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Rationale: Acute lung injury causes complex changes in protein expression in the lungs. Whereas most prior studies focused on single proteins, newer methods allowing the simultaneous study of many proteins could lead to a better understanding of pathogenesis and new targets for treatment.Objectives: The purpose of this study was to examine the changes in protein expression in the bronchoalveolar lavage fluid (BALF) of patients during the course of the acute respiratory distress syndrome (ARDS).Methods: Using two-dimensional difference gel electrophoresis (DIGE), the expression of proteins in the BALF from patients on Days 1 (n = 7), 3 (n = 8), and 7 (n = 5) of ARDS were compared with findings in normal volunteers (n = 9). The patterns of protein expression were analyzed using principal component analysis (PCA). Biological processes that were enriched in the BALF proteins of patients with ARDS were identified using Gene Ontology (GO) analysis. Protein networks that model the protein interactions in the BALF were generated using Ingenuity Pathway Analysis.Measurements and Main Results: An average of 991 protein spots were detected using DIGE. Of these, 80 protein spots, representing 37 unique proteins in all of the fluids, were identified using mass spectrometry. PCA confirmed important differences between the proteins in the ARDS and normal samples. GO analysis showed that these differences are due to the enrichment of proteins involved in inflammation, infection, and injury. The protein network analysis showed that the protein interactions in ARDS are complex and redundant, and revealed unexpected central components in the protein networks.Conclusions: Proteomics and protein network analysis reveals the complex nature of lung protein interactions in ARDS. The results provide new insights about protein networks in injured lungs, and identify novel mediators that are likely to be involved in the pathogenesis and progression of acute lung injury. [ABSTRACT FROM AUTHOR]

The ability of high-density lipoprotein (HDL) to promote cellular cholesterol efflux is a more robust predictor of cardiovascular disease protection than HDL-cholesterol levels in plasma. Previously, we found that lipidated HDL containing both apolipoprotein A-I (APOA1) and A-II (APOA2) promotes cholesterol efflux via the ATP-binding cassette transporter (ABCA1). In the current study, we directly added purified, lipid-free APOA2 to human plasma and found a dose-dependent increase in whole plasma cholesterol efflux capacity (CEC). APOA2 likewise increased the CEC of isolated HDL with the maximum effect occurring when equal masses of APOA1 and APOA2 coexisted on the particles. Follow-up experiments with reconstituted HDL corroborated that the presence of both APOA1 and APOA2 were necessary for the increased efflux. Using limited proteolysis and chemical cross-linking mass spectrometry, we found that APOA2 induced a conformational change in the N- and C-terminal helices of APOA1. Using reconstituted HDL with APOA1 deletion mutants, we further showed that APOA2 lost its ability to stimulate ABCA1 efflux to HDL if the C-terminal domain of APOA1 was absent, but retained this ability when the N-terminal domain was absent. Based on these findings, we propose a model in which APOA2 displaces the C-terminal helix of APOA1 from the HDL surface which can then interact with ABCA1 - much like it does in lipid-poor APOA1. These findings suggest APOA2 may be a novel therapeutic target given this ability to open a large, high-capacity pool of HDL particles to enhance ABCA1-mediated cholesterol efflux., Competing Interests: Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: The apolipoprotein E gene (APOE) is an established central player in the pathogenesis of Alzheimer's disease (AD), with distinct apoE isoforms exerting diverse effects. apoE influences not only amyloid-beta and tau pathologies but also lipid and energy metabolism, neuroinflammation, cerebral vascular health, and sex-dependent disease manifestations. Furthermore, ancestral background may significantly impact the link between APOE and AD, underscoring the need for more inclusive research., Methods: In 2023, the Alzheimer's Association convened multidisciplinary researchers at the "AAIC Advancements: APOE" conference to discuss various topics, including apoE isoforms and their roles in AD pathogenesis, progress in apoE-targeted therapeutic strategies, updates on disease models and interventions that modulate apoE expression and function., Results: This manuscript presents highlights from the conference and provides an overview of opportunities for further research in the field., Discussion: Understanding apoE's multifaceted roles in AD pathogenesis will help develop targeted interventions for AD and advance the field of AD precision medicine., Highlights: APOE is a central player in the pathogenesis of Alzheimer's disease. APOE exerts a numerous effects throughout the brain on amyloid-beta, tau, and other pathways. The AAIC Advancements: APOE conference encouraged discussions and collaborations on understanding the role of APOE., (© 2024 The Author(s). Alzheimer's & Dementia published by Wiley Periodicals LLC on behalf of Alzheimer's Association.)

Background: The cholesterol efflux capacity of high density lipoprotein (HDL) is negatively associated with cardiovascular risk. Small HDL particles account almost quantitatively for cholesterol efflux capacity, perhaps mediated through efflux of cholesterol and outer leaflet plasma membrane phospholipids by ABCA1 (ATP binding cassette subfamily A member 1). People with type 1 diabetes are at increased coronary artery disease (CAD) risk despite normal HDL-cholesterol concentrations. We therefore tested the hypothesis that small HDL particles (HDL-P)-rather than HDL-cholesterol-predict incident CAD in type 1 diabetes., Methods and Results: Incident CAD (CAD death, myocardial infarction, or coronary revascularization) was determined in 550 individuals with childhood-onset type 1 diabetes. HDL-P was quantified by calibrated ion mobility analysis and cholesterol efflux capacity was quantified with validated assays. During a median follow-up of 26 years, 36.5% of the participants developed incident CAD, for an incidence density of 181.3 per 10 000 person-years. In multivariable Cox models, neither HDL-cholesterol nor apolipoprotein A1 concentration was significantly associated with CAD risk. In contrast, higher extra-small HDL-P concentrations were significantly associated with decreased CAD risk (hazard ratio [HR], 0.26 [95% CI, 0.14-0.50]). Weaker associations were observed for total HDL-P (HR, 0.88 [95% CI, 0.83-0.93]), small HDL (HR, 0.83 [95% CI, 0.68-1.02]), medium HDL (HR, 0.79 [95% CI, 0.71-0.89]), and large HDL (HR, 0.72 [95% CI, 0.59-0.89]). Although cholesterol efflux capacity was negatively associated with incident CAD, this association was no longer significant after adjustment for total HDL-P., Conclusions: Lower concentrations of total HDL-P and HDL subpopulations were positively associated with incident CAD independently of HDL-cholesterol, apolipoprotein A1, and other common CVD risk factors. Extra-small HDL was a much stronger predictor of risk than the other HDLs. Our data are consistent with the proposal that extra-small HDL plays a critical role in cardioprotection in type 1 diabetes, mediated by macrophage cholesterol efflux by the ABCA1 pathway.

Altered apolipoprotein kinetics play a critical role in promoting dyslipidemia and atherogenesis. Human apolipoprotein kinetics have been extensively evaluated, but similar studies in mice are hampered by the lack of robust methods suitable for the small amounts of blood that can be collected at sequential time points from individual mice. We describe a targeted liquid chromatography tandem mass spectrometry method for simultaneously quantifying the stable isotope enrichment of several apolipoproteins represented by multiple peptides in serial blood samples (15 μl each) obtained after retro-orbital injection of 13 C 6 , 15 N 2 -lysine (Lys8) in mice. We determined apolipoprotein fractional clearance rates (FCRs) and production rates (PRs) in WT mice and in two genetic models widely used for atherosclerosis research, LDL receptor-deficient (Ldlr -/- ) and apolipoprotein E-deficient (Apoe -/- ) mice. Injection of Lys8 produced a unique and readily detectable mass shift of labeled compared with unlabeled peptides with sensitivity allowing robust kinetics analyses. Ldlr -/- mice showed slower FCRs of APOA1, APOA4, total APOB, APOB100, APOCs, APOE and APOM, while FCRs of APOA1, APOB100, APOC2, APOC3, and APOM were not lower in Apoe -/- mice versus WT mice. APOE PR was increased in Ldlr -/- mice, and APOB100 and APOA4 PRs were reduced in Apoe -/- mice. Thus, our method reproducibly quantifies plasma apolipoprotein kinetics in different mouse models. The method can easily be expanded to include a wide range of proteins in the same biospecimen and should be useful for determining the kinetics of apolipoproteins in animal models of human disease., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Competing Interests: Conflict of interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: K. E. B. serves on the scientific advisory board of Esperion Therapeutics, Inc.

Lipoproteins in cerebrospinal fluid (CSF) of the central nervous system (CNS) resemble plasma high-density lipoproteins (HDLs), which are a compositionally and structurally diverse spectrum of nanoparticles with pleiotropic functionality. Whether CSF lipoproteins (CSF-Lps) exhibit similar heterogeneity is poorly understood because they are present at 100-fold lower concentrations than plasma HDL. To investigate the diversity of CSF-Lps, we developed a sensitive fluorescent technology to characterize lipoprotein subspecies in small volumes of human CSF. We identified 10 distinctly sized populations of CSF-Lps, most of which were larger than plasma HDL. Mass spectrometric analysis identified 303 proteins across the populations, over half of which have not been reported in plasma HDL. Computational analysis revealed that CSF-Lps are enriched in proteins important for wound healing, inflammation, immune response, and both neuron generation and development. Network analysis indicated that different subpopulations of CSF-Lps contain unique combinations of these proteins. Our study demonstrates that CSF-Lp subspecies likely exist that contain compositional signatures related to CNS health.

Background: Helicobacter pylori (Hp) infects the stomach of 50% of the world's population. Importantly, chronic infection by this bacterium correlates with the appearance of several extra-gastric pathologies, including neurodegenerative diseases. In such conditions, brain astrocytes become reactive and neurotoxic. However, it is still unclear whether this highly prevalent bacterium or the nanosized outer membrane vesicles (OMVs) they produce, can reach the brain, thus affecting neurons/astrocytes. Here, we evaluated the effects of Hp OMVs on astrocytes and neurons in vivo and in vitro., Methods: Purified OMVs were characterized by mass spectrometry (MS/MS). Labeled OMVs were administered orally or injected into the mouse tail vein to study OMV-brain distribution. By immunofluorescence of tissue samples, we evaluated: GFAP (astrocytes), βIII tubulin (neurons), and urease (OMVs). The in vitro effect of OMVs in astrocytes was assessed by monitoring NF-κB activation, expression of reactivity markers, cytokines in astrocyte-conditioned medium (ACM), and neuronal cell viability., Results: Urease and GroEL were prominent proteins in OMVs. Urease (OMVs) was present in the mouse brain and its detection coincided with astrocyte reactivity and neuronal damage. In vitro, OMVs induced astrocyte reactivity by increasing the intermediate filament proteins GFAP and vimentin, the plasma membrane α V β 3 integrin, and the hemichannel connexin 43. OMVs also produced neurotoxic factors and promoted the release of IFNγ in a manner dependent on the activation of the transcription factor NF-κB. Surface antigens on reactive astrocytes, as well as secreted factors in response to OMVs, were shown to inhibit neurite outgrowth and damage neurons., Conclusions: OMVs administered orally or injected into the mouse bloodstream reach the brain, altering astrocyte function and promoting neuronal damage in vivo. The effects of OMVs on astrocytes were confirmed in vitro and shown to be NF-κB-dependent. These findings suggest that Hp could trigger systemic effects by releasing nanosized vesicles that cross epithelial barriers and access the CNS, thus altering brain cells., (© 2023. The Author(s).)

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., (© 2022. The Author(s).)

Atherosclerotic CVD is the major cause of death in patients with type 1 diabetes mellitus (T1DM). Alterations in the HDL proteome have been shown to associate with prevalent CVD in T1DM. We therefore sought to determine which proteins carried by HDL might predict incident CVD in patients with T1DM. Using targeted MS/MS, we quantified 50 proteins in HDL from 181 T1DM subjects enrolled in the prospective Coronary Artery Calcification in Type 1 Diabetes study. We used Cox proportional regression analysis and a case-cohort design to test associations of HDL proteins with incident CVD (myocardial infarction, coronary artery bypass grafting, angioplasty, or death from coronary heart disease). We found that only one HDL protein-SFTPB (pulmonary surfactant protein B)-predicted incident CVD in all the models tested. In a fully adjusted model that controlled for lipids and other risk factors, the hazard ratio was 2.17 per SD increase of SFTPB (95% confidence interval, 1.12-4.21, P = 0.022). In addition, plasma fractionation demonstrated that SFTPB is nearly entirely bound to HDL. Although previous studies have shown that high plasma levels of SFTPB associate with prevalent atherosclerosis only in smokers, we found that SFTPB predicted incident CVD in T1DM independently of smoking status and a wide range of confounding factors, including HDL-C, LDL-C, and triglyceride levels. Because SFTPB is almost entirely bound to plasma HDL, our observations support the proposal that SFTPB carried by HDL is a marker-and perhaps mediator-of CVD risk in patients with T1DM., Competing Interests: Conflict of interest Jay Heinecke is named as a coinventor on patents for using oxidation and protein markers to predict the risk of CVD. All other authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Because of its critical role in HDL formation, significant efforts have been devoted to studying apolipoprotein A-I (APOA1) structural transitions in response to lipid binding. To assess the requirements for the conformational freedom of its termini during HDL particle formation, we generated three dimeric APOA1 molecules with their termini covalently joined in different combinations. The dimeric (d)-APOA1C-N mutant coupled the C-terminus of one APOA1 molecule to the N-terminus of a second with a short alanine linker, whereas the d-APOA1C-C and d-APOA1N-N mutants coupled the C-termini and the N-termini of two APOA1 molecules, respectively, using introduced cysteine residues to form disulfide linkages. We then tested the ability of these constructs to generate reconstituted HDL by detergent-assisted and spontaneous phospholipid microsolubilization methods. Using cholate dialysis, we demonstrate WT and all APOA1 mutants generated reconstituted HDL particles of similar sizes, morphologies, compositions, and abilities to activate lecithin:cholesterol acyltransferase. Unlike WT, however, the mutants were incapable of spontaneously solubilizing short chain phospholipids into discoidal particles. We found lipid-free d-APOA1C-N and d-APOA1N-N retained most of WT APOA1's ability to promote cholesterol efflux via the ATP binding cassette transporter A1, whereas d-APOA1C-C exhibited impaired cholesterol efflux. Our data support the double belt model for a lipid-bound APOA1 structure in nascent HDL particles and refute other postulated arrangements like the "double super helix." Furthermore, we conclude the conformational freedom of both the N- and C-termini of APOA1 is important in spontaneous microsolubilization of bulk phospholipid but is not critical for ABCA1-mediated cholesterol efflux., Competing Interests: Conflict of interest The authors state no conflict of interest., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Loss-of-function mutations in the transcription factor CREB3L3 (CREBH) associate with severe hypertriglyceridemia in humans. CREBH is believed to lower plasma triglycerides by augmenting the activity of lipoprotein lipase (LPL). However, by using a mouse model of type 1 diabetes mellitus (T1DM), we found that greater liver expression of active CREBH normalized both elevated plasma triglycerides and cholesterol. Residual triglyceride-rich lipoprotein (TRL) remnants were enriched in apolipoprotein E (APOE) and impoverished in APOC3, an apolipoprotein composition indicative of increased hepatic clearance. The underlying mechanism was independent of LPL, as CREBH reduced both triglycerides and cholesterol in LPL-deficient mice. Instead, APOE was critical for CREBH's ability to lower circulating remnant lipoproteins because it failed to reduce TRL cholesterol in Apoe-/- mice. Importantly, individuals with CREB3L3 loss-of-function mutations exhibited increased levels of remnant lipoproteins that were deprived of APOE. Recent evidence suggests that impaired clearance of TRL remnants promotes cardiovascular disease in patients with T1DM. Consistently, we found that hepatic expression of CREBH prevented the progression of diabetes-accelerated atherosclerosis. Our results support the proposal that CREBH acts through an APOE-dependent pathway to increase hepatic clearance of remnant lipoproteins. They also implicate elevated levels of remnants in the pathogenesis of atherosclerosis in T1DM.

OBJECTIVE: Niacin therapy fails to reduce cardiovascular events in statin-treated subjects even though it increases plasma HDL-C (HDL [high-density lipoprotein] cholesterol) and decreases LDL-C (LDL [low-density lipoprotein] cholesterol) and triglyceride levels. To investigate potential mechanisms for this lack of cardioprotection, we quantified the HDL proteome of subjects in 2 niacin clinical trials: the CPC study (Carotid Plaque Composition) and the HDL Proteomics substudy of the AIM-HIGH trial (Atherothrombosis Intervention in Metabolic Syndrome with Low HDL/High Triglycerides). APPROACH AND RESULTS: Using targeted proteomics, we quantified levels of 31 HDL proteins from 124 CPC subjects and 120 AIM-HIGH subjects. The samples were obtained at baseline and after 1 year of statin monotherapy or niacin-statin combination therapy. Compared with statin monotherapy, niacin-statin combination therapy did not reduce HDL-associated apolipoproteins APOC1, APOC2, APOC3, and APOC4, despite significantly lowering triglycerides. In contrast, niacin markedly elevated HDL-associated PLTP (phospholipid transfer protein), CLU (clusterin), and HP/HPR (haptoglobin/haptoglobinrelated proteins; P≤0.0001 for each) in both the CPC and AIM-HIGH cohorts. CONCLUSIONS: The addition of niacin to statin therapy resulted in elevated levels of multiple HDL proteins linked to increased atherosclerotic risk, which might have compromised the cardioprotective effects associated with higher HDL-C levels and lower levels of LDL-C and triglycerides. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT00715273; NCT00880178; NCT00120289.

[Figure: see text].

Human high-density lipoproteins (HDLs) are a complex mixture of structurally related nanoparticles that perform distinct physiological functions. We previously showed that human HDL containing apolipoprotein A-I (APOA1) but not apolipoprotein A-II (APOA2), designated LpA-I, is composed primarily of two discretely sized populations. Here, we isolated these particles directly from human plasma by antibody affinity chromatography, separated them by high-resolution size-exclusion chromatography and performed a deep molecular characterization of each species. The large and small LpA-I populations were spherical with mean diameters of 109 Å and 91 Å, respectively. Unexpectedly, isotope dilution MS/MS with [ 15 N]-APOA1 in concert with quantitation of particle concentration by calibrated ion mobility analysis demonstrated that the large particles contained fewer APOA1 molecules than the small particles; the stoichiometries were 3.0 and 3.7 molecules of APOA1 per particle, respectively. MS/MS experiments showed that the protein cargo of large LpA-I particles was more diverse. Human HDL and isolated particles containing both APOA1 and APOA2 exhibit a much wider range and variation of particle sizes than LpA-I, indicating that APOA2 is likely the major contributor to HDL size heterogeneity. We propose a ratchet model based on the trefoil structure of APOA1 whereby the helical cage maintaining particle structure has two "settings"-large and small-that accounts for these findings. This understanding of the determinants of HDL particle size and protein cargo distribution serves as a basis for determining the roles of HDL subpopulations in metabolism and disease states., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

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.

Diabetes is now a pandemic disease. Moreover, a large number of people with prediabetes are at risk for developing frank diabetes worldwide. Both type 1 and type 2 diabetes increase the risk of atherosclerotic cardiovascular disease (CVD). Even with statin treatment to lower LDL cholesterol, patients with diabetes have a high residual CVD risk. Factors mediating the residual risk are incompletely characterized. An attractive hypothesis is that remnant lipoprotein particles (RLPs), derived by lipolysis from VLDL and chylomicrons, contribute to this residual risk. RLPs constitute a heterogeneous population of lipoprotein particles, varying markedly in size and composition. Although a universally accepted definition is lacking, for the purpose of this review we define RLPs as postlipolytic partially triglyceride-depleted particles derived from chylomicrons and VLDL that are relatively enriched in cholesteryl esters and apolipoprotein (apo)E. RLPs derived from chylomicrons contain apoB48, while those derived from VLDL contain apoB100. Clarity as to the role of RLPs in CVD risk is hampered by lack of a widely accepted definition and a paucity of adequate methods for their accurate and precise quantification. New specific methods for RLP quantification would greatly improve our understanding of their biology and role in promoting atherosclerosis in diabetes and other disorders., (© 2020 by the American Diabetes Association.)

Objective: A subset of people with long-standing type 1 diabetes (T1D) appears to be protected from microvascular and macrovascular complications. Previous studies have focused on improved abilities to respond to glucose and its downstream effects as protective mechanisms. It is unclear whether lipoproteins play a role in the vascular health of these people. We therefore determined whether HDL particle concentration, size, function, and/or protein composition associate with protection from vascular complications., Research Design and Methods: We studied two independent cross-sectional cohorts with T1D: the T1D Exchange Living Biobank ( n = 47) and the Joslin Medalist Study ( n = 100). Some of the subjects had vascular complications, whereas others never exhibited vascular complications, despite an average duration of diabetes in the cohorts of 45 years. We assessed HDL particle size and concentration by calibrated ion mobility analysis, the HDL proteome by targeted mass spectrometry, and HDL function ex vivo by quantifying cholesterol efflux capacity and inhibition of monocyte adhesion to endothelial cells., Results: In both cohorts, people without vascular complications exhibited significantly higher concentrations of medium-sized HDL particles (M-HDL) independently of total and HDL cholesterol levels. While no consistent differences in HDL functions were observed ex vivo, people without vascular complications had higher levels of HDL-associated paraoxonase 1 (PON1), an enzyme that inhibits atherosclerosis in animal models., Conclusions: Elevated concentrations of M-HDL particles and elevated levels of HDL-associated PON1 may contribute to long-term protection from the vascular complications of diabetes by pathways that are independent of total cholesterol and HDL cholesterol., (© 2019 by the American Diabetes Association.)

Objective: HDL (high-density lipoprotein) infusion reduces atherosclerosis in animal models and is being evaluated as a treatment in humans. Studies have shown either anti- or proinflammatory effects of HDL in macrophages, and there is no consensus on the underlying mechanisms. Here, we interrogate the effects of HDL on inflammatory gene expression in macrophages. Approach and Results: We cultured bone marrow-derived macrophages, treated them with reconstituted HDL or HDL isolated from APOA1 Tg ;Ldlr -/- mice, and challenged them with lipopolysaccharide. Transcriptional profiling showed that HDL exerts a broad anti-inflammatory effect on lipopolysaccharide-induced genes and proinflammatory effect in a subset of genes enriched for chemokines. Cholesterol removal by POPC (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine) liposomes or β-methylcyclodextrin mimicked both pro- and anti-inflammatory effects of HDL, whereas cholesterol loading by POPC/cholesterol-liposomes or acetylated LDL (low-density lipoprotein) before HDL attenuated these effects, indicating that these responses are mediated by cholesterol efflux. While early anti-inflammatory effects reflect reduced TLR (Toll-like receptor) 4 levels, late anti-inflammatory effects are due to reduced IFN (interferon) receptor signaling. Proinflammatory effects occur late and represent a modified endoplasmic reticulum stress response, mediated by IRE1a (inositol-requiring enzyme 1a)/ASK1 (apoptosis signal-regulating kinase 1)/p38 MAPK (p38 mitogen-activated protein kinase) signaling, that occurs under conditions of extreme cholesterol depletion. To investigate the effects of HDL on inflammatory gene expression in myeloid cells in atherosclerotic lesions, we injected reconstituted HDL into Apoe -/- or Ldlr -/- mice fed a Western-type diet. Reconstituted HDL infusions produced anti-inflammatory effects in lesion macrophages without any evidence of proinflammatory effects., Conclusions: Reconstituted HDL infusions in hypercholesterolemic atherosclerotic mice produced anti-inflammatory effects in lesion macrophages suggesting a beneficial therapeutic effect of HDL in vivo.

Background: Despite robust cholesterol lowering, cardiovascular disease risk remains increased in patients with diabetes mellitus. Consistent with this, diabetes mellitus impairs atherosclerosis regression after cholesterol lowering in humans and mice. In mice, this is attributed in part to hyperglycemia-induced monocytosis, which increases monocyte entry into plaques despite cholesterol lowering. In addition, diabetes mellitus skews plaque macrophages toward an atherogenic inflammatory M1 phenotype instead of toward the atherosclerosis-resolving M2 state typical with cholesterol lowering. Functional high-density lipoprotein (HDL), typically low in patients with diabetes mellitus, reduces monocyte precursor proliferation in murine bone marrow and has anti-inflammatory effects on human and murine macrophages. Our study aimed to test whether raising functional HDL levels in diabetic mice prevents monocytosis, reduces the quantity and inflammation of plaque macrophages, and enhances atherosclerosis regression after cholesterol lowering., Methods: Aortic arches containing plaques developed in Ldlr -/- mice were transplanted into either wild-type, diabetic wild-type, or diabetic mice transgenic for human apolipoprotein AI, which have elevated functional HDL. Recipient mice all had low levels of low-density lipoprotein cholesterol to promote plaque regression. After 2 weeks, plaques in recipient mouse aortic grafts were examined., Results: Diabetic wild-type mice had impaired atherosclerosis regression, which was normalized by raising HDL levels. This benefit was linked to suppressed hyperglycemia-driven myelopoiesis, monocytosis, and neutrophilia. Increased HDL improved cholesterol efflux from bone marrow progenitors, suppressing their proliferation and monocyte and neutrophil production capacity. In addition to reducing circulating monocytes available for recruitment into plaques, in the diabetic milieu, HDL suppressed the general recruitability of monocytes to inflammatory sites and promoted plaque macrophage polarization to the M2, atherosclerosis-resolving state. There was also a decrease in plaque neutrophil extracellular traps, which are atherogenic and increased by diabetes mellitus., Conclusions: Raising apolipoprotein AI and functional levels of HDL promotes multiple favorable changes in the production of monocytes and neutrophils and in the inflammatory environment of atherosclerotic plaques of diabetic mice after cholesterol lowering and may represent a novel approach to reduce cardiovascular disease risk in people with diabetes mellitus.

Type 1 diabetes mellitus (T1DM) increases the risk of atherosclerotic cardiovascular disease (CVD) in humans by poorly understood mechanisms. Using mouse models of T1DM-accelerated atherosclerosis, we found that relative insulin deficiency rather than hyperglycemia elevated levels of apolipoprotein C3 (APOC3), an apolipoprotein that prevents clearance of triglyceride-rich lipoproteins (TRLs) and their remnants. We then showed that serum APOC3 levels predict incident CVD events in subjects with T1DM in the Coronary Artery Calcification in Type 1 Diabetes (CACTI) study. To explore underlying mechanisms, we investigated the impact of Apoc3 antisense oligonucleotides (ASOs) on lipoprotein metabolism and atherosclerosis in a mouse model of T1DM. Apoc3 ASO treatment abolished the increased hepatic Apoc3 expression in diabetic mice - resulting in lower levels of TRLs - without improving glycemic control. APOC3 suppression also prevented arterial accumulation of APOC3-containing lipoprotein particles, macrophage foam cell formation, and the accelerated atherosclerosis in diabetic mice. Our observations demonstrate that relative insulin deficiency increases APOC3 and that this results in elevated levels of TRLs and accelerated atherosclerosis in a mouse model of T1DM. Because serum levels of APOC3 predicted incident CVD events in the CACTI study, inhibiting APOC3 might reduce CVD risk in T1DM patients.

Objective- Albuminuria is an important risk factor for cardiovascular disease in diabetes mellitus. We determined whether albuminuria associates with alterations in the proteome of HDL (high-density lipoprotein) of subjects with type 1 diabetes mellitus and whether those alterations associated with coronary artery calcification. Approach and Results- In a cross-sectional study of 191 subjects enrolled in the DCCT (Diabetes Control and Complications Trial)/EDIC study (Epidemiology of Diabetes Interventions and Complications), we used isotope dilution tandem mass spectrometry to quantify 46 proteins in HDL. Stringent statistical analysis demonstrated that 8 proteins associated with albuminuria. Two of those proteins, AMBP (α1-microglobulin/bikunin precursor) and PTGDS (prostaglandin-H2 D-isomerase), strongly and positively associated with the albumin excretion rate ( P<10 -6 ). Furthermore, PON (paraoxonase) 1 and PON3 levels in HDL strongly and negatively associated with the presence of coronary artery calcium, with odds ratios per 1-SD difference of 0.63 (95% CI, 0.43-0.92; P=0.018) for PON1 and 0.59 (95% CI, 0.40-0.87; P=0.0079) for PON3. Only 1 protein, PON1, associated with both albumin excretion rate and coronary artery calcification. Conclusions- Our observations indicate that the HDL proteome is remodeled in type 1 diabetes mellitus subjects with albuminuria. Moreover, low concentrations of the antiatherosclerotic protein PON1 in HDL associated with both albuminuria and coronary artery calcification, raising the possibility that alterations in HDL protein cargo mediate, in part, the known association of albuminuria with cardiovascular risk in type 1 diabetes mellitus. Visual Overview- An online visual overview is available for this article.

Apolipoprotein A1 (APOA1), the major protein of high-density lipoprotein (HDL), contains 10 helical repeats that play key roles in protein-protein and protein-lipid interactions. The current structural model for HDL proposes that APOA1 forms an antiparallel dimer in which helix 5 in monomer 1 associates with helix 5 in monomer 2 along a left-left (LL5/5) interface, forming a protein complex with a 2-fold axis of symmetry centered on helix 5. However, computational studies suggest that other orientations are possible. To test this idea, we used a zero-length chemical cross-linking reagent that forms covalent bonds between closely apposed basic and acidic residues. Using proteolytic digestion and tandem mass spectrometry, we identified amino acids in the central region of the antiparallel APOA1 dimer of HDL that were in close contact. As predicted by the current model, we found six intermolecular cross-links that were consistent with the antiparallel LL5/5 registry. However, we also identified three intermolecular cross-links that were consistent with the antiparallel LL5/4 registry. The LL5/5 is the major structural conformation of the two complexes in both reconstituted discoidal HDL particles and in spherical HDL from human plasma. Molecular dynamic simulations suggest that that LL5/5 and LL5/4 APOA1 dimers possess similar free energies of dimerization, with LL5/5 having the lowest free energy. Our observations indicate that phospholipidated APOA1 in HDL forms different antiparallel dimers that could play distinct roles in enzyme regulation, assembly of specific protein complexes, and the functional properties of HDL in humans., (© 2019 He et al.)

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., (Copyright © 2019 Pamir et al. Published by The American Society for Biochemistry and Molecular Biology, Inc.)

Objective- To assess the role of HDL (high-density lipoprotein)-mediated cholesterol mass efflux capacity (CMEC) in incident cardiovascular disease and carotid plaque progression. Approach and Results- We measured CMEC in 2 cohorts aged 45 to 84 years at baseline derived from the MESA (Multi-Ethnic Study of Atherosclerosis). Cohort 1 comprised 465 cases with incident cardiovascular disease events during 10 years of follow-up and 465 age- and sex-matched controls; cohort 2 comprised 407 cases with progression of carotid plaque measured by ultrasonography at 2 exams >10 years and 407 similarly matched controls. Covariates and outcome events were ascertained according to the MESA protocol. CMEC level was modestly correlated with HDL cholesterol ( R=0.13; P<0.001) but was not associated with age, sex, race/ethnicity, body mass index, diabetes mellitus, alcohol use, smoking status, or statin use. Higher CMEC level was significantly associated with lower odds of cardiovascular disease (odds ratio, 0.82 per SD of CMEC [95% CI, 0.69-0.98; P=0.031] in the fully adjusted model) in cohort 1 but higher odds of carotid plaque progression (odds ratio, 1.24 per SD of CMEC [95% CI, 1.04-1.48; P=0.018] in the fully adjusted model) in cohort 2 but without dose-response effect. In subgroup analysis within cohort 1, higher CMEC was associated with lower risk of incident coronary heart disease events (odds ratio, 0.72 per SD of CMEC (95% CI, 0.5-0.91; P=0.007) while no association was found with stroke events. Conclusions- These findings support a role for HDL-mediated cholesterol efflux in an atheroprotective mechanism for coronary heart disease but not stroke.

In a GM-CSF driven myeloid cell deficient mouse model (Csf2 -/- ) that has preserved insulin sensitivity despite increased adiposity, we used unbiased three-dimensional integration of proteome profiles, metabolic profiles, and gene regulatory networks to understand adipose tissue proteome-wide changes and their metabolic implications. Multi-dimensional liquid chromatography mass spectrometry and extended multiplex mass labeling was used to analyze proteomes of epididymal adipose tissues isolated from Csf2 +/+ and Csf2 -/- mice that were fed low fat, high fat, or high fat plus cholesterol diets for 8 weeks. The metabolic health (as measured by body weight, adiposity, plasma fasting glucose, insulin, triglycerides, phospholipids, total cholesterol levels, and glucose and insulin tolerance tests) deteriorated with diet for both genotypes, while mice lacking Csf2 were protected from insulin resistance. Regardless of diet, 30 mostly mitochondrial, branch chain amino acids (BCAA), and lysine metabolism proteins were altered between Csf2 -/- and Csf2 +/+ mice (FDR < 0.05). Lack of GM-CSF driven myeloid cells lead to reduced adipose tissue 2-oxoglutarate dehydrogenase complex (DHTKD1) levels and subsequent increase in plasma 2-aminoadipate (2-AA) levels, both of which are reported to correlate with insulin resistance. Tissue DHTKD1 levels were >4-fold upregulated and plasma 2-AA levels were >2 fold reduced in Csf2 -/- mice (p < 0.05). GM-CSF driven myeloid cells link peripheral insulin sensitivity to adiposity via lysine metabolism involving DHTKD1/2-AA axis in a diet independent manner.

Understanding the function of high-density lipoprotein (HDL) requires detailed knowledge of the structure of its primary protein, apolipoprotein A-I (APOA1). However, APOA1 flexibility and HDL heterogeneity have confounded decades of efforts to determine high-resolution structures and consistent models. Here, molecular dynamics simulations totaling 30 μs on two nascent HDLs, each with 2 APOA1 and either 160 phospholipids and 24 cholesterols or 200 phospholipids and 20 cholesterols, show that residues 1-21 of the N-terminal domains of APOA1 interact via strong salt bridges. Residues 26-43 of one APOA1 in the smaller particle form a hinge on the disc edge, which displaces the C-terminal domain of the other APOA1 to the phospholipid surface. The proposed structures are supported by chemical cross-linking, Rosetta modeling of the N-terminal domain, and analysis of the lipid-free ∆185APOA1 crystal structure. These structures provide a framework for understanding HDL maturation and revise all previous models of nascent HDL., Competing Interests: The authors declare no conflict of interest.

Apolipoprotein (apo)A-I is an organizing scaffold protein that is critical to high-density lipoprotein (HDL) structure and metabolism, probably mediating many of its cardioprotective properties. However, HDL biogenesis is poorly understood, as lipid-free apoA-I has been notoriously resistant to high-resolution structural study. Published models from low-resolution techniques share certain features but vary considerably in shape and secondary structure. To tackle this central issue in lipoprotein biology, we assembled a team of structural biologists specializing in apolipoproteins and set out to build a consensus model of monomeric lipid-free human apoA-I. Combining novel and published cross-link constraints, small-angle X-ray scattering (SAXS), hydrogen-deuterium exchange (HDX) and crystallography data, we propose a time-averaged model consistent with much of the experimental data published over the last 40 years. The model provides a long-sought platform for understanding and testing details of HDL biogenesis, structure and function.

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.

Context: Growing evidence challenges the concept that high-density lipoprotein-cholesterol (HDL-C) is cardioprotective after menopause. HDL particle concentration (HDL-P) and cholesterol efflux capacity (CEC) might be better predictors of cardiovascular risk., Objective: Quantify alterations in HDL-P and CEC during menopause, correlating those changes with alterations in estradiol (E2) and FSH., Design: Longitudinal study of HDL metrics before and after menopause as indexed by the final menstrual period (FMP)., Participants: Forty-six women, mean baseline age 47.1 years, 33% black, 67% white., Main Outcomes and Measures: HDL-P concentration (HDL-PIMA) by calibrated ion mobility analysis (IMA); macrophage CEC with cAMP-stimulated macrophages; ATP-binding cassette transporter A1 (ABCA1)-specific CEC with BHK cells expressing human ABCA1., Results: After a median of 2.1 years since FMP, both HDL-C (P = .03) and HDL-PIMA (P = .01) increased, with a selective increase in large HDL-PIMA (P = .01), whereas sizes of medium and small HDL-PIMA were decreased (P < .05). These changes were independent of race, body mass index, and time difference. Macrophage CEC and ABCA1-specific CEC increased after FMP (both P < .001). Greater declines in E2 correlated with larger increases in small HDL-PIMA (P = .01), whereas greater increases in FSH associated with greater reductions in the size of medium HDL-PIMA (P = .04). Macrophage CEC and ABCA1-specific CEC correlated positively with E2 levels only before menopause (P = .04 and .009, respectively)., Conclusions: Large HDL-PIMA and CEC increased significantly in the early phase of the menopausal transition. Whether patterns of these alterations differ in late postmenopause is unknown. Further exploration is needed to assess that and to determine whether the reported changes in HDL metrics associate with increased cardiovascular risk after menopause.

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 (<30% stenosis) coronary artery disease. ED was defined as <50% change in coronary blood flow in response to intracoronary infusions of acetylcholine during diagnostic coronary angiography. Cholesterol efflux capacity and HDL particle concentration (HDL-PIMA) were assessed with validated assays. Cholesterol efflux capacity and HDL-PIMA were both strong, inverse predictors of ED (P<0.001 and 0.005, respectively). In contrast, HDL-C and other traditional lipid risk factors did not differ significantly between control and ED subjects. Large HDL particles were markedly decreased in ED subjects (33%; P=0.005). After correction for HDL-C, both efflux capacity and HDL-PIMA remained significant predictors of ED status. HDL-PIMA 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-PIMA are reduced in subjects with coronary ED, independently of HDL-C. Alterations in HDL-PIMA 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., (© 2016 American Heart Association, Inc.)

Low levels of high-density lipoprotein cholesterol (HDL-C) and high triglyceride levels contribute to the excess rate of cardiovascular events seen in subjects with type 2 diabetes. Fenofibrate treatment partially reverses dyslipidemia in these subjects. However, a paradoxical marked reduction in HDL-C and HDL's major protein, apolipoprotein A-I, is a complication of fenofibrate in combination with rosiglitazone, an insulin-sensitizing agent. Risk factors for this condition, termed hypoalphalipoproteinemia, have yet to be identified. Using a case-control study design with subjects enrolled in the Action to Control Cardiovascular Risk in Diabetes (ACCORD) trial, we tested the hypothesis that alterations in HDL's protein cargo predispose diabetic subjects to fenofibrate/rosiglitazone-induced hypoalphalipoproteinemia. HDL was isolated from blood obtained from controls (no decreases or increase in HDL-C while receiving fenofibrate/rosiglitazone therapy) and cases (developed hypoalphalipoproteinemia after fenofibrate/rosiglitazone treatment) participating in the ACCORD study before they began fenofibrate/rosiglitazone treatment. HDL proteins were quantified by targeted parallel reaction monitoring (PRM) and selected reaction monitoring (SRM) with isotope dilution. This approach demonstrated marked increases in the relative concentrations of paraoxonase/arylesterase 1 (PON1), apolipoprotein C-II (APOC2), apolipoprotein C-I, and apolipoprotein H in the HDL of subjects who developed hypoalphalipoproteinemia. The case and control subjects did not differ significantly in baseline HDL-C levels or other traditional lipid risk factors. We used orthogonal biochemical techniques to confirm increased levels of PON1 and APOC2. Our observations suggest that an imbalance in HDL proteins predisposes diabetic subjects to develop hypoalphalipoproteinemia on fenofibrate/rosiglitazone therapy., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Objective: We investigated relationships between statin and niacin/statin combination therapy and the concentration of high-density lipoprotein particles (HDL-P) and cholesterol efflux capacity, 2 HDL metrics that might better assess cardiovascular disease risk than HDL-cholesterol (HDL-C) levels., Approach: In the Carotid Plaque Composition Study, 126 subjects with a history of cardiovascular disease were randomized to atorvastatin or combination therapy (atorvastatin/niacin). At baseline and after 1 year of treatment, the concentration of HDL and its 3 subclasses (small, medium, and large) were quantified by calibrated ion mobility analysis (HDL-PIMA). We also measured total cholesterol efflux from macrophages and ATP-binding cassette transporter A1 (ABCA1)-specific cholesterol efflux capacity., Results: Atorvastatin decreased low-density lipoprotein cholesterol by 39% and raised HDL-C by 11% (P=0.0001) but did not increase HDL-PIMA or macrophage cholesterol efflux. Combination therapy raised HDL-C by 39% (P<0.0001) but increased HDL-PIMA by only 14%. Triglyceride levels did not correlate with HDL-PIMA (P=0.39), in contrast to their strongly negative correlation with HDL-C (P<0.0001). Combination therapy increased macrophage cholesterol efflux capacity (16%, P<0.0001) but not ABCA1-specific efflux. ABCA1-specific cholesterol efflux capacity decreased significantly (P=0.013) in statin-treated subjects, with or without niacin therapy., Conclusions: Statin therapy increased HDL-C levels but failed to increase HDL-PIMA. It also reduced ABCA1-specific cholesterol efflux capacity. Adding niacin to statin therapy increased HDL-C and macrophage efflux, but had much less effect on HDL-PIMA. It also failed to improve ABCA1-specific efflux, a key cholesterol exporter in macrophages. Our observations raise the possibility that niacin might not target the relevant atheroprotective population of HDL., (© 2015 American Heart Association, Inc.)

Cholesterol efflux capacity associates strongly and negatively with the incidence and prevalence of human CVD. We investigated the relationships of HDL's size and protein cargo with its cholesterol efflux capacity using APOB-depleted serum and HDLs isolated from five inbred mouse strains with different susceptibilities to atherosclerosis. Like humans, mouse HDL carried >70 proteins linked to lipid metabolism, the acute-phase response, proteinase inhibition, and the immune system. HDL's content of specific proteins strongly correlated with its size and cholesterol efflux capacity, suggesting that its protein cargo regulates its function. Cholesterol efflux capacity with macrophages strongly and positively correlated with retinol binding protein 4 (RBP4) and PLTP, but not APOA1. In contrast, ABCA1-specific cholesterol efflux correlated strongly with HDL's content of APOA1, APOC3, and APOD, but not RBP4 and PLTP. Unexpectedly, APOE had a strong negative correlation with ABCA1-specific cholesterol efflux capacity. Moreover, the ABCA1-specific cholesterol efflux capacity of HDL isolated from APOE-deficient mice was significantly greater than that of HDL from wild-type mice. Our observations demonstrate that the HDL-associated APOE regulates HDL's ABCA1-specific cholesterol efflux capacity. These findings may be clinically relevant because HDL's APOE content associates with CVD risk and ABCA1 deficiency promotes unregulated cholesterol accumulation in human macrophages., (Copyright © 2016 by the American Society for Biochemistry and Molecular Biology, Inc.)

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., (Copyright © 2016 by the American Society for Biochemistry and Molecular Biology, Inc.)

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.

The physiological roles of macrophages and dendritic cells (DCs) in lean white adipose tissue homeostasis have received little attention. Because DCs are generated from bone marrow progenitors in the presence of granulocyte/macrophage colony-stimulating factor (GM-CSF), we used GM-CSF-deficient (Csf2(-/-)) mice fed a low fat diet to test the hypothesis that adipose tissue DCs regulate the development of adipose tissue. At 4 weeks of age, Csf2(-/-) mice had 75% fewer CD45(+)Cd11b(+)Cd11c(+)MHCII(+) F4/80(-) DCs in white adipose tissue than did wild-type controls. Furthermore, the Csf2(-/-) mice showed a 30% increase in whole body adiposity, which persisted to adulthood. Adipocytes from Csf2(-/-) mice were 50% larger by volume and contained higher levels of adipogenesis gene transcripts, indicating enhanced adipocyte differentiation. In contrast, adipogenesis/adipocyte lipid accumulation was inhibited when preadipocytes were co-cultured with CD45(+)Cd11b(+)Cd11c(+)MHCII(+)F4/80(-) DCs. Medium conditioned by DCs, but not by macrophages, also inhibited adipocyte lipid accumulation. Proteomic analysis revealed that matrix metalloproteinase 12 and fibronectin 1 were greatly enriched in the medium conditioned by DCs compared with that conditioned by macrophages. Silencing fibronectin or genetic deletion of matrix metalloproteinase 12 in DCs partially reversed the inhibition of adipocyte lipid accumulation. Our observations indicate that DCs residing in adipose tissue play a critical role in suppressing normal adipose tissue expansion., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: It is critical to develop new metrics to determine whether HDL is cardioprotective in humans. One promising approach is HDL particle concentration (HDL-P), the size and concentration of HDL in plasma. However, the 2 methods currently used to determine HDL-P yield concentrations that differ >5-fold. We therefore developed and validated an improved approach to quantify HDL-P, termed calibrated ion mobility analysis (calibrated IMA)., Methods: HDL was isolated from plasma by ultracentrifugation, introduced into the gas phase with electrospray ionization, separated by size, and quantified by particle counting. We used a calibration curve constructed with purified proteins to correct for the ionization efficiency of HDL particles., Results: The concentrations of gold nanoparticles and reconstituted HDLs measured by calibrated IMA were indistinguishable from concentrations determined by orthogonal methods. In plasma of control (n = 40) and cerebrovascular disease (n = 40) participants, 3 subspecies of HDL were reproducibility measured, with an estimated total HDL-P of 13.4 (2.4) μmol/L. HDL-C accounted for 48% of the variance in HDL-P. HDL-P was significantly lower in participants with cerebrovascular disease (P = 0.002), and this difference remained significant after adjustment for HDL cholesterol concentrations (P = 0.02)., Conclusions: Calibrated IMA accurately determined the concentration of gold nanoparticles and synthetic HDL, strongly suggesting that the method could accurately quantify HDL particle concentration. The estimated stoichiometry of apolipoprotein A-I determined by calibrated IMA was 3-4 per HDL particle, in agreement with current structural models. Furthermore, HDL-P was associated with cardiovascular disease status in a clinical population independently of HDL cholesterol., (© 2014 American Association for Clinical Chemistry.)