Myocardial Osteopontin Expression Is Associated With Left Ventricular Hypertrophy
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.
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 β3-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.
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.
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 β3-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.
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.
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, 5× formamide, 1× 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.
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.
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, β3-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.
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-β (TGF-β) 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 β3-integrin receptors on cells, since growth was inhibited by antibodies against either OP or β3-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 β3-integrin or RGD peptides did not block the ET-1–induced ANP mRNA increase. OP binds to αvβ1-, αvβ3-, and αvβ5-integrins.6 22 Thus far, functional β1-integrins and associated α-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 αv- and β5-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
|ANP||=||atrial natriuretic peptide|
|CHOB||=||Chinese hamster ovary cDNA B|
|LVH||=||left ventricular hypertrophy|
|MPIIIB 10||=||monoclonal antibody against osteopontin|
|NOS2||=||inducible nitric oxide synthase|
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.
- Copyright © 1997 by American Heart Association
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