AIBP protects against metabolic abnormalities and atherosclerosis

Apolipoprotein A-I binding protein (AIBP) has been shown to augment cholesterol efflux from endothelial cells and macrophages. In zebrafish and mice, AIBP-mediated regulation of cholesterol levels in the plasma membrane of endothelial cells controls angiogenesis. The goal of this work was to evaluate metabolic changes and atherosclerosis in AIBP loss-of-function and gain-of-function animal studies. Here, we show that Apoa1bp−/−Ldlr−/− mice fed a high-cholesterol, high-fat diet had exacerbated weight gain, liver steatosis, glucose intolerance, hypercholesterolemia, hypertriglyceridemia, and larger atherosclerotic lesions compared with Ldlr−/− mice. Feeding Apoa1bp−/−Ldlr−/− mice a high-cholesterol, normal-fat diet did not result in significant differences in lipid levels or size of atherosclerotic lesions from Ldlr−/− mice. Conversely, adeno-associated virus-mediated overexpression of AIBP reduced hyperlipidemia and atherosclerosis in high-cholesterol, high-fat diet-fed Ldlr−/− mice. Injections of recombinant AIBP reduced aortic inflammation in Ldlr−/− mice fed a short high-cholesterol, high-fat diet. Conditional overexpression of AIBP in zebrafish also reduced diet-induced vascular lipid accumulation. In experiments with isolated macrophages, AIBP facilitated cholesterol efflux to HDL, reduced lipid rafts content, and inhibited inflammatory responses to lipopolysaccharide.jlr Our data demonstrate that AIBP confers protection against diet-induced metabolic abnormalities and atherosclerosis. Apolipoprotein A-I binding protein (AIBP) has been shown to augment cholesterol efflux from endothelial cells and macrophages. In zebrafish and mice, AIBP-mediated regulation of cholesterol levels in the plasma membrane of endothelial cells controls angiogenesis. The goal of this work was to evaluate metabolic changes and atherosclerosis in AIBP loss-of-function and gain-of-function animal studies. Here, we show that Apoa1bp−/−Ldlr−/− mice fed a high-cholesterol, high-fat diet had exacerbated weight gain, liver steatosis, glucose intolerance, hypercholesterolemia, hypertriglyceridemia, and larger atherosclerotic lesions compared with Ldlr−/− mice. Feeding Apoa1bp−/−Ldlr−/− mice a high-cholesterol, normal-fat diet did not result in significant differences in lipid levels or size of atherosclerotic lesions from Ldlr−/− mice. Conversely, adeno-associated virus-mediated overexpression of AIBP reduced hyperlipidemia and atherosclerosis in high-cholesterol, high-fat diet-fed Ldlr−/− mice. Injections of recombinant AIBP reduced aortic inflammation in Ldlr−/− mice fed a short high-cholesterol, high-fat diet. Conditional overexpression of AIBP in zebrafish also reduced diet-induced vascular lipid accumulation. In experiments with isolated macrophages, AIBP facilitated cholesterol efflux to HDL, reduced lipid rafts content, and inhibited inflammatory responses to lipopolysaccharide.jlr Our data demonstrate that AIBP confers protection against diet-induced metabolic abnormalities and atherosclerosis. Cardiovascular disease (CVD) is a leading cause of morbidity and mortality in the United States, accounting for approximately one-third of all US deaths (1.Go A.S. Mozaffarian D. Roger V.L. Benjamin E.J. Berry J.D. Blaha M.J. Dai S. Ford E.S. Fox C.S. Franco S. Fullerton H.J. Gillespie C. Hailpern S.M. Heit J.A. Howard V.J. Huffman M.D. Judd S.E. Kissela B.M. Kittner S.J. Lackland D.T. Lichtman J.H. Lisabeth L.D. Mackey R.H. Magid D.J. Marcus G.M. Marelli A. Matchar D.B. McGuire D.K. Mohler 3rd, E.R. Moy C.S. Mussolino M.E. Neumar R.W. Nichol G. Pandey D.K. Paynter N.P. Reeves M.J. Sorlie P.D. Stein J. Towfighi A. Turan T.N. Virani S.S. Wong N.D. Woo D. Turner M.B. American Heart Association Statistics Committee, and Stroke Statistics Subcomittee Heart disease and stroke statistics–2014 update: a report from the American Heart Association.Circulation. 2014; 129: e28-e292Crossref PubMed Scopus (4494) Google Scholar). Therapy to reduce LDL cholesterol (LDL-C) has led to remarkable improvement in clinical outcomes, decreasing the incidence of acute cardiovascular events by 25–35% (2.Baigent C. Keech A. Kearney P.M. Blackwell L. Buck G. Pollicino C. Kirby A. Sourjina T. Peto R. Collins R. et al.Efficacy and safety of cholesterol-lowering treatment: prospective meta-analysis of data from 90,056 participants in 14 randomised trials of statins.Lancet. 2005; 366: 1267-1278Abstract Full Text Full Text PDF PubMed Scopus (5760) Google Scholar, 3.Steinberg D. Glass C.K. Witztum J.L. Evidence mandating earlier and more aggressive treatment of hypercholesterolemia.Circulation. 2008; 118: 672-677Crossref PubMed Scopus (89) Google Scholar). Statins, ezetimibe, and recently apoB antisense and PCSK9 monoclonal antibodies all target plasma LDL-C as a major atherogenic factor. However, improving the other arc of cholesterol homeostasis–reverse cholesterol transport and atheroprotective HDL–has proven to be a challenging task. Results of clinical trials of cholesteryl ester transfer protein inhibitors put in doubt the efficacy of simply raising HDL cholesterol (HDL-C) levels as an ultimate therapeutic goal (4.Tall A.R. Rader D.J. Trials and tribulations of CETP inhibitors.Circ. Res. 2018; 122: 106-112Crossref PubMed Scopus (138) Google Scholar). Although HDL-C clearly correlates with atheroprotection in epidemiology studies, the current consensus in the cardiovascular field underscores the added importance of improving the functionality of HDL (5.Larach D.B. deGoma E.M. Rader D.J. Targeting high density lipoproteins in the prevention of cardiovascular disease?.Curr. Cardiol. Rep. 2012; 14: 684-691Crossref PubMed Scopus (29) Google Scholar, 6.Fisher E.A. Feig J.E. Hewing B. Hazen S.L. Smith J.D. High-density lipoprotein function, dysfunction, and reverse cholesterol transport.Arterioscler. Thromb. Vasc. Biol. 2012; 32: 2813-2820Crossref PubMed Scopus (273) Google Scholar, 7.Rosenson R.S. Brewer H.B. Davidson W.S. Fayad Z.A. Fuster V. Goldstein J. Hellerstein M. Jiang X.c. Phillips M.C. Rader D.J. et al.Cholesterol efflux and atheroprotection: advancing the concept of reverse cholesterol transport.Circulation. 2012; 125: 1905-1919Crossref PubMed Scopus (684) Google Scholar, 8.Heinecke J.W. The not-so-simple HDL story: a new era for quantifying HDL and cardiovascular risk?.Nat. Med. 2012; 18: 1346-1347Crossref PubMed Scopus (52) Google Scholar). However, in vivo mechanisms regulating HDL function are not sufficiently understood. Our recent work and the work of others have highlighted apolipoprotein A-I binding protein (AIBP), which augments cholesterol efflux from endothelial cells (9.Fang L. Choi S.H. Baek J.S. Liu C. Almazan F. Ulrich F. Wiesner P. Taleb A. Deer E. Pattison J. et al.Control of angiogenesis by AIBP-mediated cholesterol efflux.Nature. 2013; 498: 118-122Crossref PubMed Scopus (135) Google Scholar, 10.Mao R. Meng S. Gu Q. Araujo-Gutierrez R. Kumar S. Yan Q. Almazan F. Youker K.A. Fu Y. Pownall H.J. et al.AIBP limits angiogenesis through gamma-secretase-mediated upregulation of Notch signaling.Circ. Res. 2017; 120: 1727-1739Crossref PubMed Scopus (33) Google Scholar) and macrophages (11.Zhang M. Li L. Xie W. Wu J.F. Yao F. Tan Y.L. Xia X.D. Liu X.Y. Liu D. Lan G. et al.Apolipoprotein A-1 binding protein promotes macrophage cholesterol efflux by facilitating apolipoprotein A-1 binding to ABCA1 and preventing ABCA1 degradation.Atherosclerosis. 2016; 248: 149-159Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar) to HDL. AIBP is a secreted protein discovered in a screen of proteins that physically associate with apoA-I (12.Ritter M. Buechler C. Boettcher A. Barlage S. Schmitz-Madry A. Orso E. Bared S.M. Schmiedeknecht G. Baehr C.H. Fricker G. et al.Cloning and characterization of a novel apolipoprotein A-I binding protein, AI-BP, secreted by cells of the kidney proximal tubules in response to HDL or ApoA-I.Genomics. 2002; 79: 693-702Crossref PubMed Scopus (63) Google Scholar). Human APOA1BP mRNA encoding AIBP is ubiquitously expressed, with the highest expression in kidney, heart, liver, thyroid gland, adrenal gland, and testis (12.Ritter M. Buechler C. Boettcher A. Barlage S. Schmitz-Madry A. Orso E. Bared S.M. Schmiedeknecht G. Baehr C.H. Fricker G. et al.Cloning and characterization of a novel apolipoprotein A-I binding protein, AI-BP, secreted by cells of the kidney proximal tubules in response to HDL or ApoA-I.Genomics. 2002; 79: 693-702Crossref PubMed Scopus (63) Google Scholar). AIBP is found in cerebrospinal fluid and urine and in plasma of patients with sepsis (12.Ritter M. Buechler C. Boettcher A. Barlage S. Schmitz-Madry A. Orso E. Bared S.M. Schmiedeknecht G. Baehr C.H. Fricker G. et al.Cloning and characterization of a novel apolipoprotein A-I binding protein, AI-BP, secreted by cells of the kidney proximal tubules in response to HDL or ApoA-I.Genomics. 2002; 79: 693-702Crossref PubMed Scopus (63) Google Scholar). The human APOA1BP gene is located at 1q21.2-1q22 on chromosome 1, which corresponds to the 1q21-q23 locus for familial combined hyperlipidemia, a common multifactorial and heterogeneous dyslipidemia predisposing to premature coronary artery disease (13.Bodnar J.S. Chatterjee A. Castellani L.W. Ross D.A. Ohmen J. Cavalcoli J. Wu C. Dains K.M. Catanese J. Chu M. et al.Positional cloning of the combined hyperlipidemia gene Hyplip1.Nat. Genet. 2002; 30: 110-116Crossref PubMed Scopus (180) Google Scholar). Yet, there are no studies directly linking AIBP polymorphism with dyslipidemia or risk of CVD. AIBP does not bind cholesterol or induce cholesterol efflux in the absence of HDL or apoA-I, but it does increase the turnover of HDL and thus accelerates cholesterol efflux (9.Fang L. Choi S.H. Baek J.S. Liu C. Almazan F. Ulrich F. Wiesner P. Taleb A. Deer E. Pattison J. et al.Control of angiogenesis by AIBP-mediated cholesterol efflux.Nature. 2013; 498: 118-122Crossref PubMed Scopus (135) Google Scholar). The goal of this work was to evaluate metabolic changes and atherosclerosis in AIBP loss-of-function and gain-of-function animal studies. We report that AIBP deficiency exacerbates weight gain, hyperlipidemia, and atherosclerosis, while overexpression of AIBP is protective against vascular lipid accumulation, atherosclerosis, and metabolic abnormalities. All animal experiments were conducted according to protocols approved by the Institutional Animal Care and Use Committee of the University of California at San Diego. Mice were housed up to five per standard cage at room temperature and maintained on a 12:12 h light:dark cycle, with lights on at 07:00. Both food and water were available ad libitum. Wild-type C57BL/6 and Ldlr−/− mice were initially purchased from the Jackson Laboratory (Bar Harbor, ME) and bred in-house for experiments. Apoa1bp−/− mice on a C57BL/6 background were generated in our group as previously described (10.Mao R. Meng S. Gu Q. Araujo-Gutierrez R. Kumar S. Yan Q. Almazan F. Youker K.A. Fu Y. Pownall H.J. et al.AIBP limits angiogenesis through gamma-secretase-mediated upregulation of Notch signaling.Circ. Res. 2017; 120: 1727-1739Crossref PubMed Scopus (33) Google Scholar) and cross-bred with Ldlr−/− mice. Apoa1bp−/−Ldlr−/− mice develop and breed normally. For metabolic studies, mice on a C57BL/6 background were fed a high-fat diet (HFD; Research Diets D12451; supplemental Table S1) containing 45% kcal from fat, starting at age 10 weeks. For metabolic and atherosclerosis studies, mice on an Ldlr−/− background were fed either a Western diet (Teklad TD.96121; supplemental Table S2) containing 42% kcal from fat (21% milkfat) and 1.25% cholesterol or a high-cholesterol, normal-fat diet (Teklad TD.97131; supplemental Table S3) containing 1% cholesterol, starting at age 8 weeks. Adult zebrafish were maintained at 28°C on a 14 h light/10 h dark cycle as previously described (14.Westerfield M. University of Oregon Press.Eugene, OR. 2007; Google Scholar) and fed brine shrimp twice a day. Zebrafish larvae were fed Golden Pearls (Brine Shrimp Direct, Ogden, UT) twice a day, starting from 5 days postfertilization (dpf). The transgenic hsp70:apoa1bp-2A-mRFP zebrafish was generated by using constructs and methods previously described (9.Fang L. Choi S.H. Baek J.S. Liu C. Almazan F. Ulrich F. Wiesner P. Taleb A. Deer E. Pattison J. et al.Control of angiogenesis by AIBP-mediated cholesterol efflux.Nature. 2013; 498: 118-122Crossref PubMed Scopus (135) Google Scholar, 15.Fang L. Green S.R. Baek J.S. Lee S.H. Ellett F. Deer E. Lieschke G.J. Witztum J.L. Tsimikas S. Miller Y.I. In vivo visualization and attenuation of oxidized lipid accumulation in hypercholesterolemic zebrafish.J. Clin. Invest. 2011; 121: 4861-4869Crossref PubMed Scopus (68) Google Scholar, 16.Kim J.H. Lee S.R. Li L.H. Park H.J. Park J.H. Lee K.Y. Kim M.K. Shin B.A. Choi S.Y. High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice.PLoS One. 2011; 6: e18556Crossref PubMed Scopus (844) Google Scholar). The self-cleavage 2A peptide allows for expression of two separate proteins in the same tissue, with mRFP serving as an indicator of successful protein expression. Transgene expression was initiated by single or repeated heat shocks (transferring zebrafish for 1 h into water warmed to 37°C) and detected via mRFP fluorescence. Expression of the zebrafish Apoa1bp protein was confirmed in Western blot with a guinea pig polyclonal anti-zebrafish Apoa1bp antibody (9.Fang L. Choi S.H. Baek J.S. Liu C. Almazan F. Ulrich F. Wiesner P. Taleb A. Deer E. Pattison J. et al.Control of angiogenesis by AIBP-mediated cholesterol efflux.Nature. 2013; 498: 118-122Crossref PubMed Scopus (135) Google Scholar). For vascular lipid deposit experiments, Golden Pearls supplemented with 4% (wt/wt) cholesterol and 1 µg/g TopFluor Cholesterol (Avanti Polar Lipids) were fed to zebrafish from 5 to 15 dpf as described (15.Fang L. Green S.R. Baek J.S. Lee S.H. Ellett F. Deer E. Lieschke G.J. Witztum J.L. Tsimikas S. Miller Y.I. In vivo visualization and attenuation of oxidized lipid accumulation in hypercholesterolemic zebrafish.J. Clin. Invest. 2011; 121: 4861-4869Crossref PubMed Scopus (68) Google Scholar). Zebrafish were imaged by using a Nikon A1 confocal microscope, and images were analyzed as described (15.Fang L. Green S.R. Baek J.S. Lee S.H. Ellett F. Deer E. Lieschke G.J. Witztum J.L. Tsimikas S. Miller Y.I. In vivo visualization and attenuation of oxidized lipid accumulation in hypercholesterolemic zebrafish.J. Clin. Invest. 2011; 121: 4861-4869Crossref PubMed Scopus (68) Google Scholar). Cohort sizes for experiments involving Apoa1bp−/− mice, which were considered the major goal of the study, were calculated based on an assumption that the difference between means would be 1.3- to 2.0-fold, with SDs of 10–50% of mean, depending on experiment; 80% power; and P < 0.05. Animals were assigned to respective groups randomly within genotype. Individuals performing atherosclerosis analysis were not informed of genetic background or hypothesis. Glucose tolerance test was performed at 8 weeks of diet feeding and 18 weeks of age. Mice on the C57BL/6 background were fasted 4 h prior to testing. Glucose was administered to mice at a dose of 1 g per kg of body weight via ip injection of 25% dextrose in sterile saline (VetOne). Blood was collected from tail at times 0, 10, 30, 60, 90, and 120 min, and blood glucose was measured on a OneTouch Ultra glucose monitor. Additional blood was collected into heparinized capillary tubes at times 0 and 10 min for measurement of plasma insulin via ELISA (Alpco; catalog no. 80-INSMSH-E01). In cohorts of mice on the Ldlr−/− background, plasma was isolated from terminal blood, and plasma glucose levels were measured by using a kit from Crystal Chem (catalog no. 81692) and insulin levels by using an ultrasensitive insulin ELISA (Alpco; catalog no. 80-INSMSU-E01). Blood was collected from mice into EDTA tubes upon euthanization, and plasma was collected following centrifugation. Lipoprotein profiles were determined by pooling plasma from each genotype and quantifying cholesterol content of each fraction via fast protein liquid chromatography with a Superose 6 column. Atherosclerosis was assessed as previously described (17.Tsimikas S. Miyanohara A. Hartvigsen K. Merki E. Shaw P.X. Chou M.Y. Pattison J. Torzewski M. Sollors J. Friedmann T. et al.Human oxidation-specific antibodies reduce foam cell formation and atherosclerosis progression.J. Am. Coll. Cardiol. 2011; 58: 1715-1727Crossref PubMed Scopus (82) Google Scholar). Briefly, en face atherosclerosis was quantified via computer-assisted image analysis (ImagePro) of Sudan-stained whole aorta. Aortic root atherosclerosis was quantified by cutting cross-sections starting from the aortic origin until the last leaflet. Sections were stained with a modified Van Gieson stain, and lesion area was quantified via computer-assisted image analysis (ImagePro). RAW264.7 cells were cultured in DMEM (Cellgro) supplemented with 10% heat-inactivated FBS (Omega Scientific) and 50 μg/ml gentamicin (Omega Scientific). Cell and tissue lysates were subjected to gel electrophoresis and immunoblot as described (18.Choi S.H. Wiesner P. Almazan F. Kim J. Miller Y.I. Spleen tyrosine kinase regulates AP-1 dependent transcriptional response to minimally oxidized LDL.PLoS One. 2012; 7: e32378Crossref PubMed Scopus (24) Google Scholar). Antibodies were purchased from Cell Signaling Technology: p65 (catalog no. 4767), phospho-p65 (catalog no. 3033), ERK1/2 (catalog no. 4695), phospho-ERK1/2 (catalog no. 9101), and GAPDH (catalog no. 2118). Total RNA was isolated by using Nucleospin RNA columns (Clontech). Isolated RNA was reverse-transcribed by using RNA to cDNA EcoDry (Clontech) following the manufacturer's protocol. Quantitative PCR (qPCR) was performed by using a KAPA SYBR FAST Universal qPCR kit (KAPA Biosystems, KK4602), with primers ordered from Integrated DNA Technologies and a Rotor Gene Q thermocycler (Qiagen). Primer sequences are listed in supplemental Table S4. AIBP was produced in a baculovirus/insect cell system to allow for posttranslational modification and to ensure endotoxin-free preparation. Human AIBP was cloned into a pAcHLT-C vector behind the polyhedrin promoter. The vector contained an N-terminal His-tag to enable purification and detection. Insect Sf9 cells were transfected with BD BaculoGold Baculovirus DNA and the AIBP vector. After 4–5 days, the supernatant was collected to afford a baculovirus stock. Fresh Sf9 cells were infected with the AIBP-producing baculovirus; cell pellets were collected after 3 days, lysed, sonicated, and cleared by centrifugation; and the supernatants were loaded onto a Ni-NTA agarose column and eluted with imidazole. Protein was dialyzed against PBS, and the concentration was measured. Aliquots were stored at −80°C. In vitro experiments were conducted with Kdo2-LipidA (Avanti Polar Lipids), a well-characterized active component of lipopolysaccharide (LPS) and a highly specific Toll-like receptor 4 (TLR4) agonist (19.Raetz C.R. Garrett T.A. Reynolds C.M. Shaw W.A. Moore J.D. Smith Jr., D.C. Ribeiro A.A. Murphy R.C. Ulevitch R.J. Fearns C. et al.Kdo2-Lipid A of Escherichia coli, a defined endotoxin that activates macrophages via TLR-4.J. Lipid Res. 2006; 47: 1097-1111Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). In text and figure legends, we refer to Kdo2-LipidA as LPS. Mouse AIBP (25–283 aa) was fused with fibronectin secretion sequence (FIB) at the N terminus and 6X-His at the C terminus (FIB-AIBP-His). FIB-AIBP-His was cloned into the pAAV-MCS vector (Agilent Technologies). All clones were sequenced to confirm the presence of the insert. Adeno-associated virus (AAV)-293 cells (Agilent Technologies) were transfected with 20 μg each of pAAV-FIB-AIBP-His, pAAV2 (Agilent Technologies), and pHelper DNA (Agilent Technologies) following the routine calcium phosphate-based protocol (Agilent Technologies). Subsequent steps of virus harvest, purification, and storage were according to published protocols (20.Huang X. Hartley A-V. Yin Y. Herskowitz J.H. Lah J.J. Ressler K.J. AAV2 production with optimized N/P ratio and PEI-mediated transfection results in low toxicity and high titer for in vitro and in vivo applications.J. Virol. Methods. 2013; 193: 270-277Crossref PubMed Scopus (42) Google Scholar). Viral DNA was extracted from purified virus, and the number of gene copies (gc) was determined by using qPCR with primers for the inverted terminal repeats (Takara Bio Inc.). Testing the FIB-AIBP-His plasmid and the AAV2-FIB-AIBP-His, we found that infected HEK 293 cells expressed AIBP of the correct molecular mass, recognized by anti-His (ThermoFisher, catalog no. MA1-21315). For AAV2-mediated AIBP expression, Ldlr−/− mice were iv injected with empty virus or AAV2-AIBP at 1 × 1012 gc per mouse. At 3 weeks postinjection, mice were fed a Western diet (Teklad TD.96121) for an additional 16 weeks. Testing for the expression of FIB-AIBP-His, liver was homogenized in RIPA buffer, and lysates were analyzed in Western blot. Flow cytometry of white adipose tissue (WAT) was performed as previously described (21.Sears D.D. Miles P.D. Chapman J. Ofrecio J.M. Almazan F. Thapar D. Miller Y.I. 12/15-Lipoxygenase is required for the early onset of high fat diet-induced adipose tissue inflammation and insulin resistance in mice.PLoS One. 2009; 4: e7250Crossref PubMed Scopus (105) Google Scholar). In brief, mice were euthanized, and tissue was perfused with PBS. WAT was minced in 5% BSA Dulbecco's PBS and centrifuged to remove erythrocytes and free leukocytes. Tissue was dissociated by using collagenase (Sigma C6885), filtered, and separated from adipocytes via centrifugation. The stromal vascular fraction was used for FACS analysis. Antibodies used were CD11b-FITC (BD 553310), F4/80-APC (Ab Serotec MCA497APC), and CD11c-PE (BD 553802). A cholesterol efflux assay was performed as described (22.Whetzel A.M. Sturek J.M. Nagelin M.H. Bolick D.T. Gebre A.K. Parks J.S. Bruce A.C. Skaflen M.D. Hedrick C.C. ABCG1 deficiency in mice promotes endothelial activation and monocyte-endothelial interactions.Arterioscler. Thromb. Vasc. Biol. 2010; 30: 809-817Crossref PubMed Scopus (44) Google Scholar, 23.O'Connell B.J. Denis M. Genest J. Cellular physiology of cholesterol efflux in vascular endothelial cells.Circulation. 2004; 110: 2881-2888Crossref PubMed Scopus (82) Google Scholar), with modifications. In brief, RAW264.7 cells were loaded with 2 µCi/ml [3H]cholesterol (American Radiolabeled Chemicals), and cholesterol efflux was initiated by the addition of efflux medium containing 25 µg/ml HDL, in the presence or absence of 0.2 µg/ml recombinant AIBP, for 4 h. Background, nonspecific release of [3H]cholesterol was measured in the absence of HDL. After incubation, the medium was collected and counted in a liquid scintillation counter LS 6500 (Beckman Coulter). The cells were extracted with 2-propanol, and the lipid extract was added to ScintiVerse BD Cocktail (Fisher) and counted. Cholesterol efflux was expressed as a percentage of [3H] counts in the medium compared with combined [3H] counts in the cells and the medium. In these experiments, to replicate in vivo conditions, cells were not treated with a liver X receptor agonist or acyl-CoA cholesterol acyltransferase inhibitor. For lipid raft measurements, RAW264.7 macrophages were incubated with FITC-conjugated cholera toxin B (CTB; Sigma) for 1 h on ice. Cells were washed two times with a FACS buffer, fixed with 3.7% formaldehyde for 15 min on ice, washed three times with a FACS buffer, and analyzed by using a FACSCanto II (BD Biosciences) flow cytometer. Results were analyzed by using Student's t-test (for differences between two groups), one-way ANOVA (for multiple groups), or two-way ANOVA with the Bonferroni post hoc test (for multiple groups time course experiments), using GraphPad Prism. Differences between groups with P < 0.05 were considered statistically significant. Values were excluded if determined to be a significant outlier via the extreme studentized deviate test. Systemic Apoa1bp knockout mice have been developed in our laboratory as described (10.Mao R. Meng S. Gu Q. Araujo-Gutierrez R. Kumar S. Yan Q. Almazan F. Youker K.A. Fu Y. Pownall H.J. et al.AIBP limits angiogenesis through gamma-secretase-mediated upregulation of Notch signaling.Circ. Res. 2017; 120: 1727-1739Crossref PubMed Scopus (33) Google Scholar). C57BL/6 and Apoa1bp−/− mice were fed a high-fat, normal-cholesterol diet (HFD) for 10 weeks starting from the age of 10 weeks. Despite having similar weights prior to diet feeding, Apoa1bp−/− mice were significantly heavier than their wild-type counterparts following the diet, despite consuming equivalent amounts of food (Fig. 1A, B). Apoa1bp−/− mice also exhibited impaired glucose clearance and increased circulating plasma insulin levels when subjected to a glucose tolerance test (Fig. 1C–E). Flow cytometry analysis of the WAT stromal vascular fraction revealed that epididymal WAT from Apoa1bp−/− mice contained a greater proportion of F4/80+CD11b+CD11c+ proinflammatory macrophages (Fig. 1F). Overall, Apoa1bp−/− mice exhibited more hallmarks of metabolic disease than their wild-type counterparts when fed a HFD. Similar to HFD-fed Apoa1bp−/− mice, Apoa1bp−/−Ldlr−/− mice fed a Western diet (high-fat, high-cholesterol) for 12 weeks became significantly heavier than Ldlr−/− mice (Fig. 2A). The livers of Apoa1bp−/−Ldlr−/− mice had increased cholesterol and triglyceride content (Fig. 2B, C). Liver expression of TNFα mRNA was increased nearly 3-fold in Apoa1bp−/−Ldlr−/− mice, suggesting increased liver inflammation (Fig. 2D). Plasma levels of glucose and insulin trended higher in terminal blood of nonfasted Apoa1bp−/−Ldlr−/− mice (supplemental Fig. S1A, B). Apoa1bp−/−Ldlr−/− mice fed a Western diet had higher plasma cholesterol and triglyceride levels, primarily due to elevated VLDL and LDL, as compared with Ldlr−/− mice (Fig. 3A–D). Importantly, atherosclerotic lesions in the aortic root were significantly larger in Apoa1bp−/−Ldlr−/− mice compared with Ldlr−/− mice (Fig. 3E–G). Overall, mice lacking AIBP had significantly increased weight gain, lipid levels in both plasma and liver tissue, and atherosclerosis when fed a high-fat, high-cholesterol diet. However, feeding Apoa1bp−/−Ldlr−/− mice a diet enriched in cholesterol (1%) but with normal fat content did not result in significant differences in weight, plasma cholesterol, triglycerides, glucose levels, or atherosclerosis when compared with Ldlr−/− mice (Fig. 4 and supplemental Fig. S1C). Plasma insulin levels in Apoa1bp−/−Ldlr−/− mice were higher than in Ldlr−/− mice, but absolute numbers remained low (supplemental Fig. S1D). These results suggest the importance of AIBP-mediated metabolic changes in the development of atherosclerosis in the context of high-fat, high-cholesterol diets.Fig. 4Unchanged weight gain, hyperlipidemia, and atherosclerosis in AIBP-deficient mice fed a high-cholesterol, normal-fat diet. Ldlr−/− and Apoa1bp−/−Ldlr−/− mice were fed a 1% cholesterol, normal-fat diet for 16 weeks starting from age 8 weeks. A: Weight at the time of euthanization (n = 10; Student's t-test). B–E: Plasma lipids at euthanization. Total cholesterol (B), cholesterol lipoprotein profile (C), total triglycerides (D), and triglyceride lipoprotein profile (E) (n = 10, Student's t-test) are shown. F: En face atherosclerotic lesions (n = 10; Student's t-test). G: Aortic root atherosclerotic lesion size as a function of distance from first leaflet appearance (n = 16; two-way ANOVA with Bonferroni's post hoc test). H: Aortic root lesion volume (area under the curve from G; n = 16, Student's t-test). Mean ± SEM; NS, not significant differences.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Next, we tested if sustained overexpression of mouse AIBP can reduce atherosclerosis. We generated an AAV2 with the FIB-AIBP-His construct. The fibronectin signal peptide (FIB) was inserted to ensure secretion of His-tagged mouse AIBP. Five-week-old Ldlr−/− mice were infected with AAV2-AIBP or the empty AAV2 (control) and, starting at 8 weeks of age, were fed a Western diet for 16 weeks. At the time of euthanization, livers were collected to confirm AIBP-His protein expression (supplemental Fig. S2); however, AIBP-His was not detectable in plasma. Both the control and AAV2-AIBP cohorts had similar weights prior to the start of the diet, but the mice overexpressing AIBP were significantly protected against diet-induced weight gain (Fig. 5A). Liver triglyceride levels were significantly lower and cholesterol levels trended lower in mice infected with AAV2-AIBP compared with the mice infected with the empty AAV2 (Fig. 5B, C). Plasma cholesterol levels had a trend toward reduction (Fig. 5D, E), and plasma triglycerides were significantly decreased (Fig. 5F, G). Importantly, there was a significant decrease in en face lesions (Fig. 5H), as well as a trend of reduced aortic root atherosclerosis in the AAV2-AIBP mice (Fig. 5I, J). Together, these data show protection against weight gain, plasma lipid increases, and atherosclerosis by AAV-delivered AIBP. We have previously described a hyperlipidemic zebrafish model in which vascular accumulation of lipid deposits was observed in live animals 10–14 days after initiation of high-cholesterol feeding (15.Fang L. Green S.R. Baek J.S. Lee S.H. Ellett F. Deer E. Lieschke G.J. Witztum J.L. Tsimikas S. Miller Y.I. In vivo visualization and attenuation of oxidized lipid accumulation in hypercholesterolemic zebrafish.J. Clin. Invest.