(Circulation. 1997;96:3063-3071.)
© 1997 American Heart Association, Inc.
Articles |
Myocardial Osteopontin Expression Is Associated With Left Ventricular Hypertrophy
From the Department of Medicine and Pathology, Division of Endocrinology, Diabetes, and Hypertension, University of Southern California School of Medicine, Los Angeles, Calif (K.G., Y.S.D., N.A., W.P.M., C.C.M., W.A.H.); the Department of Pathology, University of Washington School of Medicine, Seattle (C.M.G.); and the Department of Medicine/Cardiology, German Heart Institute and Virchow Klinikum, Humboldt Universität Berlin, Germany (K.G.).
Correspondence to Willa A. Hsueh, MD, UCLA School of Medicine, Division of Endocrinology, Diabetes, and Hypertension, Warren Hall, 2nd Floor, 900 Veteran Ave, MC 178622, Los Angeles, CA 90024.
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Background Osteopontin (OP) has been identified in cultured rat cardiac fibroblasts, where it contributes to angiotensin II (AII)-induced remodeling processes; in cultured cardiomyocytes; and in macrophages in cardiac tissues with inflammation. However, the presence of OP has not been reported in histological sections of myocardial tissue. In the present study, we investigated (1) the regulation of OP mRNA expression in cultured rat cardiomyocytes; (2) the localization of OP mRNA in neonatal and adult normal and hypertrophied rat hearts; and (3) the histology of OP expression in myocardial specimens from humans either with myocyte hypertrophy or with no pathological changes.
Methods and Results Cultured neonatal cardiomyocytes expressed OP mRNA and were immunoreactive for OP. Endothelin-1 (ET-1) and norepinephrine (NE) increased both OP and atrial natriuretic peptide (ANP) mRNA levels twofold to threefold (P<.01). OP mRNA was prominent in ventricular tissue from neonatal and adult rats with renovascular hypertension and aortic banding, whereas barely detectable levels were observed in normal adult cardiac tissue. ANP and OP mRNA levels in normal and hypertrophied ventricles correlated (r2=.87, P<.001). OP immunoreactivity and mRNA transcripts were predominantly found in cardiomyocytes not associated with inflammatory cells in sections from neonatal and adult hypertrophied hearts. No staining was detectable in normal adult hearts. Human myocardium with extensive fibrosis and cardiomyocyte hypertrophy obtained from explanted hearts with either idiopathic (n=5) or ischemic cardiomyopathy (n=7) demonstrated substantial myocyte immunoreactivity for both OP and ANP in right and left ventricles that was not associated with leukocyte infiltration. In situ hybridization identified cardiomyocytes as the major source of OP mRNA transcripts in these hearts. In contrast, OP immunoreactivity was not detectable in four of five endomyocardial biopsies with normal histology.
Conclusions The present study provides the first evidence that cardiomyocytes are a prominent source of OP in vivo and suggests that induction of OP expression is strongly associated with ventricular hypertrophy.
Key Words: hypertrophy • endothelin • adhesion protein • osteopontin
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Osteopontin has recently been identified in cultured neonatal and adult rat cardiac fibroblasts and in cultured neonatal rat cardiomyocytes.1 2 However, little is known about its distribution in the heart in vivo, particularly in humans. Immunostaining localized OP to macrophages in necrotic areas in rat heart3 and to inflammatory cells in a genetic model of dilated cardiomyopathy in Syrian hamsters,4 thus implicating a potential role for OP in inflammatory processes in the heart. In human heart this adhesion protein has been identified only in macrophages in calcified areas of aortic valvular lesions,5 where it was suggested to be responsible for laying down of calcium. We found enhanced ventricular OP mRNA levels in two models of LVH in the rat: aortic banding and 2K1C renovascular hypertension. Cardiac fibroblasts taken from both normal and hypertrophied hearts expressed OP when subjected to culture; AII potently (0.1 nmol/L) stimulated OP expression and protein production in these cultured cardiac fibroblasts.1 We found that OP played a critical role in both AII-induced DNA synthesis and adhesion to collagen by cultured cardiac fibroblasts; these effects appeared to be mediated by OP binding to ß3-integrins on the surface of the fibroblasts by RGD sequences located on the OP molecule.1 6 These results underscored the need to characterize the expression of OP in normal and hypertrophied heart. Our results suggest that the cardiac myocyte is an important source of OP in LVH in both rats and humans.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Animals
Adult Sprague-Dawley rats (Simonsen) weighing 165 to 200 g were maintained in an environmentally controlled vivarium (23±1°C, 30% to 40% humidity) with a 12-hour light/12-hour dark photocycle (lights on 7:00 am). Hypertension was induced with the use of a 2K1C preparation, as described elsewhere.7 At 6 weeks after surgery, blood pressures were measured before the rats were killed by an indirect tail-cuff method (IITC Instruments Inc). At the time of tissue harvest, animals were administered a pentobarbital overdose and decapitated. The heart and kidneys were rapidly removed and snap-frozen in liquid nitrogen. Tissues were stored at -80°C freeze before use. Aortic-banded animals and sham animals were obtained from Taconic Laboratory Animals and Services (Germantown, NY): 3.0 surgical silk was placed around the aorta between the brachycephalic and left carotid arteries. An 18.5-gauge blunt needle was placed over the aorta, the silk was tied snugly around the aorta and needle, and the needle was slid out from under the knot. The hearts were examined 3 to 5 weeks after banding.
Cell Culture
Cardiomyocyte cultures were prepared from Sprague- Dawley
rats 1 to 3 days after birth and characterized as previously
described.8 Briefly, neonatal hearts were dissected free
of atria, minced, and subjected to trypsin and DNAse II digestion. The
isolated cells were preplated for 30 minutes in MEM/5% fetal bovine
serum (FBS). During this period of time the nonmyocytes
attached to the plate while the myocytes remained floating, thus
separating the two populations. The floating myocytes were seeded in a
second dish in MEM/5% FBS. After 3 to 4 days, they were cultured in
serum-free MEM containing insulin (5 µg/mL), transferrin (5
µg/mL), and selenium (5 ng/mL) (ITS) for 24 hours and
subjected to experimental treatment. Cultured myocytes from the heart
were studied at 70% confluence. One day before treatment they were
placed in serum-free media containing ITS (Sigma). AII (1
µmol/L, Bachem), ET-1 (0.1 µmol/L, Peninsula),
NE (10 µmol/L, Sigma), or vehicle was also incubated for
24 hours. ET-1 incubations were also performed after 1-hour
pretreatment with RGD peptides; RGE peptides (both from Bachem); or
blocking antibodies against the following: OP, MPIIIB10 (University of
Iowa, Iowa City; under contract N01-HD-6-2915 from the NICHD), or
ß3-integrin, clone F11 (PharMingen), or nonspecific mouse
IgG (Zymed) as control. Each treatment was performed on three different
preparations of cells in duplicate.
Isolation and Analysis of RNA
Total RNA was isolated from myocytes with the use of the
guanidinium–sodium acetate phenol–chloroform method.9
RNA was size-fractionated by electrophoresis through a denaturing 1%
agarose gel, transferred to nitrocellulose membranes, and hybridized
with cDNA probes labeled with [32P]-dCTP (3000
Ci/mmol) by random priming. The OP probe was prepared as previously
described.10 The cDNA for rat ANP, which cross-reacts with
human ANP, was kindly provided by D. Gardner (University of California
San Francisco, School of Medicine). The hybridization signals of the
specific mRNAs of interest were normalized to those of CHOB for
differences in loading or transfer.11 CHOB cDNA is
originally isolated from Chinese hamster ovary cells that corresponds
to an RNA ubiquitously expressed in mammalian tissues, which does not
exhibit regulation as a function of growth or
development.11 Quantitation of Northern blots was
performed by densitometric analysis with the use of the Gel
Documentation System (model GDS 5000, UVP, Inc), and the results were
analyzed with the densitometric software (Gel Base UVP, Inc).
Several autoradiographic film exposures (from 12 hours to 7
days) were used to ensure that the density of the signals were linear
on each film.
Myocardial Tissues
For histological evaluation, rat heart tissues
(neonate, adult, 2K1C rat and its appropriate sham, aortic-banded rat
and its appropriate sham) were carefully washed in PBS and placed
immediately in neutral buffered formalin and were paraffin embedded.
Sections of human hearts were taken from 12 patients undergoing heart
transplantation due to a history of ischemic or idiopathic
cardiomyopathy. Right ventricular
myocardial biopsies from 5 patients with recurrent
ventricular arrhythmia or suspected idiopathic
cardiomyopathy who ultimately had normal myocardial
histology were obtained as control.
Immunohistochemistry
The avidin-biotin peroxidase complex (ABC) technique as
described by Hsu et al12 was used. For
histological localization of rat OP, OP199 at a titer
of 1:1000 (1 µg/mL) or MPIIIB10 1:500 (72 ng/mL) was
used. Macrophages were stained with the antibody ED1 (Titer
1:400, Harlan Bioproducts for Science). Myocytes were stained with
a monoclonal antibody against cardiac tropomyosin (titer 1:500, clone
No. CH1, T 9283, Sigma). Nonimmune serum was used as a control. A
rabbit anti-human ANP antibody (Peninsula Laboratories) was used at a
titer 1:400. The primary antibody incubations were performed in 1%
bovine serum albumin/2% goat serum containing PBS for 60
minutes. A biotinylated rabbit anti-goat antibody or rabbit anti-mouse
antibody (Zymed) was applied followed by an incubation with
streptavidin-peroxidase complex. Peroxidase activity was detected with
the use of amino ethyl carbazole as a chromogen (Zymed). Slides were
then counterstained with Mayer's acid hematoxylin. For
immunocytochemistry, myocytes of neonatal or adult rat hearts were
placed on gelatin-coated culture chamber slides and incubated in
culture medium for 24 hours. The cells were fixated in omnifix (Zymed)
and permeabilized in 0.05% Triton X100 (Sigma).
Further procedures were performed as mentioned above.
In Situ Hybridization
The 2B7 plasmid,10 which contains a 1.0-kb cDNA
fragment for rat OP, was linearized by digestion with either
HindIII or BamHIII. A riboprobe was generated by
transcription of linearized cDNA with T3- and T7-polymerase with the
use of digoxigenin-labeled UTP (Boehringer) as substrate. The
clone OP-30 plasmid containing 1.4 kb human OP cDNA (ATCC; Rockville,
Md) was linearized with either Pst I or Xba I to
generate digoxigenin-labeled riboprobes for detection of human OP mRNA.
Formalin-fixed, paraffin-embedded, 4-µm-thick sections of the hearts
were deparaffinized according to standard protocols. The sections were
washed with PBS and digested with proteinase K (5 µg/mL) in
Tris-EDTA, pH 8.0, for 60 minutes at 37°C. After several washes,
slides were dehydrated in ethanol (90%, 95%, 100%) and air dried.
Prehybridization buffer (0.3 mol/L NaCl, 20 mmol/L
Tris, pH 8.0, 5 mmol/L EDTA, 5x formamide, 1x Denhardt's
solution) was applied for at least 2 hours at 55°C. For
hybridization, digoxigenin-labeled riboprobe for OP (final
concentration, 20 to 40 ng/µL ) was added, and hybridizations
were performed overnight in a humidified chamber at 55°C. After
hybridization, sections were washed four times in standard saline
citrate (SSC), two times in SSC, and once in SSC with 1.0% BSA at
55°C for 20 minutes. Afterward, alkaline phosphatase–conjugated
antidigoxigenin antibody at a dilution of 1:2500 (Boehringer
Mannheim) was applied and sections were incubated for 40 minutes at
37°C in a humidified chamber. Signal was developed with nitroblue
tetrazolium salt/5-bromo-4-chloro-3-indoyl-phosphate
(Boehringer Mannheim). After development, sections were
counterstained with hematoxylin and mounted.
Statistical Analysis
Group means were compared with the use of the two-tailed
Student's t test. Differences between treatments were
analyzed statistically by ANOVA. Values of P<.05
were considered significant. Statview statistics software for Macintosh
computers was used for correlation analysis and ANOVA.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
OP Is Expressed by Cultured Rat Neonatal Cardiac Myocytes and Is Regulated by ET-1 and NE
Myocytes were identified with the use of three criteria: morphological characteristics, immunostaining for tropomyosin, and expression of ANP mRNA. Cultured neonatal myocytes are flat cells of various shape and size that have cellular fibrils and contract spontaneously. On the basis of these characteristics, our myocyte cultures contained 10% to 15% contamination of nonmyocyte cells. Immunostaining of neonatal myocytes placed on gelatin-coated slides revealed positive staining for OP of nearly all cells under quiescent conditions (Fig 1A
). Staining was present
in the cytoplasm of the cultured myocytes. No staining was seen with
nonimmune serum (not shown). Northern analysis identified a
single OP mRNA band of 1.6 kb and an ANP mRNA band of 0.8 kb in RNA
extracted from cultured myocytes (Fig 1B). Incubation of myocytes for
24 hours with AII (1 µmol/L) increased OP mRNA slightly
(P<.07 versus control) and had no significant effect on
myocyte ANP mRNA. ET-1 (0.1 µmol/L) and NE (10
µmol/L), well-known hypertrophic factors for
cardiomyocytes,13 increased cellular OP and
ANP mRNA levels twofold to threefold (P<.01 versus control)
at 24 hours after treatment, which was the maximum response observed in
several time-course experiments from 4 to 48 hours (Fig 1, B through
D). Preincubation with blocking antibodies for OP,
ß3-integrin or RGD peptides, did not interfere with the
effect of ET-1 to enhance myocyte ANP mRNA expression, a marker of
myocyte hypertrophy, which was similar to the lack of
effect of control IgG or RGE peptides (Fig 1D). These antibodies and
peptides also did not block ET-1 stimulation of OP mRNA.
|
OP mRNA and Protein Are Present in Myocardium of
Neonatal Rats
Immunohistochemical localization of OP in neonatal rat hearts
showed diffuse cytoplasmic staining in the myocardium
predominantly in the left and right ventricles (Fig 2A). No specific staining was
seen in the endocardium, leaflets, or vascular
endothelium. A similar pattern of staining was also
found in fetal myocardium (day 18, not shown). Normal adult
rat hearts had no immunohistochemically detectable OP with the use of
either a monoclonal antibody, MPIIIB10, or the polyclonal antiserum
OP199. Northern analysis of total RNA from neonatal (3 days)
and adult rat hearts (12 weeks) correlated with immunohistochemical
staining. OP mRNA was easily detected in total RNA extracted from
neonatal rat hearts, whereas only a faint signal for OP mRNA was seen
in RNA from normal adult rat hearts. The quantitation of OP mRNA after
normalization to CHOB demonstrated 2.5-fold higher levels in neonatal
rat hearts (n=5) compared with normal adult rat hearts (n=5;
P<.05, Fig 3).
|
|
Cardiomyocyte OP Is Upregulated in LVH
In contrast to the normal adult heart, OP expression was readily
detectable in hypertrophied ventricles from rats subjected to clipping
of the renal artery (2K1C) but not in ventricles from sham-operated
animals. The heart/body weight ratio (2K1C, 0.38±0.02; sham,
0.31±0.02; P<.02) and the systolic blood pressure
(2K1C, 146±4 mm Hg; sham, 101±4 mm Hg;
P<.001) were increased in the 2K1C group compared with the
sham-operated group. The ventricular OP/CHOB mRNA ratio
(2K1C, 0.51±0.07; sham, 0.22±0.06; P<.02) and
ventricular ANP/CHOB mRNA ratio (2K1C, 0.37±0.03; sham,
0.12±0.05; P<.01) were both significantly augmented in
hearts with LVH compared with hearts of the sham group (Fig 3A
).
In another model of LVH, induced after 4 weeks of aortic banding in the
rat, ventricular OP expression was also upregulated
compared with hearts of sham-operated animals (Fig 3A). The
heart–to–body weight ratio of these animals was increased (banded,
0.518±0.0.54; sham, 0.401±0.038; P<.05, n=7 each). The
presence of LVH was confirmed by an upregulation of ANP mRNA levels in
the left ventricle (banded, 0.35±0.05 ANP mRNA/CHOB mRNA), whereas
sham-operated rats showed only minimal expression of
ventricular ANP (sham, 0.12±0.02 ANP mRNA/CHOB mRNA;
P<.01). Ventricular levels of OP mRNA and ANP
mRNA were significantly correlated in both LVH models and sham-operated
rats (r2=.87, P<.001; Fig 3B). In
addition, elevated levels of OP mRNA were found in the atria from
hearts of the banded animals compared with sham, whereas ANP mRNA
levels were similar in the atrium from the banded and sham-operated
groups (Fig 3C). This observation suggests that OP and ANP expression
may be regulated by different factors in the atrial myocytes.
Immunohistochemistry from sham hearts from both LVH models demonstrated
no immunoreactivity for OP in the atrium (data not shown).
The presence of OP was immunohistochemically demonstrated in myocytes
of tissues from hypertrophied ventricles from 2K1C animals (Fig 4A),
whereas no significant staining was observed in ventricles from
sham-operated animals (Fig 4B). Little immunoreactivity
was seen in fibroblasts, whereas no immunoreactivity was seen in
vascular endothelium, endocardium, or valve leaflets.
Control staining with nonimmune IgG was negative. There was no
association with macrophage infiltration when sections were
stained with an antimacrophage antibody (ED1, data not shown).
Staining was performed in serial sections from 6 rats with LVH and
immunoreactivity for OP was consistently identified with both
antibodies, OP199 and MPIIIB 10. Furthermore, in situ hybridization
identified ventricular cardiomyocytes as the
major source of OP transcripts in hypertrophied ventricles (Fig 4C). No
signal was seen in interstitial and
endothelial cells. Control hybridization with sense
riboprobe in hypertrophied ventricles or with antisense riboprobe in
ventricular sections from sham hearts did not reveal
significant signal (Fig 4D).
|
In hearts from aortic-banded rats immunohistochemistry demonstrated a
strong expression of OP in myocytes and in vascular smooth muscle
cells, but little interstitial expression and no vascular
endothelial expression was detected (Fig 5A). Ventricles from
sham-operated animals demonstrated little immunoreactivity for OP (Fig 5B). In situ hybridization with a digoxigenin-labeled riboprobe for OP
mRNA demonstrated expression of transcripts preferentially in
ventricular myocytes of the LVH hearts (not shown), whereas
the sense riboprobe did not show a significant signal.
|
Expression of OP in Human Myocardium
To investigate OP expression in human hearts,
immunostaining was performed on hearts explanted at the
time of cardiac transplantation for idiopathic
cardiomyopathy (n=5) or ischemic heart
disease (n=7). As a control, right ventricular
endomyocardial biopsies from patients without a
history of cardiomyopathy or hypertension were
used. Increased fiber thickness and changes in shape and size of
myocyte nuclei as well as various degrees of interstitial
fibrosis were seen in the hypertrophied specimens from explanted hearts
but not in the control hearts (n=5). In hypertrophied ventricles from
cardiomyopathic and ischemic hearts, staining
for OP was present in myocytes (Fig 6A) and in the media of small
intramyocardial arteries (not
shown). ANP immunoreactivity
was also readily detected in myocytes (Fig 6B). Diffuse myocyte
staining for OP and ANP was observed in tissue sections from the right
(n=12) as well as the left (n=12) ventricle from these hearts.
Immunoreactivity was rarely seen in interstitial cells, and
no significant reactivity was seen in the endocardium or vascular
endothelium. No specific reactivity was seen in
association with ischemic areas or with inflammation related to
healing ischemic damage. Staining with control IgG was negative
(not shown). In situ hybridization in specimens from 5 patients (3
patients with idiopathic and 2 with ischemic
cardiomyopathy) with a riboprobe for human OP mRNA
identified myocytes as the major source of OP mRNA transcripts in these
end-stage hearts (Fig 6C). Control hybridization with sense riboprobes
did not show detectable signal (Fig 6D).
|
Right ventricular endomyocardial biopsies with normal histology were chosen as controls. Four of these five biopsies had no immunoreactivity either for OP or for ANP. Only one biopsy showed little reactivity for OP in a patchy and variable distribution in myocytes (not shown). About 25% of the myocytes in this specimen showed positive staining in the cytoplasm.
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
The present investigation is the first histological report demonstrating the presence of OP expression in the heart that is not related to inflammation and the appearance of macrophages. We found OP mRNA and protein in myocardial areas in neonatal rat heart that was barely detectable in the normal adult rat heart (12 weeks and older). However, OP expression was substantially upregulated with the development of LVH and correlated with ventricular expression of ANP, a known marker of myocyte hypertrophy. Immunostaining for OP was primarily detected in the muscle cells of the heart, although some staining was also noted in fibroblasts and vascular smooth muscle cells. To confirm that the presence of OP in myocytes was due to synthesis and not uptake, we performed in situ hybridization, which indicated the presence of OP mRNA in the myocytes. Thus in the growing heart of the fetus or neonate or in the hypertrophied adult heart, the cardiomyocyte is a major site of OP expression. Similar results were obtained with immunohistochemical staining and in situ hybrization of human hearts that had extensive fibrosis and myocyte hypertrophy; the pattern of distribution was similar to that of ANP. OP was present primarily in cardiomyocytes and in some vascular smooth muscle cells of smaller coronary arteries. In contrast, endomyocardial biopsies from 4 of 5 patients with normal histology did not reveal detectable OP or ANP by immunohistochemistry, consistent with our observations in normal versus hypertrophied rat ventricles.
OP has been identified in macrophages in cardiac injury models associated with inflammation in rats, humans, and hamsters with heritable cardiomyopathy.3 4 Immunostaining did not clearly identify cell types other than macrophages in the heart that contained OP. An acute model of cryoinjury in the rat that was not associated with myocardial hypertrophy and one explanted human heart with myocardial ischemia was immunostained in that study,3 and no OP was detected in the cardiomyocytes. Although the source of antibody in that investigation was identical to ours, the lack of LVH in that rodent model could explain the differences. Furthermore, we used a quick perfusion-fixation technique and formalin-based fixatives for our rat and human tissue sections, whereas a non–formalin-based approach was used in the previous study. We found that formalin fixation seems to preserve the OP antigen and that we do not consistently detect OP by immunostaining with non–formalin-based techniques. In addition, we used higher concentrations of the monoclonal antibody than used in previous studies.
To investigate regulation of OP expression in the cardiomyocyte, we treated cultured neonatal cardiac myocytes with AII, ET-1, and NE. The cultured neonatal cardiomyocytes stained positively with the OP antibody and expressed detectable message by in situ hybridization. AII modestly increased OP mRNA and had little effect on ANP mRNA levels, whereas ET-1 and NE enhanced both OP and ANP message levels by threefold in cultured cardiomyocytes. Both of the latter are well-described hypertrophic factors known to induce ANP in ventricular cardiomyocytes and to promote switching from the adult to fetal phenotype pattern of contractile protein gene expression characteristic of myocyte hypertrophy.13 The cardiac expression of ET-1 has been shown to be increased in LVH induced by overload14 or by myocardial infarction,15 and elevated ET-1 plasma levels have been measured in patients with heart failure.16 It is possible that ET-1 contributes to the upregulation of OP expression in LVH. Glucocorticoids have also been demonstrated to stimulate both OP and ANP production in cultured neonatal rat myocytes,2 17 and chronic treatment with dexamethasone, a potent synthetic glucocorticoid, enhanced cardiac hypertrophy in newborn rats.18 Taken together, our observations suggest that enhanced OP production accompanies cardiomyocyte hypertrophy both in vivo and in vitro.
Different growth factors regulate OP production in the cardiac fibroblast compared with the cardiomyocyte. In the rat cardiac fibroblast, AII (10 pmol/L) is a potent stimulus to increase OP mRNA levels and protein production; platelet-derived growth factor (PDGF) and transforming growth factor-ß (TGF-ß) moderately enhance OP message levels, whereas ET-1 and NE have no effect.1 These differences suggest separate growth factors, and possibly different mechanisms influence fibroblast and myocyte behaviors in the cardiac remodeling process. Thus the approaches to prevention and attenuation of myocyte hypertrophy and cardiac fibrosis may be different, although either process affects the development and progression of the other. OP produced by the myocyte probably contributes to growth and adhesion of the cardiac fibroblast1 and therefore may serve as an important paracrine mechanism of communication between myocyte and fibroblast. The cardiac fibroblast itself is also known to produce factors that stimulate myocyte hypertrophy.19 20 Further studies will be important to elucidate the paracrine interactions between the cardiac fibroblast and myocyte.
The role of OP in cardiomyocyte function is unknown. OP
binds to integrin receptors on the surface of cells. This binding
involves RGD sequences located on OP and activates
integrin-linked signal transduction pathways that mediate cell
functions including growth, adhesion, migration, and other
events.21 22 23 24 Adhesion is necessary for growth; in
particular, we found that OP was necessary for cardiac fibroblast
proliferation induced by AII.1 This effect involved
binding of OP to ß3-integrin receptors on cells, since
growth was inhibited by antibodies against either OP or
ß3-integrins. RGD peptides, which compete with
RGD-containing proteins such as OP for binding to integrins, also
inhibited growth in cardiac fibroblasts.1 In the
present study OP did not appear to be involved in
hypertrophy-related events in the ventricular
cardiomyocyte leading to ANP mRNA upregulation, since
antibodies against OP and ß3-integrin or RGD peptides did
not block the ET-1–induced ANP mRNA increase. OP binds to
vß1-, vß3-,
and vß5-integrins.6 22 Thus
far, functional ß1-integrins and associated -integrins
have been demonstrated on cardiomyocytes, and their
expression has been enhanced along with that of laminins and collagen
in tissue from hypertrophied rat heart.25 We found the
presence of v- and ß5-integrin mRNA in
cultured cardiomyocytes, which represent another
potential receptor for OP on these cells (K. Graf, W.A. Hsueh;
unpublished observations). Recently, Singh et al2 reported
that OP inhibits expression and activation of NOS2 in cardiac neonatal
myocytes. Nitric oxide is antiproliferative and may prevent fibrosis in
the heart, since infusion of AII and L-NAME, an inhibitor
of nitric oxide formation, leads to massive fibrosis.26
Thus OP may have secondary effects on the cardiac fibrotic process. On
the other hand, upregulation or increased activity of NOS2 has been
described in cardiac specimens from patients with heart failure and
impaired left ventricular function.27 28 In
vitro data have demonstrated that induction of NOS2 can depress
contractile function of cardiac myocytes.29 30 This
suggests a possible role of cardiac OP on the regulation of NOS2
expression and thus on the contractile response. Further investigations
of the role of OP in the response to injury in the heart are likely to
shed light on the cellular and biochemical mechanisms involved in
cardiac hypertrophy and remodeling.
|
Selected Abbreviations and Acronyms |
|---|
|
|
Acknowledgments |
|---|
This work was supported in part by National Institutes of Health grant 5R01 DK30254 to Dr Hsueh. The authors wish to thank Lilit Yegiyants for excellent technical assistance and Janie Teran and Denise Edwards for their secretarial contributions.
Received January 22, 1997; revision received May 16, 1997; accepted May 28, 1997.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Ashizawa N, Graf K, Do YS, Nunohiro T, Giachelli CM, Meehan WP, Tuan TL, Hsueh WA. Osteopontin is produced by rat cardiac fibroblasts and mediates AII-induced DNA synthesis and collagen gel contraction. J Clin Invest. 1996;98:2218-2227.[Medline] [Order article via Infotrieve]
2.
Singh K, Ballingand J, Fischer T, Smith T, Kelly
R. Glucocorticoids increase osteopontin expression in cardiac
myocytes and microvascular endothelial cells.
J Biol Chem. 1995;270:28471-28478.
3. Murry C, Giachelli CM, Schwartz S, Vracko R. Macrophages express osteopontin during repair of myocardial necrosis. Am J Pathol. 1994;145:1450-1462.[Abstract]
4.
Williams EB, Halpert L, Wickline S, Davison G, Parks
W, Rottman J. Osteopontin expression is increased in the
heritable cardiomyopathy of Syrian
hamsters. Circulation. 1995;92:705-709.
5.
O'Brien K, Kuusisto J, Reichenbach D, Ferguson M,
Giachelli CM, Alpers C, Otto C. Osteopontin is expressed in
human aortic valvular lesions. Circulation. 1995;92:2029-2032.
6. Liaw L, Skinner MP, Raines EW, Ross R, Cheresh DA, Schwartz SM, Giachelli CM. The adhesive and migratory effects of osteopontin are mediated via distinct cell surface integrins: role of alpha v beta 3 in smooth muscle cell migration to osteopontin in vitro. J Clin Invest. 1995;95:713-724.
7.
Buchanan TA, Sipos G, Gadallah S, Yip K, Marsh D,
Hsueh WA, Bergman R. Glucose tolerance and insulin actions in
rats with renovascular hypertension. Hypertension. 1991;18:341-347.
8.
Simpson P, McGrath A, Savion S. Myocyte
hypertrophy in neonatal rat heart cultures and its
regulation by serum and catecholamines. Circ
Res. 1982;51:787-801.
9. Chomczynski P, Sacchi N. Single step method of RNA isolation by acid granidium hemocyanate-phenol-chloroform extraction. Anal Biochem. 1989;162:156-159.
10. Giachelli CM, Bae N, Lombardi D, Majesky M, Schwartz SM. Molecular cloning and characterization of 2B7, a rat mRNA which distinguishes smooth muscle cell phenotypes in vitro and is identical to osteopontin (secreted phosphoprotein I, 2aR). Biochem Biophys Res Commun. 1991;177:867-873.[Medline] [Order article via Infotrieve]
11. Harpold M, Evans R, Saidtt-Georgieff M, Darnell J. Production of mRNA in Chinese hamster cells; relationship of the rate of synthesis to the cytoplasmic concentration of nine specific mRNA sequences. Cell. 1979;17:1025-1035.[Medline] [Order article via Infotrieve]
12. Hsu S, Raine L, Fanger H. The use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem. 1981;29:557-564.
13.
Shubeita H, McDonough P, Harris A, Knowlton K,
Glembotsky C, Brown J, Chien K. Endothelin induction of inositol
phospholipid hydrolysis, sarcomere assembly, and cardiac gene
expression in ventricular myocytes: a paracrine mechanism
for myocardial cell hypertrophy. J Biol
Chem. 1990;265:20555-20562.
14.
Arai M, Yoguchi A, Iso T, Takahashi T, Imai S, Murata
K, Suzuki T. Endothelin-1 and its binding sites are upregulated
in pressure overload cardiac hypertrophy.
Am J Physiol. 1995;268:H2084-H2091.
15.
Sakai S, Miyauchi T, Sakurai T, Kasuya Y, Ihara M,
Yamaguchi I, Goto K, Sugishita Y. Endogenous
endothelin-1 participates in the maintenance of cardiac
function in rats with congestive heart failure: marked increase in
endothelin-1 production in the failing heart.
Circulation. 1996;93:1214-1222.
16.
Cody R, Haas G, Binkley P, Capers Q, Kelley R.
Plasma endothelin correlates with the extent of pulmonary
hypertension in patients with chronic congestive heart failure.
Circulation. 1992;85:504-509.
17.
Argentin S, Sun Y, Lihrmann I, Schmidt T, Drouin J,
Nemer M. Distal cis-acting promoter sequences mediate
glucocorticoid stimulation of cardiac atrial natriuretic
factor gene transcription. J Biol Chem. 1991;266:23315-23322.
18. Brem A, Bina R, Klinger J, King T. Glucocorticoid metabolism in the newborn rat heart. Proc Soc Exp Biol Med. 1995;209:146-151.[Medline] [Order article via Infotrieve]
19. Long C, Henrich C, Simpson P. A growth factor for cardiac myocytes is produced by cardiac nonmyocytes. Cell Regul. 1991;2:1081-1095.[Medline] [Order article via Infotrieve]
20. Fujisaki H, Ito H, Hirata Y, Tanaka M, Hata M, Lin M, Adachi S, Akimoto H, Marumo F, Hiroe M. Natriuretic peptides inhibit angiotensin II-induced proliferation of rat cardiac fibroblasts by blocking endothelin-1 gene expression. J Clin Invest. 1995;96:1059-1065.
21. Rodan GA. Osteopontin overview. Ann N Y Acad Sci. 1995;760:1-5.
22. Giachelli CM, Schwartz SM, Liaw L. Molecular and cellular biology of osteopontin. Trends Cardiovasc Med. 1995;5:88-95.
23. Hynes R. Integrins: versatility, modulation and signaling in cell adhesion. Cell. 1992;69:11-25.[Medline] [Order article via Infotrieve]
24.
Clark E, Brugge J. Integrins and signal
transduction pathway: the road taken. Science. 1995;268:233-239.
25.
Terracio L, Rubin K, Gullberg D, Balog E, Carver W,
Jyring R, Borg T. Expression of collagen binding integrins
during cardiac development and hypertrophy.
Circ Res. 1991;68:734-744.
26. Hou J, Kato J, Cohen RA, Chobanian AV, Brecher P. Angiotensin II-induced cardiac fibrosis in the rat is increased by chronic inhibition of nitric oxide synthase. J Clin Invest. 1995;96:2469-2477.
27.
Lewis NP, Tsao PS, Rickenbacher PR, Xue C, Johns RA,
Haywood GA, von der Leyen HE, Trindale PT, Cooke JP, Hunt SA,
Billingham ME, Valentine HA, Fowler MP. Induction of nitric
oxide synthase in the human cardiac allograft is associated with
contractile dysfunction of the left ventricle.
Circulation. 1996;93:720-729.
28.
Haywood GA, Tsao PS, von der Leyen HE, Mann MJ, Keeling
PJ, Trindade PT, Lewis NP, Byrne CD, Rickenbacher PR, Bisphoric NH,
Cooke PJ, McKenna WJ, Fowler MB. Expression of inducible nitric
oxide synthase in human heart failure. Circulation. 1996;93:1087-1094.
29.
Brady AJB, Poole-Wilson PA, Harding SE, Warren
JB. Nitric oxide production within cardiac myocytes
reduces their contractility in endotoxemia.
Am J Physiol. 1992;263:H1963-H1966.
30. Ballingand JL, Ungureanu D, Kelly RA, Kobzik L, Pimental D, Michel T, Smith TW. Abnormal contractile function due to induction of nitric oxide synthesis in rat cardiac myocytes follows exposure to activated macrophage-conditioned medium. J Clin Invest. 1993;91:2314-2319.
This article has been cited by other articles:
 |
|
 |
|
  H. Yue, W. Li, R. Desnoyer, and S. S. Karnik Role of nuclear unphosphorylated STAT3 in angiotensin II type 1 receptor-induced cardiac hypertrophy Cardiovasc Res, September 16, 2009; (2009) cvp285v2. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Q. Yu, R. Vazquez, E. V. Khojeini, C. Patel, R. Venkataramani, and D. F. Larson IL-18 induction of osteopontin mediates cardiac fibrosis and diastolic dysfunction in mice Am J Physiol Heart Circ Physiol, July 1, 2009; 297(1): H76 - H85. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. P. Sheehy, S. Huang, and K. K. Parker Time-Warped Comparison of Gene Expression in Adaptive and Maladaptive Cardiac Hypertrophy Circ Cardiovasc Genet, April 1, 2009; 2(2): 116 - 124. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Caglayan, B. Stauber, A. R. Collins, C. J. Lyon, F. Yin, J. Liu, S. Rosenkranz, E. Erdmann, L. E. Peterson, R. S. Ross, et al. Differential Roles of Cardiomyocyte and Macrophage Peroxisome Proliferator-Activated Receptor {gamma} in Cardiac Fibrosis Diabetes, September 1, 2008; 57(9): 2470 - 2479. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Rosenberg, C. Zugck, M. Nelles, C. Juenger, D. Frank, A. Remppis, E. Giannitsis, H. A. Katus, and N. Frey Osteopontin, a New Prognostic Biomarker in Patients With Chronic Heart Failure Circ Heart Fail, May 1, 2008; 1(1): 43 - 49. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Zahradka Novel Role for Osteopontin in Cardiac Fibrosis Circ. Res., February 15, 2008; 102(3): 270 - 272. [Full Text] [PDF]
|
|
|
|
 |
|
 M. Scatena, L. Liaw, and C. M. Giachelli Osteopontin: A Multifunctional Molecule Regulating Chronic Inflammation and Vascular Disease Arterioscler Thromb Vasc Biol, November 1, 2007; 27(11): 2302 - 2309. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Fielitz, S. Philipp, L. R. Herda, E. Schuch, B. Pilz, C. Schubert, V. Gunzler, R. Willenbrock, and V. Regitz-Zagrosek Inhibition of prolyl 4-hydroxylase prevents left ventricular remodelling in rats with thoracic aortic banding Eur J Heart Fail, April 1, 2007; 9(4): 336 - 342. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. D. Thakker, N. G. Frangogiannis, M. Bujak, P. Zymek, J. W. Gaubatz, A. K. Reddy, G. Taffet, L. H. Michael, M. L. Entman, and C. M. Ballantyne Effects of diet-induced obesity on inflammation and remodeling after myocardial infarction Am J Physiol Heart Circ Physiol, November 1, 2006; 291(5): H2504 - H2514. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. C. Bilchick, S. K. Saha, E. Mikolajczyk, L. Cope, W. J. Ferguson, W. Yu, S. Girouard, and D. A. Kass Differential regional gene expression from cardiac dyssynchrony induced by chronic right ventricular free wall pacing in the mouse Physiol Genomics, September 14, 2006; 26(2): 109 - 115. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. M. Manso, L. Elsherif, S.-M. Kang, and R. S. Ross Integrins, membrane-type matrix metalloproteinases and ADAMs: Potential implications for cardiac remodeling Cardiovasc Res, February 15, 2006; 69(3): 574 - 584. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Satoh, M. Nakamura, T. Akatsu, Y. Shimoda, I. Segawa, and K. Hiramori Myocardial osteopontin expression is associated with collagen fibrillogenesis in human dilated cardiomyopathy Eur J Heart Fail, August 1, 2005; 7(5): 755 - 762. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Stawowy, C. Margeta, F. Blaschke, C. Lindschau, C. Spencer-Hansch, M. Leitges, G. Biagini, E. Fleck, and K. Graf Protein kinase C epsilon mediates angiotensin II-induced activation of {beta}1-integrins in cardiac fibroblasts Cardiovasc Res, July 1, 2005; 67(1): 50 - 59. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Graf and P. Stawowy Osteopontin: A Protective Mediator of Cardiac Fibrosis? Hypertension, December 1, 2004; 44(6): 809 - 810. [Full Text] [PDF]
|
|
|
|
|
|
Z. Xie, M. Singh, and K. Singh Osteopontin Modulates Myocardial Hypertrophy in Response to Chronic Pressure Overload in Mice Hypertension, December 1, 2004; 44(6): 826 - 831. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. W.M. Schellings, Y. M. Pinto, and S. Heymans Matricellular proteins in the heart: possible role during stress and remodeling Cardiovasc Res, October 1, 2004; 64(1): 24 - 31. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. S Ross Molecular and mechanical synergy: cross-talk between integrins and growth factor receptors Cardiovasc Res, August 15, 2004; 63(3): 381 - 390. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Diez Profibrotic Effects of Angiotensin II in the Heart: A Matter of Mediators Hypertension, June 1, 2004; 43(6): 1164 - 1165. [Full Text] [PDF]
|
|
|
|
|
|
Y. Matsui, N. Jia, H. Okamoto, S. Kon, H. Onozuka, M. Akino, L. Liu, J. Morimoto, S. R. Rittling, D. Denhardt, et al. Role of Osteopontin in Cardiac Fibrosis and Remodeling in Angiotensin II-Induced Cardiac Hypertrophy Hypertension, June 1, 2004; 43(6): 1195 - 1201. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. R. Collins, J. Schnee, W. Wang, S. Kim, M. C. Fishbein, D. Bruemmer, R. E. Law, S. Nicholas, R. S. Ross, and W. A. Hsueh Osteopontin modulates angiotensin II- induced fibrosis in the intact murine heart J. Am. Coll. Cardiol., May 5, 2004; 43(9): 1698 - 1705. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. Aherrahrou, S. B. Axtner, P. M. Kaczmarek, A. Jurat, S. Korff, L. C. Doehring, D. Weichenhan, H. A. Katus, and B. T. Ivandic A Locus on Chromosome 7 Determines Dramatic Up-Regulation of Osteopontin in Dystrophic Cardiac Calcification in Mice Am. J. Pathol., April 1, 2004; 164(4): 1379 - 1387. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C.-M. Cheng, H.-J. Hong, J.-C. Liu, N.-L. Shih, S.-H. Juan, S.-H. Loh, P. Chan, J.-J. Chen, and T.-H. Cheng Crucial Role of Extracellular Signal-Regulated Kinase Pathway in Reactive Oxygen Species-Mediated Endothelin-1 Gene Expression Induced by Endothelin-1 in Rat Cardiac Fibroblasts Mol. Pharmacol., May 1, 2003; 63(5): 1002 - 1011. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. R. R. Heyen, E. R. Blasi, K. Nikula, R. Rocha, H. A. Daust, G. Frierdich, J. F. Van Vleet, P. De Ciechi, E. G. McMahon, and A. E. Rudolph Structural, functional, and molecular characterization of the SHHF model of heart failure Am J Physiol Heart Circ Physiol, November 1, 2002; 283(5): H1775 - H1784. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Rocha, A. E. Rudolph, G. E. Frierdich, D. A. Nachowiak, B. K. Kekec, E. A. G. Blomme, E. G. McMahon, and J. A. Delyani Aldosterone induces a vascular inflammatory phenotype in the rat heart Am J Physiol Heart Circ Physiol, November 1, 2002; 283(5): H1802 - H1810. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Stawowy, F. Blaschke, P. Pfautsch, S. Goetze, F. Lippek, B. Wollert-Wulf, E. Fleck, and K. Graf Increased myocardial expression of osteopontin in patients with advanced heart failure Eur J Heart Fail, March 1, 2002; 4(2): 139 - 146. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Mavroidis and Y. Capetanaki Extensive Induction of Important Mediators of Fibrosis and Dystrophic Calcification in Desmin-Deficient Cardiomyopathy Am. J. Pathol., March 1, 2002; 160(3): 943 - 952. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. E. Dostal Regulation of Cardiac Collagen : Angiotensin and Cross-Talk With Local Growth Factors Hypertension, March 1, 2001; 37(3): 841 - 844. [Full Text] [PDF]
|
|
|
|
 |
|
 G.Y.H Lip, D.C Felmeden, F.L Li-Saw-Hee, and D.G Beevers Hypertensive heart disease. A complex syndrome or a hypertensive 'cardiomyopathy'? Eur. Heart J., October 2, 2000; 21(20): 1653 - 1665. [PDF]
|
|
|
|
|
|
C. Kupfahl, D. Pink, K. Friedrich, H. R. Zurbrugg, M. Neuss, C. Warnecke, J. Fielitz, K. Graf, E. Fleck, and V. Regitz-Zagrosek Angiotensin II directly increases transforming growth factor {beta}1 and osteopontin and indirectly affects collagen mRNA expression in the human heart Cardiovasc Res, June 1, 2000; 46(3): 463 - 475. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. M. Schnee and W. A. Hsueh Angiotensin II, adhesion, and cardiac fibrosis Cardiovasc Res, May 1, 2000; 46(2): 264 - 268. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. D. Sehl, J. T. N. Tai, K. J. Hillan, L. A. Brown, A. Goddard, R. Yang, H. Jin, and D. G. Lowe Application of cDNA Microarrays in Determining Molecular Phenotype in Cardiac Growth, Development, and Response to Injury Circulation, April 25, 2000; 101(16): 1990 - 1999. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Graf, M. Neuss, P. Stawowy, W. A. Hsueh, E. Fleck, and R. E. Law Angiotensin II and {alpha}v{beta}3 Integrin Expression in Rat Neonatal Cardiac Fibroblasts Hypertension, April 1, 2000; 35(4): 978 - 984. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Kawano, Y. S. Do, Y. Kawano, V. Starnes, M. Barr, R. E. Law, and W. A. Hsueh Angiotensin II Has Multiple Profibrotic Effects in Human Cardiac Fibroblasts Circulation, March 14, 2000; 101(10): 1130 - 1137. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Sodek, B. Ganss, and M.D. McKee Osteopontin Critical Reviews in Oral Biology & Medicine, January 1, 2000; 11(3): 279 - 303. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Kawano, R. J. Cody, K. Graf, S. Goetze, Y. Kawano, J. Schnee, R. E. Law, and W. A. Hsueh Angiotensin II Enhances Integrin and {alpha}-Actinin Expression in Adult Rat Cardiac Fibroblasts Hypertension, January 1, 2000; 35(1): 273 - 279. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Singh, G. Sirokman, C. Communal, K. G. Robinson, C. H. Conrad, W. W. Brooks, O. H. L. Bing, and W. S. Colucci Myocardial Osteopontin Expression Coincides With the Development of Heart Failure Hypertension, February 1, 1999; 33(2): 663 - 670. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. M. Taylor, J. D. Rovin, and J. T. Parsons A Role for Focal Adhesion Kinase in Phenylephrine-induced Hypertrophy of Rat Ventricular Cardiomyocytes J. Biol. Chem., June 16, 2000; 275(25): 19250 - 19257. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. A. Trueblood, Z. Xie, C. Communal, F. Sam, S. Ngoy, L. Liaw, A. W. Jenkins, J. Wang, D. B. Sawyer, O. H. L. Bing, et al. Exaggerated Left Ventricular Dilation and Reduced Collagen Deposition After Myocardial Infarction in Mice Lacking Osteopontin Circ. Res., May 25, 2001; 88(10): 1080 - 1087. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||