• No results found

Osteopontin is elevated during neointima formation in rat arteries and is a novel component of human atherosclerotic plaques

N/A
N/A
Protected

Academic year: 2020

Share "Osteopontin is elevated during neointima formation in rat arteries and is a novel component of human atherosclerotic plaques"

Copied!
12
0
0

Loading.... (view fulltext now)

Full text

(1)

Osteopontin is elevated during neointima

formation in rat arteries and is a novel

component of human atherosclerotic plaques.

C M Giachelli, … , C E Alpers, S M Schwartz

J Clin Invest.

1993;

92(4)

:1686-1696.

https://doi.org/10.1172/JCI116755

.

In an earlier report, we used differential cloning to identify genes that might be critical in

controlling arterial neointima formation (Giachelli, C., N. Bae, D. Lombardi, M. Majesky, and

S. Schwartz. 1991. Biochem. Biophys. Res. Commun. 177:867-873). In this study, we

sequenced the complete cDNA and conclusively identified one of these genes, 2B7, as rat

osteopontin. Using immunochemistry and in situ hybridization, we found that medial smooth

muscle cells (SMC) in uninjured arteries contained very low levels of osteopontin protein

and mRNA. Injury to either the adult rat aorta or carotid artery using a balloon catheter

initiated a qualitatively similar time-dependent increase in both osteopontin protein and

mRNA in arterial SMC. Expression was transient and highly localized to neointimal SMC

during the proliferative and migratory phases of arterial injury, suggesting a possible role for

osteopontin in these processes. In vitro, basic fibroblast growth factor (bFGF), transforming

growth factor-beta (TGF-beta), and angiotensin II (AII), all proteins implicated in the rat

arterial injury response, elevated osteopontin expression in confluent vascular SMC.

Finally, we found that osteopontin was a novel component of the human atherosclerotic

plaque found most strikingly associated with calcified deposits. These data implicate

osteopontin as a potentially important mediator of arterial neointima formation as well as

dystrophic calcification that often accompanies this process.

Research Article

Find the latest version:

(2)

Osteopontin

Is Elevated

during

Neointima Formation in Rat

Arteries

and Is

a

Novel

Component

of Human Atherosclerotic

Plaques

CeciliaM.Giachelli, Nancy Bae, ManuelaAlmeida, David T. Denhardt,* Charles E. Alpers, and Stephen M.Schwartz

Department of Pathology SJ-60 University of WashingtonSchoolofMedicine, Seattle, Washington98195; and *NelsonBiologicalLaboratories, Rutgers University, Piscataway,NewJersey08855

Abstract

In an earlier report, we used differential cloning to identify genes that might be critical in controlling arterial neointima formation (Giachelli,C., N.Bae,D. Lombardi,M.Majesky,

and S. Schwartz. 1991. Biochem.

Biophys.

Res. Commun. 177:867-873).Inthis study, wesequencedthecompletecDNA andconclusively identified oneof these genes, 2B7,as rat

os-teopontin. Using immunochemistryand in situ hybridization,

wefoundthat medial smooth muscle cells(SMC)inuninjured

arteries contained very low levels of

osteopontin

protein and mRNA. Injury to either the adult rat aorta or carotid artery

usinga balloon catheter initiatedaqualitatively similar time-dependent increase in both osteopontin proteinand mRNA in arterialSMC.Expressionwastransient andhighlylocalizedto

neointimal SMCduringtheproliferativeandmigratoryphases of arterial injury,suggestingapossibleroleforosteopontinin these processes. In vitro, basic fibroblast growth factor

(bFGF),transforming growthfactor-,@

(TGF-,8),

and

angioten-sin II (AII),all proteins

implicated

in the ratarterial

injury

response, elevatedosteopontinexpressioninconfluentvascular SMC. Finally, we found thatosteopontinwas a novel compo-nentofthe humanatheroscleroticplaque foundmoststrikingly

associated withcalcified deposits.These dataimplicate

osteo-pontinas a

potentially important

mediator of arterial neointima

formationas wellasdystrophiccalcification that often

accom-paniesthis process.(J.Clin. Invest. 1993.

92:1686-1696.)

Key

words:osteopontin-smooth muscle-angioplasty-

neointima*

atherosclerosis

Introduction

The most commoncharacteristicof the response of blood

ves-selstoinjury istheformation ofaneointima. Thisoccurs

fol-lowing angioplasty,butisalso seen inhypertension,renal and

radiationinjury, or trauma to any layerof the vessel wall ( 1). Mostimportantly,formationoftheneointimafollowinginjury is believedtobe an important model for the initial steps and

progressionofatheroscleroticlesions as well as the rapid reste-notic changes seen after angioplasty (2, 3).

Thestepsinvolvedinformationoftheneointima have been

Address correspondence to Dr. Cecilia M. Giachelli, Department of

Pathology(SJ-60),UniversityofWashington,Seattle, WA 98195.

Receivedfbrpublication 17 December 1992 and in revised form 7 Mai,1993.

studied indetail, usingthe model of balloonangioplasty

injury

in the rat artery (2). In this model, endothelial denudation combines with medial damage to induce quiescent arterial smooth muscle cells (SMC) toenterthe cell cycle, remodel theircytoskeletalcomponents, andchangeadhesiveproperties

and gene expression patterns. The net result is an activationor

modulation of medial cells to become migratory and invade a normally SMC-deficient vascular compartment: the

neoin-tima. Once there, neointimal SMC adopt a number ofspecial

propertiesthatdistinguish them from the underlying medial SMC. We might, therefore, consider the cells that form the neointima as "activated" or modulated to perform thespecial

functions associated with the repair process.

Although it is unclear what initiates SMC activation under these conditions, neointimal SMC share many ofthe properties of

developmentally

immature SMC

(

1,

4)

andit is

intriguing

to

consider thepossibilitythat similarmechanismsareused dur-ingdevelopment and during formation of the neointima. We haveexplored this hypothesisby differentiallycloning genes that wereselectively elevated inbothratneointimaland pup medial SMC compared to uninjured adult medial SMC (5, 6). As a result of this effort, we have discovered several genes

whichappear tomark an"activated"SMCphenotype both in

vitroand in vivo. Theseincluded elastin andcollagen I

(al

) (6), as well as 2B7, an mRNA that had not previously been

described in smooth muscle cells (5). When partially

se-quenced,2B7showedhighhomologytoosteopontin(secreted

phosphoprotein 1)(5). In that report, we showed that 2B7 mRNA was present in total RNA preparedfromthe uninjured aorta and carotidarteriesby Northernanalysis.As predicted by our cloning strategy, 2B7 mRNA levels increased

approxi-mately fivefoldinthe carotidartery 48 hfollowing

de-endothe-lializationbyballooncatheter,and remained elevated 7-14 d afterinjury (5).

Inthispaper we reportfull-lengthsequencedata unequivo-callyidentifying2B7 asencodingratosteopontin.Osteopontin

isa highly acidic, secreted glycoprotein associated with bone

morphogenesis (7, 8), cell transformation (9, 10), immune cellactivation( 1 1, 12), andbacterialresistance ( 12, 13).

Al-though it is Al-thoughttoplay a role in bone mineralization and to

mediatebone celladhesion to calcifiedmatrixvia an

arginine-glycine-aspartate

(RGD)-dependent mechanism (8), the

function of osteopontin inothertissuesis notyet understood. Tothisend, wehaveinvestigatedthe potential role of

osteo-pontin in arterial tissuebydetermining its cellular distribution

throughout the variousphases of neointima formation after

balloon catheter injuryto rataorta and carotidarteries.In

ad-1.Abbreviationsusedinthispaper:All, angiotensin 11; GAPD,

glycer-aldehyde 3-phosphate dehydrogenase; RGD, arginine-glycine-aspar-tate;SMC.smoothmusclecells.

1686 Giache/li, Bae, Almeida, Denhardi,Alpers,and Schwartz

J.Clin. Invest.

©TheAmericanSociety forClinicalInvestigation, Inc.

0021-9738/93/10/1686/11 $2.00

(3)

dition,we reportinvitro studies aimedatdeterminingwhether

peptide factors thoughttobe important in arterial neointima formation regulate osteopontin mRNA and protein

expres-sion. Finally,wehavefound that osteopontin isa novel

compo-nentofatheroscleroticplaque. Ourdata suggest that

osteopon-tin may play an important role in arterial neointima

forma-tion.

Methods

Animalsurgery andpreparation oftissues. Adult male Sprague-Dawley

ratsweighing- 400 g (at - 3 moof age) were purchased from either

Simonsen Laboratories (Gilroy, CA)orTyler Labs, Inc. (Bellevue, WA). Rats wereanesthetizedwith anintramuscularinjection of Inno-var-vet, 0.2 ml/kg and intraperitoneal injection of pentobarbital, 5.4 mg/kg(Pitman-Moore, Mundelein,IL).Angioplasty ofthecarotidor aorta wasperformed usingaballoon embolectomy catheteras previ-ously described ( 14). Briefly,the 2Fembolectomy catheterwas intro-ducedthroughtheleft externalcarotid, advancedintotheabdominal

aorta,inflated,andwithdrawn. Thissequence wasrepeatedthreetimes

toachieve total endothelial denudation of the thoracicaorta and the

leftcommon carotid artery(as monitored byEvan's bluestaining).

Thecontralateral, uninjured carotidarteryfromthe sameanimal,or aortas andkidneys from uninjured animals,served ascontrol tissues. Surgically treated animalswerehousedindividually in plasticcages and

fedratchow andwaterad lib.Attheappropriate times after injury, the

rats werekilled by ether inhalation, and, in some cases, tissue was

perfusion-fixedin4%paraformaldehyde,andembedded inparaffinas

previously described(15). Microtome sections (5gm)weremounted

onvectabond-coated slides (Vector Labs,Burlingame, CA). Tissues preparedinthiswayfromfourtosixratspertimepointwereused for bothimmunocytochemicalandinsituhybridizationstudies.For

isola-tionof RNA,aortasfrom foursimilarly treatedrats werepooled and processedaspreviously described (5).Humanatheroscleroticplaques

were obtainedfrom patients undergoing carotid endarterectomy sur-gery. Human coronaryarterieswereobtained afterautopsy.These

tis-sueswerefixedandembeddedaspreviously described (16).

ImmunocvtochemistrY'.

The avidin-biotin peroxidase complex (ABC) techniqueasdescribedby Hsu et al. ( 17) was used to detect the osteopontin epitopes in arterial tissue. Thereagents for thisanalysis

were purchased as a kit(VectastainElite; Vector Labs). Slides were

deparaffinized,rehydrated, and incubated 35min in3%H202 in metha-noltoblock endogenous peroxidase activity. Nonspecific bindingwas

blockedby incubating slides with normal rabbit ormouseserumin PBScontaining 1%BSA and 20,g/ml f-galactosidase(Gibco-BRL Gaithersburg, MD). 2ArC and2arNareaffinity-purified,rabbit poly-clonal antibodies that recognizetheCOOH-terminal 73-214amino acids andNH2-terminal 215-294 amino acids ofmouseosteopontin

fusedto

f-galactosidase,

respectively( 18).LF-7isapreviously

charac-terized rabbit polyclonal antibody directed against human

bone-de-rivedosteopontin(19). Theseantibodieswereused at 1:50dilutions

(for affinity-purified 2arC and 2arN) and between 1:12,000 and 1:2,000(forLF-7antiserum). Control antibodieswereusedat

equiva-lentconcentrations and included: (a) rabbit ormouse nonimmune serum; (b)anti-f-galactosidase antiserum; and(c) omission of the primary antibodies. Primary antibodiesweredilutedin PBS and incu-batedat roomtemperaturefor 1 h.Washinganddevelopingof color

wereperformed assuggested by themanufacturer. For somestudies withLF-7, NiC12was added to the alkaline phosphatasesubstrate to generateablackreactionproduct,andcounterstainingof sectionswas

performed with methyl green. Cell types in human atherosclerotic

plaque weredistinguished using singlelabelimmunoperoxidase stain-ing of serial sections with anti-CD68andHAM-56, whichrecognize

humanmacrophages (20),andHHF-35,whichrecognizes muscle

ac-tin and therefore isspecificfor smooth muscle inthiscontext(21).

Theseantibodies wereutilized as previously described ( 16). HAM-56 and HHF-35 were kindly provided by Dr. Allen Gown, University of

Washington, anti-CD68 was purchased from DAKO (Copenhagen,

Denmark),andLF-7 was a gift from Dr. Larry Fisher, National Insti-tutes of Health.

DNA sequencing. 2B7 was cloned into pBluescript S/K (Stratagene

Inc. LaJolla, CA) as previously described (5). Dideoxy sequencing was

performed using a sequenase sequencing kit (IBI/Kodak Co., New Haven, CT) and vector or insert-specific oligonucleotide primers

(Operon Technologies, Inc., Alameda, CA).PCGENE and

Intelligenet-ics Suite (IntelliGenetIntelligenet-ics, Inc., Palo Alto, CA) software wasutilizedfor sequence comparisons. The sequenceof nucleotides in2B7 was

deter-mined at least twice from both strands of the cDNA. Where differences were noted when compared to the sequence for rat bone osteopontin (22), a second SMC-derived osteopontin clone, 1B7, was also

se-quenced to rule out sequence errors due to cloning artifacts.

Insitu

hybridization.

Riboprobe in situ hybridizations were

per-formed on paraffin sections according to the method of Wilcox ( 15). The 2B7plasmid was linearized with either BglI or MamI, and anti-sense or anti-sense riboprobes were generated with T7 or T3 polymerase,

respectively, in the presence of35S-labeledUTP (DuPont & Co., Bos-ton, MA). Probes were separated from unincorporated precursors us-ing a G-50 Nick column (Pharmacia LKB Biotechnology, Piscataway, NJ). Peak radioactive fractions were pooled, precipitated in ethanol, andresuspended at300,000cpm/,ul.Hybridizations were performed at

520C overnight. Slides were washed andautoradiographed as previ-ouslydescribed (15).Exposure times were 14 d unless otherwise noted.

SMC culture,Northern, and Western blot analyses. Rat SMC were derivedfrom normal adult or 1 2-d-old (pup) rat aortic tunica media or 2-wkballooned rat carotid neointima by enzymatic dispersion as previ-ouslydescribed (23). Cells were grown in Waymouth's complete me-dia (Gibco Laboratories, Grand Island, NY) supplemented with so-dium pyruvate, nonessential amino acids and glutamate, 5% bovine serum(Hyclone Laboratories, Inc., Logan, UT), penicillin, and strep-tomycin, and used between passages 10 and 15. Factors used for osteo-pontin mRNA and protein regulation studies were bovine pituitary bFGF, human PDGF (Collaborative Research, Inc., Bedford, MA), synthetic All (Sigma Chemical Co., St. Louis, MO), porcine platelet TGFB ( R & DSystems,Minneapolis, MN), and FCS (Hyclone Labora-tories, Inc.). Peptide dosages were chosen, based on published informa-tion such that maximal ligand-receptor interactions would be facili-tated. RNA was isolated and analyzed by Northern analysis as previ-ouslydescribed using the rat osteopontin cDNA clone,2B7(5),the rat

elastin cDNA clone, 56A3 (6), and a human glyceraldehyde-3-phos-phate dehydrogenase clone (GAPD), pHcGap (24). Densitometry wasperformed using a densitometer (modelR-112; Beckman Instru-ments,Inc., Fullerton, CA) on several different film exposures to en-sure linearity of response.

Forprotein studies, cells were treated with the appropriate peptide factors and conditioned media were collected 48 h after addition. Dur-ing this time there was no evidence of cell death as judged by light microscopy. Conditioned media samples were dialyzed against PBS,

concentrated on centriprep ultrafiltration columns (Amicon Corp., Danvers, MA), and assessed for protein content using the Micro BCA protein assay kit with BSA as a standard (Pierce Chemical Co.,

Rock-ford,IL). Barium citrateadsorptionwasperformedbyadding'/iovol 0.61 M barium chloride and

'/io

vol0.13 M sodium citrate to dialyzed conditioned media samples. The resulting precipitate and adherent proteins were washed three times with water and eluted into 0.2 M sodiumcitrate.Equivalent amountsof proteinwerethendissolved in

sample buffer, boiled, and applied to a 9%polyacrylamidegel by the method ofLaemmli.Identical gels were either stained with Coomassie brilliant blue G250 or transferred to nitrocelluloseusinganelectroblot system (Bio-RadLaboratories, Richmond,CA). Western blotanalysis

was performed usingMPIIIB1O( 1)5 ng/ml and an alkaline phospha-tase detection system.MPIIIBIO(I ) is a mousemonoclonalantibody

directedagainstrat boneosteopontin obtained fromthe

(4)

talStudiesHybridoma Bank (IowaCity, IA). Insomecases,

125I-la-beled sheep anti-mouseIgGF(ab')2 (NEN, Wilmington, DE)was

uti-lizedasthesecondaryantibody, and autoradiographywasperformed

todetect radiolabeled bands.

Results

Complete

sequence

analysis ofdifferential

clone 2B7.

Complete

sequencingof the 1,025-bp2B7 cDNA andsubsequent

com-putersearchindicated that this sequence shared 99.6%identity

with thecomparable regionof thepreviously reported cDNA

forratboneosteopontin (22).Clone 2B7beginsatposition16

and ends at position 1040 relative to rat bone osteopontin

mRNAand contains the

entire coding

region.

Ofthe three base substitutions(outof1,025), onlyone(aC to T transitionat

position 101)ledtoaconservative changein the amino acid sequenceat

position

8fromaLeutoPhe. The other differences

innucleotidesequencewere anAtoG substitutionatposition 160 andaGto A

substitution

at

position 529,

neither of which

changedthe

corresponding

amino acid sequence.

Fig. 1showstheaminoacid sequence derived from

transla-tion of the

largest

open

reading

frame within the 2B7 cDNA sequence.Theratsmoothmuscle-derivedosteopontinmRNA

encodesa317amino acidproteinwith severalinteresting fea-tures.Theseareindicatedin

Fig.

1 and include:

(a)

an

NH2-ter-minal

hydrophobic signal

sequence

typical

ofmost secreted

proteins;

(b)

a

highly

acidic

aspartate-rich

domain

potentially

important for

Ca"+

orcationic

protein binding; (c)

a

putative

V 10 u 20

Met Arg Leu Ala Val Val CysPhe Cys Lou Phe GlyLouAla SerCysLeuProVal Lys

30 40

Val Ala Glu PheGlySerSer Glu Glu Lys AlaHis Tyr Ser Lys His Ser Asp Ala Val

50 60

Ala Thr TrpLouLys Pro Asp Pro SerGlnLysGlnAsnLou Leu Ala ProGln Asn Ser

70 80

Val Ser Ser Glu Glu Thr Asp Asp Phe LysGinGlu Thr Leu ProSOrAsnSer AsnGlu

90 100

SerHisAspHis MetAsp AspAspAsp Asp Asp Asp AspAspGly AspHis Ala Glu Ser

110 120

GluAspSerValAsn SerAsp Glu SerAspGluSOrHis HisSerAsp Glu Ser Asp Glu

130 140

SOrPhe Thr AlaSOrThrGinAla Asp Val Lou Thr Pro Ile Ala Pro Thr Val Asp Val ProAspGlyArgGly AspOSer LeuAla(TyrGlyLeuArgTerLysSer ArgSerPhe)Pro

170 180

Val Ser Asp GluGinTyr Pro AspAla Thr Asp Glu Asp LouThr Ser Arg Met Lys Ser

190 200

Gin Glu SerAsp Glu Ala Ile Lys Val Ile Pro Val AlaGinArgLeuSOrValProSer

210 220

LlspGinAspSOrAsnGly LysThrSerHis GluSar)Ser GinLouAsp GluProSer Val

230 240

GluThr HisSer Leu GluGinSer LysGlu Tyr LysGinArg Ala SerHisGlu Ser Thr

250 260

GluGinSOrAsp Ala sleAsp SerAla GluLys ProAsp Ala leAsp Ser Ala GluArg

270 280

SerAsp Ala IleAspSOrGin Ala SOrSerLysAlaSOrLou GluHisGinSer His Glu

290 300

PheHis SerHis GluAsp Lys LouValLouAspPro Lys Ser Lys Glu AspAsp(ArgTyr

310 317

LeuLys Phe Arg Ile)SerHisGlu Leu Glu Ser Ser Ser Ser Glu Val Asn

Figure1. Amino acidsequenceof rat smooth muscle-derived

osteo-pontin produced fromthe 2B7 cDNA nucleotide sequence. The ar-rowhead marks the single amino acid difference between rat smooth muscle and ratbone-derived osteopontin sequences. The vertical arrowindicates the putativesignal peptidecleavage site. A highly

acidic aspartate-rich domain isunderlined. An RGD adhesion motif

is boxed. Two potential heparin-bindingsites asdescribed by Prince

(25)areshown inparentheses. A thrombin cleave site (T) lies within oneofthesesites.Asequenceshowinghomology to a Ca'Ibinding

E-Fhandisshowninbrackets.

E-Fhand-like

Ca"+ binding

site similartothatseenin

throm-bospondin

and

fibronectin; (d)

an RGD cell adhesion

se-quence commontomany

integrin ligands; (e)

aconsensus

se-quenceforthrombin

cleavage (

18);

and

(f)

two

potential

hepa-rin

binding

sequences(25). These featuresarecharacteristic of

osteopontin

as

descibed

inanumber of other

species

(

18, 19, 26).

In situ

localization

of

osteopontin mRNA and protein in

normalrat arteries. As illustrated in Fig. 2

A,

uninjured rat

arteries showedverylow level

staining

with

2arC,

an

affinity-purified, polyclonal antibody

raised

against

the

COOH-termi-nal

portion

ofmouse

osteopontin expressed

in Escherichiacoli ( 18). The

staining

observed was diffuse in some areas, but

somefocal patcheswerealsoseen.Insome

sections,

extracellu-lar

staining

along elastic lamellae was evident

(Fig.

2 A, arrows).No

staining

wasobservedwith control antibodies in anyof thearterysections examined

(not

shown).

Likewise,

osteopontin

mRNA

levels,

asmeasuredby in situ

hybridization,

werelow in

uninjured

arteries

(Fig.

3

A).

Signal

value higher than that observed in arterial sections

using

the

sense probe (not shown) was seen,

however,

in several cells scattered

throughout

themedia in the carotidartery.This

find-ing

is in agreement with our

previous

Northern blot

experi-mentsshowing

osteopontin

mRNA

expression

incarotid

arter-ies(5).Noantisense probe

hybridization

wasobserved in

endo-thelial oradventitial cells. Thesense

probe

gavenolocalized

signal

inanyof thearterysections examined

(not shown).

Increased

osteopontin mRNA

and

protein

levels

in rat

neointimal

SMC.2dafter arterial balloon

angioplasty,

osteo-pontin

mRNAlevelswereelevated ina

subpopulation

of

ca-rotid and aortic SMC closesttothe lumenasshown in

Fig.

3,B

andC,

respectively.

4d after

injury,

SMC

expressing

osteopon-tin mRNA were still locatedat the luminal aspect, butcells adjacent to the external elastic laminaalso showed elevated

expression (Fig.

3

D).

By7dafter

injury,

the

highest intensity

of grains

were overcells in the

newly

forming neointima,

buta

few cells at the media-adventitial interface still showed in-creased

expression

of

osteopontin

mRNA

(Fig.

3

E).

10dafter

injury,

alllayers of neointimal SMC showed

high

level

expres-sion of

osteopontin

mRNAcomparedtothe

underlying

media in botharteries (datanotshown). By 3wkafter angioplasty,

osteopontin

mRNA haddecreased

substantially

in the neointi-mal layertolevels equaltothose inthe underlying media in

boththe carotid(not shown)and aorta

(Fig.

3F). However,a

small

population

of neointimal cells

directly adjacent

tothe

lumencontinuedtoshowhigher levels of osteopontinmRNA

than

neighboring

intimal SMC. This localized expression of

osteopontin

mRNA was evident 6 wkafter

injury,

atwhich time

osteopontin

mRNAlevels in all otherpartsof the vessel,

including

the remainder ofthe neointima, had decreased to

uninjured

levels (not shown). Corresponding sections probed with thesenseprobe showednosignal (shown in Fig. 3 G for 2-d ballooned aorta). At all time points, adjacent sections probed withthe sense probe showed nospecific signal(shown

inFig. 3 Gfor 2-d balloonedaorta).

Early afterinjury, osteopontin mRNA and protein levels correlatedwellin thedifferent layers ofthearterywall.2 dafter

injury

(not shown),only selected cellsliningthelumen were

positive

for osteopontin protein.AsshowninFig. 2,BandC,

osteopontin protein

wasincreasedintheaortaandcarotid4d

after

injury

and was

highest

intheluminal SMC.These same

regions

wereshownto havethe

highest

levelsof osteopontin

(5)

Jei

e

A

"I e w

.;$. 7

m

4

..a

I

...._ ..

.,

rw

I

A

K:.

Aot

A..

i;

.1....-.

ro

B

I

OV

F.I..94

4~~~~~~~~~~~~~C4.

"

X~~~~~~~~~~~f'~~~~~~~~.

r~~~~~~~I

El. ... , ...

m

..

~~~~~~~~~~~~~~~~.

-1-s

*o...

I

D

Figure 2. Immunohistochemicalanalysis of osteopontin protein levels in normalandballooncatheter-injuredratarteries. Aorta and carotid

ar-teriesfromuninjuredratsandratssubjectedtoballoonangioplastywerefixed, embedded,andstained for osteopontin usingtheaffinity purified, 2arCantibody (A-F). (A) Uninjuredratcarotidarteryshowingsomelocalized patchesof stainingalong theelasticlamellae (arrows); (B) 4-d

balloonedcarotidshowing three neointimal SMC(arrowheads)stronglypositive for osteopontin,aswell asdiscretepatchesof staining along elastic lamellaintheoutermedia(arrowis);(C)4-dballoonedaorta;(D-F) 3-wk balloonedaortashowing preferential osteopontinstaining in theSMC of theneointima,aswell asfociof positive cellsintheoutermedia (F,arrowheads). i, neointima;m,media;a,adventitia;e, endothe-lium; bar, 10m.

mRNAearlyafter injury (see Fig. 3, B-D). Some sections

pre-pared from 4-d ballooned rat aortasand carotid arteriesalso

showed cells ofthe outermedia

expressing

elevated levelsof

osteopontin

similartothemRNA pattern shown in

Fig.

3D. By 7d, the 2-3celllayer-thick neointimaspresentinthe aorta andcarotidwere

highly

enriched in

osteopontin protein

(data

notshown), with apattern identicaltothat observedforthe mRNA(seeFig. 3E).

Antibody staining for osteopontinat 2and 3 wkafter bal-loon angioplasty showed that the neointima was highly enriched for osteopontin (shown for3 wk inFig. 2 D). The

highestintensity of stainingwascytoplasmic, but upon higher powerexamination (Fig.2, Eand F) somestaining could be

seenextracellularly in distinctpatches throughout the

neoin-timaandmediaoverwhatappeared to be parts or edgesofcells,

or alongelastic lamellae. Although staining was observed in

selected cells

of

the media, theintensitywassubstantiallylower thanseenin the neointima. One exceptionwasseveral clusters

of SMC in theoutermedialregion whoselevelofosteopontin protein wasnearly as high as that observed in the neointimal

region (Fig.2 F, arrows). Counterstainingof theseslides with

hematoxylinshowed thatthelabeledcells werewithin the

ex-ternal elasticlamina,andnotpart of the adventitiallayer(not shown).

The localization ofosteopontin proteinatthis timewasin

strikingcontrast totheosteopontin mRNA pattern shown in

Fig. 3F.WhereasosteopontinmRNAlevels in the intima and

mediaweredecreased almosttouninjured levels,osteopontin

protein levels were still high in the intima compared to the

underlyingmedia. The accumulation ofosteopontin proteinin

cellsoftheintima, however,wastransient. In sections prepared fromarteries4and 6 wk afterinjury, osteopontin proteinlevels haddeclinedtouninjuredlevels in boththeneointima and the

media (datanot shown). Thus, the difference in osteopontin

mRNA andproteinobservedat3 wk mostlikely reflectsthe

precursor-product relationship of the molecules. Identical

staining

patternswereobservedwhenasecondaffinity-purified anti-mouse osteopontin antibody, 2arN (18), was used in-steadof2arCtostain similar sections.

Regulation of

osteopontin mRNA

and secreted

protein

lev-els incultured vascular SMC. To explore the regulation of

os-teopontin

expression in SMC,

postconfluent,

quiescentSMC weretreatedwithFCS, or severalpeptidefactorsthought to be

important in neointima formation. Asshown in Fig. 4, AII,

Osteopontin ExpressionafterVascular Injury 1689

I

.4

4,j...

7

t

J-14

W

.VI

-l.-,PT.:

" :1

s

-.1

w I

A."

Op ...:.

(6)

Figure3.Insituhybridizationanalysis of osteopontinmRNAlevels innormal andballooncatheter-injuredratarteries.Ratarterial sectionswere

hybridizedtotheantisense (A-F)riboprobesgenerated fromtranscriptionof the 2B7plasmid. (A)Uninjuredcarotidartery;(B)2-d ballooned carotidshowing increased grainsovercellsadjacenttothelumen(arrows); (C)2-d balloonedaortashowingasimilarpatternofhighintensity signal localizedtotheluminal SMC(arrowis);(D)4-d balloonedaortashowing high levels of grainsovertheluminal (arrows)andoutermedial

(arrowheads)SMC: (E)7-d balloonedaortashowinga2-3celllayer-thickaccumulation of neointimal SMC (arrows)stronglypositivefor osteopontin mRNA; (F) 3-wkballoonedaortashowingelevatedgrainsover asubpopulationof neointimalcellsclosesttothelumen (arrows), but levelsin theremainder oftheintima (i) similartothose in theunderlyingmedia(m).Arrowheadspointtotheinternal elastic lamina

whichseparates theneointimaandmedia:a,adventitia; i, neointima;m,media; bar, 10,um.

bFGF, FCS,and

TGF,3

stimulated expression of osteopontin

mRNA24 h aftertreatmentcompared tountreated controls.

FCSandAllappeared tobe mosteffective,followedby

TGF,3

and bFGF.OsteopontinmRNAremained elevatedin SMC 48 h aftertreatment with

TGFf,

bFGF, and All, but not FCS.

PDGF, on the other hand, had no effect on osteopontin mRNAlevelsatanytimestudied, despitethe presenceof both PDGF-a and

-#

subunit mRNAsin these cells as measured by Northern blot analysis (Giachelli, C. M., unpublished

observa-tion).

Interestingly, osteopontin mRNA levels in untreated,

post-confluent SMC were abouttwofold higher on day 3 than on days 0, 1, or 2. This effect was specific to osteopontin, since

elastinmRNA levels did not show the same pattern (Fig. 4 A),

and

qualitatively

similarresults wereobtained in three

addi-tional

experiments.

Thereasonfor this elevation isunclear, but the data suggest that an endogenous regulator of osteopontin mRNAsynthesis ordegradation may be activated in

postcon-fluentSMC. Further studies are required to test this possibility. Westernblot analysis wasperformed to determine whether

bFGF, All, or TGFf3 increased osteopontin protein released by SMC. A monoclonal antibody specific forratbone

osteopon-tin,MPIIIB10( 1), wasutilizedsinceitwasavailable inlarger quantitiesthan either of the rabbit polyclonal antibodiesand thus could be used moreconveniently for Westernblotting.

Fig. 5A showsthespecificity ofMPIIIB10( 1 )antibody com-pared to 2arC for rat pup SMC-derived osteopontin. Both 2arC andMPIIIBIO(

1()

detected abroad band at66 kDinrat

pup-conditioned media, consistentwiththesize ofosteopontin reported in rat bone (7). The identity ofthe 66-kD band as

osteopontinwasfurtherconfirmed byitsabilitytobeadsorbed

to barium citrate and specifically eluted into 0.2 Msodium citrate, propertiespreviously reported for osteopontin (27).

2arC alsointeractedweaklywithasmallerproteinat50 kD,

which mostlikelyrepresents a degradation productof

osteo-pontin.

Asshown inFig.5B,osteopontinproteinwasincreasedin themedia conditioned by confluentratSMC aftertreatment

with eachofthe threepeptidefactors.Amajor bandat66kD,

comparable to that seen for pup rat SMC, was present in

(7)

A treatment none PDGF All bFGF TGFp FCS

__Sa0 231 23123 12312 1--a

days 0 1 I 3 1 2 3 1 2 3 1 2 3 i 2 3 1 2 3

el e

op 4, * * 0 * *

O***

*-o

*4 O *

gapd4 ** ei* * O** * 66- i

B

CL

0

B.

=

=

v L

.f

0:< ~- .o=

none PDGF All bFGF TGFb FCS

treatment

Figure4. Effect ofpeptidefactors andserum onosteopontin and

elastin mRNA levelsincultured vascular SMC.(A)Northern blot

analysisof RNAs from SMC treated with PDGF(10ng/ml),All

(10-6M),bFGF(10ng/ml),TGFJ3 (20 ng/ml),or20% FCSorleft untreated(none).Theautoradiogramshown resultedfroma24-h

exposureof thehybridizedfilters. The sizes of the observed bands

wereestimated, basedonmigration ofthe 18and28S ribosomal

RNAs,andare asfollows:elastin,3.5kb; osteopontin, 1.6 kb;and

GAPD, 1.6kb. GAPD mRNAlevelsclosely paralleled28 and 18S

ribosomalRNAlevelsasjudged byUVshadowingofthezetaprobe

membrane(not shown). (B)Densitometricanalysisofosteopontin

mRNAlevels shown in A normalizedtoGAPDmRNAlevels. Values

aregivenasthe ratio of OPtoGAPDarbitrarydensitometric units.

treated culturescomparedtoaverylow leveldetectablein

un-treated SMC. Similar increaseswereobserved whenasecond

group of cellswastreated identically and conditioned media

proteinswereexamined 24 h after addition (not shown). The

Coomassie stainingpatterns shown inFig. 5 C indicated that

equivalent amounts of protein were loaded onto the acryl-amidegels used for Western blot analysis. Although these stud-ies donotruleoutchangesinosteopontin protein levels in the culture media duetochangesindegradationrateor

compart-mentalization, they stronglysuggestthatbFGF, AII, and TGF-allincreasesynthesis and secretion of osteopontin inratSMC.

Also shown inFig. 5 B isacomparison of osteopontin

pro-tein levelsfound in theculture media of adult andpupratSMC after 48 hofconditioning. Asexpected, basedonpreviously reported mRNA levels (5),pupSMC-conditioned media con-tained a much higher steady-state level of osteopontin than comparably collected media conditioned by adult SMC. These levels wereeven higher than peptide factor-stimulated levels

and suggestthatpupSMC might beagoodsourceof smooth muscle-derived osteopontin.

Osteopontinisanovelcomponentofhuman atherosclerotic

plaques. We examined specimens of humanartery(3

coro-nary arteriesfromautopsy and 8 carotid

endarterectomy-de-C .

co

66v, ilk

Figure5. Western blotanalysesofosteopontinreleasedbyratvascular SMC in culture.(A) Comparisonof 2arC andMPIIIBI0( 1) anti-bodies. RatpupSMC culture supernatantswerepartially purifiedby

adsorption (a)ordialyzedandconcentrated(b)and 25,ugofthe

resulting proteins processedfor Westernblotanalysiswith either 2arC (1:50)orMPIIIB10( 1) ( 1:5,000). MW,molecularweightmarkers,

whichwere runconcurrently. (B) Effectofpeptidefactorsonlevels

of osteopontin releasedbyadult medialSMC. Adult medial SMC

weretreated identicallytothose described inFig.4and theamount

of osteopontinproteininconditionedmediasamples2 d after treat-mentwasdeterminedbyWestern blotanalysis usingMPIIIBI0(1)

(1:5,000)astheprimaryantibodyand'25I-labeledsheepanti-mouse

IgGasthesecondary antibody.Theautoradiogramsobtained after

a5-dexposureareshown. Theapparentmolecularweightof

osteo-pontinestimated fromconcurrentlyrunmolecularweightmarkers is indicated inkilodaltons.(C)Coomassie bluestainingof the

sam-plesutilized for the Western blotsofB,indicating equivalent protein loadingfor each lane.

rived samples) for osteopontin proteincontentby immunocy-tochemical staining using LF-7, an antibody that has been

showntobespecific for human bone osteopontin (19).In

arte-rial sections containing atherosclerotic plaques, LF-7 staining

wasfocal and almost entirely limitedtotheintimaasshown in Fig. 6 A. While no staining was seen in the media of these

vessels,faint cellular stainingwasobservedinnervesandsome scatteredcells of theadventitia. Higher-power examinationof

the sections indicated that the LF-7 staining was associated

with both cellular and acellular regions ofthe plaque. The

stron-gestandmoststriking stainingwasobservedsurrounding

baso-philic calcified deposits, which often gave agranular

appear-ance (Figs. 6 B and 7, A and B). In addition, the matrix

surrounding cholesterol clefts showed elevated LF-7 staining compared to other matrix areaswithin the same plaque, or

comparedtonormalsamples (Figs. 6Band7,AandB).

Promi-Osteopontin Expression afterVascularInjury 1691

A.

MW

MPIO(1)

a b

2arC

(8)

} z s AOK.

Ilk*p.

(9)

nent cellular LF-7 staining was observed in selected plaque SMC and macrophages, including macrophage-derived foam cells(Fig. 6, C and D).

In two normal coronaryarteries, no cellular LF-7 staining was observedin any of the vessel layers (not shown). However, occasionalfine granular staining was observed in the matrix of theintima, but, incontrasttothe plaque, these granules were not basophilic when an adjacent section was stained with he-motoxylin and eosin (not shown). The significance of this granular staining is unknown at present, but might represent early nidi for calcification or some other extracellular accumu-lation. Thesepossibilitiesarecurrently underinvestigation.

Discussion

In a previous study, we used subtraction cloning to isolate genes thatmight be importantinarterialneointima formation (5).Inthe presentstudy, we used sequenceanalysis to

conclu-sivelyshow that oneofthese genes,2B7,encodes rat

osteopon-tin. Furthermore,wehavedemonstratedbyinsitutechniques that osteopontin mRNA andproteinaredramatically upregu-lated in vivo following rat arterial angioplasty selectively in

neointimal SMC.Neointimal osteopontin expressionwas

tran-sient, occurring over thefirst 3 wk after arterial injury. The

striking temporal pattern and localization ofosteopontin in nascent neointimalcells suggests thatosteopontinmay be im-portantintheearly processesregulating neointimaformation in the rat such asmigration, proliferation, and/or differentia-tion. Furthermore,wefoundsignificant osteopontin proteinin humanatheroscleroticplaques localizedtoselected SMCand macrophages,butmoststrikingly, inassociationwith calcium

deposits. No

osteopontin protein

wasobserved in normal hu-man arterialmedia. Basedonthese

findings,

we

speculate

that

osteopontinmay beimportantinplaque progressionaswellas

the

dystrophic

calcification commonly observed in these

le-sions.

Osteopontin is a secreted

phosphoprotein originally

de-scribed as anoncollagenous

protein

inbone matrix

(8),

but morerecentlyobservedinseveral

nonmineralizing tissues,

in-cluding

kidney

andarteries

(5).

In

bone,

osteopontin

and other

acidic

phosphoproteins

are

thought

to

play

critical roles in

facil-itatingand

regulating

calcium

deposition (28). Osteopontin

is

a low affinity,

high capacity, calcium-binding protein

(29), whichcanbindtightlyto

hydroxyapatite,

the

primary

constitu-ent of mineralized bone

(22).

In

addition, osteopontin

con-tainsan RGD celladhesionmotif that is similartothat found in manycell-matrix adhesion

molecules,

including

fibronectin,

thrombospondin,

and vitronectin

(30),

and it has been shown

tointeract with at least one integrin, the vitronectin receptor

(afvl3)

on osteoblasts (31 ). Based on theseobservations, as well ascolocalization studies, it has been suggested that osteopontin may becritical for binding of osteoblasts and osteoclasts to the bone matrix (32). In addition, a role for osteopontin in cellular movement has been proposed, since osteopontinexpression is upregulated in many tumor cell lines (9, 10) and correlates with metastatic potential ( 10). Furthermore, osteopontin (termed Eta- 1 in that report) binds with high affinity to macrophages, and subcutaneous injection of purified Eta- 1 into mice stimulates infiltration of macrophages to the site of

injection(33).

The present study supports a role for osteopontin in facili-tating cellular adhesion and/or migration during arterial neointima formation in the following ways. First, sequence analysis of 2B7 verified that rat smooth muscle can make a

form ofosteopontin mRNA that includes an RGD-domain,

previouslyshown to be important for cellular adhesion to

os-teopontin(22). Consistent with this, we have recently found that purified smooth muscle-derived osteopontin facilitates RGD-dependent adhesion and migration of rat vascular SMC in vitro (Liaw, L., and C. M. Giachelli, unpublished data).

Second, in a rat modelof neointima formation, osteopontin mRNA andprotein levels were elevated specifically in SMC at theluminal surface of rat vessels at early times (between 2 and 7 dafter angioplasty) when SMC leave the media and enter newlyformingintima (2). Osteopontin levels were decreased

touninjured levelsby 4 wkafter injury,when SMCmigration

isat aminimumand the rateofintimal thickening has declined

(2).Last,osteopontin protein was enriched in focal regions of human atherosclerotic intima and not found in the underlying

media, providingevidence that gradients of osteopontin may

exist in diseased vessels. Although correlative, these data sug-gest that atleast one roleforosteopontin in neointimal

forma-tionmightbeinfacilitatingcellular movement orpreferential

adhesion in theneointima. However, osteopontin might also

beimportantintheproliferativeordifferentiationevents that also occurintheearly phases of neointima formation ( 1, 2). In

this regard,osteopontin has recently been shown to correlate with cell cycle entry in cultured SMC (34). Development of

specificinvivoinhibitors of osteopontinwill help us greatly to

distinguishbetween thesepossibilities.

Recentevidencehas shown that growthfactorsplay a role ininitiatingandregulating neointima formation following

bal-loonangioplasty. Medial SMC proliferationhas been shown to

be due in large part to endogenous release of bFGF by the

injured arterial media (35).Both bFGF and PDGFarethought

tostimulatemigration ofthemedialSMCintotheneointima

Figure6. Osteopontin isanovel componentof humanatherosclerotic plaques. (A)Humanatherosclerotic coronaryarterysection

immunos-tainedwithanti-humanosteopontinantibody, LF-7,and counterstained withmethylgreen. Arrowshighlightareasof theplaqueshowing

ele-vatedLF-7staining.These correlatewithareasoflipiddeposition (L),calciumdeposition(C),macrophage(M),SMC(S),and foam cells(F),

asevidenced by eosinandhematoxylinstainingand celltype-specificantibody staining performedon anadjacentsection,asdescribed in the

Methods section (notshown).(B-D).Humancarotidendarterectomysamplesimmunostained with LF-7 and counterstained withmethylgreen.

(B)Aregion of plaque showing extensive calciumdeposition(C)embeddednear aregionof cholesterol andlipidaccumulation(L).Note the

pronouncedstaining that outlinesthecalcifieddepositandsomecholesterolclefts,and existsthroughoutthesurroundingmatrix. At lowerright

areregions containing granularcalciumdeposits (g). (C)Aregionofplaque containingboth SMC(S)andmacrophages(M)thatwere

LF-7-positive. Severalstriking examplesof each celltypeareindicatedwitharrows.Note that thepatternofstaininginmacrophageswas polar-ized overaportion ofthecellcomparedtoSMCwhoseprofileswerehighlighted bytheantibodies,thusallowingthe visualization of thelong

processesofthese cells.(D)Aregionofplaquecontaininganinflammatoryinfiltrate. The cells in thisregion, includingaprominentfoam cell (F), arestronglypositive for LF-7staining, showingpolarizationto oneside ofthecytoplasm.The LF-7

staining

wascontained in

HAM-56-andCD68-positivecells,asdeterminedbystainingofanadjacentsection with these antibodies(not shown).Bar, 10,M.

(10)

0

d

0

0

E

10

Co

E

0~ 0

~C-c

o^ t

, .-0

-CC

.~._

o

00 00

00

.Q ._

<Cu3

0)

s0

-. 0

_Cd

crio

0C

a)

.-0 CA ) 00

a .C.

Ce

C

0

e

(11)

(35, 36). Factors initiating intimal proliferation are less well understood, but TGF3 has been shown to increase in arteries during wound repair (37), and both All and TGF/ selectively stimulated neointimal SMC replication when given exoge-nously to animals in vivo ( 14, 37). Our in vitro studies suggest that several of these growth factors may alsobe important in regulating osteopontin expression in SMC. TGFI, All, and bFGF, but not PDGF,elevated osteopontin mRNA and pro-tein levels in confluent, quiescent adult medial aortic SMC. Although bFGF, TGFf, and PDGF have previously been shown to regulate osteopontin mRNA expression in other cell systems(38,39), thisisthefirstreportof their action on vascu-lar SMC. Furthermore, this is the first report ofAllmodulation of osteopontin mRNAlevels in any cell type. Whether these

factorsareimportantinvivo regulators of osteopontin

synthe-sis duringneointima formation is currently being examined. One of the most exciting findings resulting from this study was thefocal osteopontin accumulation in human atheroscle-roticplaques and its strikingcolocalization with calcium

de-posits. One possibility isthat the osteopontin found in these

areas wasderived from plaque SMC and macrophages. Consis-tent with this idea,weobservedintracellular osteopontin stain-ing inboth plaque SMC and macrophages (Fig. 6) and prelimi-naryinsituhybridization studiesindicate that these cell types

synthesize osteopontin mRNA in human coronary atherec-tomy specimens (O'Brien, E., and C. M. Giachelli, unpub-lished data).Alternatively, osteopontin might be derived from theblood, since human plasma was shown to contain low

lev-els ofosteopontin protein ( 10). Regardless of the source, the close association of osteopontin protein with calcifiedregions, togetherwithitsbiochemicalproperties(7) and proposed role

inbiomineralization(28), tempt us to speculate that

osteopon-tin mightalso play a role in the calcification commonly ob-served in atheroscleroticlesions. Since calcification may

con-tributesignificantlytoluminalocclusion and arterial

dysfunc-tionin atherosclerotic vessels (40), determining the possible rolesof osteopontinin plaqueformationanddystrophic

calci-fication should behigh prioritiesfor furtherresearch.

Acknowledgments

Theauthorsthank Dr. LarryFisher for thegift of anti-human

osteo-pontin antiserum, LF-7. We are indebted to Donna Lombardi and

MarinaFergusonfor help with in situ hybridizationand immunohisto-chemistry, respectively. We also thank Drs. Earl Benditt, Edward

O'Brien,JoanLemire,ChuckMurry,and DavidGraingerforhelpful

discussions,andHolly Kabinoff forexpertpreparationof the

manu-script.

This workwassupported byNIH grantsHL-03174 and HL-47 151

andgrant3124of the Council for Tobacco Research-USA.

References

1.Schwartz,S.M..R.L.Heimark,and M. W.Majesky. 1990. Developmental mechanismsunderlying pathology of arteries. Physiol.Rev.70:1177-1209.

2.Reidy,M.A.,J.Fingerle,and M. W.Majesky.1988.Proliferationof vascu-larsmoothmuscle cells in vivo. InHyperlipidaemiaandAtherosclerosis. K. E.

Sucklingand P. H. E.Groot, editors. Academic Press,London. 149-164. 3.Ross,R. 1988. Thepathogenesis of atherosclerosis.In Heart Disease:A

Textbook ofCardiovascular Medicine. 3rd ed. E. Braunwald, editor. W.B.

SaundersCo..Philadelphia. 1135-1152.

4.Majesky,M. W.,andS. M.Schwartz. 1990. Smoothmusclediversityin

arterialwoundrepair. Toxicol.PathoL. 18:554-559.

5.Giachelli, C..N. Bae, D.Lombardi,M.Majesky, andS.Schwartz.1991.

Molecularcloningandcharacterizationof2B7,aratmRNA whichdistinguishes smoothmuscle cellphenotypes in vitro and is identicaltoosteopontin(secreted

phosphoproteinI,2ar).Biochem. Biophvs.Res.Commnun.177:867-873. 6.Majesky,M. W., C. M.Giachelli,M. A.Reidy,and S. M.Schwartz.1992. Ratcarotid neointimalsmooth musclecellsre-express adevelopmentally

regu-lated mRNAphenotype during repair ofarterialinjury.Circ. Res.71:759-768.

7.Butler. W. T.1989.The nature andsignificanceofosteopontin.Connect.

TissueRes.23:123-136.

8. Heinegard. D.,K.Hultenby, A.Oldberg, F. Reinholt, and M. Wendel.

1989. Macromolecules in bone matrix.Connect.Tissue Res.21:3-11.

9.Craig. A. M., M. Nemir, B. B. Mukherjee. A. F.Chambers,and D. T.

Denhardt. 1988. Identification ofthemajorphosphoproteinsecreted by many

rodent cell lines as2ar/osteopontin: Enhanced expression in H-ras-transformed

3T3cells.Biochemn.Biophvs. Res. Commun. 157:166-173.

10.Senger.D. R., C. A.Perruzzi,and A.Papadopoulos. 1989.Elevated

ex-pression ofsecreted phosphoproteinI (osteopontin,2ar) as aconsequence of neoplastic transformation. Anticancer Res. 9:1291-1299.

1 1. Lampe, M. A., R.Patarca,M. V.Iregui,and H.Cantor. 1991.Polyclonal

Bcellactivationbythe Eta- Icytokineand thedevelopment ofsystemic

autoim-munedisease.J.Imnnunol. 147:2902-2906.

12. Patarca, R.,G. J.Freeman,R. P.Singh,F-Y.Wei,T.Durfee,F.Blattner,

D.C. Regnier,C. A. Kozak, B. A. Mock, H. C.MorseIII et al. 1989. Structural and functional studies oftheearlyTlymphocyte activation I (Eta-I) Gene:

Definition ofa novel Tcell-dependent response associated withgeneticresistance

tobacterial infection.J.Exp.MeId. 170:145-161.

13. Fet, V., M. E.Dickinson, and B. L. Hogan. 1989. Localization ofthe

mouse genefor secreted phosphoproteinI(Spp- I )(2ar, osteopontin, bone

sialo-protein 1, 44-kDa bone phosphoprotein, tumor-secreted phosphoprotein) to

chromosome5.closely linkedtoRic (Rickettsia resistance).Genomnics.

5:375-377.

14.Daemen,M. J. A. P., D. M.Lombardi,F. T.Boxman, andS.M.Schwartz. 1991.AngiotensinIIinduces smooth musclecellproliferation in thenormaland injured arterialwall.Circ.Res.68:450-456.

15. Wilcox,J. N., K. M. Smith. L. T. Williams, S. M. Schwartz, and D.

Gordon. 1988. Platelet-derivedgrowth factormRNAdetection inhuman

athero-sclerotic plaquesbyinsitu hybridization.J.Clin.Invest.82:1134-1143. 16. O'Brien,K.D.,D.Gordon, S. Deeb,M.Ferguson, and A.Chait. 1992. Lipoprotein lipaseissynthesizedby macrophage-derived foam cells inhuman

coronary atheroscleroticplaques.J.C/in. Invest.89:1544-1550.

17.Hsu,S. M., L. Raine, and H.Fanger. 1981. Theuseof

avidin-biotin-per-oxidase complex(ABC) inimmunoperoxidase techniques: A comparison

be-tweenABCand unlabeledantibody (PAP) procedures.J.Histochein.Cttochem.

29:577.

18.Craig, A. M., J. H. Smith, and D. T. Denhardt. 1989. Osteopontin. a

transformation-associatedcelladhesionphosphoprotein, is induced by1 2-O-tet-radecanoylphorbol 13-acetate in mouseepidermis. J. Biol. (hein. 264:9682-9689.

19. Young,M.F.,J. M. Kerr. J. D. Termine, U. M.Wewer,M.GeWang, 0.W.McBride,and L. W.Fisher. 1990.cDNAcloning,mRNAdistributionand

heterogeneity, chromosomal location,and RFLPanalysis ofhumanosteopontin (OPN). Genoinics. 7:491-502.

20.Gown,A.M., T.Tsukada,and R. Ross.1986.Humanatherosclerosis. II. Immunocytochemical analysis ofthe cellularcomposition ofhuman

atheroscle-roticlesions..4m.J. Pathol. 125:191-207.

21.Tsukada,T., D.Tippens.D.Gordon, R.Ross,and A. M.Gown. 1987. HHF-35,amuscle-actin-specificmonoclonalantibody.1.Immunocytochemical andbiochemical characterization. Anm.J Pathol. 126:51-60.

22.Oldberg.A.. A.Franzen,and D.Heinegard. 1986. Cloningand sequence

analysis ofratbonesialoprotein (osteopontin)cDNA reveals anArg-Gly-Asp cell-bindingsequence. Proc.Natl.Acad. Sci. USA. 83:8819-8823.

23.Giachelli.C.M., M. W.Majesky,andS. M. Schwartz. 1991. Developmen-tally regulated cytochromeP45OIAI expressionin culturedratvascularsmooth

musclecells.J.Biol. Chem. 266:3981-3986.

24.Tso,J.Y..X-H.Sun.T-H.Kao, K.S.Reece, and R. Wu.1985. Isolation

andcharacterization ofratandhumanglyceraldehyde-3-phosphate

dehydroge-nase cDNAs:Genomiccomplexityandmolecularevolution ofthe gene.Nucleic

.4cidsRes. 13:2485.

25.Prince,C. W. 1989.Secondarystructurepredictionsforratosteopontin. Connect.Tissue Res. 21:15-20.

26. Kerr. J.M.,J. D.Fisher,and M.Young. 1991. The cDNAcloningand

RNAdistribution ofbovine osteopontin. Gene(Amst.). 108:237-243.

27.Senger.D.R.,C.A.Perruzzi,A.Papadopoulos,and D. G. Tenen. 1989.

Purification ofahumanmilkproteinclosely similartotumor-secreted

phospho-proteinsandosteopontin. Biochim.Biophvs.Acta.996:43-48.

28.Gorski,J. P. 1992.Acidicphosphoproteinsfrombonematrix:Astructural rationalizationof their role inbiomineralization.CalcifTissue Int.50:391-396.

29.Chen, Y.,B.S.Bal,and J. P.Gorski. 1992. Calcium andcollagenbinding propertiesof

osteopontin.

bonesialoprotein,and boneacidicglycoprotein 75

frombone.J.Biol.Chem.267:24871-24878.

(12)

30.Ruoslahti, E., and M. D.Pierschbacher. 1987. New perspectives in cell adhesion: RGD andintegrins.Science(Wash.DC).238:491-497.

31. Oldberg, A., A. Franzen, D.Heinegard, M.Pierschbacher, and E. Ruos-lahti. 1988.Identification ofabonesialoproteinreceptor in osteosarcoma cells. J. Biol.Chem. 263:19433-19436.

32.Reinholt, F. P., K. Hultenby, A. Oldberg, and D. Heinegard. 1990. Osteo-pontin-apossible anchorofosteoclasts to bone. Proc.Natl.AcadSci. USA. 87:4473-4475.

33.Singh, R. P., R. Patarca, J. Schwartz, P. Singh,and H.Cantor. 1990. Definitionof aspecificinteraction between the early T lymphocyte activation 1 (Eta-l ) protein and murine macrophages in vitro and its effect upon macro-phagesin vivo. J. Exp.Med. 171:1931-1942.

34. Gadeau, A-P., M.Campan, D. Millet, T. Candresse, and C. Desgranges. 1992.Osteopontinoverexpression is associated with arterial smooth muscle cell proliferation in vitro. Arterioscler.Thromb. 13:120-125.

35.Lindner, V., R. A.Majack,and M. A.Reidy.1990.Basicfibroblastgrowth factorstimulates endothelialregrowth andproliferationin denuded arteries. J. Clin.Invest.85:2004-2008.

36.Jawien, A., D. F. Bowen-Pope, V. Lindner, S. M. Schwartz, and A. W. Clowes. 1992.Platelet-derived growth factor promotes smooth muscle migration and intimalthickening in a rat model of balloon angioplasty. J. Clin. Invest. 89:507-511.

37.Majesky,M.W.,V.Lindner,D. R.Twardzik,S. M.Schwartz,andM. A.

Reidy. 1991. Productionoftransforminggrowth factorf,duringrepair of arterial injury. J. Clin. Invest. 88:904-910.

38.Noda, M., and G. A. Rodan. 1989. Type ,3 transforming growth factor regulatesexpression of genes encoding bone matrix proteins. Connect. Tissue Res.21:71-75.

39. Rodan,S. B., G.Wesolowski, K. Yoon, and G. A. Rodan. 1989. Opposing effects of fibroblast growth factor and pertussis toxin on alkaline phosphatase, osteopontin, osteocalcin, and type I collagen mRNA levels in ROS17/2.8cells. J.Biol.Chem. 264:19934-19941.

40.Fleckenstein-Grun, G., and A. Fleckenstein. 1991. Calcium-A neglected key factor in arteriosclerosis. The pathogenic role ofarterial calcium overload and its prevention by calcium antagonists.Ann.Med. 23:589-599.

References

Related documents

The results using this alternative truncation point were nearly identical to the estimates from the main text (table A1, figure A4).. However, the estimated coefficients were

Keywords : Moringa oleifera gum, Bionanofibers, Olopatadine hydrochloride, Entrapment efficien- cy, In-vitro drug release etc.. Because of their unique characteristics such

The local SMO felt strongly that orthodontic care raised the tone of dental treatment as a whole, and hoped that it would be copied in other LEAs.28 One dentist based in Croydon

In patients who do not meet the above criteria, the screening test should consist of a 50 g oral glucose load (Glucose challenge test or GCT) followed by a plasma sugar level

This manuscript describes and demonstrates a continuous model for nucleation and growth using the well-established Johnson-Mehl-Avrami-Kolmogorov model, and provides a method

In the conventional control approach, the aim is to follow the zero displacement as a reference value for the body-bounce motion. But when this is realized, it is found that

Objectives: The present study aimed to compare the effects of mother’s voice and a stranger’s voice on the heart rate of preterm infants hospitalized in a neonatal intensive care