Lipoprotein Particles in Cerebrospinal Fluid.

The brain is the most lipid-rich organ in the body, and the intricate interplay between lipid metabolism and pathologies associated with neurodegenerative disorders is being increasingly recognized. The brain is bathed in cerebrospinal fluid (CSF), which, like plasma, contains lipid-protein complexes called lipoproteins that are responsible for extracellular lipid transport. Multiple CSF lipoprotein populations exist, some of which are produced de novo in the central nervous system and others that appear to be generated from protein constituents that are produced in the periphery. These CSF lipoproteins are thought to play key roles in maintaining lipid homeostasis in the central nervous system, while little else is known due to their limited accessibility and their low abundance in CSF. Recent work has provided new insights into the compositional complexity of CSF lipoprotein families and their metabolism in cerebral circulation. The purpose of this review is to summarize our current state of knowledge on the composition, origin, and metabolism of CSF lipoproteins.

A fast and sensitive size-exclusion chromatography method for plasma extracellular vesicle proteomic analysis.

Extracellular vesicles (EVs) carry diverse biomolecules derived from their parental cells, making their components excellent biomarker candidates. However, purifying EVs is a major hurdle in biomarker discovery since current methods require large amounts of samples, are time-consuming and typically have poor reproducibility. Here we describe a simple, fast, and sensitive EV fractionation method using size exclusion chromatography (SEC) on a fast protein liquid chromatography (FPLC) system. Our method uses a Superose 6 Increase 5/150, which has a bed volume of 2.9 mL. The FPLC system and small column size enable reproducible separation of only 50 µL of human plasma in 15 min. To demonstrate the utility of our method, we used longitudinal samples from a group of individuals who underwent intense exercise. A total of 838 proteins were identified, of which, 261 were previously characterized as EV proteins, including classical markers, such as cluster of differentiation (CD)9 and CD81. Quantitative analysis showed low technical variability with correlation coefficients greater than 0.9 between replicates. The analysis captured differences in relevant EV proteins involved in response to physical activity. Our method enables fast and sensitive fractionation of plasma EVs with low variability, which will facilitate biomarker studies in large clinical cohorts.

APOA2 Increases Cholesterol Efflux Capacity to Plasma HDL by Displacing the C-terminus of Resident APOA1.

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.

Systematic review of type 1 diabetes biomarkers reveals regulation in circulating proteins related to complement, lipid metabolism, and immune response.

BACKGROUND: Type 1 diabetes (T1D) results from an autoimmune attack of the pancreatic ß cells that progresses to dysglycemia and symptomatic hyperglycemia. Current biomarkers to track this evolution are limited, with development of islet autoantibodies marking the onset of autoimmunity and metabolic tests used to detect dysglycemia. Therefore, additional biomarkers are needed to better track disease initiation and progression. Multiple clinical studies have used proteomics to identify biomarker candidates. However, most of the studies were limited to the initial candidate identification, which needs to be further validated and have assays developed for clinical use. Here we curate these studies to help prioritize biomarker candidates for validation studies and to obtain a broader view of processes regulated during disease development. METHODS: This systematic review was registered with Open Science Framework ( https://doi.org/10.17605/OSF.IO/N8TSA ). Using PRISMA guidelines, we conducted a systematic search of proteomics studies of T1D in the PubMed to identify putative protein biomarkers of the disease. Studies that performed mass spectrometry-based untargeted/targeted proteomic analysis of human serum/plasma of control, pre-seroconversion, post-seroconversion, and/or T1D-diagnosed subjects were included. For unbiased screening, 3 reviewers screened all the articles independently using the pre-determined criteria. RESULTS: A total of 13 studies met our inclusion criteria, resulting in the identification of 266 unique proteins, with 31 (11.6%) being identified across 3 or more studies. The circulating protein biomarkers were found to be enriched in complement, lipid metabolism, and immune response pathways, all of which are found to be dysregulated in different phases of T1D development. We found 2 subsets: 17 proteins (C3, C1R, C8G, C4B, IBP2, IBP3, ITIH1, ITIH2, BTD, APOE, TETN, C1S, C6A3, SAA4, ALS, SEPP1 and PI16) and 3 proteins (C3, CLUS and C4A) have consistent regulation in at least 2 independent studies at post-seroconversion and post-diagnosis compared to controls, respectively, making them strong candidates for clinical assay development. CONCLUSIONS: Biomarkers analyzed in this systematic review highlight alterations in specific biological processes in T1D, including complement, lipid metabolism, and immune response pathways, and may have potential for further use in the clinic as prognostic or diagnostic assays.

Lipoprotein subfraction patterns throughout gestation in The Gambia: changes in subfraction composition and their relationships with infant birth weights.

BACKGROUND: Lipoprotein subfraction concentrations have been shown to change as gestation progresses in resource-rich settings. The objective of the current study was to evaluate the impact of pregnancy on different-sized lipoprotein particle concentrations and compositions in a resource-poor setting. METHOD: Samples were collected from pregnant women in rural Gambia at enrollment (8-20 weeks), 20 weeks, and 30 weeks of gestation. Concentrations of different-sized high-density, low-density, and triglyceride-rich lipoprotein particles (HDL, LDL, and TRL, respectively) were measured by nuclear magnetic resonance in 126 pooled plasma samples from a subset of women. HDL was isolated and the HDL proteome evaluated using mass spectroscopy. Subfraction concentrations from women in The Gambia were also compared to concentrations in women in the U.S. in mid gestation. RESULTS: Total lipoprotein particles and all-sized TRL, LDL, and HDL particle concentrations increased during gestation, with the exception of medium-sized LDL and HDL particles which decreased. Subfraction concentrations were not associated with infant birth weights, though relationships were found between some lipoprotein subfraction concentrations in women with normal versus low birth weight infants (< 2500 kg). HDL's proteome also changed during gestation, showing enrichment in proteins associated with metal ion binding, hemostasis, lipid metabolism, protease inhibitors, proteolysis, and complement activation. Compared to women in the U.S., Gambian women had lower large- and small-sized LDL and HDL concentrations, but similar medium-sized LDL and HDL concentrations. CONCLUSIONS: Most lipoprotein subfraction concentrations increase throughout pregnancy in Gambian women and are lower in Gambian vs U.S. women, the exception being medium-sized LDL and HDL particle concentrations which decrease during gestation and are similar in both cohorts of women. The proteomes of HDL also change in ways to support gestation. These changes warrant further study to determine how a lack of change or different changes could impact negative pregnancy outcomes.

Proteomic Determinants of Variation in Cholesterol Efflux: Observations from the Dallas Heart Study.

High-density lipoproteins (HDLs) are promising targets for predicting and treating atherosclerotic cardiovascular disease (ASCVD), as they mediate removal of excess cholesterol from lipid-laden macrophages that accumulate in the vasculature. This functional property of HDLs, termed cholesterol efflux capacity (CEC), is inversely associated with ASCVD. HDLs are compositionally diverse, associating with >250 different proteins, but their relative contribution to CEC remains poorly understood. Our goal was to identify and define key HDL-associated proteins that modulate CEC in humans. The proteomic signature of plasma HDL was quantified in 36 individuals in the multi-ethnic population-based Dallas Heart Study (DHS) cohort that exhibited persistent extremely high (>=90th%) or extremely low CEC (<=10th%) over 15 years. Levels of apolipoprotein (Apo)A-I associated ApoC-II, ApoC-III, and ApoA-IV were differentially correlated with CEC in high (r = 0.49, 0.41, and -0.21 respectively) and low (r = -0.46, -0.41, and 0.66 respectively) CEC groups (p for heterogeneity (pHet) = 0.03, 0.04, and 0.003 respectively). Further, we observed that levels of ApoA-I with ApoC-III, complement C3 (CO3), ApoE, and plasminogen (PLMG) were inversely associated with CEC in individuals within the low CEC group (r = -0.11 to -0.25 for subspecies with these proteins vs. r = 0.58 to 0.65 for subspecies lacking these proteins; p < 0.05 for heterogeneity). These findings suggest that enrichment of specific proteins on HDLs and, thus, different subspecies of HDLs, differentially modulate the removal of cholesterol from the vasculature.

Apolipoprotein A-I modulates HDL particle size in the absence of apolipoprotein A-II.

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 [15N]-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.

Pregnancy is accompanied by larger high density lipoprotein particles and compositionally distinct subspecies.

Pregnancy is accompanied by significant physiological changes, which can impact the health and development of the fetus and mother. Pregnancy-induced changes in plasma lipoproteins are well documented, with modest to no impact observed on the generic measure of high density lipoprotein (HDL) cholesterol. However, the impact of pregnancy on the concentration and composition of HDL subspecies has not been examined in depth. In this prospective study, we collected plasma from 24 nonpregnant and 19 pregnant women in their second trimester. Using nuclear magnetic resonance (NMR), we quantified 11 different lipoprotein subspecies from plasma by size, including three in the HDL class. We observed an increase in the number of larger HDL particles in pregnant women, which were confirmed by tracking phospholipids across lipoproteins using high-resolution gel-filtration chromatography. Using liquid chromatography-mass spectrometry (LC-MS), we identified 87 lipid-associated proteins across size-speciated fractions. We report drastic shifts in multiple protein clusters across different HDL size fractions in pregnant females compared with nonpregnant controls that have major implications on HDL function. These findings significantly elevate our understanding of how changes in lipoprotein metabolism during pregnancy could impact the health of both the fetus and the mother.

Modified sites and functional consequences of 4-oxo-2-nonenal adducts in HDL that are elevated in familial hypercholesterolemia.

The lipid aldehyde 4-oxo-2-nonenal (ONE) is a highly reactive protein crosslinker derived from peroxidation of n-6 polyunsaturated fatty acids and generated together with 4-hydroxynonenal (HNE). Lipid peroxidation product-mediated crosslinking of proteins in high-density lipoprotein (HDL) causes HDL dysfunction and contributes to atherogenesis. Although HNE is relatively well-studied, the role of ONE in atherosclerosis and in modifying HDL is unknown. Here, we found that individuals with familial hypercholesterolemia (FH) had significantly higher ONE-ketoamide (lysine) adducts in HDL (54.6 ± 33.8 pmol/mg) than healthy controls (15.3 ± 5.6 pmol/mg). ONE crosslinked apolipoprotein A-I (apoA-I) on HDL at a concentration of > 3 mol ONE per 10 mol apoA-I (0.3 eq), which was 100-fold lower than HNE, but comparable to the potent protein crosslinker isolevuglandin. ONE-modified HDL partially inhibited HDL's ability to protect against lipopolysaccharide (LPS)-induced tumor necrosis factor α (TNFα) and interleukin-1ß (IL-1ß) gene expression in murine macrophages. At 3 eq, ONE dramatically decreased apoA-I exchange from HDL, from ∼46.5 to ∼18.4% (p < 0.001). Surprisingly, ONE modification of HDL or apoA-I did not alter macrophage cholesterol efflux capacity. LC-MS/MS analysis revealed that Lys-12, Lys-23, Lys-96, and Lys-226 in apoA-I are modified by ONE ketoamide adducts. Compared with other dicarbonyl scavengers, pentylpyridoxamine (PPM) most efficaciously blocked ONE-induced protein crosslinking in HDL and also prevented HDL dysfunction in an in vitro model of inflammation. Our findings show that ONE-HDL adducts cause HDL dysfunction and are elevated in individuals with FH who have severe hypercholesterolemia.

Studies in genetically modified mice implicate maternal HDL as a mediator of fetal growth.

Studies in humans have shown a direct association between maternal plasma cholesterol concentrations and infant birthweight. Similarly, previous studies in our laboratory have shown that chow-fed mice lacking apolipoprotein (apo) A-I, the major protein in HDL, have low HDL-cholesterol (HDL-C) concentrations and smaller fetuses in midgestation. In the current study, we measured fetal weights in mice with varying levels of apoA-I gene dose (knockout, wild-type, and transgenic) and examined metabolic pathways known to affect fetal growth. As expected, we found the differences in apoA-I expression led to changes in HDL particle size and protein cargo as well as plasma cholesterol concentrations. Fetal masses correlated directly with maternal plasma cholesterol and apoA-I concentrations, but placental masses and histology did not differ between groups of mice. There was no significant difference in glucose or amino acid transport to the fetus or in expression levels of the glucose (glucose transporter 1 and 2) or amino acid (sodium-coupled neutral amino acid transporter 1 and 2) transporters in whole placentas, although there was a trend for greater uptake of both nutrients in the whole fetal unit (fetus + placenta) of mice with greater apoA-I levels; significant differences in transport rates occurred when mice without apoA-I (knockout) vs. mice with apoA-I (wild-type and transgenic) were compared. Glucose tolerance tests were improved in the mice with the highest level of apoA-I, suggesting increased insulin-induced uptake of glucose by tissues of apoA-I transgenic mice. Thus, maternal HDL is associated with fetal growth, an effect that is likely mediated by plasma cholesterol or other HDL-cargo, including apolipoproteins or complement system proteins. A direct role of enhanced glucose and/or amino acid transport cannot be excluded.-Rebholz, S. L., Melchior, J. T., Davidson, W. S., Jones, H. N., Welge, J. A., Prentice, A. M., Moore, S. E., Woollett, L. A. Studies in genetically modified mice implicate maternal HDL as a mediator of fetal growth.

Distinct Proteomic Signatures in 16 HDL (High-Density Lipoprotein) Subspecies.

Objective- HDL (high-density lipoprotein) in plasma is a heterogeneous group of lipoproteins typically containing apo AI as the principal protein. Most HDLs contain additional proteins from a palate of nearly 100 HDL-associated polypeptides. We hypothesized that some of these proteins define distinct and stable apo AI HDL subspecies with unique proteomes that drive function and associations with disease. Approach and Results- We produced 17 plasma pools from 80 normolipidemic human participants (32 men, 48 women; aged 21-66 years). Using immunoaffinity isolation techniques, we isolated apo AI containing species from plasma and then used antibodies to 16 additional HDL protein components to isolate compositional subspecies. We characterized previously described HDL subspecies containing apo AII, apo CIII, and apo E; and 13 novel HDL subspecies defined by presence of apo AIV, apo CI, apo CII, apo J, α-1-antitrypsin, α-2-macroglobulin, plasminogen, fibrinogen, ceruloplasmin, haptoglobin, paraoxonase-1, apo LI, or complement C3. The novel species ranged in abundance from 1% to 18% of total plasma apo AI. Their concentrations were stable over time as demonstrated by intraclass correlations in repeated sampling from the same participants over 3 to 24 months (0.33-0.86; mean 0.62). Some proteomes of the subspecies relative to total HDL were strongly correlated, often among subspecies defined by similar functions: lipid metabolism, hemostasis, antioxidant, or anti-inflammatory. Permutation analysis showed that the proteomes of 12 of the 16 subspecies differed significantly from that of total HDL. Conclusions- Taken together, correlation and permutation analyses support speciation of HDL. Functional studies of these novel subspecies and determination of their relation to diseases may provide new avenues to understand the HDL system of lipoproteins.

A thumbwheel mechanism for APOA1 activation of LCAT activity in HDL.

APOA1 is the most abundant protein in HDL. It modulates interactions that affect HDL's cardioprotective functions, in part via its activation of the enzyme, LCAT. On nascent discoidal HDL, APOA1 comprises 10 α-helical repeats arranged in an anti-parallel stacked-ring structure that encapsulates a lipid bilayer. Previous chemical cross-linking studies suggested that these APOA1 rings can adopt at least two different orientations, or registries, with respect to each other; however, the functional impact of these structural changes is unknown. Here, we placed cysteine residues at locations predicted to form disulfide bonds in each orientation and then measured APOA1's ability to adopt the two registries during HDL particle formation. We found that most APOA1 oriented with the fifth helix of one molecule across from fifth helix of the other (5/5 helical registry), but a fraction adopted a 5/2 registry. Engineered HDLs that were locked in 5/5 or 5/2 registries by disulfide bonds equally promoted cholesterol efflux from macrophages, indicating functional particles. However, unlike the 5/5 registry or the WT, the 5/2 registry impaired LCAT cholesteryl esterification activity (P < 0.001), despite LCAT binding equally to all particles. Chemical cross-linking studies suggest that full LCAT activity requires a hybrid epitope composed of helices 5-7 on one APOA1 molecule and helices 3-4 on the other. Thus, APOA1 may use a reciprocating thumbwheel-like mechanism to activate HDL-remodeling proteins.

Obesity is associated with an altered HDL subspecies profile among adolescents with metabolic disease.

We aimed to determine the risk factors associated with the depletion of large HDL particles and enrichment of small HDL particles observed in adolescents with T2D. Four groups of adolescents were recruited: 1) lean insulin-sensitive (L-IS), normal BMI and no insulin resistance; 2) lean insulin-resistant (L-IR), normal BMI but insulin resistance (fasting insulin levels ≥ 25 mU/ml and homeostatic model assessment of insulin resistance ≥ 6); 3) obese insulin-sensitive (O-IS), BMI ≥ 95th percentile and no insulin resistance; and 4) obese insulin-resistant (O-IR), BMI ≥ 95th percentile and insulin resistance. Plasma was separated by using gel-filtration chromatography to assess the HDL subspecies profile and compared with that of obese adolescents with T2D (O-T2D). Large HDL subspecies were significantly lower across groups from L-IS > L-IR > O-IS > O-IR > O-T2D (P < 0.0001); small HDL particles were higher from L-IS to O-T2D (P < 0.0001); and medium-sized particles did not differ across groups. The contributions of obesity, insulin resistance, and diabetes to HDL subspecies profile were between 23% and 28%, 1% and 10%, and 4% and 9%, respectively. Obesity is the major risk factor associated with the altered HDL subspecies profile previously reported in adolescents with T2D, with smaller contributions from insulin resistance and diabetes.

High density lipoproteins selectively promote the survival of human regulatory T cells.

HDLs appear to affect regulatory T cell (Treg) homeostasis, as suggested by the increased Treg counts in HDL-treated mice and by the positive correlation between Treg frequency and HDL-cholesterol levels in statin-treated healthy adults. However, the underlying mechanisms remain unclear. Herein, we show that HDLs, not LDLs, significantly decreased the apoptosis of human Tregs in vitro, whereas they did not alter naïve or memory CD4+ T cell survival. Similarly, oleic acid bound to serum albumin increased Treg survival. Tregs bound and internalized high amounts of HDL compared with other subsets, which might arise from the higher expression of the scavenger receptor class B type I by Tregs; accordingly, blocking this receptor hindered HDL-mediated Treg survival. Mechanistically, we showed that HDL increased Treg ATP concentration and mitochondrial activity, enhancing basal respiration, maximal respiration, and spare respiratory capacity. Blockade of FA oxidation by etoxomir abolished the HDL-mediated enhanced survival and mitochondrial activity. Our findings thus suggest that Tregs can specifically internalize HDLs from their microenvironment and use them as an energy source. Furthermore, a novel implication of our data is that enhanced Treg survival may contribute to HDLs' anti-inflammatory properties.

Apolipoprotein A-II alters the proteome of human lipoproteins and enhances cholesterol efflux from ABCA1.

HDLs are a family of heterogeneous particles that vary in size, composition, and function. The structure of most HDLs is maintained by two scaffold proteins, apoA-I and apoA-II, but up to 95 other "accessory" proteins have been found associated with the particles. Recent evidence suggests that these accessory proteins are distributed across various subspecies and drive specific biological functions. Unfortunately, our understanding of the molecular composition of such subspecies is limited. To begin to address this issue, we separated human plasma and HDL isolated by ultracentrifugation (UC-HDL) into particles with apoA-I and no apoA-II (LpA-I) and those with both apoA-I and apoA-II (LpA-I/A-II). MS studies revealed distinct differences between the subfractions. LpA-I exhibited significantly more protein diversity than LpA-I/A-II when isolated directly from plasma. However, this difference was lost in UC-HDL. Most LpA-I/A-II accessory proteins were associated with lipid transport pathways, whereas those in LpA-I were associated with inflammatory response, hemostasis, immune response, metal ion binding, and protease inhibition. We found that the presence of apoA-II enhanced ABCA1-mediated efflux compared with LpA-I particles. This effect was independent of the accessory protein signature suggesting that apoA-II induces a structural change in apoA-I in HDLs.

An Evaluation of the Crystal Structure of C-terminal Truncated Apolipoprotein A-I in Solution Reveals Structural Dynamics Related to Lipid Binding.

Apolipoprotein (apo) A-I mediates many of the anti-atherogenic functions attributed to high density lipoprotein. Unfortunately, efforts toward a high resolution structure of full-length apoA-I have not been fruitful, although there have been successes with deletion mutants. Recently, a C-terminal truncation (apoA-I(Δ185-243)) was crystallized as a dimer. The structure showed two helical bundles connected by a long, curved pair of swapped helical domains. To compare this structure to that existing under solution conditions, we applied small angle x-ray scattering and isotope-assisted chemical cross-linking to apoA-I(Δ185-243) in its dimeric and monomeric forms. For the dimer, we found evidence for the shared domains and aspects of the N-terminal bundles, but not the molecular curvature seen in the crystal. We also found that the N-terminal bundles equilibrate between open and closed states. Interestingly, this movement is one of the transitions proposed during lipid binding. The monomer was consistent with a model in which the long shared helix doubles back onto the helical bundle. Combined with the crystal structure, these data offer an important starting point to understand the molecular details of high density lipoprotein biogenesis.

The effects of apolipoprotein B depletion on HDL subspecies composition and function.

HDL cholesterol (HDL-C) efflux function may be a more robust biomarker of coronary artery disease risk than HDL-C. To study HDL function, apoB-containing lipoproteins are precipitated from serum. Whether apoB precipitation affects HDL subspecies composition and function has not been thoroughly investigated. We studied the effects of four common apoB precipitation methods [polyethylene glycol (PEG), dextran sulfate/magnesium chloride (MgCl2), heparin sodium/manganese chloride (MnCl2), and LipoSep immunoprecipitation (IP)] on HDL subspecies composition, apolipoproteins, and function (cholesterol efflux and reduction of LDL oxidation). PEG dramatically shifted the size distribution of HDL and apolipoproteins (assessed by two independent methods), while leaving substantial amounts of reagent in the sample. PEG also changed the distribution of cholesterol efflux and LDL oxidation across size fractions, but not overall efflux across the HDL range. Dextran sulfate/MgCl2, heparin sodium/MnCl2, and LipoSep IP did not change the size distribution of HDL subspecies, but altered the quantity of a subset of apolipoproteins. Thus, each of the apoB precipitation methods affected HDL composition and/or size distribution. We conclude that careful evaluation is needed when selecting apoB depletion methods for existing and future bioassays of HDL function.

Targeted Knockdown of Hepatic SOAT2 With Antisense Oligonucleotides Stabilizes Atherosclerotic Plaque in ApoB100-only LDLr-/- Mice.

OBJECTIVE: To test the hypothesis that the attenuation of cholesterol oleate packaging into apoB-containing lipoproteins will arrest progression of pre-existing atherosclerotic lesions. APPROACH AND RESULTS: Atherosclerosis was induced in apoB-100 only, LDLr(-/-) mice by feeding a diet enriched in cis-monounsaturated fatty acids for 24 weeks. A subset of mice was then euthanized to quantify the extent of atherosclerosis. The remaining mice were continued on the same diet (controls) or assigned to the following treatments for 16 weeks: (1) a diet enriched in n-3 polyunsaturated fatty acids, (2) the cis-monounsaturated fatty acid diet plus biweekly injections of an antisense oligonucleotide specific to hepatic sterol-O-acyltransferase 2 (SOAT2); or (3) the cis-monounsaturated fatty acid diet and biweekly injections of a nontargeting hepatic antisense oligonucleotide. Extent of atherosclerotic lesions in the aorta was monitored morphometrically in vivo with magnetic resonance imaging and ex vivo histologically and immunochemically. Hepatic knockdown of SOAT2 via antisense oligonucleotide treatment arrested lesion growth and stabilized lesions. CONCLUSIONS: Hepatic knockdown of SOAT2 in apoB100-only, LDLr(-/-) mice resulted in remodeling of aortic atherosclerotic lesions into a stable phenotype, suggesting SOAT2 is a viable target for the treatment of atherosclerosis.

The structure of human apolipoprotein A-IV as revealed by stable isotope-assisted cross-linking, molecular dynamics, and small angle x-ray scattering.

Apolipoprotein (apo)A-IV plays important roles in dietary lipid and glucose metabolism, and knowledge of its structure is required to fully understand the molecular basis of these functions. However, typical of the entire class of exchangeable apolipoproteins, its dynamic nature and affinity for lipid has posed challenges to traditional high resolution structural approaches. We previously reported an x-ray crystal structure of a dimeric truncation mutant of apoA-IV, which showed a unique helix-swapping molecular interface. Unfortunately, the structures of the N and C termini that are important for lipid binding were not visualized. To build a more complete model, we used chemical cross-linking to derive distance constraints across the full-length protein. The approach was enhanced with stable isotope labeling to overcome ambiguities in determining molecular span of the cross-links given the remarkable similarities in the monomeric and dimeric apoA-IV structures. Using 51 distance constraints, we created a starting model for full-length monomeric apoA-IV and then subjected it to two modeling approaches: (i) molecular dynamics simulations and (ii) fitting to small angle x-ray scattering data. This resulted in the most detailed models yet for lipid-free monomeric or dimeric apoA-IV. Importantly, these models were of sufficient detail to direct the experimental identification of new functional residues that participate in a "clasp" mechanism to modulate apoA-IV lipid affinity. The isotope-assisted cross-linking approach should prove useful for further study of this family of apolipoproteins in both the lipid-free and -bound states.

A novel assay to measure low-density lipoproteins binding to proteoglycans.

BACKGROUND: The binding of low-density lipoprotein (LDL) to proteoglycans (PGs) in the extracellular matrix (ECM) of the arterial intima is a key initial step in the development of atherosclerosis. Although many techniques have been developed to assess this binding, most of the methods are labor-intensive and technically challenging to standardize across research laboratories. Thus, sensitive, and reproducible assay to detect LDL binding to PGs is needed to screen clinical populations for atherosclerosis risk. OBJECTIVES: The aim of this study was to develop a quantitative, and reproducible assay to evaluate the affinity of LDL towards PGs and to replicate previously published results on LDL-PG binding. METHODS: Immunofluorescence microscopy was performed to visualize the binding of LDL to PGs using mouse vascular smooth muscle (MOVAS) cells. An in-cell ELISA (ICE) was also developed and optimized to quantitatively measure LDL-PG binding using fixed MOVAS cells cultured in a 96-well format. RESULTS: We used the ICE assay to show that, despite equal APOB concentrations, LDL isolated from adults with cardiovascular disease bound to PG to a greater extent than LDL isolated from adults without cardiovascular disease (p<0.05). CONCLUSION: We have developed an LDL-PG binding assay that is capable of detecting differences in PG binding affinities despite equal APOB concentrations. Future work will focus on candidate apolipoproteins that enhance or diminish this interaction.