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European Journal of Heart Failure 2005 7(5):755-762; doi:10.1016/j.ejheart.2004.10.019
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© 2004 European Society of Cardiology

Myocardial osteopontin expression is associated with collagen fibrillogenesis in human dilated cardiomyopathy

Mamoru Satoh*, Motoyuki Nakamura, Tomonari Akatsu, Yudai Shimoda, Ikuo Segawa and Katsuhiko Hiramori

Second Department of Internal Medicine Iwate Medical University School of Medicine, 19-1 Uchimaru, Morioka 020-8505, Iwate, Japan E-mail address: m_satoh{at}imu.ncvc.go.jp

* Corresponding author. Tel.: +81 19 651 5111; fax: +81 19 651 0401.


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 
Background: Osteopontin (OPN), an extracellular matrix (ECM) protein, plays an important role in myocardial remodeling by promoting collagen synthesis and accumulation in experimental animal models.

Aims: We hypothesized that OPN could be expressed in myocardial tissues and contribute to collagen accumulation and myocardial dysfunction in human dilated cardiomyopathy (DCM).

Methods and results: Endomyocardial biopsy tissues were obtained from 51 patients with DCM and 15 controls by right ventricular endomyocardial biopsy. OPN, collagen types I (Col I) and III (Col III) mRNA levels were measured by real-time reverse transcriptase polymerase chain reaction (RT–PCR). The cellular source of OPN was analyzed using immunohistochemistry and in situ hybridization. Myocardial collagen volume fraction (CVF) was determined by digital planimetry. OPN, Col I and Col III mRNA levels were higher in DCM patients than in controls (P<0.01). OPN mRNA levels were positively correlated with Col I levels and CVF in DCM patients (OPN vs. Col I: r=0.60, P<0.01; OPN vs. CVF: r=0.52, P<0.001). Immunostaining of OPN was present in cardiomyocytes from DCM patients. In situ hybridization identified cardiomyocytes as the major source of OPN mRNA transcription in DCM patients. OPN and Col I mRNA levels were highly expressed in the DCM subgroup with large left ventricular (LV) end-systolic diameter (LVESD≥54.5 mm) or low LV ejection fraction (LVEF<29.5%). There was a weak positive correlation between OPN mRNA levels and LV end-systolic diameter (r=0.39, P<0.01). Levels of OPN mRNA were also negatively correlated with LV ejection fraction (r=–0.43, P<0.01).

Conclusions: These results suggest that OPN may play a pivotal role in the development of Col-I-induced cardiac fibrosis and dysfunction in human DCM.

Key Words: Collagen type I • Immunohistochemistry • In situ hybridization • Real-time polymerase chain reaction

Received July 20, 2004; Revised July 20, 2004; Accepted October 20, 2004


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 
The myocardial extracellular matrix (ECM) is composed of a collagen network that consists mainly of collagen types I (Col I) and III (Col III), which maintains the structural and functional integrity of the heart [1]. Alteration of the collagen network, such as a differential increase in Col I or Col III, may play an important role in myocardial dysfunction in the ischemic and nonischemic failing heart [2,3]. Several studies of dilated cardiomyopathy (DCM) have demonstrated that changes in collagen content were characterized primarily by an increase in the percentage of Col I [4,5]. While Col III forms an elastic network which stores kinetic energy as elastic recoil, Col I represents a stiff fibrillar protein which provides tensile strength. An increase in Col I protein levels may therefore impose increasing myocardial stiffness, compromising diastolic and systolic ventricular function in DCM [6].

Increased expression of osteopontin (OPN) in the heart has recently been shown to play an important role in myocardial remodeling by promoting collagen synthesis and accumulation due to Col I expression [7]. An experimental animal model has demonstrated that increased expression of OPN, an ECM protein, coincides with the development of heart failure [8]. OPN is an adhesive glycoprotein with an arginine–glycine–aspartic acid sequence [9]. Although first isolated from mineralized bone matrix, OPN has been shown to be synthesized by several cardiovascular cells, such as cardiac myocytes, microvascular endothelial cells and fibroblasts [10–12]. It has also been reported that immunoactivity of OPN was predominantly found in cardiac myocytes, and its levels were related to impaired left ventricular (LV) function in human DCM [13]. OPN can bind directly to Col I and interact with Col II, III, IV and V and with fibronectin [14,15]. Furthermore, OPN can affect the expression and activity of matrix metalloproteinase (MMP) [16]. It has therefore been proposed that myocardial OPN expression regulates the collagen network and may be involved in the process of cardiac remodeling by collagen synthesis and accumulation. However, no reports have yet investigated the relationship between myocardial OPN expression and alteration of collagen fibrillogenesis in human DCM.

In this study, we examined the expression of OPN and Col I and Col III mRNAs and analyzed myocardial fibrosis in myocardial tissues obtained from DCM patients and controls. We also examined the relationship between these mRNA expression levels and myocardial fibrosis and clinical severity of DCM.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 5. Conclusions
 References
 
2.1. Subjects
Endomyocardial tissues were obtained from 51 patients with DCM by right ventricular endomyocardial biopsy (39 males and 12 females; mean age=48.9±2.2 years). All patients underwent coronary angiography at the time of biopsy to exclude ischemic heart disease and other secondary cardiac diseases. The clinical diagnosis of DCM was made according to the World Health Organization/International Society and Federation of Cardiology Task Force criteria [17]. DCM patients had had New York Heart Association (NYHA) class IV heart failure over the 2 months prior to biopsy and were in NYHA class II (n=27) or III (n=24) at the time of examination. Echocardiography (Acuson Sequoia Echo 256, Siemens) was used to determine LV end-systolic diameter (LVESD) and LV ejection fraction (LVEF) at the time of biopsy. LVESD was measured by two-dimensionally directed M-mode echocardiography, and LVEF was derived from LV volumes using the area-length method. The DCM patients were receiving loop diuretics (n=41), digitalis (n=27), spironolactone (n=26, 25 to 50 mg/day), angiotensin-converting enzyme inhibitors (ACEIs: enalapril, 5 mg/day, n=19; benazepril, 5 mg/day, n=8; temocapril, 5 mg/day, n=1), angiotensin II type 1 receptor blockers (ARBs: candesartan, 8 mg/day, n=12; valsartan, 80 mg/day, n=6; losartan, 50 mg/day, n=1) or β blockers (n=21, carvedilol, 12.5 mg/day).

Control myocardial tissues were obtained by right ventricular endomyocardial biopsy from 15 subjects (12 male and 3 female; mean age=41.7±2.7 years) with suspected cardiac disorders such as myocardial deposition disease, hypertensive heart disease and hypertrophic cardiomyopathy on the basis of arrhythmia and echocardiographic changes such as premature beats and a slight increase in ventricular wall thickness. The resulting pathologic findings and close clinical examination failed to show any evidence of myocardial disease, including ischemic heart disease and hypertensive heart disease, or functional abnormality, and these subjects were thus designated as the control group. The clinical characteristics of the two groups are compared in Table 1. The study protocol was approved by our hospital ethics committee, and written informed consent was obtained from all subjects.


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  		Table 1 Baseline clinical characteristics of patients

 
2.2. Real-time quantitative reverse transcriptase polymerase chain reaction (RT–PCR)
Total RNA was extracted from biopsy samples by the acid guanidinium thiocyanate–phenol–chloroform method and treated with DNase I (GIBCO BRL) [18]. The purity of the extracted RNA was estimated by determining the ratio of absorbance at 260 nm to that at 280 nm. The sequences for primers and probes used were as follows: OPN: forward primer 5'-TCA ATT CAT CTG GAA TAC ATC CA-3', reverse primer 5'-TCA GCT TCC TTT TGC CAA AT-3' and probe 5'-AGA CAT ATT TCC CCC TAC CAA ATG TTC ATG-3', Col I: forward primer 5'-TTC CTT TGC ATT CAT CTC TCA A-3', reverse primer 5'-CAA GTG GAC CAA GCT TCC TT-3' and probe 5'-TTT TTA TCT TTG ACC AAC CGA ACA TGA CC-3', Col III: forward primer 5'-GTT CTC GTA AAA ACC CCG CT-3', reverse primer 5'-ATG CAT GTT TCC CCA GTT TC-3' and probe 5'-TTG ACC CTA ACC AAG GAT GCA AAT TGG-3'. These oligonucleotide primers and probes for OPN, Col I and Col III were chosen from published cDNA coding sequences (GenBank Nos D14813 [GenBank] , Z74615 [GenBank] and X14420 [GenBank] , respectively) [19–21]. For all samples, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was amplified using TaqMan GAPDH control reagents as an internal control (PE Biosystem, Foster City, CA, USA). The probe contained a fluorescent reporter (6-carboxyfluorescein) at the 5' end and fluorescent quencher (6-carboxytramethylrhodamine) at the 3' end. We analyzed mRNA expression levels using a real-time quantitative RT–PCR method with an ABI PRISM 7700 sequence detector (PE Applied Biosystems Division) as previously described [22]. The cDNA was synthesized and amplified by reverse RT–PCR using TaqMan EZ RT–PCR kit (PE Applied Biosystems Division). Amplification of human GAPDH on 10-fold serial diluted human control RNA (RNA concentration: 50 ng/µL to 5 pg/µL) using TaqMan GAPDH control reagents (PE Applied Biosytsems) were used to built in the standard curve. The cDNA was synthesized from extracted total RNA (100 ng) by reverse transcription at 60 °C for 30 min and then amplified with 40 cycles of PCR, with each cycle consisting of denaturation at 94 °C for 20 s and annealing and extension at 62 °C for 1 min. A quantitative PCR method was developed using detection and 5' nuclease assay by an ABI PRISM 7700 sequence detector (PE Applied Biosystems Division). To improve the accuracy of the real-time PCR method for quantification of OPN, Col I, Col III and GAPDH mRNAs, amplifications were performed by triplicate for each RNA sample. To account for variations in input RNA and RT efficiency, OPN, Col I and Col III levels were normalized to GAPDH expression in each sample. To account for PCR amplification of contaminating genomic DNA, a control without RT was included.

2.3. Immunohistochemistry
Immunostaining of OPN was performed on paraffin sections to determine the cellular source of OPN. Monoclonal antihuman osteopontin antibody (R&D system) was used as a primary antibody. The tissue sections were deparaffined and thoroughly dehydrated. After inhibition of endogenous peroxidase and blocking of nonspecific reactions, monoclonal antihuman osteopontin antibody was applied. Biotinylated mouse immunoglobulin was used as a secondary antibody. Peroxidase-labeled streptavidin (Histofine, MAX-PO kit, Nichiren) was applied and visualized using diaminobenzidine as a chromogen.

The specificity of the immunohistochemistry was confirmed by substituting the primary antibody with mouse IgG isotype control (Dako) on sections from patients with DCM.

2.4. In situ hybridization
In situ hybridization was performed on paraffin sections. Antisense oligonucleotide probe of OPN (5'-GGG CTG CTC TAG GAG CCA GAC CGT GG-3') was used for in situ hybridization [19]. Sense oligonucleotide probe of OPN was used as negative control. The probes were labeled with a 3'-biotinylated tail (Britati tail). For every specimen, we used 20-base poly-T oligonucleotide probe (Research Genetics) to examine the retention of mRNA in biopsy samples. Hybridization was performed with a MicroProbe staining system (Fisher Scientific). Tissue sections were placed on Probe ON Plus microscope slides (Fisher Scientific) and were rapidly dewaxed, cleared with alcohol, thoroughly rehydrated with Tris-based buffer at pH 7.4 (Universal Buffer, Research Genetics) and then digested with pepsin (1.25 mg/mL; Research Genetics) for 3 min at 105 °C. The probes were applied in a formamide-free diluent, and the slides were heated to 105 °C for 3 min and allowed to hybridize at 40 °C for 30 min. The sections were then washed with 2x SSC buffer (300 mmol/L NaCl and 30 mmol/L trisodium citrate, pH 7.0) and detected with alkaline phosphatase-conjugated streptavidin (Research Genetics). After hybridization, the products were washed in AP chromogen buffer, pH 9.5 (Research Genetics), and then visualized with fast red. The slides were counterstained with hematoxylin, air dried and then coverslipped for microscopic examination.

2.5. Collagen morphometry
Serial paraffin sections (5 µm thick) were stained with collagen-specific Sirius red stain (0.5% in saturated picric acid). Collagen volume fraction (CVF) was measured as a percentage of the circumference of each specimen using digital planimetry with a computer-associated morphometric program (KS 300, Kontron Elektronik, Munchen, Germany). In each section, CVF was analyzed in 9.2±0.7 fields (magnificationx50.0). The histomorphologic study was performed by two pathologists blinded to the other characteristics of the patients under study.

2.6. Statistical analysis
All values are presented as mean±S.E.M. The difference in mRNA expression was analyzed by unpaired t test. Pearson's correlation coefficients were used to examine the relationship between mRNA levels and CVF. A value of P<0.05 was considered statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 5. Conclusions
 References
 
3.1. Expressions of OPN, Col I and Col III mRNAs
Myocardial OPN mRNA levels were significantly greater in DCM patients than in controls (OPN/GAPDH ratio: 10.87±0.90 vs. 0.52±0.09, P<0.01). Levels of Col I and Col III mRNA expressions in DCM patients were also greater than these levels in controls (Col I/GAPDH ratio: 7.25±0.71 vs. 0.13±0.05, P<0.01; Col III/GAPDH ratio: 0.53±0.07 vs. 0.05±0.02, P<0.01). In DCM patients, myocardial Col I mRNA levels were higher than Col III mRNA levels (P<0.01). A positive correlation was found between OPN mRNA and Col I mRNA levels in DCM patients (r=0.60, P<0.01) (Fig. 1). There was no statistical correlation between Col III mRNA and OPN mRNA or Col I mRNA levels in DCM patients (Col III vs. OPN: r=0.01, P=0.94; Col I vs. Col III: r=–0.07, P=0.61).


Figure 1
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  		Fig. 1 Relationship between OPN mRNA and Col I mRNA levels in myocardium obtained from patients with DCM. Significant correlation: r=0.60, p<0.01.

 
3.2. Cellular source of OPN in myocardial tissues
To investigate OPN expression in myocardial tissues, immunohistochemistry was performed on specimens obtained from DCM patients and controls. Diffuse immunostaining of OPN was dominantly present in the cytoplasm of cardiomyocytes from DCM patients (Fig. 2a). No significant staining of OPN was seen in the interstitial space, endocardium or vascular endothelium. There was no evidence of nonspecific immunostaining in myocardium obtained from patients with DCM (Fig. 2b). OPN immunostaining was not present in any specimens from control subjects.


Figure 2
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  		Fig. 2 (a) OPN staining of myocardial tissue in DCM patients. OPN immunostaining was found in cardiac myocytes (arrows). (b) Immunostaining of isotype controls in myocardial tissues of DCM patients. (c) OPN mRNA hybridization signals were present in cardiomyocytes (arrows). (d) Poly-T massages were identified in cardiomyocytes in all specimens. Magnification: x100.

 
In situ hybridization in specimens from DCM patients with oligonucleotide probe for human OPN mRNA identified cardiac myocytes as the major source of OPN mRNA transcriptions in myocardial tissues (Fig. 2c). Poly-T message was also identified in all specimens (Fig. 2d). Control sections that were hybridized with sense oligonucleotide probe exhibited no hybridization signals.

3.3. Collagen morphometry
Myocardial CVF was significantly greater in patients with DCM than in controls (7.0±0.5% vs. 0.6±0.1%, P<0.01). A positive correlation was found between CVF and OPN mRNA or Col I mRNA levels (CVF vs. OPN: r=0.52, P<0.001; CVF vs. Col I: r=0.63, P<0.001) (Fig. 3). There was no statistical correlation between CVF and Col III mRNA levels (r=–0.12, P=0.39).


Figure 3
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  		Fig. 3 Relationship between CVF and (a) OPN or (b) Col I mRNA levels in patients with DCM. Significant correlation: CVF vs. OPN, r=0.52, P<0.001; CVF vs. Col I, r=0.63, P<0.001. CVF—collagen volume fraction.

 
3.4. Clinical outcomes
When DCM patients were divided into two subgroups by mean value of LV end-systolic diameter (LVESD=54.5 mm) and LV ejection fraction (LVEF=29.5%), both OPN and Col I mRNA levels were higher in the DCM subgroups with large LVESD (≥54.5 mm) or low LVEF (<29.5%) (Fig. 4). Levels of Col III mRNA expression did not differ significantly between the two DCM subgroups. There was a weak positive correlation between OPN mRNA levels and LVESD (r=0.39, P<0.01). Levels of OPN mRNA were also negatively correlated with LVEF (r=–0.43, P<0.01).


Figure 4
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  		Fig. 4 The levels of OPN and Col I mRNA expression in two subgroups of DCM. LVEF—left ventricular ejection fraction, LVESD—left ventricular end-systolic diameter.

 
When DCM patients were divided into two subgroups by NYHA class II or III, OPN, Col I and Col III mRNA levels did not differ between both subgroups (OPN/GAPDH ratio; 11.97±1.17 vs. 9.97±1.38, P=0.35: Col I/GAPDH ratio; 7.21±1.06 vs. 7.30±0.96, P=0.95: Col III/GAPDH ratio; 0.54±0.10 vs. 0.52±0.11, P=0.94). In the DCM subgroup (n=20), receiving combination treatment with spironolactone and either ACEIs or ARBs, OPN and Col I mRNA levels and CVF were significantly lower than in the subgroup not receiving this drug combination (n=31; OPN/GAPDH ratio; 7.21±0.79 vs. 13.23±1.22, P<0.001: Col I/GAPDH ratio; 4.97±0.79 vs. 8.73±0.98, P<0.01: CVF; 5.5±0.7% vs. 8.0±0.6%, P=0.01).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
5. Conclusions
 References
 
The most important findings of the present study are the following: (1) myocardial expression of OPN, Col I and Col III mRNAs was higher in DCM patients than in controls; (2) OPN mRNA levels were correlated with Col I mRNA levels and CVF in DCM patients; (3) OPN immunostaining and its mRNA transcription were localized in cardiomyocytes; (4) OPN and Col I mRNA levels were closely related to LV dimension and systolic dysfunction in DCM.

4.1. Myocardial expression of OPN, Col I and Col III
OPN has been shown to play an important role in regulating the synthesis and turnover of ECM proteins, including collagen, in an experimental mouse model [23]. In addition, an alteration in ECM protein expression may stimulate cardiac fibrosis and remodeling, leading to increased myocardial stiffness and dysfunction in the failing heart [2,3]. This study has demonstrated that expression of OPN mRNA was significantly elevated in myocardium obtained from patients with DCM and was related to Col I mRNA and CVF levels. An OPN knockout mouse model after myocardial infarction has shown a lack of collagen accumulation due to a marked decrease of Col I mRNA expression compared with wild-type mice [7]. OPN can directly increase binding to collagen, notably Col I, and regulate tissue remodeling [14]. Increased myocardial expression of OPN may therefore induce collagen synthesis and accumulation through an increase in Col I levels and may lead to progression of cardiac fibrosis and dysfunction in DCM. In our study, immunostaining for OPN was localized in cardiac myocytes in the myocardium from DCM patients. To confirm that this localization of OPN in cardiac myocytes was due to synthesis and not uptake, we performed in situ hybridization, which indicated the presence of OPN mRNA transcription in cardiac myocytes. Graf et al. have reported that cardiac myocytes are a prominent source of OPN expression in hypertrophied rat heart [24]. In accordance with our observations, Stawowy et al. also demonstrated immunoreactivity of OPN in cardiac myocytes in human DCM [13]. Thus, the cardiac myocyte is likely to be a major site of OPN expression in the heart in DCM.

This study has demonstrated a shift in the Col 1/Col III mRNA ratio for DCM patients (Col I/Col III, 13.7) compared with controls (Col I/Col III, 3.3). Moreover, Col I mRNA levels were positively correlated with CVF in myocardium of DCM patients, but Col III was not correlated with CVF. Several studies have shown that a differential increase in Col I and Col III leads to an increased Col I/Col III ratio (Col I>Col III levels) and induces myocardial collagen remodeling in DCM patients [4–6]. It may be possible that a shift in the myocardial ECM balance towards a predominance of Col I neosynthesis is involved in the increased myocardial strength and stiffness in human DCM.

4.2. OPN expression and LV function
Another important finding of our study is the high level of OPN mRNA expression with advanced LV dilatation and dysfunction in DCM patients. Another recent study has demonstrated a significant correlation between increased OPN immunoreactivity in cardiac myocytes and impaired LV function in DCM patients, although this study did not measure OPN mRNA levels [13]. These findings suggest that myocardial expression of OPN may be related to impaired LV function in human DCM. The stimulus for OPN expression in the myocardium in DCM remains unclear on the basis of this study. However, several stimuli, including inflammatory cytokines and angiotensin II, can stimulate OPN expression in cardiovascular cells [11,12]. Xie et al. have recently reported that tumor necrosis factor-{alpha} (TNF-{alpha}) acts synergistically with angiotensin II to increase OPN mRNA in rat cardiac fibroblasts [25]. Our previous studies have demonstrated that activation of the tumor necrosis factor-{alpha} (TNF-{alpha}) cascade against viral infection was associated with LV dysfunction in human DCM [22,26,27]. Ashkar et al. have reported that OPN was identified as a key cytokine that activates an immune response through viral infection [28]. This report is therefore consistent with a role for OPN in fibrosis or healing in response to injury, such as viral infection. Our study has also shown that clinical severity of DCM is related to Col I expression and CVF levels. These observations suggest that myocardial expression of OPN induces Col I-mediated cardiac fibrosis and leads to progressive ventricular dilatation and cardiac dysfunction in the DCM heart.

An additional finding from our study was the lower expression of OPN and Col I mRNAs and myocardial CVF in the DCM subgroup treated with a combination of spironolactone and either ACEIs or ARBs compared to the subgroup not receiving these drugs. An OPN knockout mouse model has recently demonstrated that OPN plays an important role in aldosterone-induced remodeling with effects on LV dilation, fibrosis and apoptosis [29]. It has also been reported that angiotensin II up-regulated OPN mRNA expression in cardiac fibroblasts and that administration of an anti-OPN antibody and ARB completely blocks the effect of angiotensin II [12]. In a hypertensive rat model, it has been shown that treatment with an ACEI prevented myocardial necrosis and fibrosis, thereby preventing OPN expression [8]. These observations suggest that the decrease in myocardial fibrosis with suppression of myocardial OPN expression may be enhanced by combined administration of spironolactone and either ACEIs or ARBs. Further prospective studies are needed to confirm this hypothesis.

4.3. Hypothetical role of OPN in DCM
Based on data from our previous reports and this present report, we speculate that the mechanism underlying OPN-induced fibrillogenesis in human DCM is as follows. Our previous study reported that the toll-like signaling pathway is triggered by viral pathogen and may be involved in activation of TNF-{alpha} cascade in DCM hearts [27]. Activated TNF-{alpha} cascade, through its converting enzyme, may be related to the clinical severity of DCM [22]. Cardiac-specific overexpressing TNF-{alpha} transgenic mouse models have demonstrated that TNF-{alpha} plays an important role in the process of LV remodeling, including alternation of ECM [30,31]. It has also been reported that myocardial ECM may be mainly regulated by OPN production in DCM hearts [8,13]. In vitro models have reported that OPN is time-dependently increased by TNF-{alpha} stimulation, suggesting a closed interaction between OPN and TNF-{alpha} synthesis [28,32]. In the present study, we have suggested that OPN-induced collagen fibrillogenesis may characterize LV stiffness and dysfunction in DCM patients. Therefore, our previous reports combined with the present report lead us to speculate that OPN production via activated TNF-{alpha} cascade may play an important role in the process of collagen fibrillogenesis in DCM hearts. Further studies will be required to confirm the cross-sectional relationship between TNF-{alpha}-induced OPN and collagen fibrillogenesis in human DCM.

4.4. Limitations
Although our study did not confirm that right ventricular OPN mRNA expression and collagen accumulation were directly reflected in left ventricular changes, no significant difference in OPN immunoreactivity between right and left ventricular tissues has been reported in failing human hearts [24]. It has also been reported that myocardial collagen proliferation induced by chronic aldosterone or angiotensin II infusion did not differ between the right and left ventricles [33]. These studies therefore show that left ventricular OPN mRNA expression and collagen volume may be reflected by these findings in the right ventricle.

Although OPN and Col I levels and myocardial CVF were found to be lower in DCM patients treated with spironolactone and either ACEIs or ARBs, these treatments have not undergone randomized prospective analysis, in addition, there were no data on drug-induced hemodynamic and clinical improvement in DCM patients leading to lower OPN and Col I expressions. Therefore, this study cannot confirm the capacity of these medications to decrease OPN, Col I and CVF levels in the myocardium.


   5. Conclusions
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
References
 
This study has shown that increased myocardial expression of OPN mRNA is associated with Col-I-induced cardiac fibrosis and with clinical severity in human DCM. These results suggest that OPN may play a pivotal role in cardiac fibrosis and dysfunction in human DCM.


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

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