The Function and Distribution of Apolipoprotein A1 in the ... 10.1161/CIRCULATIONAHA.113.002624 1 The Function and Distribution of Apolipoprotein A1 in the Artery Wall are Markedly

DOI: 10.1161/CIRCULATIONAHA.113.002624

1

The Function and Distribution of Apolipoprotein A1 in the Artery Wall are

Markedly Distinct from those in Plasma

Running title: DiDonato et al.; Artery wall apoA1 is dysfunctional and not in HDL

Joseph A. DiDonato, PhD1*; Ying Huang, PhD1*; Kulwant Aulak, PhD1; Orli Even-Or, PhD2;

Gary Gerstenecker, PhD1,3; Valentin Gogonea, PhD1,3; Yuping Wu, PhD4; Paul L. Fox, PhD1;

W.H. Wilson Tang, MD1,5; Edward F. Plow, PhD6; Jonathan D. Smith, PhD1,5;

Edward A. Fisher, MD2; Stanley L. Hazen, MD PhD1,3,5

1Dept of Cellular & Molecular Medicine; 6Dept of Molecular Cardiology, Lerner Research

Institute; 5Dept of Cardiovascular Medicine, Heart and Vascular Institute, Cleveland Clinic,

Cleveland, OH; 2Dept of Medicine, New York University, New York, NY; 3 Dept of Chemistry; 4Dept of Mathematics, Cleveland State University, Cleveland, OH

*Co-first authors

Address for Correspondence:

Joseph DiDonato, PhD Stanley L Hazen, MD, PhD

Cleveland Clinic Cleveland Clinic

9500 Euclid Avenue, NE-10 9500 Euclid Avenue, NE-10

Cleveland, OH, 44195 Cleveland, OH, 44195

Tel: 216-445-2174 Tel: 216-445-9763

Fax: 216-636-0392 Fax: 216-444-9404

E-mail: [email protected] E-mail: [email protected]

Journal Subject Codes: Atherosclerosis:[90] Lipid and lipoprotein metabolism, Vascular biology:[96] Mechanism of atherosclerosis/growth factors, Atherosclerosis:[137] Cell biology/structural biology

Edward A. Fisher, MD2; Stanley L. Hazen, MD PhD1,3,5

1Dept of Cellular & Molecular Medicine; 6Dept of Molecular Cardiology, Lerner Research

InInInstststiititutte;e;; 55DeDeptptpt oof Cardiovascular Medicine, HHHeaaart and Vascullaraa Insnstitititutute, Cleveland Clinic,

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Abstract

Background—Prior studies show apolipoprotein A1 (apoA1) recovered from human

atherosclerotic lesions is highly oxidized. Ex vivo oxidation of apoA1 or high density lipoprotein

(HDL) cross-links apoA1 and impairs lipid binding, cholesterol efflux and lecithin cholesterol

acyltransferase (LCAT) activities of the lipoprotein. Remarkably, no studies to date directly

quantify either the function or HDL particle distribution of apoA1 recovered from the human

artery wall.

Methods and Results—A monoclonal antibody (mAb 10G1.5) was developed that equally

recognizes lipid-free and HDL-associated apoA1 in both native and oxidized forms. Examination

of homogenates of atherosclerotic plaque-laden aorta showed >100-fold enrichment of apoA1

compared to normal aorta (P<0.001). Surprisingly, buoyant density fractionation revealed only a

minority (<3% of total) of apoA1 recovered from either lesions or normal aorta resides within an

HDL-like particle (1.063 d 1.21). In contrast, the majority (>90%) of apoA1 within aortic

tissue (normal and lesions) was recovered within the lipoprotein-depleted fraction (d>1.21).

Moreover, both lesion and normal artery wall apoA1 is highly cross-linked (50-70% of total),

and functional characterization of apoA1 quantitatively recovered from aorta using mAb 10G1.5

showed ~80% lower cholesterol efflux activity and ~90% lower LCAT activity relative to

circulating apoA1.

Conclusions—The function and distribution of apoA1 in human aorta are quite distinct from

those found in plasma. The lipoprotein is markedly enriched within atherosclerotic-plaque,

predominantly lipid-poor, not associated with HDL, extensively oxidatively cross-linked, and

functionally impaired.

Key words: plaque, apolipoproteins, arteriosclerosis, cardiovascular diseases

g p p

of homogenates of atherosclerotic plaque-laden aorta showed >100-fold enrichmemeentntnt ooof f apapapoAoAoA111

compared to normal aorta (P<0.001). Surprisingly, buoyant density fractionation revealed only a

mimiinononorirityt ((<3<3<3%%% ofof ttototall)) ofof aapopoA1A rece ovo erered fror mm eeiitherr llesesions ooorr r noormrmalal aorrtaa resideses wwithin an

HDHDH LL-like particlclle (1.000633 ddd 11.22211)1). InIn connntrraast, thhhe mmajajajorororityyy (>>>9900%) offf apappooAA1 wwwitththininn aaaorttiic

iisss ueuee (((nononormrmrmalal aaandndnd lllesesesioioionsnsns)) wawaass s rerereccocovevererered d d wiwiwithththininin ttthehehe llipipipopopoprorooteteteinin-d-d-depepepleleletetetedd d frfracacctititiononon (((d>d>>111.212121).).)

Moreover, bobooththth lllesesesioioionn n anana d d nononormrmalalal aaartrtererry yy wawaalllll aaapppoAoAoA111 isis hhhigighlhlhly y y crcrossss-s-lililinknknkededed ((50500-7-770%0%0% ooof total)),



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Introduction

The poor performance of several recent clinical trials targeting elevation of high density lipoprotein

(HDL) cholesterol1-3, and the recent Mendelian genetic studies questioning a causal link between

genetic variants controlling HDL cholesterol levels and cardiovascular disease risk4, argue for a

reappraisal of our understanding of HDL. Such a reappraisal demands that we question

assumptions about the pathobiology of the lipoprotein, particularly where direct investigation is

lacking. Much of what is known biologically about apoA1 in human studies comes from

investigations employing isolated lipoprotein particles from the circulation (plasma or serum)

using buoyant density ultracentrifugation. A known exchangeable lipoprotein, it is widely

recognized that the vast majority of apoA1 within the circulation resides on spherical HDL

particles where it serves as the major structural protein of a complex macromolecular assembly of

lipoprotein particles with defined buoyant density (1.063 d 1.21)5. An unproven assumption is

that the numerous biological functions observed with HDL or apoA1 recovered from the

circulation will mirror what occurs elsewhere in vivo.

The functional properties of apoA1 and HDL within the circulation, however, may not

faithfully reflect what occurs within the artery wall. Early studies identified that lipoproteins

isolated from the artery wall, particularly LDL, undergo assorted alterations including proteolysis,

various oxidative modifications and lipolysis to varying extents6, 7. Several years ago we reported

that apoA1 recovered from human atherosclerotic arterial lesions was selectively targeted for

oxidative modification by myeloperoxidase (MPO)-generated and nitric oxide (NO)-derived

oxidants, and that oxidative modification of apoA1 and HDL ex vivo to a comparable extent

resulted in loss of cholesterol efflux activity of the lipoprotein8. Parallel functional characterization

and mass spectrometry studies of circulating HDL isolated by buoyant density ultracentrifugation

ecognized that the vast majority of apoA1 within the circulation resides on sphericiccalal HHDLDLDL

particles where it serves as the major structural protein of a complex macromolecular assembly of

ipopooprprprototot ieieinnn papaparticccleleless with defined buoyant density y y (1(1.063 d 1.221)11 5. AnAnAn uunproven assumption is

hhhattt tthe numerrououus bbibiololologoggicicicaalal fffuununctctioioi nnns oobbbserveveved wwiiththh HHHDLDDL ooor apapoAoAoAr 11 reeccocoveverreredd d frfromomom tthehehe

ciircrcculululatatatioionn wiwiwillll mmmirrrrororr wwhwhata oooccccc ururursss elele seseewhwhwherereree e ininn vviivivooo.

The fufuuncncnctitionononalal ppprorr pepepertrtrtieies s s ofofo aaapopooA1A1A1 aaandndnd HHHDLDLDL wiwithththininn ttthehehe ccciriri cucuulalaatititiononon,,, hohoh wewewevevever,r,r, mmmay not



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revealed that higher apoA1 content of oxidative modifications specifically formed by MPO- and

NO-derived oxidants was associated with impairment in plasma membrane transporter ATP-

binding cassette A1 (ABCA1)-dependent cholesterol efflux function of the lipoprotein8, lecithin

cholesteryl acyl transferase (LCAT) activity and acquisition of pro-inflammatory activity9, 10.

Similar findings have been replicated by other groups11, 12, and numerous additional proteomics

studies have since mapped site-specific oxidative modifications to apoA1 recovered from the

human artery wall13-16. These studies collectively reveal that apoA1 is extensively oxidatively

modified within an atherosclerotic-laden artery wall, and similar oxidative modifications to the

lipoprotein ex vivo are associated with pro-atherogenic changes in apoA1 function. Of note,

however, no studies to date have directly examined the functional properties or the particle

distribution of apoA1 recovered from human artery wall. The paucity in direct functional

characterization studies is likely a result of the significant challenges that exist in obtaining

sufficient quantities of fresh human arterial tissue for such biochemical and biological studies.

Herein we sought to examine both the distribution and the functional properties of apoA1

recovered from the human artery wall. The present studies demonstrate multiple remarkable

findings, including direct evidence that the biological function and HDL particle distribution of

apoA1 within both normal and atherosclerosis-laden human aortic tissues is markedly distinct from

that of circulating apoA1 and HDL. These studies suggest that the historical focus thus far on

circulating HDL cholesterol levels may not adequately reflect what is going on with regard to

apoA1 function and HDL particle distribution within the artery wall.

Materials and Methods

Materials

D2O was purchased from Cambridge Isotopes, Inc (Andover, MA). Chelex-100 resin, fatty acid-

however, no studies to date have directly examined the functional properties or thhee e paaarrticiciclelele

distribution of apoA1 recovered from human artery wall. The paucity in direct functional rr

chharararacacacteteteriririzazazatititioon ssstututudid es is likely a result of the signgngniffficant challenggesee thahaatt t eeexist in obtaining

uufffficicient quantntitititiees ofofof ffrerereshshsh hhhuumumanann aaartrtereriial titiissssue fffoorr suucuchhh bbiioocchehemimicacalll ananand d bibibioloologogicicicalalal ssttutudididieses..

HeHererereininin wwe sosos uguughhtht ttoo exexexamammininineee bobooththh ttthhehe dddisisi trtrribbubutitiiononn aaandndnd ttthehehe ffunununctctiiionnanal l prprp opopopererrtitiiees ooof f apapoAoAoA11

ecovered fromomm ttthehee hhhumummanaa aaartrtterere y y y wawawalllll . ThThT eee prprpresese enenent t stststududu ieiees ss dededemomomonsnsn trrratata ee e mumumultlttipipi lelee rrremememarararkak ble



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free bovine serum albumin (BSA) and crystalline catalase (from bovine liver; thymol-free) were

purchased from Boehringer-Mannheim (Ridgefield, CT). Sodium phosphate, H2O2 and NaOCl

were purchased from Fisher Chemical Company (Pittsburgh, PA). Commercial apoA1

antibodies were from Abcam (Cambridge, MA), Santa Cruz Biotechnologies (South San

Francisco, CA), and Genway/Sigma (St. Louis, MO). 1,2-dimyristoyl-sn-glycero-3-

phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD-PE), was purchased from

Avanti Polar Lipids. All other materials were purchased from Sigma Chemical Company (St.

Louis, MO) except where indicated.

Methods

General Procedures

Circulating HDL and plasma-derived apoA1 purified were obtained from healthy volunteer

donors who gave written informed consent and the Institutional Review Board of the Cleveland

Clinic approved the study protocol. Mouse studies involving monoclonal antibody generation

were performed under protocols approved by the Institutional Animal Care and Use Committee

at the Cleveland Clinic. Lipoproteins, including HDL and HDL-like particles (1.063 d 1.21)

from plasma and tissue homogenates, respectively, were isolated by sequential buoyant density

ultracentrifugation at low salt concentrations using D2O/sucrose17. Protein concentrations were

determined by Markwell modified protein assay with bovine serum albumin as standard. Human

apoA1 used as control for cholesterol efflux and LCAT activity assays was purified as

described18. Reconstituted HDL (rHDL) from isolated apoA1 was prepared by cholate dialysis

method19 employing a molar ratio of apoA1:POPC:cholesterol of 1:100:10.HDL particles were

further purified by gel filtration chromatography using a Sephacryl S300 column (GE

Healthcare,Waukesha, WI) on a Bio-Rad Biologics DuoFlo FPLC (Bio-Rad, Hercules, CA).

General Procedures

Circulating HDL and plasma-derived apoA1 purified were obtained from healthy volunteer ff

doonononorsrsrs wwhohoho gggavvee e wwrwritten informed consent and ttthhhe Institutional RRReve ieew w w BBoard of the Cleveland

CCClinnnici approveeddd thhhe ststtududdyy prprprotototococololol.. MMoouuuse ssstuuudieess iinvovoollvlvininng momoonooocclolonnanall ananntititibbobodydydy gggenennerrrataatiooion n

weweererer pppererfoformrmrmeded uunndndererr pprorototoccocolslsls aaapppppproroveveved dd bbyby tttheheh IInnsnstiiitutuutititionononalall AAAninin mamamal l CaCaarere aaandndnd UUUsesee CComomommimiittteeeee

at the Clevelalaandndnd CCClililininin c.c.c Lipippopopoproooteteteininns,ss iiinnnclclcludududinining g g HDHDHDL L L anannd d d HDHDHDL-L-L lill kekeke pppararartititiclclclesese (((1.1.1.060606333 d 1.21)



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Myeloperoxidase (MPO) (donor: hydrogen peroxide, oxidoreductase, EC 1.11.1.7) was isolated

(final A430 /A280 ratio of 0.6) as described8, 13, 14and its concentration was determined

spectrophotometrically ( 430 = 170 mM-1cm-1)8, 13, 14 H2O2 and OCl concentrations were each

determined spectrophotometrically ( 240 = 39.4 M-1cm-1;20 and 292 = 350 M-1cm-1;8, 13, 14,

respectively) prior to use. Peroxynitrite (ONOO-) was purchased from Cayman Chemicals (Ann

Arbor, MI), and quantified spectrophotometrically prior to use ( 302=1.36 mM 1 cm 1)13, 14. All

buffers used were passed through a Chelex-100 column and supplemented with 100 M

diethylenetriaminepentaacetic acid (DTPA) to remove any trace levels of redox-active metals.

All glassware used was rinsed with 100 M DTPA, pH 7.4, then Chelex-100 treated distilled

deionized H2O, and baked at 500°C prior to use. Sodium dodecyl sulfate-polyacrylamide gel

electrophoresis (SDS-PAGE) was performed as described13.

Tissue Collection

Fresh surgical specimens of human aortic tissue were obtained as discarded material, both at

time of organ harvest from transplant donors and during valve/aortic arch ("elephant trunk")

replacement surgery. Tissue was immediately rinsed in ice-cold normal saline until free of

visible blood, submerged in argon-sparged 65 mM sodium phosphate buffer (pH 7.4)

supplemented with 100 M DTPA and 100 M butylated hydroxytoluene (BHT), and stored at -

80°C in screw cap specimen containers in which headspace was purged with argon. BHT was

omitted from buffer in specimens where apoA1 was isolated for functional activity assays.

Monoclonal antibody (mAb) 10G1.5 generation, specificity and labeling

Hybridoma cell lines were generated by immunizing apoA1-/- mice with purified delipidated

human apoA1 isolated from HDL recovered from healthy donors. Amongst the positive clones,

subclones were screened until a monoclonal antibody (mAb) with desired binding specificity for

All glassware used was rinsed with 100 M DTPA, pH 7.4, then Chelex-100 treaeaateteted d d dididistststilililleleled d d

deionized H2O, and baked at 500°C prior to use. Sodium dodecyl sulfate-polyacrylamide gel

elecctrtrropopophohohorereresisis s (S(SSDSDD -PAGE) was performed as dededesscs ribed13.

TTiTissssue Collelectctioioionn

FrFrresese h hh susurggiciccalalal sspppeccicimmeennns oof f huhuumamann n aaoaortrticici ttiisssuuue e wewewerere obbtbtaiaineneed d aaas ddisscaaardddededd mmmatatereriaiaialll, bbbotth h aatt

iiimememe ooofff orororgagagannn hahaharvrvrvesesesttt frfrfromomom tttrararansnsnsplplplanananttt dododonononorsrsrs aaandndnd dddurururininingg g vavavalvlvlve/e/e/aoaoaortrtrticicic aaarrrchchch ((("e"eelelelephphphanananttt trtrtrunununk"k"k )))



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equal recognition of all forms of apoA1 (see below) was identified. The subclone, mAb 10G1.5,

was selected by screening for equal recognition of lipid-free and lipidated (in rHDL) apoA1

under native conditions, as well as following oxidation by exposure to multiple different systems

including MPO/H2O2/Cl-, MPO/H2O2/NO2-, and CuSO4 (oxidized as outlined below). To

produce sufficient levels of 10G1.5 for immune-affinity purification of apoA1 from arterial

tissues, hybridoma clones were injected into pristane-treated male BALB/c mice (8 weeks of

age). Ascites fluid was collected, precipitated with ammonium sulfate, then bound and eluted

from a protein A/G column (Thermo Scientific Pierce, Rockford, IL) to purify mouse

monoclonal antibodies. Isotypes of the mAbs were determined with the mouse mAb isotyping kit

(Catalog# 26179, Pierce Rapid Antibody Isotyping Strips plus Kappa and Lambda – Mouse,

Thermo Scientific Pierce, Rockford, IL).

Specificity of mAb 10G1.5 was tested using apoA1 or rHDL either in native form, or

following incubation at 37°C in 60 mM Na[PO4] buffer (pH 7.4) with multiple different

oxidation systems. The different MPO systems consisted of 19 nM MPO, 100 �M DTPA, 40

�M H2O2 and either 100 mM NaCl or 1 mM KBr or 1mM NaNO2 as indicated. Horseradish

peroxidase (HRP 19 nM) was used with 40 μM H2O2. ApoA1 and rHDL were exposed to MPO

and HRP for 90 min at 37°C and the reactions were stopped by addition of 2mM methionine and

300 nM catalase. All other oxidation reactions were carried out for 24 hrs at 37°C. Final

concentrations of oxidants used were: H2O2, 40 μM; ONOO-, 40 �M; ONOO-/HCO3- (40 μM

each); CuSO4, 10 μM; CuSO4/H2O2 (10 μM and 40 μM, respectively); FeCl3, 10 μM;

FeCl3/H2O2 (10 μM and 40 μM, respectively). ApoA1 or rHDL (prepared from apoA1 or their

various oxidized versions) were coated at 0.5 μg/ml into EIA plates and probed with 10 ng/mL

anti-totalapoA1 monoclonal antibody 10G1.5 at room temperature for 1 h. For Western blot

Catalog# 26179, Pierce Rapid Antibody Isotyping Strips plus Kappa and Lambbddada ––– MMMouuousesese,,,

Thermo Scientific Pierce, Rockford, IL).

SSSpepepeccicifficiitytyty oof mAb 10G1.5 was tested usisisingngg apoA1 or rHHDLDD eieiithththere in native form, or

ffoollloowo ing incuubababatiionon aaat 373737°C°C°C iiin n 60600 mmmMMM NNa[[POOO4] bbuuffffererer ((pHpHpH 77.4.44)) wwiwiththh mmmulultititiplplple e dididiffffferere eennt t t

oxxidididatatatioioi nn sysysystsstememms.. TThhehe dddififfeererer ntntnt MMMPPOPO sysysystetetemsmsms ccoononssissttededd ooofff 191919 nnnM MM MPMPMPOO,O, 11000000 ��MM M DTDTDTPAAA, , 4440

�M H2O2 andndd eeeititheheherr r 10101000 mMmMmM NNNaCaCaCl l l oror 111 mmmMMM KBKBKBr r ororr 1mMmMmM NNNaNaNaNOOO22 aaasss ininndididicacacateteed.d.d. HHHorororseses radish



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analyses, mAb 10G1.5 was IRDye labeled (Li-COR Biosciences, Lincoln NE) by using LI-COR

IRDye 800CW high molecular kit at a dye/protein ratio at 4:1, and visualized by infrared

imaging. The IRDye 800CW dye bears an N-hydroxysuccinimide ester reactive group that

couples to free amino groups on the antibody, forming a stable conjugate with antibody.

Coupling was performed as per the manufacturer’s instructions.

Human apoA1 quantification

Human apoA1was quantified by an FDA approved apoA1 immunoassay on the Abbott

ARCHITECT ci8200 Integrated Analyzer System (Abbott Labs, Abbott Park, IL). All other

apoA1 was quantified by quantitative immunoblot analysis using mAb 10G1.5 as the detecting

antibody, as determined against a standard curve of known purified apoA1 standard. Immuno-

reactive bands were quantified using Image Studio software (version 2, LI-COR) or Image J

(version 1.46,http://rsbweb.nih.gov/). All nascent HDL particles, and isolated human HDL2 and

HDL3, were further purified by gel filtration chromatography using a Sephacryl S300 column

(GE Healthcare, Waukesha, WI) on a Bio-Rad Biologics DuoFlo FPLC.

Aortic tissue homogenization

Atherosclerotic lesions from aortic tissues were from subjects (n>20) of an average age of 83yr

+/- 3yr. Normal human aortic tissues were obtained from transplant donors (n=5) from Cleveland

Clinic and had an average age of 23yr +/- 7yrs. All tissue homogenization and lipoprotein

fractionation procedures were performed within a cold room to ensure maintaining tissue and

sample temperatures under 4°C. Frozen tissue blocks (submerged in 65 mM sodium phosphate

buffer, pH 7.4, under argon, within screw cap containers) were thawed by placement of the

containers in ice/water bath. Immediately before complete thaw, ice-cold Ca2+ and Mg2

+-free

Chelex-100 treated PBS supplemented with 100 M DTPA, pH 7.4 was added to rinse the tissue

antibody, as determined against a standard curve of known purified apoA1 standdaaard.d.d ImImImmumumunonon --a

eactive bands were quantified using Image Studio software (version 2, LI-COR) or Image J

vvererrsisisiononon 111.4.4.46,6,6 httptpp:/:/://r/ sbweb.nih.gov/). All nascennnt tt HHHDL particles, aaand iiisososollated human HDL2 and

HDHDLL3, were ffururthtt eeer ppururifififieieied d d bybyby ggelele ffililtrtraaationnn ccchrooommmatoooggrgrapapphyyy uusssinngng aa SSSeepphahaaccrcrylyl SSS303030000 cooolullumnmnmn

GGGEE E HeHeHealalththhcacacarere, ,, WWaWaukukkeesshaha, WIWIWI))) ononon aa BiBiBio-o-o-RRRad d d BiBiBiolologogogicicsss DDuDuoFoFoFloloo FFFPLPLP CCC.

Aortic tissuee hohohomomomogegegeninin zaaatitit onoo



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five times to remove any residual blood from tissue. The aorta segment was cleaned of

adventitial fat and again rinsed three times with ice-cold PBS supplemented with 100 μM DTPA.

Wet weigh of the aorta was determined, the tissue cut into small pieces, and then suspended in

ice-cold Ca2+ and Mg2+-free PBS supplemented with both 100 M DTPA, (pH 7.4) and a

protease inhibitor cocktail (Sigma-Aldrich, catalog number P8340), which was included in all

subsequent solutions used for homogenation and lipoprotein isolation. Aortic tissues were

homogenized in ice/water bath with a motor-driven Brinkmann homogenizer for 30sec intervals

five times with 2 minutes rest between homogenizations. Care was taken throughout

homogenization to maintain a temperature at or close to 0°C by keeping the homogenization

vessel submerged within slush (ice/water bath). The crude homogenate was centrifuged at low

speed 15,000 ×g for 30 min at 0°C and the pellet discarded. This low speed supernatant (lesion

homogenate) was then used for buoyant density isolation of LDL/VLDL (d<1.063), HDL (1.063

d 1.21) and lipoprotein-depleted (LPD) fractions (d>1.21). Fractions were dialyzed at 4°C

four times against 4 liters of 5 mM ammonium bicarbonate, with 50 μM DTPA (pH 7.4) and 25

μM BHT changed every four hours. A last change of buffer was against 4 liters of ice-cold

Chelex-100 treated 1x PBS, pH7.4.

Immuno-affinity isolation of apoA1 from aortic tissue homogenate

Immuno-affinity resin was generated by covalently coupling mAb 10G1.5 to AminoLink Plus

(Pierce Chemical, Rockford, IL) resin at a density of 1.5 mg antibody per ml of resin in an

amine-free buffer (PBS, pH 7.4) as per the manufacturer’s instruction. Reactive non-antibody-

bound sites on the resin were blocked with addition of excess ethanolamine. The affinity gel was

drained and antibody concentration in the flow through determined in order to calculate cross-

linking efficiency, which was greater than 90%. The gel was then extensively rinsed with 1M

vessel submerged within slush (ice/water bath). The crude homogenate was centrtrrififuguu ededed aaat tt lololow w

peed 15,000 ×g for 30 min at 0°C and the pellet discarded. This low speed supernatant (lesion

hoommomogegegenanaatetete))) waasss tththen used for buoyant densityy iiisosollation of LDL/L//VLVLDLDLDL (d<1.063), HDL (1.063

d 1.21) andndd lllipppopprrooteteeininin-d-d-depepepleleteteteddd (LLLPPPD) frfraactiononons (ddd>>1>1.2.2211)). FFFrraactctioioionss wwweerrere ddiaiaialylyyzezed ataat 444°CCC

fooururur tttimimimeses aaagaggaininssst 44 lliittererrs s ofof 555 mmmMM M amamammomomonininiumumum bbiicicaararboboonananatetete,, wiwiwiththh 55500 0 μMμMM DDTTPTPA A A (p(p(pHHH 7...4)4)) aandndd 2225 5mmm

μM BHT chaangngngededd eeeveveveryryy ffououour r r hoooururu s.s.. A A A lalalaststst ccchahahanngegege ooof f bubuuffffffererer wwwasasas aagagagainininststst 444 lllititerere sss ofofof iicecec -cold



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TRIS, pH 7.4, 1M NaCl, and then equilibrated in 1x PBS, pH 7.4 prior to use or storage (0.002%

sodium azide was added if stored). Individual one-time use affinity columns (1 ml, drained

resin) were prepared with immobilized 10G1.5, and artery wall apoA1 was purified from

individual samples of aortic homogenates under conditions that quantitatively recovered apoA1,

as confirmed using Western blot analyses of column fractions.

LCAT activity

Human recombinant LCAT was prepared and purified from culture medium of a CHO cell line

(generously provided by John Parks, Wake Forest University, NC) with stable expression human

LCAT21. LCAT activity was determined by calculating the conversion efficiency of

[3H]cholesterol to [3H]cholesteryl ester after lipid extraction of the reaction mixture followed by

thin-layer chromatography22. Fractional cholesterol esterification was calculated as dpm in

cholesterol esters divided by dpm in cholesterol esters plus free cholesterol. The fractional

cholesterol esterification rate was expressed as Units of activity (nanomoles of cholesterol ester

formed per hour per ng of LCAT) per g apoA1 protein.

Cholesterol efflux activity

Cholesterol efflux experiments were performed according to established procedures23. The

cholesterol efflux was calculated as the total radioactivity in the medium / (medium radioactivity

plus cell radioactivity). Results are expressed as a percentage relative to cholesterol efflux

measured using human apoA1 isolated from healthy donors (n=5) as control.

HDL imaging

HDL was dual-labeled and incubated with mouse peritoneal macrophages to individually

monitoring the fate of phospholipid versus protein components of the particle as follows. Briefly,

the protein component of isolated human HDL was first labeled with Alexa Fluor 633 reactive

3H]cholesterol to [3H]cholesteryl ester after lipid extraction of the reaction mixtutuureee ffololollololowewewedd d byby

hin-layer chromatography22. Fractional cholesterol esterification was calculated as dpm in

chholololesessteteterorooll l esesstersrss dddivi ided by dpm in cholesteroll eeestteers plus free cchohh leestststerererolo . The fractional

hchhoollese terol esteteriririfiicaatititiononn rrratatateee wwawas s exexxprprressseddd aas Unninitts ooof f acacttitivviityty (n(nnannomommooolesess oooff chchholololesese tteteroroolll eseestteer

foormrmrmededed pppererr hhhouour r peeer r ngnng ooof f LCLCCATATAT))) pepper r g gg apapapoAAA11 pprproooteeiein.n.

Cholesterol efefefflflfluxuxx aaactcttivivvittyyy



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dye kit (Molecular Probes, Eugene, OR) according to the manufacturer instructions. HDL lipid

was next labeled by first forming a phospholipid film of NBD-PE by evaporation of a 1:4,

methanol:chloroform solution overnight under vacuum, and then rehydrating the film with Alexa

Fluor 633-labeled HDL in pre-filtered PBS and 4 cycles of alternating rounds of sonication at

0 C for 1 min, followed by a 1 min interval on ice. Dual-labeled HDL was centrifuge filtered and

washed numerous times with PBS prior to incubation with macrophages. Thioglycolate-elicited

peritoneal macrophages from C57Blk/6J mice were collected and cultured as described24. Dual

labeled HDL (125 μg protein/ml) was incubated with cells at 37°C for 1 hour, and then images

were captured on a Zeiss LSM 510 Meta confocal microscope.

Proteomic analyses

To confirm the protein recovered following immuno-affinity isolation (using mAb 10G1.5) from

normal aortic tissues homogenate was apoA1, the major SDS-PAGE gel bands at molecular

weight 25kDa and 50kDa were excised. The samples were first treated with

dithiotheotol/iodoacetamide (Sigma, St. Louis, MO) in order to carbamidomethylate any

cysteines in the protein(s) and then proteins were digested using Mass Spec grade trypsin

(Promega, Madison, WI) at 37°C overnight. Tryptic peptides were loaded onto an IntegraFrit

sample trap (ProteoPep C18, 300 Å, 150 m X 2.5 cm, New Objective, Woburn MA) at 1

L/min with 5% acetonitrile and 0.1% formic acid in order to desalt the samples. The peptides

were subsequently eluted through a column (75 m X 15 cm) packed in-house with XperTek

218TP, C18, 300 Å pore size, 150 m particle size (Cobert Associates, St. Louis, MO) at 200

nL/min using a Proxeon Easy-nLC II system (Thermo Scientific, Waltham MA) with a gradient

of 5-65% acetonitrile, 0.1% formic acid over 120 minutes into a LTQ-Orbitrap Velos mass

spectrometer (Thermo Scientific, Waltham MA). Peak lists were generated using Proteome

Proteomic analyses

To confirm the protein recovered following immuno-affinity isolation (using mAb 10G1.5) from

nonormrmrmalalal aororrtititicc tisssssuueues homogenate was apoA1, thhheee mmmajor SDS-PAAGEGG gggelelel bbands at molecular

wweiigght 25kDaa aandndnd 50k0kkDDaDa wwerereree exexxcicicisesed.d. Thhe ssammmplles wewwereree ffiriri stst trreeatededd wwwitithhh

didithththioioiothththeoeoeotototol/l/l/ioiododdoaacacetettamammidide (S(S(Sigigigmamama,, StStSt. LoLoLouuuis,s,s, MMMO)O)) iinn n ororordededer r tototo cccararrbabab mmmiddodomememethththylylylatatate e ananany y y

cysteines inn ttthehehe ppprororoteteteininin(s(s( ) aaandndnd tthehehen n prprp otototeieieinsnsns wwwererere e e didid gegeg ststtededed uuusisis ngngng MMMasasa ss s SpSpSpececec ggrararadedede tttryryrypspp in ddd



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Discoverer 1.1 (Thermo Fischer Scientific, Waltham, MA). The resulting Unified Search Files

(*.srf) were searched against the Uniprot FASTA of all apolipoproteins and also against a human

protein database downloaded from the European Bioinformatic Institute (EBI, release: 2013_02).

Modifications used for searches included carbamidomethylated cysteine (fixed), oxidized

methionine and tryptophan (variable), 3-chloro and 3-nitro tyrosine (variable). Only strictly tryptic

peptides with a maximum of 2 missed cleavage site were allowed in the database searches.

Monoisotopic precursor ions were searched with a tolerance of 100 ppm with 0.8 Da for the

fragment ions on the data obtained from the hybrid LTQ-Orbitrap Velos mass spectrometer.

Unidentified fragment ions in all fragmentation spectra were manually validated using Protein

Prospector (University of California, San Francisco).

Statistical Analysis

Nonparametric statistical methods were used to determine statistical differences due to sampling

numbers. Wilcoxon rank sum test was used for two-group comparisons and the Kruskal-Wallis

test was used for multiple-group comparisons (> two groups). In cases where Kruskal-Wallis test

was performed for multiple group comparisons and found to be significant (p<0.05), multiple

comparison procedures such as the Dunn’s test was used for pairwise comparison between groups

and controls. The Wilcoxon rank sum test was also used for pairwise comparison. Where

indicated, the one-sample robust Hotelling T2 test was used to determine statistical significance

when comparing enzymatic activity between the control group and experimental group.

Results

Monoclonal antibody 10G1.5 recognizes apoA1 equally well in its lipid-free or HDL-

associated, native and oxidized forms.

We initially sought to accurately quantify and immuno-affinity isolate apoA1 from artery wall

Prospector (University of California, San Francisco).

Statistical Analysis

NoNonpnpnparararamammetetetririric ststtatatatisistical methods were used to dededeteterrmine statisticacaall difffffferererene ces due to sampling

nnummbmbers. Wilclcoxoxxooon rraaankkk sssumumum tttesest tt wwawass uussed ffookkk rr twwwo--grouououp p ccommpmpaaariissononss aannd d thththe e KrKrrususu kakakal-ll WWaWallllllisss

eeststst wwwasasa uuseseed d d fofor r mmumultltiipipllee-g-groooupupup cococompmmpaararisisisononons s (>(>(> ttwowowo ggrror upupups)s)s). IIInn ccasasasesese wwwhhehereree KKKrurusssn kakakal-WaWaWallllisis teesst t

was performemed d d fofof r r r mumumultltl ipipi leee gggroror upupup cocoompmpmparararisisisonononss s ananand d d fofofoununnddd tttooo bebebe sssigigi nininififif cacacantntntdd (((p<p<p 0.00 050505),),, mmmulu tiple ttt



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tissue homogenate (from both normal and atherosclerotic lesions). We reasoned this would

require a sufficiently tight binding antibody that demonstrated minimal recognition bias between

lipidated versus non-lipidated forms of the lipoprotein, as well as oxidized versus non-

oxidatively modified forms. Examination of every commercially available antibody we could

find (both monoclonal and polyclonal) showed significant bias in recognizing one form or

another (typically recognition of oxidized forms preferentially, and with inadequate affinity).

Figure 1a illustrates the biases observed with three characteristic commercial antibodies (two

polyclonal and one mouse monoclonal antibody). Note that despite equal mass of protein loaded

into adjacent lanes from native vs. oxidized apoA1, and HDL vs. oxidized HDL, the commercial

antibodies show varied intensity of staining (e.g. Commercial Ab 1 shows oxHDL>ox-

apoA1>>apoA1 or HDL; Commercial Ab 2 shows ox-apoA1>oxHDL>>apoA1 or HDL; and

Commercial Ab 3 shows ox-apoA1 or oxHDL>>apoA1 or HDL). We therefore sought initially

to develop a suitable antibody that met our strict apoA1 recognition criteria. Purified delipidated

human apoA1 (isolated from plasma HDL) was injected into several apoa1-/- mice. After

screening over 5,000 hybridoma clones for their ability to recognize apoA1 forms equally well, a

small number (four) met our screening program requirements. One mAb, 10G1.5, was selected

based on specific activity of recognition by ELISA, immunoblot analysis, its ability to immuno-

precipitate apoA1, as well as the growth characteristics of the hybridoma clone. Figure 1b

illustrates mAb 10G1.5 recognizes native apoA1 and apoA1 reconstituted into HDL particles

equally well. Furthermore, mAb 10G1.5 recognizes apoA1 in native vs. oxidized forms

equivalently, using a wide variety of oxidation schemes (Figure 1b). We further examined the

ability of mAb 10G1.5 to quantify different concentrations of purified apoA1 (lipid-poor) versus

equivalent amounts of total apoA1 in either isolated human HDL (total), or the individual HDL

antibodies show varied intensity of staining (e.g. Commercial Ab 1 shows oxHDDLDL>>o> x-x-x

apoA1>>apoA1 or HDL; Commercial Ab 2 shows ox-apoA1>oxHDL>>apoA1 or HDL; and

CoCommmmmmeerercicicialalal AAAb b 333 shshows ox-apoA1 or oxHDL>>>a>>apppoA1 or HDL).).. WeWee ttthheherefore sought initially ff

oo dddeve elop a ssuiuiittatabbblee aaantititibobobodydydy tthahaat tt mmemett ooour ssstrrrict aapapoAoAA11 rerecccoggngnitittioioon n crcritii eeriaiaa. PPuPuririfififiededed dddellipipipiddidattted

huhumamamann n apappoAoAoA11 (i(isososolalateteedd frfromomm ppplalalasmsmsmaa HDHDHDL)L)L) wwasasas iinnnjeeectteted dd inininttto sssevevverrralalal apapapooaoa1-1-/-/-/ mmmicicce.e.e. AAffteerer

creening ovvererer 555,0,00000000 hhhybyby ririidododomamama cccloloonenn s s s fofofor r thththeieieir r abababililliityty ttto o o rererecococogngngnizzzee e apapapoAoAoA111 fofoormrmrms ss eqeqequau lly well, aaa



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subfractions HDL2, or HDL3 (Figure 1c). As can be seen, mAb10G1.5 displayed nearly

identical ability to quantify apoA1 in its varied lipid free and lipidated forms over a range of

masses. Based upon the observed unbiased recognition of all apoA1 forms, we refer to this

antibody as "anti-total" apoA1. This mAb was used throughout the studies described below to

detect, immuno-affinity purify and quantify apoA1 recovered from plasma, atherosclerotic lesion

homogenates and normal artery wall homogenates.

The vast majority of apoA1 isolated from lesions is highly cross-linked and not HDL-

associated

The particle distribution of apoA1 within human atherosclerotic lesions has not been reported.

We therefore homogenized human aortic atherosclerotic lesions (n=10 different subjects) and

used sequential buoyant density ultracentrifugation to initially remove the VLDL/LDL- like

fraction (d<1.063), and then recover both HDL-like fraction (1.063 d 1.21) and the

lipoprotein-depleted (LPD) fraction (density >1.21), as described under Methods. Samples were

first examined on gradient (5-15%) SDS-polyacrylamide (SDS-PAGE) separations using Sypro

Ruby Red protein staining, which shows minimal protein-to-protein differences in staining,

equally stains lipoproteins, glycoproteins, and other difficult to stain proteins, and also does not

interfere with subsequent mass spectrometry analyses. Visual inspection shows a complex

protein mixture, with an unknown band migrating at ~27 kDa, the molecular weight of apoA1

(Figure 2a). Western analysis with anti-total apoA1 antibody (mAb 10G1.5) of a membrane

containing transferred proteins from a parallel-run duplicate gel readily detected within lesion

homogenates a band at the molecular weight of the apoA1 monomer (Figure 2b). Remarkably,

the vast majority of apoA1 within the aortic lesion was observed to be present not within the

HDL-like fraction, but rather, within the LPD fraction (Figure 2b). After substantial increase in

We therefore homogenized human aortic atherosclerotic lesions (n=10 different t sssubjbjb eccttsts))) ananand dd

used sequential buoyant density ultracentrifugation to initially remove the VLDL/LDL- like

frracacctititiononon (((d<d<d<1.1.1 0663)3)3), aand then recover both HDL-lililikekee fraction (1.063636 ddd 1.21) and the

iiipoooprp otein-deeplplleete eeded (((LPLPPD)D)D) fffraraactctioioonn n (d(ddenennsityyy >>>1.221))), asss dddesescrcribibeedd uundnddeeer MMMetetethohoh dsds. SaSaSammmpleleles s wewewere

fiirsrsstt t exexexamamininnedeed oonnn ggrgradaddieeentnt (55-5-15155%)%)%) SSDSDSDS-p-p-poololyyacacacryryrylaaammiidedee (((SDSDSDS-S-S PAPAPAGEGEG )) sseepapaarararatitit ononnsss ussiiingg g SySyyprrroo

Ruby Red pprorooteteteininn ssstatat ininninini g,g,, wwwhihichchch sshohoh wswsw mmminininimimimalalal prprprototo eieiin-n-n tototo-p-pprororotetet ininn dddiiifffffferererenenenceceesss ininin ssstatat ining,



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exposure of the immunoblot, apoA1 was detected within the HDL-like particle fraction (Figure

2c). Also notable within the immunoblots were prominent slower migrating forms of immuno-

reactive apoA1-containing protein bands at molecular weights of ~50, ~75 and ~100 kDa present

in particular within the starting material (homogenate) and the LPD fraction (density > 1.21), but

noticeably diminished in the HDL- like particle fraction (1.063 d 1.21). The sizes of these

slower migrating apoA1-immuno-reactive bands are consistent with the sizes of oxidatively

cross-linked dimeric and multimeric apoA1 forms.

Quantification of the distribution of protein and apoA1 forms recovered within

homogenates from multiple distinct human atherosclerotic plaque-laden aorta (n=10) is shown in

Figure 3. The majority (81.0 ± 5.6%) of the total protein in the lesion homogenate was found in

the LPD fraction, while the HDL-like fraction contained only 1.7 ± 0.2% (Figure 3a).

Quantitative analysis of the anti-total apoA1-specific immunoblots indicated that nearly all of the

apoA1 isolated from lesions was lipid-poor, and found within the LPD fraction (density > 1.21)

where 0.7 mg ± 0.4 mg apoA1 per gram wet weight of lesion material was recovered (Figure

3b), corresponding to 92.4 ± 4.1% of total apoA1 in the artery wall (Figure 3c). Surprisingly,

only a nominal amount (< 3%) of apoA1 within the artery wall (lesions) was recovered in the

HDL-like particle fraction (Figure 3b,c). Yet another remarkable finding is the abundance of

slower migrating immuno-reactive apoA1-containing bands migrating with molecular masses of

dimeric, trimeric and tetrameric forms of apoA1 within the lesion homogenates. Quantification

of these apoA1-immuno-reactive bands revealed that approximately two thirds (66 ± 4%) of

apoA1 within lesions is oxidatively cross-linked, and the cross-linked forms are preferentially

present in the LPD fraction (Figure 3d). Proteomics analyses of anti-total apoA1 (mAb 10G1.5)

immuno-precipitated higher molecular weight apoA1 forms confirmed these bands too were

Figure 3. The majority (81.0 ± 5.6%) of the total protein in the lesion homogenatatte wawawas s fofofoununund dd in

he LPD fraction, while the HDL-like fraction contained only 1.7 ± 0.2% (Figure 3a).

QuQuananantitititatatatititivevev aaanaalylylysisis of the anti-total apoA1-specececifffiic immunoblototts s inndididiccacated that nearly all of the

appooAA1 isolateded fffroroom m lelel sisiiononnss wawwas s lililippipidd--ppooor,, aannd fffouuunddd wwwitithhih nn n ththe LPLPDD D frfrracactititioonon ((deded nnsnsititity >>> 1.11 2211)

whwhherere eee 0.0.77 mgmgmg ±± 00.444 mmmgg apapa oAoAA11 pepeper rr ggrgramamm wwwetetet wwweeeigigghthtt ooff f leleesisisiononon mmmaaaterereriaiai l l wawass rererecococoveveerereedd (((FiFiigugurrre

3b), corresppononndidid ngngng ttto o 92922.444 ±±± 444.111%%% ofofo ttoootataall l apapapoAoAoA111 ininin tthehee aaartrtrtererery y y wawaw llll (((FiFiF gugugurerere 33ccc).).). SSSurururprpp isingly,



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predominately comprised of apoA1 (see below). It should be noted that total apoA1 present in

the aortic tissues reported are actually modestly underestimated as we know that under the

conditions employed, approximately 15-20% of the total apoA1 remains unrecovered in the

“pellet” from the initial tissue homogenate. This modest loss appeared acceptable since control

studies with repeated homogenization of the pellet and fractionation of the recovered material

revealed, within both crude homogenate and subsequent buoyant density isolated fractions,

virtually identical banding pattern and results to the original homogenate and fractions, based

upon both protein staining and Western blot analyses (data not shown).

ApoA1 is markedly enriched in lesions, and normal aortic tissue apoA1 similarly is lipid-

poor and highly cross-linked.

Our initial studies focused on apoA1 within atherosclerotic lesions. However, given the

surprising finding that virtually all apoA1 within aortic lesions was not on an HDL particle, and

fully two-thirds of all apoA1 within lesions was cross-linked, we decided to examine apoA1

within normal aortic tissue for comparison. Normal aortic tissue was obtained at time of organ

harvest from transplant donors. For illustrative purposes, an image of a typical normal aortic

specimen, and a typical atherosclerotic plaque-laden aortic specimen, are shown in Figure 4a.

Homogenates of normal aortic tissue (n=5) were prepared and fractionated by buoyant density

ultracentrifugation as described in Methods. Fractionation of protein from normal artery and

lesion homogenates on (5-15%) gradient reducing SDS-PAGE gels stained with Sypro Ruby Red

for protein reveal that although there are similarities in the protein banding pattern, the pattern is

noticeably different in normal vs. lesion homogenates (Figure 4b). Notably, immunoblot

probing with apoA1-specific anti-total apoA1 (mAb 10G1.5) of parallel SDS-PAGE gels

transferred to membranes showed that compared to lesion-derived homogenates, there is very

poor and highly cross-linked.

Our initial studies focused on apoA1 within atherosclerotic lesions. However, given the

uurprprpriririsisisingngg fffininindingngg tthah t virtually all apoA1 within n n aoaoortic lesions wwaaas noottt oonon an HDL particle, and

ffuullly y two-thirdsds oooff f alalllll apappoAoAoA111 wiwwiththhininin lleessiioons wwas ccroooss--l-lininnkekeedd,, wweee ddedeciciidded ddd totoo eeexaxammiminnene aaapoooA1A1A1

wiwiithththininin nnorormamamall aoaoorttticic ttiisssusuee fofoor r cococompmpmpararrisisononon.. NoNoormrmrmall aooortititiccc ttitissssueueu wwwasasas ooobtbtaiaineneed d d atat tttimimime oofof oorgrgganann

harvest fromm tttrararansnssplplplanannt t dodonononorsrr . FoFoFor r ilili luuuststs rararatititiveveve pupupurprprpososo esess, , ananan iimamamagegeg ooof f f a a a tytyypipipicacalll nononormrmmalaa aortic



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little immune-reactive apoA1 in homogenates prepared from normal aortic tissue (Figure 4c).

Since extremely low levels of apoA1 were observed within normal artery wall tissue, and

especially the HDL-like fraction, an increased protein amount was loaded onto SDS-PAGE gels

to permit visualization by Western and comparison of HDL-like and LPD fractions from the

normal artery wall homogenate (Figure 4d). The majority of protein in the normal artery wall

homogenate was found to be in the LPD fraction (Supplemental Figure 1). As observed for

aortic lesion apoA1, a significant portion of apoA1 was lipid-poor and recovered within the LPD

fraction. Further, there was a high degree of immuno-reactive apoA1 forms in the normal artery

wall that migrate at higher molecular weights, consistent with that of oxidatively cross-linked

apoA1 dimer and higher multimeric forms, particularly within the LPD fraction (Figure 4d).

Quantification of apoA1-specific immuno-blots (and taking into account volumes of

starting homogenate and density cut fractions recovered) revealed that the total apoA1 recovered

per gram of wet weight aortic tissue from normal artery wall was 120-fold less than that

recovered from lesion-laden aorta (Figure 4e, note log scale for Y axis). Remarkably, the

distribution of apoA1 within normal artery resembled those found in the same fractions from

atherosclerotic lesions, with only 3% of apoA1 being HDL-associated, and approximately 92%

of the total apoA1 present in the normal artery wall residing in the LPD fraction (Figure 4f).

Similar to that observed within lesions, nearly half (43.4 ± 13.9%) of the total apoA1 within the

normal artery wall was cross-linked and observed to reside not on the HDL-like particle (3.6 ±

2.6%), but in the LPD fraction (61.4 ± 23.0%; Figure 4g).

Since the levels of apoA1 in the normal artery wall-derived homogenates were so low,

we wanted to verify that the bands detected on Westerns using mAb 10G1.5 were in fact apoA1.

We therefore immuno-purified apoA1 from normal aortic tissue homogenates (n=5) using

apoA1 dimer and higher multimeric forms, particularly within the LPD fraction (((FiFiFigugugurere 444ddd).).).

Quantification of apoA1-specific immuno-blots (and taking into account volumes of

ttararrtititingngng hhhomomomooogenennaatatee and density cut fractions reeecococ vvvered) revealed d d thatatt ttthhehe total apoA1 recovered

per grg am of wewett weweigighhth aaaororortititicc ttitissssueueue ffrroommm nooormmmal aarrterrry y y wawaalll wwaas 1120200-f-f- oolld d lelelesssss tthahahan nn thththata

eecococovevevererer dd frfrfroomom llesssioion-n-laaadeden aoaoa rtrtrtaaa (((FFiFiggugurerere 444e, nonon ttte llogogg ssscacacallele fffororo YY aaaxixix sss).. ReReemamamarkrkababablyly, , thhhe e

distribution oof f f apapapoAoAoA11 wiwiw thhhininin nnnororormamamall l arrrteteteryryry rrresesesemememblblb ededed tthohohosesese fffouououndndn ininin ttthehehe sssamamameee frfrfracacactitit onono s from



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10G1.5 (as described in Methods), and immuno-affinity isolated proteins were separated on a

gradient non-reducing (5-15%) SDS-PAGE gel and stained for protein. The major protein bands

observed corresponded to doublet bands migrating at ~25 kDa and ~50 kDa (Supplemental

Figure 2). These were individually excised and digested with trypsin for mass spectrometry

analyses as described in Methods. Tandem MS analyses of tryptic peptides for each excised band

revealed apoA1 as the dominant protein within each, with 40-62% peptide coverage in each of

the gel-excised bands (Supplemental Tables 1-4).

Plasma apoA1 is predominantly HDL-associated, and has decreased apoA1 cross-links in

the HDL-like fraction compared to the LPD fraction.

Given the surprising HDL particle distribution and cross-link prevalence of apoA1 found in the

artery wall (Figures 2-4), and the known preponderance of apoA1 within the HDL fraction in

plasma, we examined the distribution of apoA1 in plasma using the same anti-total apoA1 mAb

(10G1.5). Plasma from normal healthy consenting donors was fractionated by sequential buoyant

density ultracentrifugation as described in Methods. The indicated amount of protein from the

starting material plasma, HDL-rich fraction and lipoprotein depleted fractions were run on

gradient (5-15%) reducing SDS-PAGE gels and stained with Coomassie Blue (Figure 5a), or

transferred for immunoblot analyses with anti-apoA1 mAb 10G1.5 (Figure 5b). As expected, the

dominant immuno-reactive band observed migrates at ~27 kDa, corresponding to the apoA1

monomer, and is predominantly recovered within the HDL fraction (~83%; Figure 5b,d). Unlike

apoA1 recovered from the artery wall, there is little noticeable higher molecular weight cross-

linked apoA1 in the starting plasma, particularly within the circulating HDL fraction. Of note,

however, the content of apoA1 cross-linked within the LPD fraction was found to be 3-fold

higher than that observed within the HDL fraction, but still represented only a small minority

Given the surprising HDL particle distribution and cross-link prevalence of apoAAA1 fofof ununund d d ininin tthhe

artery wall (Figures 2-4), and the known preponderance of apoA1 within the HDL fraction ina

pllasassmamama,, wewewe eexxxamimiminnened the distribution of apoA1 ininin ppplasma using ththhe saamememe anti-total apoA1 mAb

1110GGG1.5). Plassmmaa ffroommm nononormrmrmalalal hheaeaealtltlthyhyy ccconssennntinggg ddonnnororrs s wawawass frfrraccctitiononnatteed d bybyby sseqeqqueueentntiaii lll bububuoyoyoyan

dedennsnsititityy y ulultrtrracacacenenttrtrifffuguggaaatiioon n asss dddesesescrcrcribiibededd iiinn n MMeMethththododdss.. TTThehee iiinndndiciccatata ededed aaamomomouunnt t oofof ppproroteteteiinin fffrorom m ththhee

tarting matererriaiaial l l plplplasasa mamama,, HDHDHDL-L-L riririchchc fffraractctctioioon n n aaandndnd lllipippopopoproooteteteininin dddepepepleleleteeed dd frfrfracacactititionono s s s wewewererere rrrunu on



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(6.0±1.8%) of the total apoA1 within that fraction (Figure 5e, and longer exposure of plasma

and LPD fractions are shown in Supplemental Figure 3).

ApoA1 isolated from atherosclerotic lesions is dysfunctional

In a final series of experiments, apoA1 was isolated from additional atherosclerotic plaque-laden

aortic tissues (n=10 subjects) by individual immuno-affinity columns comprised of immobilized

anti-total apoA1 (mAb 10G1.5), as described under Methods. Immuno-isolated apoA1 was

quantified and equivalent amounts of apoA1 protein recovered from lesions from each sample, or

apoA1 purified from plasma HDL (as a control) were incubated with cholesterol-loaded murine

macrophage RAW264.7 cells to quantify cholesterol efflux activity. Remarkably, every apoA1

sample recovered from plaque-laden aorta demonstrated lower cholesterol acceptor activity,

showing 78.9% ± 6.0% less total cholesterol efflux capacity compared to control apoA1 purified

from plasma HDL from healthy donors (Figure 6). In parallel studies, each of the lesion apoA1

were incorporated into reconstituted HDL by cholate dialysis method, further purified by gel

filtration FPLC, and then comparable amounts (5 μg apoA1 mass) of each rHDL was examined

for LCAT activity and compared with rHDL generated from apoA1 purified from plasma HDL

from healthy volunteers. rHDL formed with lesion apoA1 demonstrated significantly less (89.6%

± 5.0%) LCAT activity compared to rHDL formed from control apoA1 (Figure 6). Thus, apoA1

in atherosclerotic plaque-laden aorta is markedly functionally impaired (i.e. “dysfunctional”)

with respect to both cholesterol acceptor and LCAT activities.

Discussion

The present studies reveal multiple remarkable findings about apoA1 within the artery wall.

First, the vast majority of apoA1 within both normal and atherosclerotic human arterial tissue, in

ample recovered from plaque-laden aorta demonstrated lower cholesterol accepptottor rr acctititivivivitytyty, ,,

howing 78.9% ± 6.0% less total cholesterol efflux capacity compared to control apoA1 purified

frromomm ppplalalasmsmma a HDHDDLL L frf om healthy donors (Figuree 666).. In parallel stuudidd ess, , eaeaeachc of the lesion apoA1

wwerrere incorporarateteed inntotoo rrrecececonononssttititututeteteddd HDHDHDL bbyy ccholalatte dddiaiaalylyssis sss mmeeethhohod,dd fuurrththhererer ppururrifififieieieddd byyy ggelele

fiiltlttrarar tititionono FFPLPLPLC,C, anndnd tthhehen n n ccommpmparararabababllele aaamomomounununtstss ((55 μμggg apapapoAoAoA11 mmamassss)) ofofo eeeaccch h rHrHrHDLDLD wwwaas eeexxaamimiineneed d

for LCAT actctivivivititi y y y ananand d d cocc mpmpmparaa ededed wwwititith h rHrHrHDLDLDL gggeeenenenerarar tetet d d frfrfromomom aaapopopoA1A11 pppurururififfieieied d d frfrfromomom ppplalalasma HDL



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contrast to within the circulation, is lipid-poor (i.e., in the LPD fraction, d>1.21), and does not

reside on an HDL-like (1.063 d 1.21) particle. Second, the content of apoA1 in

atherosclerotic lesions is over 100-fold higher than that observed within normal artery wall.

Third, the majority of apoA1 within arterial tissues (both normal and atherosclerotic) is

oxidatively cross-linked. Fourth, apoA1 within arterial tissues is “dysfunctional”, with ~80%

reduction in cholesterol acceptor activity and ~90% reduction in capacity to activate LCAT.

Fifth, the majority of oxidatively cross-linked apoA1 within the circulation is not HDL particle

associated, but rather, resides within the lipoprotein-depleted fraction (d>1.21). Collectively, the

present studies thus argue that examination of total apoA1 and HDL function within the

circulation may not adequately represent what is occurring within the artery wall, especially with

respect to cholesterol acceptor and LCAT activities. Moreover, the overall strategy applied in the

present studies may prove useful when examining other post-translational modifications to

apoA1, such as glycation, and site-specific oxidative modifications, as antibodies become

available. Finally, the present studies suggest that the common practice of isolating circulating

HDL for study of its biological properties, and discarding the lipid-poor (non-HDL associated)

apoA1, may in fact be throwing out the very fraction (lipid-poor) that more closely reflects the

environment within the artery wall. Of note, the extent of oxidatively cross-linked apoA1 within

the lipid-poor (lipoprotein-depleted) fraction of plasma was approximately 3-fold enriched

relative to the HDL-like fraction (Figure 5e). Based upon the cumulative results herein one

might speculate that "dysfunctional" forms of HDL monitored within the circulation will most

likely reside not on the HDL particle itself, but as lipid-poor forms in the LPD fraction (d>1.21).

It is notable that the apoA1 found within the artery wall, which was predominantly within the

LPD fraction in both atherosclerotic lesions and normal artery wall tissue, is remarkably highly

circulation may not adequately represent what is occurring within the artery walll,l,, eespss ecececiaiaalllllly y y wwith

espect to cholesterol acceptor and LCAT activities. Moreover, the overall strategy applied in the

prresessenenenttt tststudududieieies mamamay y prove useful when examininnng gg ooother post-transnsslatiiooonananal l modifications to

appooAA1, such asas ggglyyycacatitit ononn, ananand d d sisitetee-s-sspepeciciffic ooxoxidatttivvve momomodidiffif ccaatitioonons,s, asas anntntibibbodododieies ss bebebecocoomememe

avvaiaiailalaablblb e.e. FFFinininalallylyly, ththee prrresesennntt t stststudududieieiess ssusuggggggesesest thththatatt tthhhe ccomomommomomon nn pprpracacactitit cecee ooof f isissolololatata ininng g g cirrcrcuululatatiining g g

HDL for studdy y y ofofo iiitstss bbbioioologigigicacac l prprpropoppere tititieseses,, ananand dd didiiscsccararardidingngng ttthehehe lllipipipidid-p-p-poooooor r r (n(n(nonono -H-HHDLDLDL aaasss ociated)



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cross-linked (50-70%). This value is even higher than previously observed in Western blots

(though cross-linking was not quantified per se) in studies examining apoA1 oxidation levels in

the artery wall based upon buoyant density recovery of HDL-like particles8. Through use of

stable isotope dilution mass spectrometry based approaches, these studies suggested an upper

boundary of up to one of every two HDL-like particles recovered from the artery wall carried an

oxidative modification from either MPO- or NO-derived oxidants8.

One question the current studies raise is how apoA1 becomes over 100-fold enriched

within atherosclerotic plaques compared to normal artery. A second question is how artery wall

apoA1 is rendered lipid-poor, when the vast majority of apoA1 that diffuses into the artery wall

tissue from the circulation resides within the HDL particle. To address these questions we

performed preliminary confocal microscopy studies where HDL was isolated from peripheral

blood of healthy volunteers and subsequently doubly labeled (protein with AF 633 (red), and

phospholipid with NBD-PE (green) as described in Methods). Brief incubation of the doubly-

labeled HDL with macrophages led to virtually all of the phospholipid fluorophore being rapidly

taken up within the macrophage, whereas the protein (predominantly apoA1) remained on the

cell surface (data not shown). Such preliminary results provide rationale to speculate that

selective uptake of lipids may contribute as a mechanism for rapidly depleting the HDL particle

of not just cholesterol but also phospholipid, leaving the lipid-poor apoA1 behind in the

extracellular space. Lipid-poor or lipid -free forms of apoA1 are recognized as the preferred

substrate of ABCA15, 25-27. So a question that logically follows is why the lipid-poor apoA1 form

within the artery wall is such a poor cholesterol acceptor (Figure 6). In past studies we have

shown that oxidative modification of apoA1 by MPO-generated oxidants markedly impairs its

ABCA1-dependent cholesterol efflux activity8-10, 13. Other studies have also reported that

issue from the circulation resides within the HDL particle. To address these queessstioioonsss wwweee

performed preliminary confocal microscopy studies where HDL was isolated from peripheral

blloooood d d ofofof hhheaeae ltlthyy vvvololo unteers and subsequently dooouubublly labeled (protottein n wiwiwithth AF 633 (red), and

phhoosospholipid wwititith NBNBBD-D--PEPEPE (((ggrgreeeen)n)n) aass ddescrrribbbed iinin Meetethhohoddds).).) BBrBriieief f ininincuuubabaattitioonon ooof f f thththee dooo bubublylyl ---

aabebebeleleedd d HDHDDL L L wiwitthth mmacacroroophphagaggesss lllededed tto o viviirtrtrtuauaualllly y y aallll ooof ththt eee phphphosososphphpholollipipipididd flluluororropopophohoh rerere bbeieiinggg rrapapapidddlyly

aken up witthihiin n n ththhe e e mamamacrcrc opopphahahagegee, ,, whwhwherereaeaeasss ththhe e e prprprototo eieie nn n (ppprereredododomimiminananantttlylyly aaapopopoA1A1A1) ) rereremamamainininede on the



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carboxy-terminal proteolized (ie“clipped”) apoA1 can be recovered from aortic tissue that fails

to efficiently bind lipid28, 29. In the present studies, however, we did not see substantial levels of

lower molecular weight forms of apoA1 in tissue homogenates once we exercised extreme care

in keeping tissues ice-cold, and more importantly, included an extensive cocktail of protease

inhibitors during tissue dissection, homogenization and fractionation as outlined under Methods.

Given the extensive oxidative cross-linking observed in total apoA1 within lesions, oxidative

post-translational modifications of apoA1 likely explain in large part why the lipoprotein, at least

within lesions, remains lipid-poor. It should also be noted, however, that Parks and colleagues

reported that interaction of apoA1 with ABCA1 results in lipid-poor "pre-beta HDL" migrating

forms of apoA1 that themselves are poor substrates for subsequent repeat or further lipidation by

ABCA1, suggesting the requirement of an additional (non-ABCA1-mediated) process for further

maturation into an HDL particle26. Whether this contributes to the current results is unclear.

Similarly, if an additional process is needed for further lipidation of lipid-poor apoA1 after initial

interaction with ABCA1, whether this process is somehow lacking or inhibited within the artery

wall requires further study. It also would have been interesting to test if reconstituted HDL made

with apoA1 isolated from the vessel wall was also defective in either ABCG1 or passive efflux

compared to HDL made with plasma derived apoA1. However, given the limitations in the

amount of apoA1 able to be purified from human arterial tissues, these studies will need to await

further investigation.

As for the mechanism(s) for apoA1 accumulation within lesions compared to normal

artery, we have no clear answers from the present studies. We can speculate that the extensive

oxidative cross-linking noted here, and through mass spectrometry studies in the past8-10, 13, may

help provide an answer. Oxidatively modified proteins tend to be less soluble, relatively

forms of apoA1 that themselves are poor substrates for subsequent repeat or furtrthheher rr lil pipipidadadatititionono byy

ABCA1, suggesting the requirement of an additional (non-ABCA1-mediated) process for further

mamatututurararatititiononn iiintntn o ananan HHDL particle26. Whether thisss cocoontributes to thhe ee cuurrrrrenenent results is unclear.

SSSimmimilarly, if anan aaaddddittioioionanaalll prprprocococesessss isiss nneeeededd fooor fuuurrttherr r llilipipidadadatitiononn oofof llipipipiddd-ppoooooorr apappoAoAoA111 afaffteteter r inini iiitia

nnteteteraraactctc ioionn wiwiw thth AABBCBCA1A1A1, , whwhhetete heheherrr thththisis pprororocececessss iiis s sososomemeehohooww w laaackckc iining gg ororo iiinhhhibibitititededd wwwititithhhin n thhhe e ararrteeryry rr

wall requiress fufufurtrtr heheherr r stststududu y.y.. IIt t t alllsososo wwwououuldldld hhhavavavee e bebebeenenn iiintn erereresesestititingngng ttto oo teeeststst iif f f rererecococonsnsstitititutututeteted d d HDL madeeen



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protease resistant, and might thus be "retained" within the subendothelial space, particularly

within the hydrophobic environment of the atherosclerotic plaque. The phenomenon of

lipoprotein retention within the subendothelial compartment of the artery wall has previously

been suggested. Originally proposed for apoB-lipoproteins, retention or trapping of LDL

particles in the initial stages of atherosclerosis is suggested to cultivate formation of modified

LDL, which may incite biological and inflammatory responses that initiate or progress the

atherosclerotic process30. Progressive apoB lipoprotein retention through the actions of secretory

acid sphingomyelinase and lipoprotein lipase are thought to lead to accelerated lesion

progression31, 32. While we have no data to implicate lipase activation at present in depleting

lipids from HDL within the artery wall leading to lipid-depleted forms, the presence of similar

phospholipids on HDL suggests a similar retention scheme for apoA1 lipoprotein retention is

feasible, and could thus contribute to the observed accumulation of apoA1 in artery wall lesions

over time, in addition to enhanced lipid and sterol uptake into macrophages producing foam

cells. Other studies have also shown that there is increased endothelial cell permeability in

atherosclerotic lesions which could lead to increased LDL and HDL migration into the diseased

vessel wall and accelerate the retention process33-35.

While multiple studies have noted extensive oxidative modification of apoA1 recovered

from the artery wall8-11, 13-16, 36, it should be noted that there has been disagreement as to which

residues are the main sites of oxidation. Of note, our prior proteomic mapping studies employed

polyclonal antibodies (chicken anti-apoA1 or anti-HDL) to immuno-precipitate apoA1 from

arterial tissue homogenates13, 16. In contrast, proteomic mapping studies from alternative groups

typically have used buoyant density isolation to recover the HDL-like particle fraction within

lesion homogenates 11, 15. Based on the present studies using the mAb 10G1.5, which was

ipids from HDL within the artery wall leading to lipid-depleted forms, the preseennnce ee ofofof ssimimimilililarar

phospholipids on HDL suggests a similar retention scheme for apoA1 lipoprotein retention is

feeasassibibiblelele, ananandd d ccoulululddd tht us contribute to the observeveedd aaccumulation ooof appoAoAoA1 in artery wall lesions

overer time, in adaddddid ttiionon tto o enennhahahanncncededd llipipi iidd aandd sttterolll uuuptakakakee ininntooo mmacccroophphphagggeses pproroduduuciciingngn fooaoamm m

ceelllllls.s. OtOtO heheerr r ststududdieees s hhaavvee aalssoo o shshshowowownn ththhatatat ttthhehereree iis s innncrcreaeaeasesesed dd enenndododothhheleleliaiaall cceellll pppererermemeeabababilliiity y y inin

atherosclerotiticc c lelel sisisionono s s s whww icicich h h cocooulululd d lelel adadad tttooo ininincrcrcreaeaeaseseed d d LDLDDL L L ananand d d HDHDH LLL mmmigigigrararatititiononn iiintntntooo thththe diseased



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developed specifically to allow both recovery and equal quantification of apoA1 in lipid-free vs.

lipidated, and native vs. oxidized forms, it is now clear that analysis of recovered HDL-like

particles from arterial tissues only examines a very small fraction (<3%) of the total apoA1

within the artery wall. Whether the small amount of apoA1 (and its associated proteome)

recovered in an HDL-like particle from the artery wall provides a “snapshot” of the apoA1 on its

way to particle "disintegration" and formation of the lipid-poor apoA1 that is the predominant

form remains to be determined. The present studies suggest that results which focus on HDL-like

particles recovered from the artery wall need to be interpreted within the context of recognizing

their minor quantitative contribution to total apoA1 in the artery wall. An interesting question,

though not examined in the present studies, is whether changes in the HDL-associated proteome

within the circulation observed in subjects with cardiovascular disease, or within subjects at

heightened risk for cardiovascular disease37-41, have any relevance to the marked changes in the

environment of apoA1 observed within the artery wall.

The complexity surrounding the role of the HDL particle in the pathogenesis of

cardiovascular disease has been highlighted recently because of several high profile clinical trial

failures targeting raising of HDL cholesterol, and recent Mendelian randomization studies on

HDL cholesterol levels1, 2, 4, 42-47. The present studies suggest that traditional HDL measured

parameters within the circulation, such as HDL cholesterol, and apoA1 mass, may not adequately

reflect the biology of apoA1 occurring within the artery wall. Recent studies suggest that

functional measures of cholesterol efflux activity within apolipoprotein B-depleted serum may

serve as a superior surrogate for HDL function48. However, this too has recently been

questioned because the HDL particle was found to only account for a minority of the cholesterol

acceptor activity in the cholesterol efflux activity assays performed49. Further, despite the

hough not examined in the present studies, is whether changes in the HDL-assococciaiatetet dd d prprprotototeoeoeomem

within the circulation observed in subjects with cardiovascular disease, or within subjects at

heeigigghththteenenededed rrisisk fofoforrr ccardiovascular disease37-41, hahahavevee any relevancecee to ththhee e mam rked changes in the

ennvviviror nment ofoff aaappooA1A1A1 ooobsbsbserererveveved d wwiwiththt iinn tthe aarrtteryy wwwallll..

TThehee cccomompplplexexe itittyy susus rrr ouououndndndininingg g ththhe e rororolelele oooff f ththhee HDHDHDL LL papapartrticiciclelee in nn ththt eee papaaththhogogogenene eesesiisis ooff

cardiovascullararar dddisisseaeaeasesee hhhass bbbeeeee n n n hihih ghghghlighghghteteted d d rererececec ntntntlylyly bebecacacaususu e ee ofofof sssevvverereralalal hhhigigigh h h prprprofofofililile ee clcc inical trialll



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reported inverse association between cholesterol efflux activity and prevalent cardiovascular

disease, in a separate large clinical study of similar patients (i.e. sequential subjects undergoing

elective diagnostic coronary angiography), enhanced cholesterol efflux activity was observed to

paradoxically associate with increased prospective cardiovascular event risk49. It is remarkable

that what we now recognize as HDL was first described nearly a century ago50. And yet, studies

focusing on HDL still continue to surprise, and reveal how little we know about its complex

biology. The present and recent studies suggest that measurement in the circulation of HDL

cholesterol, apoA1, or even cholesterol efflux activity, may not adequately reflect what is

happening within the artery wall. Rather, development of "dysfunctional HDL" assays that

detect structurally specific modified forms of apoA1 formed in the artery wall but which diffuse

back out into the circulation may be what is needed to provide insights into the processes

happening within the artery wall.

Funding Sources: This study was supported by National Institutes of Health grants

P01HL098055, P01HL076491, and HL17964.This work was also supported in part by a grant

from the LeDucq Fondation. Dr. Hazen was also partially supported from a gift of the Leonard

Krieger Foundation.

Conflict of Interest Disclosures: Dr. Tang has previously received research grant support from

Abbott Laboratories. Drs. Hazen and Smith report being listed as co-inventor on pending and

issued patents held by the Cleveland Clinic relating to cardiovascular diagnostics. Dr. Hazen

reports having been paid as a consultant for the following companies: AstraZeneca

Pharmaceuticals LP, Cleveland Heart Lab, Esperion, Lilly, Liposcience Inc., Merck & Co.,

Inc., Pfizer Inc., and Takeda. Dr. Hazen reports receiving research funds from Abbott, Cleveland

Heart Lab, and Liposcience Inc. Dr. Smith reports having the right to receive royalty payments

for inventions or discoveries related to cardiovascular diagnostics from Cleveland Heart Lab and

being paid as a consultant for Esperion. Dr. Hazen reports having the right to receive royalty

detect structurally specific modified forms of apoA1 formed in the artery wall bubuutt whwhw icicich h h dididiffffffususe

back out into the circulation may be what is needed to provide insights into the processes

haappppppeneneniining gg wiwiwithininn tttheh artery wall.

FuFuundndndinining gg SoSoSouururcecees::: ThThhiss sstutudydydy wwwasasas sssupupppopoportrtrteeded bbby y y NaNaNatit oononalalal IIInsnstitititutuutetesss ofofo HHeaeae ltltth h h grgrgrananntststs

P0P0P01H1H1HL0L0L0989898050505555, PPP010101HLHLHL070707646464919191, anananddd HLHLHL171717969696444.ThThThisisis wwworororkkk wawawasss alalalsososo sssupupuppopoportrtrtededed iiinnn papapartrtrt bbbyyy aaa grgrgranananttt

frfromom tthehe LLeDeDucucqq FoFondndatatioionn DrDr HaHazezenn wawass alalsoso papartrtiaiallllyy susupppporortetedd frfromom aa ggififtt ofof tthehe LLeoeonanardrd



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26

payments for inventions or discoveries related to cardiovascular diagnostics and the companies

shown below: Cleveland Heart Lab., Frantz Biomarkers, LLC, Liposcience Inc., and Siemens.

The remaining authors have no disclosures to report.

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3. Schwartz GG, Olsson AG, Abt M, Ballantyne CM, Barter PJ, Brumm J, Chaititmamaman n n BRBRBR,, HoHoHolmlmlmeM, Kallend D, Leiter LA, Leitersdorf E, McMurray JJ, Mundl H, Nicholls SJ, SShhahah h h PKPKPK, , TaTaTardrdrdififi

JC, Wright RS. Effects of dalcetrapib in patients with a recent acute coronary syndrome. N Engl J Med. 2012;367:2089-2099.

4.4. VVVooioigght BFBFBF,, Peelolososo GGM,M, OOrhho-o Meelaandndere M, , FrFriikkkkke-Scchmhmidt R,R,R, BBarrbabalilic M,M JJene sen n MKMK, HindyGGG, HHHolm H, DDDininngg g ELELEL,,, JoJoJohhnhnsososon n n T,TT, SSSchchchununu kekeertrtrt HHH, Saaammmaninini NNNJ,J,J, CCClalaarrkrke e e R,R,R, HHHooopewewwelelell l l JCJCJC,,, ThThThomommpspsp oonon JFFJF, LLi M, Thorleeiffsssonnn GGG, NNeNewwtwtonn C-C-Chheehh h C, MMMusuunnuuuru u K,K,K, PPPirrrruccceelllo JJP,P, SSaalaleeheheenn n DD,D, CCChhehen LL,L, StSttewewewarart t A,, SSSchchc ilillelertrt AAA, TTThorrrststeieie nsnsdododottttiiri UU,, TTThhohorgrgeieirrsssoonn n G,G, AAAnananddd S, , EnEnEngegerrtr JJJC,C, MMMooorgggann n T,T, SpSperertututusss J,J,J SSSttotollll MMM,, BeBeBergrgrgererer K,, MaMaMartrtrtiininelellilili NNN, , GiGiGirrerelllllliii DD,D, MMMcKcKcKeoeoeowwnwn PPPPP,P, PPPatatattetetersrsononn CCCC,C,C EEpspspsteteteininin SSSEE,E, DeDeDevavavaneneneyyy JJJ, BBBurururnenenetttttt MMMSSS, MMMooooooseseserrr VVV, RRRipipipatatattititi SSS, SuSuSurararakkkkkkaaa III, NNNieieiemimiminenenennn MSMSMS, SiSiSinininisasasalololo JJJ, LoLoLokkkkkkiii MLMLML, Perola M, HaHaavuvuvulilil nnnnn aaa A,AA ddeee FaFaFaire e UU,U, GGigigiganantetete BBB, InInIngeggelslsl soonnn EE,E, ZZZeeellllllerrr TTT, WiWiWildldld PP, dedede BBBakakakker PI,



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11. Pennathur S, Bergt C, Shao B, Byun J, Kassim SY, Singh P, Green PS, McDoDonananalldld TTTOOO, Brunzell J, Chait A, Oram JF, O'Brien K, Geary RL, Heinecke JW. Human atherosclerotic ntima and blood of patients with established coronary artery disease contain high density ipopooprprprotototeieieinnn dadadamamaagegeged d by reactive nitrogen specieseses.. JJJ Biol Chem. 2020004;2;22797979:42977-42983.

1122. HHadfield KKAA,A PPPatattitit sooon nn DIDIDI,, BrBrowowown n BEBEE, HHHouuu L,, RRRyee KKKA,A,, DDDavaviiesss MJMJMJ, HHaHawkwkwkinins s CLCLCL.MMMyeeeloperoxxididaseee-dderiiveveed oxoxxidddannts mmmoododiifi y aapapoolippoopprprototteieieinn n aaa-iii annd ggeneeeraaatete dyysysfuf ncctiiiononnalll higgghh--dedennsnsititityy y lilipopopoprpprototeeeinnsns:: CCoCompmpm ararrisisononon ooofff hyhyypopopothththiioiocccyayayanononousus acicicidd d (h(hhososo ccncn))) wiwiw ttth hhhypyppococochlhloororooous s acccidid hococl)l). BiBiococheem m J. 2020113;44494 :5:53131-5-542. JJ

1313 ZhZhenengg LL SSetettltlee MM BBrurubabakekerr GG SSchchmimitttt DD HHazazenen SSLL SSmimithth JJDD KKininteterr MM LLococalalizizatatioionn ofof



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2444. PiPiPiaoaoa ZZH,H,H, KKimimm MMSSS, JJJeoeonggg MMM, YuYuYun n S,S,S LLLeeeeee SHSHSH,,, SuSuun HNHNHN,, SoSoongngn HHHY,Y,Y SSSuhuhh HHHW,W,W, JJunununggg HHH, YYYooooonSRR, KiKim TDTD, LeLee YHYH, ChChooi II. VdVdupu 1 exxacacererbabatetes baactctererememicic sshoockck iin n miicece iinfnfece ted d wiw thth pseudomonaass aeaeaeruruugigiginonoosasas . CeCeCellll IIImmmmmmunuu ololol. 20202012122;2;22808080:1:11-9- .



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3355. RRozenberg g I,I,I, SSSluukakak SSSHH,H, RRRoohohrereerr LL,L HHofmmmannnn JJ,, BBBecchcherer BBB, AkAkkhmhmmededdoovov AA,,, SoSSoliliz z J,J, MMMococchahharlrlr aa a P, BBBorereen J, Johhananseeen P, SSSteeeffelell JJ,,, WaW tataananaabebee T, LLuuscheheher r TFTFTF,, TaTaTannnnerrr FFC. HHHissstatammiminene h111 rreecceepptor prpromomomotototeses aaathththererosossclcllereroototiccc llese ioioionn fofoformrmrmatattioioonn n bybyby iiinnncrcrreaeaasis nngng vvvaasascucuulalal rrr pepepermrmrmeaeaabibililiitytyty ffororr llloowow--d-deenensisiityty ipooprprototeieinsns. ArArtet ririososclc er TThrromombb VaV sc BBioioll.. 20201010;30:0:92923-3-93930.0

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44. Schwartz GG, Olsson AG, Ballantyne CM, Barter PJ, Holme IM, Kallend D, Leiter LA, Leitersdorf E, McMurray JJ, Shah PK, Tardif JC, Chaitman BR, Duttlinger-Maddux R, Mathieson J. Rationale and design of the dal-outcomes trial: Efficacy and safety of dalcetrapib in patients with recent acute coronary syndrome. Am Heart J. 2009;158:896-901 e893. 45. McKenney J, Bays H, Koren M, Ballantyne CM, Paolini JF, Mitchel Y, Betteridge A, Kuznetsova O, Sapre A, Sisk CM, Maccubbin D. Safety of extended-release niacin/laropiprant in patients with dyslipidemia. J Clin Lipidol. 2010;4:105-112 e101. 46. Nicholls SJ. Is niacin ineffective? Or did aim-high miss its target? Cleve Clin J Med. 2012;79:38-43. 47. Rader DJ, Tall AR. The not-so-simple hdl story: Is it time to revise the hdl cholesterol hypothesis? Nat Med. 2012;18:1344-1346. 48. Khera AV, Cuchel M, de la Llera-Moya M, Rodrigues A, Burke MF, Jafri K, French BC, Phillips JA, Mucksavage ML, Wilensky RL, Mohler ER, Rothblat GH, Rader DJ. Cholesterol efflux capacity, high-density lipoprotein function, and atherosclerosis. N Engl J Med. 2011;364:127-135. 49. Li X, Tang WH, Mosier MK, Huang Y, Wu Y, Matter W, Gao V, Schmitt D, DiDonato JA, Fisher EA, Smith JD, Hazen SL. Paradoxical association of enhanced cholesterol efflux with increased incident cardiovascular risks. Arterioscler Thromb Vasc Biol. 2013;33:1696-1705. 50. Macheboeuf M. Recherches sur les phosphoaminolipides et les sterids du serum et du plasma sanguins. Ii etude physiochimique de la fraction proteidique la plus riche en phospholipids et in sterides.Proteidique la plus riche en phospholipids et in steridesproteidique la plus riche en phospholipids et in sterides. Bull. Soc. Chim. Biol. 1929;11:485-50351.

44. Schwartz GG, Olsson AG, Ballantyne CM, Barter PJ, Holme IM, Kallend D,D, LLeieie teeerr r LALALA,,,Leitersdorf E, McMurray JJ, Shah PK, Tardif JC, Chaitman BR, Duttlinger-Maddduduuxxx RR, Mathieson J. Rationale and design of the dal-outcomes trial: Efficacy and safety of dalcetrapib inpatients with recent acute coronary syndrome. Am Heart J. 2009;158:89696-901 e893. JJ

45455. MMcMcKennneeey JJ, , , BaBaB ysysy HHH,, , KoKoK rereennn M,M,M BBalallalantntynyny e e CMCMCM, PaPaolololininini i JFF,, MiMiM tctcheheell l Y,, BBetetetteteteriridgdgee A,A,A, KKuzznznetsova OO, SSaS pprpre e A,A,, SSSisisiskk k CCMCM, , , MMaMacccccubbbbinnn D. SSaafetettyyy ofoff extxtenennddeded-d-rreeleeasaseee nniniacacininin/l/l/ ararropppiipiprarannnt inpapaatiieene ts with h dyd sssliippidemememia. JJJ CCClinn LLipipi iiddoool. 220110;444:110505-1-1-1121212 e11011.

46. NiNichholollsls SSJ.J. Is niniaca in iineneffffece titive? OrOr ddidid aaimim-higgh h mimissss iitsts tarargeget?t? Cleveve e ClClinin J MMede ..2012;79:38-443.3.3



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Figure Legends:

Figure 1. ApoA1 or apoA1 in reconstituted HDL were either left untreated or oxidized at a 5:1

molar ratio of oxidant to apoA1 as described in Methods. a, Equal amounts of apoA1 were

separated on 5-15% reducing SDS-PAGE gels and proteins transferred to membranes for

Western blot detection using three distinct commercial antibodies (Ab), as indicated, or the

monoclonal (mAb) 10G1.5. Monomeric and multimeric apoA1 immuno-reactive bands are

apparent and Molecular weight markers are indicated. b, Demonstration that apoA1-specific

monoclonal antibody 10G1.5 recognizes all forms of apoA1 (lipid-free and HDL-associated,

non-oxidized and oxidized) equally well.ApoA1 (open bars) or reconstituted HDL (rHDL, filled

bars) prepared from apoA1 or their oxidized versions using the oxidation systems as indicated

were coated at 0.5 μg/ml into ELISA plates in triplicate and probed with 10 ng/mL anti-apoA1

monoclonal antibody 10G1.5. ELISA assays were performed as described in Methods.

Reactions were stopped with 0.1N HCl, and absorbance at 450nm was determined. c, Lipid-free

apoA1, isolated human plasma HDL, or the sub-HDL populations HDL2 and HDL3 were each

loaded (in triplicate for each data point) onto 5-15% reducing SDS-PAGE gels at the indicated

amounts, and then apoA1 was quantified by immuno-blot using mAb 10G1.5 as described in

Methods. All values represent the average of triplicate determinations; error bars indicate the

standard deviation.

Figure 2. ApoA1 from human atherosclerotic lesions is not located on HDL-like particles and is

heavily cross-linked. Proteins in atherosclerotic lesion homogenate or after buoyant density

ultracentrifugation fractionation into HDL-like particle and lipoprotein depleted fractions from

non-oxidized and oxidized) equally well.ApoA1 (open bars) or reconstituted HDDLLL (r(r( HDHDHDL,L,L, fffiililleled

bars) prepared from apoA1 or their oxidized versions using the oxidation systems as indicated

wewererere cccooaoateteted d d aaat 00.55.5 μμg/ml into ELISA plates in tririripplp iicate and probbedede wwititithh h 10 ng/mL anti-apoA1

mmonnonoclonal antntibibibooody y 10100G1G1G1.5.55.. EELLILISSASA aasssayyys werrre pperrrfofoormrmmeddd aasss ddedescscririr bbeed d ininin MMetetethohohodsdsds.

ReReacaca tititionono ss wewewerere ssstoooppppppeeded wwiti h hh 0.0.1N1N1N HHHCCll, aaandndnd aabsbsbsororrbbaancnccee atatat 44505050nmnmm wwwasass ddetete ererrmimiminenedd.d. c,c, Liiipipidd-d-frfrreee

apoA1, isolaateteed d d huhuumamam nnn plpp asasasmamm HHHDLDLDL,, ororor ttthehehe sususub-b-b HDHDHDL LL popopopupupulalalatititiononons HDHDHDL2L2L2 aaandndn HHHDLDLDL333 wew re each



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the indicated density ranges were separated on 5-15% reducing SDS-PAGE gels. a, Sypro Ruby

stained gel of the indicated protein samples (10 μg) from homogenate and the indicated density

ranges obtained from different atherosclerotic lesion tissue samples (n=5). b, Western blot

membrane of a duplicate run gel as in panel a with 2.4μg, 0.4μg and 2.4μg of homogenate, HDL-

like and LPD fraction proteins respectively, probed with anti-total apoA1 mAb 10G1.5. c,

Overexposure of western blot in panel b to show apoA1 in HDL-like fraction. Monomeric and

dimeric apoA1 immuno-reactive bands and Molecular weight markers are indicated.

Figure 3. Particle distribution of apoA1 obtained from human atherosclerotic artery wall. a,

Percent of total protein in starting homogenate and in buoyant density ultracentrifugation

fractions LDL-like, HDL-like and LPD determined by BCA protein assay are indicted. b,

ApoA1 (mg) recovered per gram of lesion tissue (wet weight) in the HDL-like and LPD

fractions; c, percent apoA1 to total apoA1 from atherosclerotic lesion homogenate present in the

HDL-like and LPD fractions; d, cross-linked apoA1/total apoA1 within each fraction as a

percentage was determined by quantitative western blot analysis of apoA1 immuno-reactive

bands in Figure 2b and additional blots (not shown). Values were determined from samples

(n=10), error bars represent S.D. Mean values are indicated by a heavy horizontal line. Kruskal-

Wallis test was used in panels (a) and (d) and found to be significant (P=0.0001 and P=0.0003

respectively). Dunn’s test was used to adjust for multiple comparisons and Wilcoxon Rank Sum

test was used in pairwise comparisons. Actual P values are listed when P<0.05.

Figure 4. ApoA1 obtained from the artery wall is not on an HDL-like particle and behaves

similarly to that isolated from atherosclerotic lesions. a, Representative aortic tissue from

Percent of total protein in starting homogenate and in buoyant density ultracentririfuffugagg ttitiononon

fractions LDL-like, HDL-like and LPD determined by BCA protein assay are indicted. b,

ApApoAoAoA111 (m(m(mg)g)g) reccovovovere ed per gram of lesion tissue e e (w(wwet weight) in ththt e HDHDHDLL-L like and LPD

ffrracccttions; c, peercrccennnt apappoAoAA111 tototo tttototalalal aaapopooAAA1 frfrrommm atththeeroosscclclererototticic llessiionon hhhoommoogogeenenatatte ee pprpresesenenntt ininin ttthe

HDHDDL-L-L-lililikeke aaandndnd LLPDPDPD ffraractcttioionss;;; ddd, crcrcrosooss-s-lilinknknkededed aaapopopoA1A1A /tototo alalal aaapopopoA1A1A1 wwwititithihiinn eeaeachchch fffrarar ctctctioioon aaas aa

percentage wwasasas ddeteteterere mimim nenn d d d bybyby qqquauauantntntiti atatativiviveee weweweststs erere n nn blblb otott aaanananalylylysisisis s s offf aaapopopoA1A1A1 iiimmmmmunununo-o-o rerer active



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normal and atherosclerotic human artery wall. b, Sypro Ruby stained 5-15% SDS-PAGE gel of

normal and atherosclerotic lesion homogenate (n=5) proteins (5 μg). c, Western blot of duplicate

gel as in panel a with the indicated amount of protein per lane (n=5) probed with anti-total

apoA1 mAb 10G1.5. d, Western blot of normal artery wall proteins (30 μg) from homogenate

(sample 1, pooled samples 2&3 and pooled samples 4&5), HDL-like and LPD fractions probed

with -total apoA1 mAb 10G1.5 to show apoA1 and cross-linked apoA1. e, ApoA1 (mg)

recovered per gram of normal aortic tissue (wet weight) in the HDL-like and LPD fractions; f,

apoA1 to total apoA1 as a percentage from normal aortic tissue homogenate present in the HDL-

like and LPD fractions; g, cross-linked apoA1/total apoA1 within each fraction as a percentage

was determined by quantitative western blot analysis of apoA1 immuno-reactive bands in panels

(c) and (d). Monomeric and dimeric apoA1 are indicated. Values were determined from

samples (n=5) in (c) and n=3 in (d) and additional blots (not shown), error bars represent S.D.

Mean values are indicated by a heavy horizontal line. Kruskal-Wallis test was used in panel (g)

and found to be significant (P=0.006). Wilcoxon Rank Sum test was used in pairwise

comparisons to determine statistical differences. Actual P values are listed when P<0.05.

Figure 5. ApoA1 in plasma is on HDL particles and behaves differently than artery wall derived

apoA1. Plasma from normal healthy human volunteers (n=5) was fractionated by buoyant

density gradient centrifugation. a, Coomassie Blue stained 5-15% SDS-PAGE gel of plasma,

HDL- and LPD fraction proteins and b, Western blot analysis of proteins on membrane from a

duplicate fractionated protein gel probed with anti-total apoA1 mAb 10G1.5 shows apoA1

predominately in the HDL fraction and not cross-linked. c, Percentage of total protein recovered

in starting material, and in the VLDL + LDL, HDL-like and LPD fractions. Quantitative

was determined by quantitative western blot analysis of apoA1 immuno-reactivee babaandndds s ininin pppanana eels

c) and (d). Monomeric and dimeric apoA1 are indicated. Values were determined from

aampmpmpleleless (n(n(n=5=5=5) innn (((c)c)c and n=3 in (d) and additionnnaaall bbblots (not shownwnw ), eerrrrrrooor bars represent S.D.

MMeaanan values araree innndidiiccaateteeddd bybyby aaa hheaeaeavvyvy hhooorizononontal lllinnne. KKKrurusksks alal-W-WWalallilis s tetestst wwwaasas uuseseseddd inini ppaananelelel (((gg)

anndd d fofofoununu dd tototo bbee ssiggngnififficicanana t t (P(PP=0=0=0 00.0060606).). WWWilililccocoxoxoxon n aRaRanknkk SSSumumum tttesesest t wawawas s s uussededd iinn n papaairirwiwiwissse

comparisons s tototo ddetetetererermimim nenn ssstatatatitt ststticicicalall ddififffefefererer nnncececes.s.s AcAcActutut alalal PPP vvvalalalueueues ss ararare e e lilil stststededed wwhehehen n n P<P<P<0.00 05.



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analyses of panel B and overexposed panel B reveal percentage of apoA1 to total apoA1 present

in the plasma HDL and LPD fractions in d, and cross-linked apoA1/total apoA1 within plasma,

HDL and LPD fractions in e. Values (c,d) were determined from samples (n=5). Error bars

represent S.D., and the mean is indicated by a heavy horizontal line. Kruskal-Wallis test was

used to determine statistical differences for data presented in panels (c) and (e), which was found

to be significant (P=0.0002 and P=0.01, respectively). Dunn’s test was used to adjust for

multiple comparisons in panel c and Wilcoxon Rank Sum test was then used for pairwise

comparisons. Actual P values are listed.

Figure 6. Functional characterization of lesion apoA1. Macrophage cholesterol efflux activity,

and LCAT activity, were measured in apoA1 immuno-affinity purified from human

atherosclerotic-laden plaque (n=10 different subjects) as described under Methods. ApoA1

isolated from plasma HDL recovered from healthy donors (n=3) and rHDL formed from these

apoA1 served as controls for total efflux activity, and LCAT activity, respectively. Bars

represent triplicate determinations; error bars represent S.D. P values represent comparison

between subject samples versus control apoA1 and were determined using the one-sample robust

Hotelling T2 test.

Figure 6. Functional characterization of lesion apoA1. Macrophage cholesterol eefefflflluuxux aactctctivivivititityy,

and LCAT activity, were measured in apoA1 immuno-affinity purified from human

attheheerororosscsclelelerororotititic-lalaaddedenn plaque (n=10 different subjbjbjecece tts) as describededd undnddererer Methods. ApoA1

ssollaated from plpllaaasmmama HHHDLDLDL rrecececovovererereeded ffrooom hhheeaalthhyy ddonnnoorors s (n(n( =3=3)) ) aannd d rHrHrHDDLDL fffoorormemeed d frfrfromomm tttheeheseee

apapoAoAoA111 seservrvvededed aass cocoontntrrrollsls fforr ttotototalalal eeefffffluluux xx acacactttivivivitytyt , ,, anannd LCLCCAATAT aaactctivivititity,y,y rrressspepep ctctc ivivvelely.y.y. BBBararrs

epresent tripipplililicacac tetee dddetettererrmimiinananatit ononons;s;s eeerrr ororor bbbararars s s rerereprprpresese enene t t S.SS D.D.D. P P P vavav luuueseses rrrepepeprereresesentntnt cccomomompapp rison



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A. Fisher and Stanley L. HazenEdwardGogonea, Yuping Wu, Paul L. Fox, W. H. Wilson Tang, Edward F. Plow, Jonathan D. Smith,

Joseph A. DiDonato, Ying Huang, Kulwant Aulak, Orli Even-Or, Gary Gerstenecker, Valentinfrom those in Plasma

The Function and Distribution of Apolipoprotein A1 in the Artery Wall are Markedly Distinct

Print ISSN: 0009-7322. Online ISSN: 1524-4539 Copyright © 2013 American Heart Association, Inc. All rights reserved.

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Supplemental Material

The function and distribution of apolipoprotein A1 in the artery wall are markedly distinct from those in plasma

Joseph A. DiDonato, PhD1*, Ying Huang, PhD1*, Kulwant Aulak, PhD1, Orli Even-Or, PhD2, Gary Gerstenecker, PhD1,3, Valentin Gogonea, PhD1,3, Yuping Wu, PhD4, Paul L. Fox, PhD1, W.H. Wilson Tang, MD1,5, Edward F. Plow, PhD6, Jonathan D. Smith, PhD1,5, Edward A. Fisher, MD2,and Stanley L. Hazen, MD PhD1,3,5

1Department of Cellular & Molecular Medicine, Lerner Research Institute, Cleveland Clinic,

Cleveland, Ohio 2 Department of Medicine, New York University, New York, New York. 3 Department of Chemistry, Cleveland State University, Cleveland, Ohio 4 Department of Mathematics, Cleveland State University, Cleveland, Ohio 5 Department of Cardiovascular Medicine, Heart and Vascular Institute, Cleveland Clinic,

Cleveland, Ohio 6 Department of Molecular Cardiology, Lerner Research Institute, Cleveland Clinic, Cleveland,

Ohio

*Co-first authors

2

Supplemental Table 1: Peak Lists of Apolipoprotein A1 Peptides in Lower ~25 kDa band verified by MS/MS

~25 kDa MW Lower Band Protein Identification

apolipoprotein A1 preproprotein Sequence coverage 52%

Observed Peptide Sequence XCorr Charge m/z [Da] ΔM

[ppm]

AELQEGAR 1.82 2 437.2241 -1.85

ARAHVDALR 2.13 3 336.8608 -1.99

ATEHLSTLSEK 3.56 3 405.8789 0.46

DLATVYVDVLK 3.51 2 618.348 0.43

DYVSQFEGSALGK 4.16 2 700.8381 -0.30

EQLGPVTQEFWDNLEK 4.86 2 966.9692 -1.45

LEALKENGGAR 2.18 3 386.5468 -1.21

LLDNWDSVTSTFSK 5.09 2 806.8943 -2.54

QGLLPVLESFK 2.25 2 615.8572 -1.81

VQPYLDDFQK 2.00 2 626.8133 -1.25

ApoA1 was immuno-precipitated from normal artery wall homogenates and run on SDS-PAGE as described. The lower band migrating at ~25 kDa was excised (Supplemental Figure 2), digested with trypsin, and analyzed via nanospray-ESI on an Orbitrap-LTQ Velos. The resultant spectra were searched using Proteome Discoverer 1.1 against the Uniprot human database 2013_2. Shown is a listing of tryptic peptides confirmed by MS/MS for apoA1 with their respective SEQUEST correlation scores (XCorr), charge state, precursor mass and mass shift of the observed peptide from the theoretical mass (ΔM [ppm]).

3

Supplemental Table 2: Peak Lists of Apolipoprotein A1 Peptides in Upper~25 kDa band verified by MS/MS ~25 kDa MW Upper Band Protein Identification


Observed Peptide Sequence XCorr Charge m/z [Da] ΔM [ppm]

AKPALEDLR 3.25 3 338.19705 -2.00

ATEHLSTLSEK 3.49 3 405.87778 -2.32

DLATVYVDVLK 3.54 2 618.34631 -2.33

DYVSQFEGSALGK 4.64 2 700.83600 -3.26

EQLGPVTQEFWDNLEK 3.64 2 966.96796 -2.71

LLDNWDSVTSTFSK 4.75 2 806.89410 -2.77

QGLLPVLESFK 2.58 2 615.85687 -2.31

VQPYLDDFQK 2.39 2 626.81274 -2.12

VSFLSALEEYTK 5.02 2 693.85925 -2.84

ApoA1 was immuno-precipitated from normal artery wall homogenates and run on SDS-PAGE as described. The upper band migrating at ~25 kDa was excised (Supplemental Figure 2), digested with trypsin, and analyzed via nanospray-ESI on an Orbitrap-LTQ Velos. The resultant spectra were searched using Proteome Discoverer 1.1 against the Uniprot human database 2013_2. Shown is a listing of tryptic peptides confirmed by MS/MS for apoA1 with their respective SEQUEST correlation scores (XCorr), charge state, precursor mass and mass shift of the observed peptide from the theoretical mass (ΔM [ppm]).

4

Supplemental Table 3: Peak Lists of Apolipoprotein A1 Peptides in Lower ~50 kDa band verified by MS/MS ~50 kDa MW Lower Band Protein Identification



AKPALEDLR 3.37 3 338.19766 -0.20

AKPALEDLRQGLLPVLESFKVSFLSALEEYTK 4.06 5 719.19965 0.47

ATEHLSTLSEK 3.18 3 405.87866 -0.14

DLATVYVDVLK 3.36 2 618.34692 -1.34

DSGRDYVSQFEGSALGK 5.47 3 605.95453 -1.01

DYVSQFEGSALGK 3.71 2 700.83752 -1.08

LEALKENGGAR 2.20 3 386.54700 -0.81

LLDNWDSVTSTFSK 4.20 2 806.89502 -1.63

LREQLGPVTQEFWDNLEK 6.07 3 734.70935 -2.53

LREQLGPVTQEFWDNLEKETEGLR 6.30 4 722.62006 -0.07

QGLLPVLESFK 2.70 2 615.85760 -1.12

VKDLATVYVDVLK 4.27 3 488.28836 -0.75

VQPYLDDFQK 2.50 2 626.81378 -0.47

VSFLSALEEYTK 4.70 2 693.86029 -1.35

ApoA1 was immuno-precipitated from normal artery wall homogenates and run on SDS-PAGE as described. The lower band migrating at ~50 kDa was excised (Supplemental Figure 2), digested with trypsin, and analyzed via nanospray-ESI on an Orbitrap-LTQ Velos. The resultant spectra were searched using Proteome Discoverer 1.1 against the Uniprot human database 2013_2. Shown is a listing of tryptic peptides confirmed by MS/MS for apoA1 with their respective SEQUEST correlation scores (XCorr), charge state, precursor mass and mass shift of the observed peptide from the theoretical mass (ΔM [ppm]).

5

Supplemental Table 4: Peak Lists of Apolipoprotein A1 Peptides in Upper ~50 kDa band verified by MS/MS ~50 kDa MW Upper Band Protein Identification



AKPALEDLR 2.60 3 338.19690 -2.46

ATEHLSTLSEK 2.58 3 405.87784 -2.17

DLATVYVDVLK 3.22 2 618.34631 -2.04

DSGRDYVSQFEGSALGK 2.60 3 605.95453 -1.01

LLDNWDSVTSTFSK 2.58 2 806.89478 -1.93

LSPLGEEMR 3.22 2 516.26184 -2.58

QGLLPVLESFK 2.57 2 615.85699 -2.11

THLAPYSDELR 3.17 3 434.55347 -2.02

VKDLATVYVDVLK 2.97 3 488.28790 -1.62

VSFLSALEEYTK 4.33 2 693.85999 -1.79

ApoA1 was immuno-precipitated from normal artery wall homogenates and run on SDS-PAGE as described. The upper band migrating at ~50 kDa was excised (Supplemental Figure 2), digested with trypsin, and analyzed via nanospray-ESI on an Orbitrap-LTQ Velos. The resultant spectra were searched using Proteome Discoverer 1.1 against the Uniprot human database 2013_2. Shown is a listing of tryptic peptides confirmed by MS/MS for apoA1 with their respective SEQUEST correlation scores (XCorr), charge state, precursor mass and mass shift of the observed peptide from the theoretical mass (ΔM [ppm]).

6

Supplemental Figure 1

Supplemental Figure 1. The distribution of protein recovered in normal artery wall

homogenate before (100%) and following fractionation by ultracentrifugation. Normal

artery wall homogenates (n=5) were fractionated by sequential buoyant density

ultracentrifugation using D2O/Sucrose mixtures to produce the indicated density fractions, as

described under Methods, and then total protein was determined from starting material and

LDL-like, HDL-like and lipoprotein-depleted (LPD) fractions. The percentage of total protein

relative to homogenate (100%) in each sub-fraction is indicated. Error bars indicate standard

deviation. Note that the majority of protein recovered from normal artery wall homogenate is

within the lipoprotein-depleted fraction.

7


Supplemental Figure 2. ApoA1 is present in the normal artery wall and is cross-linked.

ApoA1 was immuno-precipitated from normal artery wall homogenates (n=5) and separated

on a non-reducing (5-15%) SDS-PAGE gradient gel and stained with Coomassie Blue.

Indicated bracketed bands (upper and lower ~25 kDa and ~50 kDa protein bands) were

excised from the gel and subjected to trypsin digestion and mass spectrometry analysis. The

major peptides recovered and sequenced from each indicated excised regions of the gel

were observed by LC-MS/MS to be from apoA1 (Supplemental Tables 1-4).

8


Supplemental Figure 3. Plasma contains both monomeric (major) and slower

migrating (minor) oxidized and cross-linked apoA1 forms. Indicated amounts of plasma

and LPD fraction proteins (n=4) from those plasma samples were run on a reducing 12%

SDS-PAGE gel. The proteins were then transferred to membrane for Western blot probing

with mAb 10G1.5. The major bands noted within both plasma and the LPD fractions appear

at the anticipated molecular weight of the apoA1 monomer (~25 kDa). Longer exposures

(shown) of the Western blot reveal the presence of slower migrating apoA1 immuno-reactive

bands (indicated by arrowheads), consistent with oxidatively cross-linked forms of apoA1.

Documents

The Function and Distribution of Apolipoprotein A1 in the ... 10.1161/CIRCULATIONAHA.113.002624 1 The Function and Distribution of Apolipoprotein A1 in the Artery Wall are Markedly