.6-fold increase in apoAI-mediated cholesterol efflux (all P Conclusions: EC transduced with HDAdAnti release exosomes containing anti-miR-33a-5p. These exosomes transfer anti-miR-33a-5p to SMC and Mϕ, increasing cholesterol efflux. Application of this approach in vivo may accelerate vessel wall cholesterol efflux, preventing and reversing atherosclerosis.

Atherosclerosis, a disease of blood vessels, is driven by cholesterol accumulation and inflammation. Gene therapy that removes cholesterol from blood vessels and decreases inflammation is a promising approach for prevention and treatment of atherosclerosis. In previous work, we reported that helper-dependent adenoviral (HDAd) overexpression of apolipoprotein A-I (apoAI) in endothelial cells (ECs) increases cholesterol efflux in vitro and reduces atherosclerosis in vivo. However, the effect of HDAdApoAI on atherosclerosis is partial. To improve this therapy, we considered concurrent overexpression of ATP-binding cassette subfamily A, member 1 (ABCA1), a protein that is required for apoAI-mediated cholesterol efflux. Before attempting combined apoAI/ABCA1 gene therapy, we tested whether an HDAd that expresses ABCA1 (HDAdABCA1) increases EC cholesterol efflux, whether increased cholesterol efflux alters normal EC physiology, and whether ABCA1 overexpression in ECs has anti-inflammatory effects. HDAdABCA1 increased EC ABCA1 protein (∼3-fold; p

Rationale: Coronary endothelial dysfunction (ED)—an early marker of atherosclerosis—increases the risk of cardiovascular events. Objective: We tested the hypothesis that cholesterol efflux capacity and high-density lipoprotein (HDL) particle concentration predict coronary ED better than HDL-cholesterol (HDL-C). Methods and Results: We studied 80 subjects with nonobstructive (IMA ) were assessed with validated assays. Cholesterol efflux capacity and HDL-P IMA were both strong, inverse predictors of ED ( P P =0.005). After correction for HDL-C, both efflux capacity and HDL-P IMA remained significant predictors of ED status. HDL-P IMA explained cholesterol efflux capacity more effectively than HDL-C ( r =0.54 and 0.36, respectively). The efflux capacities of isolated HDL and serum HDL correlated strongly ( r =0.49). Conclusions: Cholesterol efflux capacity and HDL-P IMA are reduced in subjects with coronary ED, independently of HDL-C. Alterations in HDL-P IMA and HDL itself account for a much larger fraction of the variation in cholesterol efflux capacity than does HDL-C. A selective decrease in large HDL particles may contribute to impaired cholesterol efflux capacity in ED subjects. These observations support a role for HDL size, concentration, and function as markers—and perhaps mediators—of coronary atherosclerosis in humans.

Background High-density lipoproteins (HDL) modified by glycation have been reported to be dysfunctional. Little is known regarding the anti-inflammatory effects on adipocytes of glycated HDL. Aims We tested whether modification of HDL in vitro by glycolaldehyde (GAD), malondialdehyde (MDA) or glucose affected HDL's anti-inflammatory properties and ability to promote cholesterol efflux. To determine whether similar changes occur in vivo, we examined modifications of apolipoprotein A1 (APOA1) and APOA2 and anti-inflammatory and cholesterol efflux properties of HDL isolated from subjects with type 1 diabetes in poor glycemic control. Results In vitro modification with both GAD and MDA blunted HDL's ability to inhibit palmitate-induced inflammation and cholesterol efflux in adipocytes. Modification of HDL by glucose had little impact on HDL function, like the response using HDL isolated from subjects with diabetes. Mass spectrophotometric analysis revealed that lysine residues in APOA1 and APOA2 of HDL modified by GAD and MDA in vitro differed from those modified by glucose, which resembled that seen with HDL from patients with type1 diabetes. Conclusions Modification of lysine residues in HDL by GAD and MDA in vitro does not mirror the HDL glycation in vivo in patients with diabetes, but resembles HDL modified in vitro by glucose.

High-density lipoproteins (HDL) are nanoparticles with >80 associated proteins, phospholipids, cholesterol and cholesteryl esters. A comprehensive genetic analysis of the regulation of proteome of HDL isolated from a panel of 100 diverse inbred strains of mice, Hybrid Mouse Diversity Panel (HMDP), revealed widely varied HDL protein levels across the strains. Some of this variation was explained by local, cis-acting regulation, termed cis-protein quantitative trait loci. Variations in apolipoprotein A-II and apolipoprotein C-3 affected the abundance of multiple HDL proteins indicating a coordinated regulation. We identified modules of co-varying proteins and define a protein-protein interaction network describing the protein composition of the naturally occurring subspecies of HDL in mice. Sterol efflux capacity varied up to 3-fold across the strains and HDL proteins displayed distinct correlation patterns with macrophage and ABCA1 specific cholesterol efflux capacity and cholesterol exchange, suggesting that subspecies of HDL participate in discrete functions. The baseline and stimulated sterol efflux capacity phenotypes associated with distinct QTLs with smaller effect size suggesting a multi genetic regulation. Our results highlight the complexity of HDL particles by revealing high degree of heterogeneity and intercorrelation, some of which is associated with functional variation, supporting the concept that HDL-cholesterol alone is not an accurate measure of HDL’s properties such as protection against CAD.

Background: ABCA1 removes cholesterol from vascular wall cells via apoAI-mediated efflux, generating HDL that transports cholesterol to the liver for excretion. This process of reverse cholesterol transport is atheroprotective; therefore, strategies that increase vascular wall ABCA1 may prevent or reverse atherosclerosis. Cholesterol efflux mediated by ABCA1 and apoAI can also have anti-inflammatory effects; however, excess depletion of cellular cholesterol can cause cell stress and apoptosis. We tested whether transducing endothelial cells (EC) with a helper-dependent adenoviral vector that expresses ABCA1 (HDAdABCA1) enhances apoAI-mediated cholesterol efflux and reduces inflammatory markers without causing cellular toxicity. Methods: We cloned rabbit ABCA1, constructed HDAdABCA1, transduced bovine aortic EC (BAEC) with either HDAdABCA1 or empty vector (HDAdNull). We measured ABCA1 protein by immunoblotting and apoAI-mediated cholesterol efflux by loading EC with 3 [H] cholesterol, then adding apoAI protein to serum-free medium. We assessed EC phenotype using MTT (metabolic activity), BrdU (proliferation), wound-healing assay (migration), and flow cytometry (apoptosis). We measured ICAM-1, VCAM-1, IL-6, and TNFα mRNA in transduced EC by qRT-PCR both under basal conditions and after serum-starvation, addition of apoAI, and LPS challenge. Results: We observed a ~3-fold increase in ABCA1 protein in EC transduced with HDAdABCA1 and a ~2-fold increase in apoAI-mediated cholesterol efflux (P Conclusions: HDAdABCA1 increases EC ABCA1 expression and enhances apoAI-mediated cholesterol efflux, but does not cause toxicity or increase inflammatory markers. In contrast, ABCA1 overexpression appears to have anti-inflammatory effects. Future studies will test if HDAdABCA1 decreases lipid rafts, and whether overexpression of ABCA1 in EC in vivo is atheroprotective.

Objective: Niacin and omega-3 fatty acids are two therapeutic agents that have been extensively studied for their ability to reduce cardiovascular disease risk, but their effectiveness has more recently been called into question. In this study, we investigate whether these agents alone and in combination alter HDL function, in particular, HDL-apolipoprotein A-I exchange (HAE), a measure of HDL dynamics, and serum cholesterol efflux capacity (CEC). Approach: Fifty-six subjects with metabolic syndrome (MetSyn) were recruited to a double-blind trial and randomized to 16 weeks of treatment with dual placebo, extended release niacin (ERN, 2g/day), prescription omega-3 ethyl esters (P-OM3, 4g/day), or combination. HDL function was assessed at baseline and following 16 weeks of treatment by measuring HAE, macrophage CEC, and ABCA1-specific CEC. Results: Compared to placebo, ERN and P-OM3 alone significantly increased HAE by 15.1 [8.2, 22.0] (pP P =0.002). When evaluated by HAE:apoA-I ratio (a measure of apoA-I specific activity), ERN increased apoA-I specific activity by 20.1% [4.8, 36.9] ( P =0.008), P-OM3 by 30.1% [14.4, 45.9] ( P< 0.0001), however with combination there was no increase, 9% [-6.6, 26.6] ( P =0.34). Triglyceride-adjusted macrophage CEC showed marginally significant increases with P-OM3 therapy ( P =0.05). No therapy significantly improved ABCA1-specific CEC. Conclusions: Much of the effect of ERN on HDL function can be attributed to this therapy raising apoA-I levels, but P-OM3 raises HDL function by independent means, increasing apoA-I specific activity. Future investigation is needed to determine whether interaction between ERN and P-OM3 therapies in combination reduces their overall effectiveness.

Low-grade chronic inflammation plays an important role in the pathogenesis of obesity-induced insulin resistance. ABCA1 is essential for reverse cholesterol transport and HDL synthesis, and protects against macrophage inflammation. In the present study, the effects of ABCA1 deficiency in hematopoietic cells on diet-induced inflammation and insulin resistance were tested in vivo using bone marrow transplanted (BMT)-WT and BMT-ABCA1(-/-) mice. When challenged with a high-fat high-carbohydrate diabetogenic diet with added cholesterol (HFHSC), BMT-ABCA1(-/-) mice displayed enhanced insulin resistance and impaired glucose tolerance as compared with BMT-WT mice. The worsened insulin resistance and impaired glucose tolerance in BMT-ABCA1(-/-) mice were accompanied by increased macrophage accumulation and inflammation in adipose tissue and liver. Moreover, BMT-ABCA1(-/-) mice had significantly higher hematopoietic stem cell proliferation, myeloid cell expansion, and monocytosis when challenged with the HFHSC diet. In vitro studies indicated that macrophages from ABCA1(-/-) mice showed significantly increased inflammatory responses induced by saturated fatty acids. Taken together, these studies point to an important role for hematopoietic ABCA1 in modulating a feed-forward mechanism in obesity such that inflamed tissue macrophages stimulate the production of more monocytes, leading to an exacerbation of inflammation and associated disease processes.

HDL from healthy humans and lean mice inhibits palmitate-induced adipocyte inflammation; however, the effect of the inflammatory state on the functional properties of HDL on adipocytes is unknown. Here, we found that HDL from mice injected with AgNO3 fails to inhibit palmitate-induced inflammation and reduces cholesterol efflux from 3T3-L1 adipocytes. Moreover, HDL isolated from obese mice with moderate inflammation and humans with systemic lupus erythematosus had similar effects. Since serum amyloid A (SAA) concentrations in HDL increase with inflammation, we investigated whether elevated SAA is a causal factor in HDL dysfunction. HDL from AgNO3-injected mice lacking Saa1.1 and Saa2.1 exhibited a partial restoration of antiinflammatory and cholesterol efflux properties in adipocytes. Conversely, incorporation of SAA into HDL preparations reduced antiinflammatory properties but not to the same extent as HDL from AgNO3-injected mice. SAA-enriched HDL colocalized with cell surface–associated extracellular matrix (ECM) of adipocytes, suggesting impaired access to the plasma membrane. Enzymatic digestion of proteoglycans in the ECM restored the ability of SAA-containing HDL to inhibit palmitate-induced inflammation and cholesterol efflux. Collectively, these findings indicate that inflammation results in a loss of the antiinflammatory properties of HDL on adipocytes, which appears to partially result from the SAA component of HDL binding to cell-surface proteoglycans, thereby preventing access of HDL to the plasma membrane.

Recent studies demonstrate that HDL's ability to promote cholesterol efflux from macrophages associates strongly with cardioprotection in humans independently of HDL-cholesterol (HDL-C) and apoA-I, HDL's major protein. However, the mechanisms that impair cholesterol efflux capacity during vascular disease are unclear. Inflammation, a well-established risk factor for cardiovascular disease, has been shown to impair HDL's cholesterol efflux capacity. We therefore tested the hypothesis that HDL's impaired efflux capacity is mediated by specific changes of its protein cargo. Humans with acute inflammation induced by low-level endotoxin had unchanged HDL-C levels, but their HDL-C efflux capacity was significantly impaired. Proteomic analyses demonstrated that HDL's cholesterol efflux capacity correlated inversely with HDL content of serum amyloid A (SAA)1 and SAA2. In mice, acute inflammation caused a marked impairment of HDL-C efflux capacity that correlated with a large increase in HDL SAA. In striking contrast, the efflux capacity of mouse inflammatory HDL was preserved with genetic ablation of SAA1 and SAA2. Our observations indicate that the inflammatory impairment of HDL-C efflux capacity is due in part to SAA-mediated remodeling of HDL's protein cargo.

Cardiovascular disease is the leading cause of death in end-stage renal disease (ESRD) patients treated with hemodialysis. An important contributor might be a decline in the cardioprotective effects of high-density lipoprotein (HDL). One important factor affecting HDL's cardioprotective properties may involve the alterations of protein composition in HDL. In the current study, we used complementary proteomics approaches to detect and quantify relative levels of proteins in HDL isolated from control and ESRD subjects. Shotgun proteomics analysis of HDL isolated from 20 control and 40 ESRD subjects identified 63 proteins in HDL. Targeted quantitative proteomics by isotope-dilution selective reaction monitoring revealed that 22 proteins were significantly enriched and 6 proteins were significantly decreased in ESRD patients. Strikingly, six proteins implicated in renal disease, including B2M, CST3, and PTGDS, were markedly increased in HDL of uremic subjects. Moreover, several of these proteins (SAA1, apoC-III, PON1, etc.) have been associated with atherosclerosis. Our observations indicate that the HDL proteome is extensively remodeled in uremic subjects. Alterations of the protein cargo of HDL might impact HDL's proposed cardioprotective properties. Quantifying proteins in HDL may be useful in the assessment of cardiovascular risk in patients with ESRD and in assessing response to therapeutic interventions.

Using genetic and biochemical approaches, we investigated proteins that regulate macrophage cholesterol efflux capacity (CEC) and ABCA1-specific CEC (ABCA1 CEC), 2 functional assays that predict cardiovascular disease (CVD). Macrophage CEC and the concentration of HDL particles were markedly reduced in mice deficient in apolipoprotein A-I (APOA1) or apolipoprotein E (APOE) but not apolipoprotein A-IV (APOA4). ABCA1 CEC was markedly reduced in APOA1-deficient mice but was barely affected in mice deficient in APOE or APOA4. High-resolution size-exclusion chromatography of plasma produced 2 major peaks of ABCA1 CEC activity. The early-eluting peak, which coeluted with HDL, was markedly reduced in APOA1- or APOE-deficient mice. The late-eluting peak was modestly reduced in APOA1-deficient mice but little affected in APOE- or APOA4-deficient mice. Ion-exchange chromatography and shotgun proteomics suggested that plasminogen (PLG) accounted for a substantial fraction of the ABCA1 CEC activity in the peak not associated with HDL. Human PLG promoted cholesterol efflux by the ABCA1 pathway, and PLG-dependent efflux was inhibited by lipoprotein(a) [Lp(a)]. Our observations identify APOA1, APOE, and PLG as key determinants of CEC. Because PLG and Lp(a) associate with human CVD risk, interplay among the proteins might affect atherosclerosis by regulating cholesterol efflux from macrophages.

Objective Oxidative stress and oxidized high-density lipoprotein (HDL) are implicated as risk factors for cardiovascular disease (CVD) in systemic lupus erythematosus (SLE). Yet, how HDL is oxidized and rendered dysfunctional in SLE remains unclear. Neutrophil extracellular traps (NETs), the levels of which are elevated in lupus, possess oxidant-generating enzymes, including myeloperoxidase (MPO), NADPH oxidase (NOX), and nitric oxide synthase (NOS). We hypothesized that NETs mediate HDL oxidation, impairing cholesterol efflux capacity (CEC). Methods Plasma MPO levels and CEC activity were examined in controls and lupus patients, and 3-chlorotyrosine (MPO specific) and 3-nitrotyrosine (derived from reactive nitrogen species) were quantified in human HDL. Multivariable linear models were used to estimate and test differences between groups. HDL was exposed to NETs from control and lupus neutrophils in the presence or absence of MPO, NOX, NOS inhibitors, and chloroquine (CQ). Murine HDL oxidation was quantified after NET inhibition in vivo. Results SLE patients displayed higher MPO levels and diminished CEC compared to controls. SLE HDL had higher 3-nitrotyrosine and 3-chlorotyrosine content than control HDL, with site-specific oxidation signatures on apolipoprotein A-I. Experiments with human and murine NETs confirmed that chlorination was mediated by MPO and NOX, and nitration by NOS and NOX. Mice with lupus treated with the NET inhibitor Cl-amidine displayed significantly decreased HDL oxidation. CQ inhibited NET formation in vitro. Conclusion Active NOS, NOX, and MPO within NETs significantly modify HDL, rendering the lipoprotein proatherogenic. Since NET formation is enhanced in SLE, these findings support a novel role for NET-derived lipoprotein oxidation in SLE-associated CVD and identify additional proatherogenic roles of neutrophils and putative protective roles of antimalarials in autoimmunity.

Rationale: Macrophage accumulation in adipose tissue associates with insulin resistance and increased cardiovascular disease risk. We previously have shown that generation of reactive oxygen species and monocyte chemotactic factors after exposure of adipocytes to saturated fatty acids, such as palmitate, occurs via translocation of NADPH oxidase 4 into lipid rafts (LRs). The anti-inflammatory effects of apolipoprotein AI (apoAI) and high-density lipoprotein (HDL) on macrophages and endothelial cells seem to occur via cholesterol depletion of LRs. However, little is known concerning anti-inflammatory effects of HDL and apoAI on adipocytes. Objective: To determine whether apoAI and HDL inhibit inflammation in adipocytes and adipose tissue, and whether this is dependent on LRs. Methods and Results: In 3T3L-1 adipocytes, apoAI, HDL, and methyl-β-cyclodextrin inhibited chemotactic factor expression. ApoAI and HDL also disrupted LRs, reduced plasma membrane cholesterol content, inhibited NADPH oxidase 4 translocation into LRs, and reduced palmitate-induced reactive oxygen species generation and monocyte chemotactic factor expression. Silencing ATP-binding cassette A-1 abrogated the effect of apoAI, but not HDL, whereas silencing ATP-binding cassette G-1 or scavenger receptor B-1 abrogated the effect of HDL but not apoAI. In vivo, apoAI transgenic mice fed a high-fat, high-sucrose, cholesterol-containing diet showed reduced chemotactic factor and proinflammatory cytokine expression and reduced macrophage accumulation in adipose tissue. Conclusions: ApoAI and HDL have anti-inflammatory effects in adipocytes and adipose tissue similar to their effects in other cell types. These effects are consistent with disruption and removal of cholesterol from LRs, which are regulated by cholesterol transporters, such as ATP-binding cassette A-1, ATP-binding cassette G-1, and scavenger receptor B-1.

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.

Objective Phagocyte-derived myeloperoxidase (MPO) and pro-inflammatory high density lipoprotein (HDL) associate with rheumatoid arthritis (RA), but the link between MPO and HDL has not been systematically examined. In this study, we investigated whether MPO can oxidise HDL and determined MPO-specific oxidative signature by apoA-1 by peptide mapping in RA subjects with and without known cardiovascular disease (CVD). Methods Two MPO oxidation products, 3-chlorotyrosine and 3-nitrotyrosine, were quantified by tandem mass spectrometry (MS/MS) in in vitro model system studies and in plasma and HDL derived from healthy controls and RA subjects. MPO levels and cholesterol efflux were determined. Site-specific nitration and chlorination of apoA-1 peptides were quantified by MS/MS. Results RA subjects demonstrated higher levels of MPO, MPO-oxidised HDL and diminished cholesterol efflux. There was marked increase in MPO-specific 3-chlorotyrosine and 3-nitrotyrosine content in HDL in RA subjects consistent with specific targeting of HDL, with increased nitration in RA subjects with CVD. Cholesterol efflux capacity was diminished in RA subjects and correlated inversely with HDL 3-chlorotyrosine suggesting a mechanistic role for MPO. Nitrated HDL was elevated in RACVD subjects compared with RA subjects without CVD. Oxidative peptide mapping revealed site-specific unique oxidation signatures on apoA-1 for RA subjects with and without CVD. Conclusions We report an increase in MPO-mediated HDL oxidation that is regiospecific in RA and accentuated in those with CVD. Decreased cholesterol efflux capacity due to MPO-mediated chlorination is a potential mechanism for atherosclerosis in RA and raises the possibility that oxidant resistant forms of HDL may attenuate this increased risk.

ATP-binding cassette transporter A1 (ABCA1) mediates formation of disc-shaped high-density lipoprotein (HDL) from cell lipid and lipid-free apolipoprotein A-I (apo A-I). Discoidal HDL particles are heterogeneous in physicochemical characteristics for reasons that are understood incompletely. Discoidal lipoprotein particles similar in characteristics and heterogeneity to cell-formed discoidal HDL can be reconstituted from purified lipids and apo A-I by cell-free, physicochemical methods. The heterogeneity of reconstituted HDL (rHDL) is sensitive to the lipid composition of the starting lipid/apo A-I mixture. To determine whether the heterogeneity of cell-formed HDL is similarly sensitive to changes in cell lipids, we investigated four compounds that have well-established effects on cell lipid metabolism and ABCA1-mediated cell cholesterol efflux. 2-Bromopalmitate, D609, monensin and U18666A decreased formation of the larger-sized, but dramatically increased formation of the smaller-sized HDL. 2-Bromopalmitate did not appear to affect ABCA1 activity, subcellular localization or oligomerization, but induced dissolution of the cholesterol-phospholipid complexes in the plasma membrane. Arachidonic and linoleic acids shifted HDL formation to the smaller-sized species. Tangier disease mutations and inhibitors of ABCA1 activity wheat germ agglutinin and AG 490 reduced formation of both larger-sized and smaller-sized HDL. The effect of probucol was similar to the effect of 2-bromopalmitate. Taking rHDL formation as a paradigm, we propose that ABCA1 mutations and activity inhibitors reduce the amount of cell lipid available for HDL formation, and the compounds in the 2-bromopalmitate group and the polyunsaturated fatty acids change cell lipid composition from one that favors formation of the larger-sized HDL particles to one that favors formation of the smaller-sized species.

The effects of androgens on cardiovascular disease (CVD) risk in men remain unclear. To better characterize the relationship between androgens and HDL, we investigated the effects of testosterone replacement on HDL protein composition and serum HDL-mediated cholesterol efflux in hypogonadal men. Twenty-three older hypogonadal men (ages 51–83, baseline testosterone < 280 ng/dl) were administered replacement testosterone therapy (1% transdermal gel) with or without the 5α-reductase inhibitor dutasteride. At baseline and after three months of treatment, we determined fasting lipid concentrations, HDL protein composition, and the cholesterol efflux capacity of serum HDL. Testosterone replacement did not affect HDL cholesterol (HDL-C) concentrations but conferred significant increases in HDL-associated paraoxonase 1 (PON1) and fibrinogen α chain (FGA) (P = 0.022 and P = 0.023, respectively) and a decrease in apolipoprotein A-IV (apoA-IV) (P = 0.016). Exogenous testosterone did not affect the cholesterol efflux capacity of serum HDL. No differences were observed between men who received testosterone alone and those who also received dutasteride. Testosterone replacement in older hypogonadal men alters the protein composition of HDL but does not significantly change serum HDL-mediated cholesterol efflux. These effects appear independent of testosterone conversion to dihydrotestosterone. Further research is needed to determine how changes in HDL protein content affect CVD risk in men.

Mifepristone is a glucocorticoid and progestin antagonist under investigation for the treatment of Cushing's syndrome. Mifepristone decreases high-density lipoprotein (HDL) cholesterol (HDL-C) levels in treated patients, but the clinical significance of this is unclear because recent studies suggest that functional properties of HDL predict cardiovascular disease status better than does HDL-C concentration.The aim of the study was to characterize the impact of mifepristone administration on HDL particle concentration and function.We conducted a double-blind, randomized, placebo-controlled trial at a single-site, clinical research center.Thirty healthy postmenopausal female volunteers participated in the study.Individuals were randomized to receive daily oral mifepristone (600 mg) or placebo for 6 wk.We measured HDL-C, serum HDL particle concentration, and HDL-mediated cholesterol efflux by treatment group.As expected, ACTH, cortisol, estradiol, and testosterone levels increased in the mifepristone group. Mifepristone treatment decreased HDL-C and HDL particle concentration by 26 and 25%, respectively, but did not alter pre-β HDL concentration. In contrast, the serum HDL-mediated cholesterol efflux decreased with mifepristone treatment by only 12%, resulting in an effective increase of the efflux capacity per HDL particle. No changes were observed in cholesterol ester transfer protein or lecithin:cholesterol acyltransferase activity.Treatment with mifepristone reduced HDL-C, HDL particle concentration, and serum HDL cholesterol efflux in postmenopausal women. However, on a per particle basis, the efflux capacity of serum HDL increased. These observations support the concept that a decrease in HDL-C may not represent proportional impairment of HDL function.

Exogenous androgens can lower HDL-cholesterol (HDL-C) concentrations, yet men with low serum testosterone have elevated rates of cardiovascular disease (CVD). HDL function may better predict CVD risk than absolute HDL-C quantity. We evaluated the acute effects of medical castration in men on HDL-C, cholesterol efflux capacity and HDL protein composition. Twenty-one healthy men, ages 18–55, received the GnRH antagonist acyline and one of the following for 28 days: Group 1: placebo, Group 2: transdermal testosterone gel and placebo, Group 3: transdermal testosterone gel and an aromatase inhibitor. Sex steroids, fasting lipids, and cholesterol efflux to apoB-depleted serum were measured in all subjects. The HDL proteome was assessed in Group 1 subjects only. In Group 1, serum testosterone concentrations were reduced by >95%, and HDL-C and cholesterol efflux capacity increased (p=0.02 and p=0.04 vs. baseline, respectively). HDL-associated clusterin increased significantly with sex steroid withdrawal (p=0.007 vs. baseline). Testosterone withdrawal in young, healthy men increases HDL-C and cholesterol efflux capacity. Moreover, sex steroid deprivation changes HDL protein composition. Further investigation of the effects of sex steroids on HDL composition and function may help resolve the apparently conflicting data regarding testosterone, HDL-C, and CVD risk.

Diabetes and insulin resistance increase the risk of cardiovascular disease caused by atherosclerosis through mechanisms that are poorly understood. Lipid-loaded macrophages are key contributors to all stages of atherosclerosis. We have recently shown that diabetes associated with increased plasma lipids reduces cholesterol efflux and levels of the reverse cholesterol transporter ABCA1 (ATP-binding cassette transporter A1) in mouse macrophages, which likely contributes to macrophage lipid accumulation in diabetes. Furthermore, we and others have shown that unsaturated fatty acids reduce ABCA1-mediated cholesterol efflux, and that this effect is mediated by the acyl-CoA derivatives of the fatty acids. We therefore investigated whether acyl-CoA synthetase 1 (ACSL1), a key enzyme mediating acyl-CoA synthesis in macrophages, could directly influence ABCA1 levels and cholesterol efflux in these cells. Mouse macrophages deficient in ACSL1 exhibited reduced sensitivity to oleate- and linoleate-mediated ABCA1 degradation, which resulted in increased ABCA1 levels and increased apolipoprotein A-I-dependent cholesterol efflux in the presence of these fatty acids, as compared with wildtype mouse macrophages. Conversely, overexpression of ACSL1 resulted in reduced ABCA1 levels and reduced cholesterol efflux in the presence of unsaturated fatty acids. Thus, the reduced ABCA1 and cholesterol efflux in macrophages subjected to conditions of diabetes and elevated fatty load may, at least in part, be mediated by ACSL1. These observations raise the possibility that ABCA1 levels could be increased by inhibition of acyl-CoA synthetase activity in vivo. 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).