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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: didonaj@ccf.org E-mail: hazens@ccf.org
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,
CCCleeveveland, OOH;H;H; 22DeDeDepptpt ooof ff MeMeMedididi icicinnene,,, NNeN www YYoYorrkrk UUUniiverrrsititity,y, NNNeweww YYYorork,k,k, NNNY;;; k 333 DDeDeptptpt ooof f f ChChChemememisisi ttrt y;4 eDeD pt ooff f MaMathththememe aticiccs,s CCCleveeelaaand Statatetee UUUniniveveversittty, Cleeveveelaandndnd, , OHOH
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DOI: 10.1161/CIRCULATIONAHA.113.002624
2
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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DOI: 10.1161/CIRCULATIONAHA.113.002624
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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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DOI: 10.1161/CIRCULATIONAHA.113.002624
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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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DOI: 10.1161/CIRCULATIONAHA.113.002624
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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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DOI: 10.1161/CIRCULATIONAHA.113.002624
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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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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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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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DOI: 10.1161/CIRCULATIONAHA.113.002624
34
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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Figure 1
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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.
is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Circulation published online August 22, 2013;Circulation.
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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
apolipoprotein A1 preproprotein Sequence coverage 44%
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
apolipoprotein A1 preproprotein Sequence coverage 54%
Observed Peptide Sequence XCorr Charge m/z [Da] ΔM [ppm]
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
apolipoprotein A1 preproprotein Sequence coverage 44%
Observed Peptide Sequence XCorr Charge m/z [Da] ΔM [ppm]
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
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
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.
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