European Journal of Heart Failure

European Journal of Heart Failure 2002 4(2):139-146; doi:10.1016/S1388-9842(01)00237-9

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© 2002 European Society of Cardiology

Increased myocardial expression of osteopontin in patients with advanced heart failure

Philipp Stawowy^a, Florian Blaschke^a, Peter Pfautsch^a, Stephan Goetze^a, Frank Lippek^b, Brigitte Wollert-Wulf^a, Eckart Fleck^a and Kristof Graf^a,*

^a Department of Medicine/Cardiology, German Heart Institute Berlin Augustenburger Platz 1, D-13353 Berlin, Germany
^b Department of Pathology, Charité, Humboldt-University Berlin Schumannstraße 20-21, D-10117 Berlin, Germany

^* Corresponding author. Tel.: +49-30-4593-2400; fax: +49-30-4593-2500. E-mail address: graf{at}dhzb.de

	Abstract

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
The expression of the adhesion protein osteopontin (OP) is associated with cardiac hypertrophy and is significantly increased after transition to heart failure in experimental animal models. We, therefore, hypothesized that OP could be upregulated in heart failure in humans. In the present study, we investigated the expression of OP in myocardial biopsies obtained from patients with heart failure due to dilated cardiomyopathy (mean LVEF = 30.3±4.4%, mean±S.D., n = 10, group A) compared to patients with a normal left-ventricular ejection fraction (mean LVEF = 61±11.2%, n = 9; group B). Myocardial immunoreactivity for OP was examined using two different antibodies against OP. The expression of cardiac myocyte OP was significantly upregulated in group A in comparison to group B (P<0.0001). Both groups also displayed OP immunoreactivity in non-myocytes, including vascular smooth muscle cells and cardiac fibroblasts (P = not significant). Statistical analysis revealed a significant correlation of increased OP immunoreactivity in cardiac myocytes of patients with impaired left ventricular function, assessed by hemodynamic data (LVEF, RVEF, LVESVI, LVEDVI and LVEDP, R = –0.828, –0.671, 0.751, 0.685 and 0.461, respectively; all P<0.05). Furthermore, OP expression correlated with cardiac myocyte hypertrophy (mean diameter 21.0±1.8 µm in group A and 16.6±2.1 µm in group B; P<0.0001). In conclusion, the present study indicates, that factors and/or mechanisms involved in heart failure in patients with dilated cardiomyopathy, lead to induction of OP expression in humans.

Key Words: Dilated cardiomyopathy • Myocardial biopsy • Adhesion molecules • Heart failure

Received June 29, 2001; Revised August 17, 2001; Accepted October 23, 2001

	1. Introduction

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
Heart failure is associated with the activation of multiple molecular and cellular changes in the circulation and heart, including the activation of several signaling pathways, re-expression of fetal gene programs in the myocardium, as well as apoptosis. These events contribute to consecutive alterations in the contractile apparatus, changes in calcium homeostasis and the expression of growth factors, chemokines and hormones [1]. Increased expression of extracellular matrix components [2,3], adhesions molecules and proteins [4,5] and integrin receptors [6] are important intermediates in the processes involved in heart failure progression. Ultimate histopathological changes include fibroblast proliferation and fibrosis. Increased myocardial workload leads to increased wall stress, which results in cardiac myocyte hypertrophy. This involves rearrangement and upregulation of sarcomers, rearrangement of stress fibers and remodeling of the interstitial matrix. It is still unclear whether the reactive fibrosis observed in some animal models is, in part, due to defective cell–extracellular matrix interactions, changes in collagen expression, myocardial ischemia or the action of trophic peptides [7]. Important components of this signaling processes are focal adhesion sites, connecting extracellular proteins with integrins, which in turn transduce signals from the cell surface to the nucleus (outside-in signaling) [8].

Osteopontin (OP) is an adhesion molecule which was first identified in bone tissue [9]. It has been implicated as a major mediator of the profibrotic changes induced by angiotensin II (Ang II) during heart failure. Osteopontin has been identified in cultured neonatal and adult rat cardiac fibroblasts [10] and is found in macrophages in necrotic areas in rat hearts [11]. Furthermore, inflammatory cells express OP in a genetic animal model of dilated cardiomyopathy [12], indicating a potential role for OP during inflammatory processes in the heart. We have demonstrated that cardiac myocytes are an important source of OP in left ventricular hypertrophy in both man and rodents [4]. Interestingly, Singh and co-workers [13] showed that OP is dramatically increased in the left ventricular myocardium of a heart failure model in rats, after transition from left ventricular hypertrophy to heart failure. These studies suggest that OP might be upregulated in patients with heart failure due to dilated cardiomyopathy.

We, therefore, examined the expression of OP in myocardial biopsies from patients with heart failure to investigate whether its appearance correlates with the impairment of hemodynamic parameters.

	2. Methods

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
2.1. Subject studied
Right ventricular biopsies from 19 patients were included in this study (January 1998 to August 1998). Based on hemodynamic data, the subjects were assigned to two groups. Group A consisted of patients diagnosed with dilated cardiomyopathy (DCMP) with a left ventricular ejection fraction (LVEF) <50%; patients in group B had a normal LVEF (>50%). Diagnosis of DCMP was made according to the definition and classification of the World Health Organization/International Society and Federation of Cardiology task force [14]. Patients in group B included patients clinically suspected to suffer from cardiomyopathy, including hypertrophic non-obstructive cardiomyopathy (HNOC) and hypertensive heart disease. Major complaints at time of presentation were dyspnea and fatigue (12 patients) and/or angina pectoris (eight patients). The duration of symptoms was at least >6 months in all subjects, NYHA stages are given in Table 1. Patients’ medications were as follows: in group A, 7/10 patients received ACE-inhibitors, 6/10 received diuretics, 2/10 received β-blockers and 4/10 received digitalis. In group B, 6/9 received ACE-inhibitors, 5/9 received diuretics and 4/9 received β-blockers. One patient in group B received calcium antagonists.

View this table: [in this window] [in a new window]   Table 1 Patients’ characteristicsa

 
All subjects underwent cardiac right and left heart catheterization, including hemodynamic measurements, biplane ventriculography and selective coronary angiography. Coronary heart disease was excluded in all subjects. Left and right ventricular end-diastolic and end-systolic volume indices and ejection fractions were calculated from ventricular cineangiograms. All patients received routine two-dimensional echocardiography. The results of hemodynamic and echocardiographic studies are given in Table 1.

2.2. Immunohistochemical analysis
Two to three right ventricular biopsies were obtained during routine cardiac catherization from the right side of the interventricular septum. Biopsies were immediately fixed in 4% buffered formalin at 4 °C overnight. The biopsies were then washed in 70% ethanol for 24 h and embedded in paraffin using an automated system. Three-µm-thick paraffin sections were cut on poly-lysine-precoated slides (10%; Sigma, FRG). Deparaffinization and rehydration was done using three charges of xylene and a graded series of ethanol (100%–50%), followed by several washings in phosphate-buffered saline (PBS, containing 0.01% Triton-X100). Immunohistochemistry was performed as described [15] using the ABC Zymed Histostain-Plus kit (Zymed Laboratories, San Francisco, CA, USA, #85-9743) and the DAB Zymed Histostain-Plus kit (Zymed Laboratories, #85-9843). Briefly, incubation with the primary antibody was done over night at 4 °C, followed by incubation with a secondary antibody. Immunoreactions were revealed with horseradish peroxidase activity producing brown (3,3'-diaminobenzidine, DAB) and red (3-amino-9-ethycarbazol, AEC) stainings. For monoclonal mouse antibodies and co-localization studies, localization of alkaline phosphatase activity was performed with an azo dye coupling method (Alkaline Phosphatase Substrate Kit, cat. #SK-5300 Vector Red). The following antibodies (and dilutions) were used: rabbit anti-human osteopontin (Chemicon, Temecula, CA, USA; 1:500), mouse monoclonal anti-osteopontin [MPIIIB10(1), Developmental Hybridoma Bank, Univ. of Iowa], mouse anti -tropomyosin (Sigma, FRG; 1:50), rabbit anti-CD3 (Dako, FRG; 1:1000), mouse anti HLA-DR (Dako FRG; 1:100), mouse anti leucocyte common antigen (Dako FRG; 1:1000) and mouse anti CD 68 (Dako FRG; 1:50). Specificity controls were done by omission of the first antibody and/or incubation with non-immune IgGs [16]. As positive controls for osteopontin stainings, rat kidney was used [17]. Photomicrographs were taken with a digital camera (Olympus PD10) on a Zeiss microscope (Axioskop S100, Zeiss, FRG). Tissue sections were examined in a blinded manner by two independent investigators. Three tissue sections of OP staining were studied for each patient. The percentage of positive cells in four high power fields is reported. The diameter of myocytes in each section was measured as the shortest length crossing a nucleus at a magnification of 400x.

2.3. Statistical analysis
Results are expressed as mean±S.D. Statistical analysis of comparisons between the groups was performed with Mann–Whitney's U-test, and analysis of correlation with Spearman's rank correlation coefficient. Statistical significance was designated at a probability value of less than 0.05.

	3. Results

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
3.1. Hemodynamic and echocardiographic differences in subjects studied
Based on clinical presentation, chest X-ray and electrocardiograms, all patients were suspected to suffer from cardiomyopathy. Coronary heart disease was excluded in all subjects studied. Based on clinical data obtained, subjects were assigned to two groups. Group A consisted of subjects with a LVEF significantly less than 50% (mean 30.3±4.4; mean±S.D.), whereas the LVEF was above 50% (mean 61.0±11.2; mean±S.D.) in group B (P<0.001). Both groups also differed significantly with regard to other characteristic hemodynamic parameters, confirming severe heart failure in group A (Table 1).

All myocardial biopsies were subject to routine histopathological analysis. Myocarditis was excluded in all patients according to the Dallas criteria [18]. Using specific antibodies against macrophage CD68, HLA-DR and leucocyte common antigen (LCA), we did not detect any significant mononuclear infiltrate and/or myocytolysis in the biopsies studied. Myocardial storage diseases were also excluded. Mean cardiac myocyte diameter was 21.0±1.8 µm in group A and 16.6±2.1 µm in group B (P<0.0001).

3.2. Immunolocalization of osteopontin
3.2.1. Cardiac myocytes
Cardiac myocyte OP immunoreactivity (Fig. 1C) was detected in 13 out of 19 (68%) biopsies of all subjects studied. Two different antibodies against OP were used in this study. Both antibodies gave equal results. Cells expressing OP immunoreactivity were identified as cardiac myocytes by staining of consecutive sections with an anti--tropomyosin antibody. All biopsies in group A revealed cardiac myocyte OP expression, with a mean expression of 82% (range 54–98%). Osteopontin expression in cardiac myocytes was not associated with the presence of inflammatory cells, fibrosis or other anatomical structures (e.g. endocardium, blood vessels). Compared to group A, expression of OP in group B was significantly lower (P<0.0001). Only three of the nine subjects studied in this group showed cardiac myocyte OP immunoreactivity (patients 12, 14 and 17; Table 1).

View larger version (224K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Immunolocalization of osteopontin (OP) in human right ventricular biopsies. A and B are representative photomicrographs of biopsies from subjects of the control group B. Fig. 1A depicts expression of OP (red staining; AEC) in vascular smooth muscle cells (VSMCs) of intramural coronary arteries. The arrow denotes OP immunoreactivity in a presumptive cardiac fibroblasts. OP was also found in mononuclear cells (arrows; Fig. 1B). No significant OP immunoreactivity is found in cardiac myocytes in this biopsy. Fig. 1C demonstrates increased expression of OP (brown staining with DAB as chromogen) in cardiac myocytes in a patient with heart failure (subject 1, Table 1) and cardiac myocyte hypertrophy (inset). Fig. 1D is a serial section from the same patient incubated with non-immune IgGs used as negative control. Nuclei are stained with hematoxyline. Magnification 200x.

 
3.2.2. Non-myocytes
OP was found in macrophages, mononuclear cells and vascular smooth muscle cells of intramural coronary arteries (if present on tissue sections) in 16 out of 19 biopsies (84%) studied (Fig. 1A,B). In contrast to cardiac myocyte OP expression, no significant difference in OP staining was found in the non-myocyte cell population (Fig. 2).

View larger version (10K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 The percentage of cells positive for immunoreactive osteopontin in the myocyte and non-myocyte population of group A (heart failure) and group B (control subjects). Bars show mean (±S.D.). NS=not significant.

 
3.3. Cardiac myocyte OP expression correlates with hemodynamic data
Statistical analysis revealed a close correlation of OP expression in cardiac myocytes with the severity of heart failure as determined by hemodynamic parameters. Comparative analysis demonstrated that OP immunoreactivity in cardiac myocytes correlates significantly with the left- (LVEF; Fig. 3a) and right- (RVEF; Fig. 3b) ventricular ejection fraction as well as with the left ventricular end-systolic- (LVESVI; Fig. 3c) and left ventricular end-diastolic- (LVEDVI; Fig. 3d) volume index and the left-ventricular end-diastolic pressure (LVEDP; Fig. 3e) (R=–0.828, –0.671, 0.751, 0.685 and 0.461, respectively; all P<0.05). Furthermore, a close correlation between OP cardiac myocyte expression and the mean cardiac myocyte diameter was observed (Fig. 3f; R=0.731, P<0.0001). OP immunoreactivity in non-myocyte cells did not correlate with clinical data, which is in line with its equal distribution in these cells in both groups.

View larger version (16K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Correlation between the percentage of osteopontin positive cardiac myocytes and the LVEF, RVEF, LVESVI, LVEDVI, LVEDP and MD. Closed circles indicate data from patients with heart failure (group A), were as open circles group B patients. (LVEF=left ventricular ejection fraction; RVEF=right ventricular ejection fraction; LVESVI=left ventricular end-systolic volume index; LVEDVI=left ventricular end-diastolic volume index; LVEDP=left ventricular end-diastolic pressure; MD mean measurement of myocyte diameter).

 

	4. Discussion

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
In the present study, we report a significant increase in osteopontin (OP) immunoreactivity in cardiac myocytes from patients with severe heart failure due to dilated cardiomyopathy (DCMP). Statistical analysis demonstrates that cardiac myocyte OP expression correlates significantly with right- and left-ventricular ejection fractions, end-systolic and end-diastolic volume indices and left-ventricular end-diastolic pressure in heart failure patients. Furthermore, OP expression correlates with cardiac myocyte hypertrophy obtained by histomorphometry. These results suggest that factors and/or mechanisms involved in heart failure are associated with increased OP expression in humans.

Osteopontin is an adhesion molecule, which binds to integrins via its RGD (arginin–glycine–aspartate) motif [9]. We, and others, have reported the expression and regulation of OP in human and rodent cardiac myocytes [4,13]. Osteopontin mRNA levels were significantly upregulated in rat cardiac myocytes after treatment with endothelin-1 (ET-1) and norepinephrine (NE) [4]. This OP induction coincides with the induction of atrial natriuretic peptide (ANP) [4]. The natriuretic peptides ANP and brain natriuretic peptide (BNP) are released from cardiac myocytes in response to increased pressure and have been identified as valuable markers for cardiac myocyte hypertrophy [19]. Heart failure involves the upregulation and activation of several neuroendocrine pathways, including the release of ANP, BNP, ET-1, catecholamines and the activation of the renin–angiotensin system. Clinical studies have demonstrated that elevated levels of catecholamines correlate with the severity of heart failure in patients with DCMP [20].

These neuroendocrine factors are known to promote a phenotypic switch from the adult to a neonatal pattern of gene programs, associated with cardiac myocyte hypertrophy in heart failure [21]. We demonstrate that heart failure is associated with increased OP expression, which potentially exerts paracrine effects on cardiac fibroblasts. This hypothesis is supported by in vitro studies, showing that OP and Ang II induce contraction of three-dimensional collagen gels and fibroblast proliferation [10]. Induction of fibroblast proliferation is associated with a change in their gene expression and extensive synthesis of ECM [1,22]. Pauschinger et al. [23] have recently demonstrated that DCMP in humans is associated with a significant increase in the collagen type I/III mRNA ratio (6.4 in LVEF >50% vs. 8.2 in LVEF <50%). Even though they detected increased mRNA levels for collagens I and III in biopsies from patients with a LVEF <50%, they found an increase only in type I collagen protein in these patients, whereas type III collagen protein was decreasing in patients with an LVEF <50%. This is in accordance with others, who found that changes in collagen content are mostly due to accumulation of collagen type I [24,25]. Whereas collagen type III serves as an elastic recoil of kinetic energy, type I collagen increases chamber stiffness [1]. Thus, the expression of collagens has a major impact on the systolic and diastolic function of the heart. Furthermore, the pattern of collagens potentially influences cardiac myocyte phenotype [26].

An essential link between cardiac myocytes, fibroblasts and the extracellular matrix (ECM) are the integrin receptors, which are involved in the mechanotransduction in heart failure. Activation of integrin receptors leads to cytoskeletal reorganization and induction of various intracellular signaling cascades [8]. The integrin superfamily consists of heterodimeric transmembrane proteins that comprise at least 15 and 8 β subunits that heterodimerize in more than 20 combinations. Changes in integrin expression have been found in cardiac hypertrophy [6] and it has recently been shown, that integrins control cell cycle progression in cardiac myocytes [27]. The integrin vβ3 (vitronectin receptor) has been identified as the major receptor for OP [28,29]. We have recently demonstrated, that integrin vβ3 participates in cardiac remodeling via growth factor induced chemotaxis and adhesion of cardiac fibroblasts [30]. Furthermore, OP- and Ang II-induced contraction of three-dimensional collagen gels and cardiac fibroblast growth was inhibited by a monoclonal antibody to β3 integrin [10], indicating that these actions of OP and Ang II are mediated via β3 integrin receptors. Although integrin β3 is expressed on cardiac myocytes (Stawowy and Graf; unpublished data), the interaction of OP with this integrin and its precise role requires further investigation.

A recent study [13] has shown that upregulation of OP in animal models could be inhibited by treatment with the ACE-inhibitor captopril. In rat cardiac fibroblasts, Ang II significantly increases OP levels [10], proliferation, ECM synthesis and integrin expression [31,32]. Singh et al. [13] found a predominant expression of OP in non-myocytes in rat hearts. Osteopontin was only minimally increased in both spontaneously hypertensive rats with compensated hypertrophy (no heart failure) and non-failing aortic-banded rats with pressure-overload hypertrophy, but markedly increased in both spontaneously hypertensive rats with heart failure and banded rats with heart failure [13]. Consistent with this, OP up-regulation in cardiac myocytes correlates with heart failure in our study, but not with cardiac hypertrophy (mean interventricular septal wall thickness (IVS) 10 mm in patient group A and 12 mm in control group B; P=n.s.; Table 1). The differences in cellular localization of OP reported by Singh et al. [13] compared to our results, could result from species differences, as we used human tissue samples. Another possible explanation for the different cellular localization of OP might be that the animals used by Singh et al. [13] were in heart failure for month, whereas DCMP in humans in our study was probably present for several years.

We also detected OP in a low but substantial number of non-myocyte cells, such as macrophages, vascular smooth muscle cells and cardiac fibroblasts. However, the appearance of OP in these cells was not statistically linked to heart failure in the patients examined in this study.

4.1. Study limitations
In this study, we used immunohistochemistry on tissue samples derived from routine clinical practice. Even though immunohistochemistry is only semi-quantitative in nature, it is a rapid and widely used method in clinical practice. Larger tissue samples for immunoblotting or Northern blot analysis are often not available for routine studies. Even though tissue from end-stage explanted heart has been used for quantitative analysis, the appropriate control tissue would not be available for the purpose of our study. A further limitation is that this study did not include patients with ischemic cardiomyopathy, the major cause of advanced heart failure. However, this report demonstrates the clinical evidence that upregulation of myocardial OP expression correlates with the impairment of hemodynamic parameters and cardiac myocyte hypertrophy in patients with DCMP.

	Acknowledgements

 
The study was supported by the Deutsche Forschungsgemeinschaft (GR 1368/2-2) to K. Graf.

	References

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 

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