Proteomic Analysis of HDL from Inbred Mouse Strains Implicates APOE Associated with HDL in Reduced Cholesterol Efﬂ Ux Capacity Via the ABCA1 Pathway

Supplemental Material can be found at: http://www.jlr.org/content/suppl/2015/12/15/jlr.M063701.DC1 .html ˔ Author’s Choice Proteomic analysis of HDL from inbred mouse strains implicates APOE associated with HDL in reduced cholesterol effl ux capacity via the ABCA1 pathway Nathalie Pamir , 1, * Patrick Hutchins , * Graziella Ronsein , * Tomas Vaisar , * Catherine A. Reardon , † Godfrey S. Getz , † Aldons J. Lusis , § and Jay W. Heinecke * Downloaded from Department of Medicine,* University of Washington , Seattle, WA ; Department of Pathology, † University of Chicago , Chicago, IL ; and Department of Genetics, § University of California at Los Angeles , Los Angeles, CA Abstract Cholesterol effl ux capacity associates strongly Supplementary key words atherosclerosis • cardiovascular risk • mass and negatively with the incidence and prevalence of human spectrometry • high density lipoprotein • apolipoprotein E • ATP bind- CVD. We investigated the relationships of HDL’s size and ing cassette transporter A1 www.jlr.org protein cargo with its cholesterol effl ux capacity using APOB-depleted serum and HDLs isolated from fi ve inbred mouse strains with different susceptibilities to atherosclero- Clinical and epidemiological studies show a robust at Univ of Washington Health Sciences Library SB-55, on February 5, 2016 sis. Like humans, mouse HDL carried >70 proteins linked to inverse association of HDL-cholesterol (HDL-C) levels lipid metabolism, the acute-phase response, proteinase inhi- with CVD risk ( 1 ). In randomized clinical trials, how- bition, and the immune system. HDL’s content of specifi c ever, two drugs, CETP inhibitors and niacin, that elevate proteins strongly correlated with its size and cholesterol effl ux capacity, suggesting that its protein cargo regulates its HDL-C levels by different mechanisms, failed to reduce function. Cholesterol effl ux capacity with macrophages CVD risk in statin-treated humans with established ath- strongly and positively correlated with retinol binding pro- erosclerosis ( 2, 3 ). These observations indicate that rais- tein 4 (RBP4) and PLTP, but not APOA1. In contrast, ing HDL-C levels does not necessarily improve HDL’s ABCA1-specifi c cholesterol effl ux correlated strongly with cardioprotective functions. It is therefore critical to de- HDL’s content of APOA1, APOC3, and APOD, but not velop new metrics for assessing HDL’s functionality in RBP4 and PLTP. Unexpectedly, APOE had a strong nega- humans ( 2 ). tive correlation with ABCA1-specifi c cholesterol effl ux ca- Many lines of evidence support the proposal that me- pacity. Moreover, the ABCA1-specifi c cholesterol effl ux diation of cholesterol effl ux from macrophages is one of capacity of HDL isolated from APOE-defi cient mice was signifi cantly greater than that of HDL from wild-type mice. HDL’s important functions ( 4 ). This process is the fi rst Our observations demonstrate that the HDL-associated step of reverse cholesterol transport, in which HDL APOE regulates HDL’s ABCA1-specifi c cholesterol effl ux accepts cholesterol from cells and carries it back to the capacity. These fi ndings may be clinically relevant be- liver for excretion. Two cellular pathways for choles- cause HDL’s APOE content associates with CVD risk and terol effl ux involve the membrane ABC transporters, ABCA1 defi ciency promotes unregulated cholesterol accu- ABCA1 and ABCG1, which are highly induced when mulation in human macrophages. —Pamir, N., P. Hutchins, macrophages accumulate excess cholesterol ( 5, 6 ). ABCA1 G. Ronsein, T. Vaisar, C. A. Reardon, G. S. Getz, A. J. Lusis, exports phospholipids and cholesterol from the cell and J. W. Heinecke. Proteomic analysis of HDL from inbred mouse strains implicates APOE associated with HDL in re- membrane to lipid-poor apolipoproteins ( 6 ) and to duced cholesterol effl ux capacity via the ABCA1 pathway. small dense HDL ( 7 ), while ABCG1 exports cholesterol J. Lipid Res. 2016. 57: 246–257. to mature HDL particles (5, 6 ). Humans with Tangier disease, who are defi cient in ABCA1, accumulate cholesterol-loaded macrophages in many different tissues ( 8, This work was supported by grants from the National Institutes of Health (R01 9 ). Moreover, mouse studies demonstrate that defects HL112625, R01 HL108897, P30 DK017047, and P01 HL09296) and the American Heart Association (13SDG16940064). MS experiments were per- formed by the Mass Spectrometry Resource, Department of Medicine, University of Washington, and the Quantitative and the Functional Proteomics Core of the Diabetes Research Center, University of Washington. The authors have no fi - nancial confl icts of interest to disclose. The content is solely the responsibility of Abbreviations: HDL-C, HDL-cholesterol; HDL-Pima , HDL particle the authors and does not necessarily represent the offi cial views of the National concentration and size quantifi ed by calibrated ion mobility analysis; Institutes of Health. H2-Q10, histocompatibility 2 Q region locus 10; IMA, ion mobility anal- ˔ Author’s Choice —Final version free via Creative Commons CC-BY license. ysis ; RBP4, retinol binding protein 4. 1 To whom correspondence should be addressed. Manuscript received 11 September 2015 and in revised form 15 December 2015. e-mail: [email protected] Published, JLR Papers in Press, December 15, 2015 The online version of this article (available at http://www.jlr.org) DOI 10.1194/jlr.M063701 contains a supplement. Copyright © 2016 by the American Society for Biochemistry and Molecular Biology, Inc. 246 Journal of Lipid Research Volume 57, 2016 This article is available online at http://www.jlr.org Supplemental Material can be found at: http://www.jlr.org/content/suppl/2015/12/15/jlr.M063701.DC1 .html in the apolipoprotein/ABCA1 pathway are important METHODS determinants of cholesterol accumulation by macrophages ( 5 ). Mice It has been diffi cult to assess the clinical relevance All studies were approved by the Animal Care and Use Com- of cholesterol effl ux. However, recent studies of cAMP- mittees of the University of Washington and the University of stimulated J774 macrophages radiolabeled with choles- California, Los Angeles. Mice were housed (three to fi ve per cage) in a specifi c pathogen-free barrier facility (22°C) with a terol, which measures cellular effl ux by multiple pathways 12 h light/dark cycle with free access to food and water. All the (ABCA1, ABCG1, SR-B1, and diffusion) ( 10 ), demonstrate strains were a fed low-fat diet (Wayne Rodent BLOX 8604; Har- strong inverse correlations between cholesterol effl ux ca- lan Teklad Laboratory) until they were 8 weeks old. Then mice pacity of serum HDL (serum depleted of APOB-containing that were fasted for 4 h in the morning were bled from the retro- lipoproteins) and prevalent coronary artery disease ( 11 ). orbital sinus into tubes containing EDTA (fi nal concentration Moreover, macrophage effl ux capacity remained a strong 1 mM) and euthanized by isofl uorane inhalation. Plasma was col- predictor of prevalent coronary artery disease after adjust- lected and stored at Ϫ 80°C until analysis. ment for HDL-C levels ( 11 ). Plasma lipid and APOA1 measurements The cholesterol effl ux capacity of serum HDL with Downloaded from cAMP-stimulated J774 macrophages can also be assessed Triglycerides and phospholipids (Wako Diagnostics) and cholesterol levels (Invitrogen) were determined biochemically. with fl uorescently labeled cholesterol, which primarily APOA1 levels were determined by immunoblot analysis with a measures ABCA1-mediated effl ux from these cells (12 ). A goat anti-mouse APOA1 antibody (US Biological). recent study of a population-based cohort that was free of CVD demonstrated that ABCA1-specifi c cholesterol effl ux Cholesterol effl ux assays www.jlr.org capacity assessed by this method associates strongly and Macrophage cholesterol effl ux capacity was assessed with J774 negatively with the risk of future cardiac events ( 13 ). This macrophages labeled with [ 3 H]cholesterol and stimulated with a association persisted after multivariate adjustment, sug- cAMP analog, as described by Rothblat and colleagues ( 10 ). Ef- gesting that altered HDL function affects cardiovascular fl ux via the ABCA1 or ABCG1 pathways was measured with BHK at Univ of Washington Health Sciences Library SB-55, on February 5, 2016 cells expressing mifepristone-inducible human ABCA1 or ABCG1 risk by processes distinct from those involving HDL-C or 3 3 traditional lipid risk factors. Impaired cholesterol effl ux that were radiolabeled with [ H]cholesterol ( 20 ). Effl ux of [ H] cholesterol was measured after a 4 h incubation in medium with capacity with J774 macrophages radiolabeled with choles- APOB-depleted serum HDL (2.8% v/v) or isolated HDL (30 ␮ g terol also predicted incident cardiac events in a healthy protein per milliliter). ABCA1-specifi c cholesterol effl ux capacity cohort ( 14 ). Taken together, these observations provide was calculated as the percentage of total [ 3 H]cholesterol (me- strong evidence that cholesterol effl ux capacity is a better dium plus cell) released into the medium of BHK cells stimu- measure of HDL’s cardioprotective effects than HDL-C. lated with mifepristone after the value obtained with cells The molecular factors that control HDL’s serum effl ux stimulated with medium alone was subtracted. capacity are poorly understood ( 10, 12 ). For example, HDL-C predicts only 34% of serum HDL’s macrophage HDL isolation cholesterol effl ux capacity ( 11 ); HDL-C fails to predict Serum HDL was prepared by adding calcium (2 mM fi nal con- HDL’s ABCA1-specifi c cholesterol effl ux capacity with centration) to plasma and using polyethylene glycol (8 kDa; Sigma) to precipitate lipoproteins containing APOB (VLDL, cells labeled with fl uorescent cholesterol ( 12, 13 ). Macro- IDL, LDL). After centrifugation at 10,000 g for 30 min at 4°C, phage cholesterol effl ux capacity of serum HDL also cor- serum HDL was harvested from the supernatant. HDL was iso- relates poorly with plasma APOA1 levels ( 11 ), even though lated from serum or EDTA-anticoagulated plasma using se- APOA1 is HDL’s major structural protein, accounting for quential ultracentrifugation (d = 1.063–1.21 mg/ml) ( 15, 21).

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