myocardial osteopontin expression coincides with the development of heart failure

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Myocardial Osteopontin Expression Coincides With theDevelopment of Heart Failure

Krishna Singh, Geza Sirokman, Catherine Communal, Kathleen G. Robinson, Chester H. Conrad,Wesley W. Brooks, Oscar H.L. Bing, Wilson S. Colucci

AbstractTo identify genes that are differentially expressed during the transition from compensated hypertrophy to failure,

myocardial mRNA from spontaneously hypertensive rats (SHR) with heart failure (SHR-F) was compared with that

from age-matched SHR with compensated hypertrophy (SHR-NF) and normotensive Wistar-Kyoto rats (WKY) by

differential display reverse transcriptasepolymerase chain reaction. Characterization of a transcript differentially

expressed in SHR-F yielded a cDNA with homology to the extracellular matrix protein osteopontin. Northern analysis

showed low levels of osteopontin mRNA in left ventricular myocardium from WKY and SHR-NF but a markedly

increased (10-fold) level in SHR-F. In myocardium from WKY and SHR-NF, in situ hybridization showed only scant

osteopontin mRNA, primarily in arteriolar cells. In SHR-F, in situ hybridization revealed abundant expression of

osteopontin mRNA, primarily in nonmyocytes in the interstitial and perivascular space. Similar findings for osteopontin

protein were observed in the midwall region of myocardium from the SHR-F group. Consistent with the findings in

SHR, osteopontin mRNA was minimally increased (1.9-fold) in left ventricular myocardium from nonfailing

aortic-banded rats with pressure-overload hypertrophy but was markedly increased (8-fold) in banded rats with failure.

Treatment with captopril starting before or after the onset of failure in the SHR reduced the increase in left ventricular

osteopontin mRNA levels. Thus, osteopontin expression is markedly increased in the heart coincident with the

development of heart failure. The source of osteopontin in SHR-F is primarily nonmyocytes, and its induction is

inhibited by an angiotensin-converting enzyme inhibitor, suggesting a role for angiotensin II. Given the known

biological activities of osteopontin, including cell adhesion and regulation of inducible nitric oxide synthase gene

expression, these data suggest that it could play a role in the pathophysiology of heart failure. (Hypertension.

1999;33:663-670.)

Key Words:osteopontin heart heart failure rats, inbred SHR hypertrophy

The spontaneously hypertensive rat (SHR) has beenproven to be a valuable model for studying the transitionfrom myocardial hypertrophy to failure.14 In these animals,

a period of several months of compensated hypertrophy is

followed by the development of heart failure (SHR-F). We

have shown that in comparison to SHR with compensated

hypertrophy (SHR-NF), the transition is associated with

pathological evidence of cardiac decompensation, ie, pres-

ence of pleuropericardial effusions, atrial thrombi, and right

ventricular (RV) hypertrophy with reduced contractile func-

tion and increased stiffness of papillary muscle, increased

interstitial fibrosis, and increased expression of atrial natri-

uretic peptide (ANP), collagen, fibronectin, and transforminggrowth factor-1 mRNAs.3,58

In an attempt to identify genes that are differentially

expressed coincident with the transition from hypertrophy to

failure, we used differential display reverse transcriptase

polymerase chain reaction (RT-PCR) to compare RNA iso-

lated from the left ventricles (LV) of SHR-F with that from

age-matched SHR-NF and Wistar-Kyoto rats (WKY).9 One

of the transcripts that was differentially expressed in myocar-

dium from SHR-F was for osteopontin, an extracellular

matrix protein that can act as an adhesion molecule, affect

cellular function by interacting with integrins, and modulate

the expression of inducible nitric oxide synthase.1012

Although it has recently been shown that osteopontin can

be expressed in myocardium in response to necrotic injury13

or short-term pressure overload,14,15 its expression has not

previously been associated with the development of myocar-

dial failure. This report describes the differential expression

and localization of osteopontin in the myocardium of bothSHR and aortic-banded rats coincident with the development

of pathological evidence of cardiac decompensation. Since

angiotensin II (Ang II) stimulates osteopontin expression by

cardiac fibroblasts14 and angiotensin-converting enzyme

(ACE) inhibitor can prevent many of the features of myocar-

Received June 24, 1998; first decision July 14, 1998; revision accepted October 16, 1998.From the Myocardial Biology Unit and Cardiovascular Division, Department of Medicine, Boston Medical Center, Boston Veterans Affairs Medical

Center and Boston University School of Medicine, Boston, Mass.Correspondence to Krishna Singh, PhD, Myocardial Biology Unit, Boston University School of Medicine, 80 E Concord St, Boston, MA 02118. E-mail

[email protected]

1999 American Heart Association, Inc.Hypertension is available at http://www.hypertensionaha.org

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dial failure in SHR,1,2 we also examined the effect of

treatment with the ACE inhibitor captopril on the expression

of osteopontin.

Methods

Animal ModelsMale SHR and nonhypertensive control WKY were purchased(Taconic, Germantown, NY) at the age of 6 to 9 months and boardeduntil the time of study. Beginning at 18 months of age, all animals

were observed twice per week for tachypnea and labored respira-tion.2,5,7,8 SHR were studied within 7 days of the observation ofrespiratory difficulties. Age-matched WKY and SHR without these

signs were studied at the same time.The aortic-banded rats and sham-operated WKY were purchased

from Taconic, Germantown, NY (surgeries were performed atTaconic). A loop of 3.0 surgical silk was placed around the aorticarch between the brachiocephalic and left carotid artery. A blunted18.5-gauge needle was placed over the aorta, and the silk was tiedaround the needle and aorta.16 The needle was then carefullyremoved, resulting in a nonconstricting band. The development ofheart failure was suggested by the observation of respiratory diffi-

culties and documented by pathological examination, as describedfor the SHR.2,5,7,8 The failing and nonfailing banded animals andtheir age-matched nonbanded controls were studied at 11 months ofage, 9 months after banding.

Captopril was added to the drinking water (2.0 g/L) of 9 SHR, aspreviously described.2,17 In 4 animals, it was added before the onset

of failure at either 12 (n1), 18 (n1), or 21 (n2) months of ageand continued until the age of 24 months. In 5 rats, captopril wasadministered after the onset of failure and continued for 2 to 4months before euthanasia at the age of 24 months.

After the rats were killed, the hearts were quickly removed andplaced in Krebs-Henseleit buffer. The RV and LV were carefullydissected, weighed, and immediately frozen in liquid nitrogen forRNA isolation. For in situ hybridization and immunohistochemicalstudies, hearts were perfusion-fixed with freshly prepared 4%paraformaldehyde.6

RNA IsolationTotal RNA was isolated from the LV according to the method ofChomczynski and Sacchi.18 Briefly, samples (100 to 200 mg) werehomogenized in guanidinium thiocyanate solution (4 mol/L guani-

dinium thiocyanate, 25 mmol/L Na citrate [pH 7.0], 0.5% sarcosyl,and 0.1 mol/L 2-mercaptoethanol) with a tissue homogenizer. After

the RNA was extracted with phenol-chloroform, the RNA wasprecipitated with ethanol at 20C. For differential display RT-PCR, RNA was treated with RNase-free DNase for 30 minutes at37C. The RNA was extracted again with phenol-chloroform andprecipitated with ethanol.

Differential Display and Sequence AnalysisDifferential display and sequence analysis were performed as de-scribed earlier.9 W it h t he u se o f a nc ho re d p ri me rs 5-AAGCTTTTTTTTTTTG-3, 5-AAGCTTTTTTTTTTTC-3, or 5-AAGCTTTTTTTTTTTA-3 and Moloney murine leukemia virusreverse transcriptase (GeneHunter Co), the cDNAs were prepared

Figure 1. Representative autora-diograph showing differential dis-play RT-PCR of RNA samples iso-lated from the LV of age-matchedWKY and SHR-F or SHR-NF. Theoligonucleotides consisted of an

anchor primer5-AAGCTTTTTTTTTTTC-3 and anupstream primer5-AAGCTTCGACTGT. The arrowindicates a band that is increasedin SHR-F compared with SHR-NFor WKY. The band from the SHR-Fwas excised from the gel, cloned,and sequenced.

Body and Ventricular Weights

WKY (n6) Nonfai ling SHR (n5) Failing SHR (n8)

SHR

Body weight, g 56623 37832* 38318*

LV weight, g 1.270.1 1.330.12 1.390.06

RV weight, g 0.30.03 0.240.02 0.460.02*

LV/body weight, mg/g 2.240.09 3.510.17* 3.630.04*

RV/body weight, mg/g 0.530.04 0.630.02 1.200.08*

WKY (n5) Nonfailing Banded (n5) Failing Banded (n5)

Aorticbanded rats

Body weight, g 57347 60331 59423

LV weight, g 1.030.05 1.540.04* 1.70.07*

RV weight, g 0.270.01 0.310.01 0.560.02*

LV/body weight, mg/g 1.800.07 2.550.13* 2.860.02*

RV/body weight, mg/g 0.470.02 0.510.03 0.940.05*

*P0.01 vs WKY.

P0.01 vs nonfailing SHR or banded rat.

664 Myocardial Osteopontin

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from mRNA of total RNA isolated from the LV. The reverse

transcription mixture was then amplified by random priming PCR

containing [-35S]dATP with the appropriate anchored primer and 24

different arbitrary 13-mer primers (GeneHunter Co). The products

were separated on a polyacrylamide sequencing gel containing 6

mol/L urea. Autoradiographs were evaluated by visual comparison

of the intensities of individual bands run side by side.

The differentially expressed cDNAs were excised from the dried

gel and extracted with hot water. The recovered DNA was reampli-

fied and cloned in a pCR 2.1 vector (Invitrogen). Purified plasmid

DNA fromEscherichia coliwas then used for sequencing according

to the dideoxy chain termination method with Sequanase 2.0 (Am-

ersham). Sequences were compared with a sequence database with

the use of the Geneworks program (Intelligenetics).

Northern Blot AnalysisTotal RNA was size fractionated on 1.0% formaldehyde agarose gels

containing 2.2 mol/L formaldehyde and transferred to nylon mem-branes (Gene Screen Plus; NEN DuPont) with a transblot apparatus

(Bio-Rad). Blots were hybridized overnight with the radiolabeled

osteopontin cDNA probe,19 as described previously.10 The blots

were then deprobed and hybridized with radiolabeled ANP cDNA(courtesy of Dr C. Seidman) probe. To normalize for loading

differences, the blots were then probed with an 18S oligonucleotide

(30-mer) end-labeled by T4 polynucleotide kinase.10 Differences inmRNA signal intensity were determined with the use of a phospho-

imager (PhosphoImager, Bio-Rad).

In Situ HybridizationIn situ hybridization was performed as previously described.6 Hearts

were perfusion fixed with 4% paraformaldehyde/phosphate-bufferedsaline. Parallel slices 1 to 2 mm thick, encompassing both RV and

LV, were dehydrated and embedded in Paraplast Plus embedding

medium (Oxford). Sections 4 m thick (from WKY, SHR-NF, and

SHR-F) were hybridized with single-stranded sense or antisenseRNA probes transcribed from a linearized20 full-length osteopontin

cDNA19 using [-35S]UTP. Probes were extracted with phenol-

chloroform and precipitated with ethanol.

ImmunohistochemistrySections (4 m thick) from WKY, SHR-NF, and SHR-F were

deparaffinized and stained with the use of monoclonal anti-osteopontin antibodies (MPIIIB10; developmental studies, hybrid-

oma bank) and Vectastain avidin-biotin peroxidase kit (Vector

Laboratories). Briefly, nonspecific binding was minimized by incu-bation for 20 minutes with 1.5% normal horse serum. The sections

were then incubated for 30 minutes with MPIIIB10 (1:250). After

they were washed, the sections were incubated with biotinylatedsecondary antibodies. Detection was performed with the use of

Vectastain ABC-AP reagent and Vector Red alkaline substrate kit

(Vector Laboratories). The sections were visualized and photo-graphed under epifluorescence microscopy with the use of rhoda-

mine excitation and emission filters.

Statistical AnalysisAll data are expressed as meanSEM. Two-tailed Students t test

was used to compare the group means. Probability values of0.05

were considered significant.

Results

Pathophysiologic DataThe body, LV, and RV weights for each experimental group

are shown in the Table. SHR is well documented to develop

cardiac decompensation between 18 and 24 months.24,6,9 The

LV/body weight ratio was increased to a similar degree

(50% to 60%) in the SHR-F and SHR-NF groups. The

RV/body weight ratio was higher in SHR-F than in SHR-NF,consistent with prior findings in these animals. Among the

Figure 2. Alignment of osteopontin cDNAsequence from rat (accession No. M99252)19 andthe 129-bp fragment sequenced from the 500-bpcDNA identified by differential display RT-PCR of

LV myocardial mRNA from SHR-F.

Figure 3. Increased expression of osteopontin mRNA in the LVof SHR-F. A, Representative autoradiograph of a Northernhybridization with a cDNA for osteopontin in LV myocardiumobtained from WKY, SHR-NF, and SHR-F. ANP mRNA wasmoderately increased in SHR-NF and further increased inSHR-F. B, Mean data for osteopontin mRNA in LV from WKY(n6), SHR-NF (n5), SHR-F (n8), and SHR in which captopriltreatment was begun before (CPT-1) ( n4) or after (CPT-2)

(n5) the development of clinical failure. *P0.05 vs WKY;P0.05 vs SHR-NF; #P0.05 vs SHR-F; P0.067 vs SHR-F.

Singh et al February 1999 665



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SHR-F, all animals also showed pleuropericardial effusions

and atrial thrombi, whereas these findings were absent among

the SHR-NF and WKY groups.

In aortic-banded animals, the LV/body weight ratio was

increased to a similar degree (40% to 60%) in both the

nonfailing and failing banded animals (versus nonbanded

control animals), whereas the RV/body weight ratio was

increased only in failing banded animals, similar to the

findings in SHR.

Differential Display RT-PCRTo identify genes with differential expression in LV myocar-

dium from SHR-F (versus SHR-NF and age-matched WKY),

3 hearts from each group were subjected to differential

display RT-PCR. An autoradiograph of a differential display

gel showed the increased expression of a cDNA in SHR-F

versus SHR-NF and WKY (Figure 1). This cDNA (500 bp)

was isolated and cloned, and 129 bp were sequenced, reveal-

ing 98% homology to the rat osteopontin cDNA sequence

(Figure 2).19

Northern Hybridization

The differential expression of osteopontin mRNA in SHR-Fwas confirmed by performing Northern hybridization with

total RNA isolated from the LV of age-matched SHR-F,

SHR-NF, and WKY. Hybridization with a 1.5-kb osteopontin

cDNA identified a 1.6-kb transcript similar in size to that

reported for osteopontin.10 Osteopontin mRNA was ex-

pressed at similar low levels in WKY and SHR-NF (Figure

3). In striking contrast, osteopontin mRNA was increased

10.22.57-fold (P0.05; n8) in LV from SHR-F versus

Figure 5. Immunohistochemical staining of LV myocardiumusing MPIIIB10. A shows the midwall area from SHR-NF, and Band C show the midwall area from SHR-F at magnifications of300 and 600, respectively. The orange staining forosteopontin (arrows) was observed mainly in the interstitialspace.

Figure 6. Expression of osteopontin mRNA in LV of rats withfailure caused by chronic aortic banding. A, Representativeautoradiograph of Northern hybridization with a cDNA forosteopontin in LV myocardium obtained from control WKY with-out aortic banding (WKY), banded rats without signs of failure(AOB-NF), and banded rats with signs of failure (AOB-F).Osteopontin mRNA was slightly increased in nonfailing bandedrats and markedly increased in failing rats. ANP mRNA wasmoderately increased in nonfailing banded rats and furtherincreased in failing rats. B, Mean data from 5 WKY, 5 AOB-NF,and 5 AOB-F. *P0.1 vs WKY; #P0.05 vs WKY; P0.05 vs

AOB-NF.

Figure 4. In situ hybridization with an antisense osteopontin cRNA probe. Sections from SHR-NF (A and C) and SHR-F (B and D) werehybridized with an antisense probe and photographed under darkfield illumination (magnification 100). A and B show the endocardial

area, and C and D show the midwall area. E through H are sections from SHR-F hybridized with the antisense probe, stained withhematoxylin and eosin, and photographed (magnification 400).




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SHR-NF (Figure 3). One of 8 hearts in the SHR-F group had

osteopontin mRNA values that were similar to those in the

SHR-NF group. ANP mRNA was increased 10-fold in

SHR-NF versus WKY. In SHR-F, ANP mRNA was further

increased to 70-fold versus WKY and 7-fold versus

SHR-NF (Figure 3).

In Situ HybridizationIn sections from SHR-NF hearts, in situ hybridization with

the antisense probe for osteopontin showed scant expression,

which was limited to blood vessels, possibly in endothelial

and/or smooth muscle cells (Figure 4A and 4C). Similarly,

WKY hearts showed scant expression of osteopontin mRNA

(not shown). In striking contrast, sections from SHR-F hearts

revealed intense expression of osteopontin, primarily in the

interstitial and perivascular space (Figure 4B and 4D). He-

matoxylin and eosin staining of adjacent sections confirmed

that osteopontin expression in SHR-F was associated primar-

ily with nonmuscle cells infiltrating between the myocytes

(Figure 4E through 4H). In some areas, the expression ofosteopontin was diffuse within the interstitial space (eg,

Figure 4F and 4H). In other areas, the expression was more

focal and appeared to be associated with degenerating myo-

cytes (eg, Figure 4E and 4G). Increased expression of

osteopontin mRNA was also detected in the RV of SHR-F, in

a distribution similar to that observed in the LV. No grains

were visible with the sense osteopontin probe (not shown).

ImmunohistochemistryIn sections from SHR-NF hearts, immunohistochemical anal-

ysis demonstrated low levels of immunoreactivity for os-

teopontin in the interstitial cells of the midwall region of LV(Figure 5A). Similar staining for osteopontin was observed in

sections from WKY (not shown). In sections from SHR-F,

intense staining was observed in the midwall region of LV,

primarily in the interstitial space (Figure 5B). Most of the

staining was observed in the areas of fibrosis and seemed to

be present around the myocytes (Figure 5C). The papillary

muscles from the hearts of WKY, SHR-NF, and SHR-F all

showed intense fibrosis associated with marked staining for

osteopontin.

Effect of Captopril on Osteopontin Expressionin SHRThe effect of treatment with the ACE inhibitor captopril on

LV osteopontin mRNA expression was assessed by Northern

hybridization in 9 SHR at 24 months of age. Captopril

prevented the increase in osteopontin expression in 4 of 4

hearts treated before failure, while it reduced osteopontin

expression in 4 of 5 hearts from animals treated after the onset

of failure (Figure 3B).

Osteopontin Expression in Aortic-Banded RatsCompared with age-matched WKY controls, osteopontin

mRNA was increased 1.90.6-fold (P0.1 versus WKY;

n5) in banded animals without failure and 7.72.9-fold

(P0.05 versus nonfailing banded; n5) in banded animalswith signs of failure (Figure 6). ANP was increased 3.40.9-

fold and 5.21.1-fold in nonfailing and failing banded

animals, respectively.

DiscussionUsing differential display RT-PCR and Northern analysis, we

found that the expression of osteopontin mRNA was mark-

edly increased coincident with appearance of pathophysio-logical evidence of cardiac decompensation in 2 models of

chronic pressure overload, the SHR and the aortic-banded rat.

These results, confirmed by in situ hybridization and immu-

nohistochemistry, indicated that (1) there was only low basal

expression of osteopontin in control WKY rats; (2) in the

absence of failure, basal expression of osteopontin was

unchanged or only slightly increased in SHR or aortic-banded

rats with well-established LV hypertrophy; and (3) in both

models there was a marked increase in osteopontin expres-

sion in age-matched animals that had manifestations of

cardiac decompensation. In situ hybridization and immuno-

histochemistry demonstrated that in failing animals the pri-

mary source of osteopontin mRNA was nonmyocytes andthat the protein is mainly localized in the interstitium.

Osteopontin is an arginine-glycine-aspartatecontaining

adhesive glycoprotein. Although first isolated from mineral-

ized bone matrix, osteopontin can be synthesized by several

cell types, including cardiac myocytes, microvascular endo-

thelial cells, and fibroblasts.1012,14,21,22 Osteopontin is ex-

pressed in several tissues in response to injury, suggesting a

role in the reparative process, and appears capable of medi-

ating diverse biological functions, including cell adhesion,

chemotaxis, and signaling.12,13,23,24

Several groups have recently demonstrated that osteopon-

tin can be expressed in the myocardium. Murry et al13

showedthe expression of osteopontin by macrophages in response to

myocardial necrosis caused by transdiaphragmatic freezing.

Likewise, in the cardiomyopathic Syrian hamster, Williams et

al23 found expression of osteopontin in areas of necrosis

associated with inflammation, also apparently in tissue mac-

rophages. Recently, Graf et al15 demonstrated that osteopon-

tin mRNA was increased approximately 2-fold and 3-fold,

respectively, in LV from rats with 2-kidney, 1 clip hyperten-

sion or aortic banding. In these 2 models, cardiac myocytes

were identified as the predominant source of osteopontin by

immunohistochemistry and in situ hybridization.15

In contrast to Graf et al,15 we did not find osteopontin

mRNA to be increased in the LV of SHR-NF as assessed by

differential display PCR, Northern hybridization, or in situ

hybridization, despite clear evidence of LV hypertrophy and

the expression of ANP mRNA. Likewise, we found only a

modest (1.9-fold) increase in osteopontin in nonfailing

aortic-banded rats, despite marked LV hypertrophy and

increased ANP mRNA expression. A possible explanation for

differences in findings between our studies and those of Graf

et al15 may relate to the markedly different time periods

studied. Whereas Graf et al15 examined animals relatively

soon (4 to 6 weeks) after surgery, our studies were

performed in animals that had been exposed to LV pressure

overload for many months (18 to 24 months for SHR; 9months for aortic-banded rats).




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Consistent with the in situ hybridization, immunohisto-

chemistry revealed increased expression of osteopontin in the

midwall region of SHR-F (compared with SHR-NF and

WKY). Similar to the in situ hybridization, most of the

staining was observed in the interstitial space. In contrast to in

situ hybridization, which showed increased expression of

osteopontin mRNA in the papillary muscles of only SHR-F,immunoreactivity for osteopontin protein was increased in

the papillary muscles of all 3 groups (ie, WKY, SHR-NF, and

SHR-F). This may reflect (1) the extensive fibrosis in

age-related papillary muscles from all 3 groups and/or (2) that

secretory osteopontin, once incorporated into extracellular

matrix, has a lower turnover rate and/or higher stability.

In SHR with cardiac decompensation, the major source of

osteopontin expression appears to be nonmyocytes in the

interstitium and perivascular space. Thus, our data differ from

those of Graf et al,15 who found that myocytes were the major

source of osteopontin in LV from animals with relatively

short-term hypertrophy. However, our findings are consistent

with those of Murry et al13

and Williams et al,23

who foundinterstitial expression of osteopontin by macrophages in rats

with thermal injury or cardiomyopathic hamsters, respec-

tively. Murry et al13 suggested that osteopontin is synthesized

in response to injury. Our findings suggest that, at least with

regard to osteopontin, the appearance of cardiac decompen-

sation and the transition to failure in these rat models is

associated with an injury response. Taken together with the

data of Graf et al,15 it appears reasonable to suggest that the

source of osteopontin mRNA may be myocytes early in LV

hypertrophy but shifts to interstitial cells and focal areas of

myocyte injury in late hypertrophy with the development of

heart failure.

Ang II has been shown to cause focal myocyte necrosis inrats infused with Ang II or in models of renovascular

hypertension.25 Ang II also induces osteopontin in cardiac

fibroblasts in vitro,14 and infusion of Ang II increased

osteopontin expression in kidney.26 In our study, focal ex-

pression of osteopontin mRNA appeared to be associated

with degenerating myocytes (Figure 4E and 4G). We also

found that captopril treatment of SHR reduced osteopontin

mRNA levels. These findings are thus consistent with a role

for Ang II in the induction of osteopontin in areas of myocyte

necrosis and fibrosis during the transition to failure. How-

ever, it is also possible that rats treated with captopril before

the development of heart failure remained compensated and

therefore showed no increase in osteopontin, while captopril

treatment after the development of heart failure might have

prevented the additional necrosis and healing of lesions,

thereby preventing osteopontin expression. Another possibil-

ity is that captopril suppressed the expression of osteopontin

by increasing bradykinin, which might act through nitric

oxide to attenuate the expression of growth-related genes.27

The role of osteopontin during the transition to heart failure

is not known. Indeed, relatively little is known about the role

of osteopontin in the myocardium. Osteopontin can act as an

adhesion molecule, as a chemotactic factor, and as a substrate

for the migration of macrophages, smooth muscle cells, and

endothelial cells.12

Osteopontin can also act as a cytokine tostimulate lymphocyte immunoglobulin production.11,28 These

activities are consistent with a role for osteopontin in fibrosis

or healing in response to injury.

We have also shown that osteopontin can suppress the

cytokine-induced expression of inducible nitric oxide syn-

thase in cardiac myocytes and microvascular endothelial cells

isolated from adult rat hearts.10 It is therefore of interest that

high levels of nitric oxide produced by inducible nitric oxidesynthase can impair myocyte function29 and may be toxic to

cardiac myocytes.30 Since it appears that myocardial induc-

ible nitric oxide synthase expression is increased with fail-

ure,31,32 osteopontin has the potential to modulate the effects

of inflammatory cytokines on the myocardium by attenuating

the expression of inducible nitric oxide synthase.

Thus, osteopontin expression is markedly increased in the

myocardium coincident with the development of evidence of

cardiac decompensation in 2 models of chronic pressure

overload. Given the importance of the extracellular matrix in

the pathophysiology of myocardial failure33,34 and the known

biological activities of osteopontin, it is possible that this

interstitial matrix protein plays an important role in thepathogenesis of heart failure.

AcknowledgmentsThis study was supported in part by grants R29 HL-57947 (Dr Singh)and HL-52320 and HL-42539 (Dr Colucci) from the NationalInstitutes of Health and by Medical Research Funds from theDepartment of Veterans Affairs (Drs Singh, Bing, Conrad, andBrooks). Dr Communal is supported by a fellowship from theAmerican Heart Association, Massachusetts Affiliate.

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Conrad, Wesley W. Brooks, Oscar H. L. Bing and Wilson S. ColucciKrishna Singh, Geza Sirokman, Catherine Communal, Kathleen G. Robinson, Chester H.

Myocardial Osteopontin Expression Coincides With the Development of Heart Failure

Print ISSN: 0194-911X. Online ISSN: 1524-4563Copyright 1999 American Heart Association, Inc. All rights reserved.

is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Hypertension

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myocardial osteopontin expression coincides with the development of heart failure

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