Diastolic dysfunction in the hypertensive heart is an important clinical problem, partly as a result of the rigidity caused by myocardial fibrosis. Such fibrosis has been classified into replacement (secondary) and reactive (primary) types.1,2 In replacement fibrosis, necrosis of myocytes elicits acute inflammation and myocardial dropout is subsequently replaced by collagen fibers. In contrast, reactive fibrosis is characteristically observed in pressure overloaded hearts, in which collagen fibers increase in perivascular regions without loss of cells and eventually extend among individual cardiomyocytes.3 Recently, inflammation mediated by the renin-angiotensin II (Ang II)-aldosterone system, especially involvement of aldosterone, has received much attention as a trigger of reactive fibrosis4-7; however, the molecular pathways remain to be detailed.
Fibrotic lesions do not form by abrupt deposition of collagen molecules; they form through multiple steps of synthesis and degradation of various matrix proteins, including tenascin-C. Tenascin-C is an extracellular glycoprotein with strong bioactivity, transiently expressed during embryonic development, wound healing, and cancer invasion.8-10 Accumulating evidence suggests that tenascin-C may be a key regulator in an early step of the fibrotic process in various tissue.11-13 In the heart, tenascin-C is sparsely detected in normal adults but becomes expressed in the pathological myocardium closely associated with inflammation and tissue remodeling.14-21 Based on this specific expression, we recently reported that tenascin-C can be a clinical marker for active inflammation18,19 and ventricular remodeling.21,22
The aim of the present study was to clarify the involvement of tenascin-C in the progression of reactive fibrosis in the hypertensive heart and the regulatory mechanism of tenascin-C expression, focusing on the angiotensin II-aldosterone system. We used a mouse model of hypertensive cardiac fibrosis with infusion of Ang II in which reactive fibrosis develops in perivascular regions of myocardium without necrosis of cardiomyocytes or scar formation. First, we examined histologic changes and gene expression of collagen and tenascin-C in the mouse myocardium with immunohistochemistry, in situ hybridization, and quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Next, to study the involvement of aldosterone and inflammation in regulation of tenascin-C synthesis, the effect of an aldosterone receptor blocker, eplerenone, and expression of proinflammatory/fibrotic mediators, transforming growth factor beta (TGFβ), interleukin-1 beta (IL-1β), and platelet-derived growth factor alpha (PDGF) in the model mouse were examined. Furthermore, the direct effects of these factors on tenascin-C synthesis were studied using cultured cells.
MATERIALS AND METHODS
Female 7-week-old BALB/c mice were used. All (n = 95) were given 1% NaCl drinking water and assigned to 1 of the following 5 groups: (1) vehicle control mice (n = 25); (2) Ang II-treated mice (n = 20); (3) Ang II/eplerenone-treated mice (n = 20); (4) eplerenone-treated mice (n = 19); and (5) aldosterone-treated mice (n = 11). A microosmotic pump (model 1002; Durect Co, Cuperino, CA) containing 0.1 mL of the vehicle (0.9% NaCl: 99.7% acetic acid = 15:1), 2.83 mg/mL of Ang II (SIGMA, St. Louis, MO), or 1 mg/mL of aldosterone (ACROS ORGANICS, NJ) was subcutaneously inserted under the back skin of each mouse for treatment for 4 weeks. The approximate doses of Ang II and aldosterone administered were 560 and 200 ng/kg body weight/min, respectively. The microosmotic pumps were replaced every 2 weeks. Eplerenone (Pfizer, New York), an aldosterone receptor blocker, was orally administered in the diet at 1.67 g/kg chow (the estimated dose of eplerenone was 250 mg/kg body weight/day). Body weights and blood pressure were examined every week. Blood pressure measurements were performed with the BP-98A (Softron, Tokyo, Japan) tail cuff system while the animals were conscious. All experimental protocols conformed to international guidelines and were approved by the Mie University Animal Experiment and Care Committee.
After the 4 week infusion treatment period, the hearts were excised, fixed in 4% paraformaldehyde, and embedded in paraffin. For histopathological analysis, sections were cut at 3 μm.
Immunostaining of tissue sections was performed as previously described.16 In brief, after treatment with pepsin for 10 minutes or heating in an autoclave for antigen retrieval, sections were incubated with either an antitenascin-C polyclonal rabbit antibody,16 an anti-Mac-3 rat monoclonal antibody (Pharmingen, San Diego, CA; working dilution 1:10) for identification of macrophages, an anti-PDGF-A polyclonal rabbit antibody (Santa Cruz Biotechnology, Santa Cruz, CA; working dilution 1:20), an anti-PDGF-B polyclonal rabbit antibody (Santa Cruz Biotechnology, Santa Cruz, CA; working dilution 1:20), a rat monoclonal antimurine PDGFR-α antibody (Clone APA5),23 or a rat monoclonal antimurine PDGF receptor-β antibody (Clone APB5).24 Three independent fields in perivascular regions of myocardium from each mouse were examined under a 20× objective lens, and Mac-3-positive cells, PDGF-A, and PDGF-B positive cells were counted.
Sirius red-stained slides were used to quantify myocardial collagen with an optical microscope (BH2, Olympus, Tokyo, Japan). Three independent perivascular fields from each mouse were visualized under a 20× objective lens and photographed with a Fujix Digital Camera HC 300Z/OL (Olympus). The images were analyzed using NIH Image, and percentage areas of perivascular fibrosis were calculated.
Quantitative Real-Time RT-PCR
Total RNA was extracted from fresh mouse left-ventricular tissues using ISOGEN (NipponGene, Toyama, Japan), and single-strand complementary DNA (cDNA) synthesis was performed by oligo (dT)15 priming from 1 mg aliquots in a final volume of 20 mL with a single-strand cDNA synthesis kit for RT-PCR (Roche Diagnostics, Germany) according to the manufacturer's instructions. Quantitative analysis of target messenger RNA (mRNA) expression was performed with the TaqMan real-time RT-PCR and a relative standard curve method using Light Cycler Software Ver. 3.5 (Roche). The GAPDH mRNA level was quantified as an internal control. The primers and probes for mice are listed in Table 1.
In Situ Hybridization
Preparation of digoxigenin (DIG)-labeled mouse tenascin-C cRNA probes and in situ hybridization were performed as previously described.16
Cardiac fibroblasts were obtained from ventricles of Balb/c mice and grown in Iscove's modified Dulbecco's media (IMDM) with 10% fetal bovine serum as previously described.20 Experiments were performed on secondary cultures. Cells (3 × 105 cells/well) were plated in MULTIWELL 6-well plates (Becton Dickinson, Franklin Lakes, NJ) for 48 hours in serum-free IMDM media, then treated with Ang II (0 to 10−5 mol/L), aldosterone (0 to 10−6 mol/L), IL-1β (R&D Systems, Oxon, UK; 0 to 30 ng/mL), TGF-β1 (Roche Diagnostics; 0 to 10 ng/mL), or PDGF-BB (R&D Systems; 0 to 100 ng/mL) for 6 hours. Some cells were pretreated with eplerenone (10−8, 10−7, 10−6 mol/L) for 1 hour and then stimulated with Ang II (10−7 mol/L). To assess the combined effects of cotreatment with Ang II and aldosterone, cardiac fibroblasts were pretreated with aldosterone (10−6 mol/L) for 6 hours and then incubated with Ang II (10−7 mol/L) for 6 hours. Total RNA was isolated using ISOGEN, and the relative tenascin-C mRNA levels were determined by quantitative real-time RT-PCR.
All data are expressed as means ± standard deviations (SDs). Numeric data were statistically evaluated by 1-way analysis of variance, followed by the Tukey-Kramer method for multiple comparisons. A P value less than 0.05 was considered to be statistically significant.
Systolic Blood Pressure and Body Weight
Blood pressure was elevated within 1 week after the onset of Ang II treatment and remained significantly increased compared with that of the control mice for up to 4 weeks (Fig. 1A). No significant difference was observed between Ang II-treated and Ang II/eplerenone-treated groups. Blood pressure remained normal in vehicle-control, aldosterone-receiving, and eplerenone-alone mice. Increase in body weight was similar in all experimental groups during the study, and no statistically significant intergroup differences were observed at any time (Fig. 1B).
Myocardial Fibrosis and Expression of Tenascin-C
In Ang II-treated and aldosterone-treated mice, the volume of perivascular collagen fibers in myocardium was clearly increased, extending into spaces between individual myocytes. Eplerenone treatment almost completely abolished Ang II-induced perivascular fibrosis, and aldosterone treatment induced fibrotic changes (Fig. 2A). Necrosis of cardiomyocytes and scar formation were not found in our models.
Cardiac expression of tenascin-C was immunohistochemically detected in perivascular regions in Ang II-treated mice myocardium where collagen fibers had accumulated, whereas no immunostaining was observed in the control mice. Eplerenone reduced the tenascin-C expression induced by Ang II treatment, and aldosterone treatment induced tenascin-C expression (Fig. 2B).
In situ hybridization analysis for tenascin-C mRNA in Ang II-treated mice demonstrated signals in interstitial fibroblasts residing around the vascular tunica adventitia (Fig. 2C, D). Vascular endothelial cells, vascular smooth muscle cells, and cardiomyocytes were negative for tenascin-C mRNA.
Quantitative analysis of percentage fibrotic areas (Fig. 2E) confirmed a significant increase in Ang II-treated (12.9 ± 2.5%, P < 0.001) and aldosterone-treated (7.2 ± 2.0%, P < 0.001) mice compared with control mice (3.2 ± 0.9%). Fibrotic areas in eplerenone/Ang II mice were significantly attenuated (4.3 ± 1.8%, P < 0.001) as compared with Ang II-alone. Real-time RT-PCR analysis (Fig. 2F) showed that both Ang II and aldosterone significantly upregulated the mRNA levels for collagen type Iα2 (2.6 ± 1.0 and 1.8 ± 0.3-fold, P < 0.01, respectively) and for IIIα1 (3.2 ± 2.1-fold, P < 0.05 and 2.8 ± 0.4-fold, P < 0.001, respectively) when compared with the control mice (1.0 ± 0.4 and 1.0 ± 0.3-fold, respectively). Eplerenone treatment significantly reduced this upregulation of collagen Iα2 and IIIα1 gene expression (to 1.3 ± 0.3-fold, P < 0.05 and 1.0 ± 0.3 fold, P < 0.05), when compared with Ang II-alone mice (2.6 ± 1.0 fold, 3.2 ± 2.0-fold, respectively). Parallel to the change in collagen gene expression, tenascin-C mRNA levels in Ang II-treated and aldosterone-treated groups were significantly upregulated (11.2 ± 5.8-fold increase for Ang II, P < 0.05 and 2.6 ± 0.6-fold for aldosterone, P < 0.001) compared with the control mice (1.0 ± 0.2). Eplerenone treatment significantly abrogated the induction of tenascin-C expression by Ang II (2.3 ± 1.3-fold, P < 0.05).
Immunohistologic analysis demonstrated many Mac 3 positive macrophages to have accumulated in the perivascular spaces in Ang II-treated mice, whereas only a few macrophages were observed in control mice (14.3 ± 3.4 vs 1.8 ± 0.9 cells/optic field, P < 0.05; Fig. 3A). In Ang II/eplerenone-treated mice, the number of macrophages was significantly reduced (4.5 ± 2.9 cells/optic field, P < 0.05) as compared with the Ang II-alone case. Aldosterone treatment also caused an increase of the number of macrophages (6.7 ± 1.25 cells/optic field, P < 0.01).
Expression of Cytokines and Growth Factors
Immunostaining demonstrated that Ang II treatment significantly increased the numbers of PDGF-A-(Fig. 3B) and PDGF-B-(Fig. 3C) positive cells in the perivascular region as compared with the controls (17.83 ± 3.37 vs 7.11 ± 1.67 cells/optic field, P < 0.01, 32.3 ± 7.2 vs 8.23 ± 3.35 cells/optic field, P < 0.001, respectively). Aldosterone treatment also significantly increased the number of PDGF-A and -B positive cells (12.72 ± 0.97 vs 7.11 ± 1.67 cells/optic field, P < 0.01, 17.7 ± 3.86 vs 8.23 ± 3.35 cells/optic field, P < 0.001, respectively). In Ang II/eplerenone-treated mice, the numbers of PDGF-A and PDGF-B positive cells were significantly reduced (8.39 ± 1.46 cells/optic field, P < 0.01, 10.7 ± 1.63 cells/optic field, P < 0.001, respectively) as compared with the Ang II-alone case. Perivascular fibroblasts in Ang II- or aldosterone-treated mouse heart upregulated the expression of PDGF receptor α (Fig. 3D) but did not express PDGF receptor β (Fig. 3E). No expression of either receptor was detected in the control group.
Real-time RT-PCR analysis (Fig. 4) showed that Ang II and aldosterone treatment significantly upregulated the mRNA levels for TGF-β1 (1.74 ± 0.20-fold, P < 0.05 and 1.49 ± 0.18-fold, P < 0.05, respectively; Fig. 4A) in the mouse myocardium when compared with the control mice (1.0 ± 0.35-fold). Eplerenone treatment significantly reduced TGF-β1 gene upregulation induced by Ang II treatment (0.67 ± 0.11-fold, P < 0.05). In contrast, no significant change of mRNA level of IL-1β, another major inflammatory mediator from macrophages, was observed in any of the groups (Fig. 4B).
Effects of Ang II, Aldosterone, and Inflammatory/Fibrotic Cytokines on Tenascin-C Synthesis by Cardiac Fibroblasts in Culture
The direct effects of Ang II and aldosterone on tenascin-C gene expression of cardiac fibroblasts were examined in culture by quantitative real-time RT-PCR (Fig. 5). Ang II (0 to 10−5 mol/L) significantly increased tenascin-C mRNA levels in a dose-dependent manner, and the expression level reached a peak at the concentration of 10−7 mol/L (2.1 ± 0.7-fold increase, Fig. 5A). In contrast, addition of aldosterone (0 to 10−6 mol/L) did not significantly affect tenascin-C expression levels at any of the concentrations examined (Fig. 5B). Eplerenone did not significantly influence Ang II-induced tenascin-C expression, and no synergism was evident with Ang II (10−7 mol/L) in the presence of aldosterone (10−8 mol/L, Fig. 5C).
To identify possible mediators of upregulation of tenascin-C expression in cardiac fibroblasts, cells were treated with PDGF-BB (Fig. 5D) and TGF-β (Fig. 5E). These factors caused a significant increase of tenascin-C expression in a dose-dependent manner.
Possible Involvement of Tenascin-C in Reactive Fibrosis of the Hypertensive Heart
Tenascin-C has been proposed to promote fibrosis because its expression is upregulated in various fibrogenic processes such as liver fibrosis,25 lung fibrosis,12 skin wound healing,11 and scar formation after myocarditis.17,18 Directly supporting this possibility, we have recently reported that locally applied tenascin-C accelerates collagen fiber formation in aneurysmal cavities in a rat model26 and that deficiency of tenascin-C significantly attenuates liver fibrosis in an immune-mediated chronic hepatitis mouse model.13 In the present study, we demonstrated that tenascin-C is not detected in the normal myocardium but becomes markedly upregulated in perivascular fibrosing areas in the Ang II-induced hypertensive mouse heart and that the expression level parallels the extent of fibrosis. These findings suggest that tenascin-C may be involved in the progression of reactive fibrosis, and its elevated expression might be a marker for active progression of the fibrosis in the hypertensive heart.
MECHANISM OF UPREGULATION OF TENASCIN-C GENE EXPRESSION
Previous studies demonstrated that expression of tenascin-C is upregulated with vascular remodeling in pulmonary hypertension27,28 and spontaneous hypertensive rats29 and that mechanical stress is an important tenascin-C-inducing factor (reviewed in reference 8). In this study, we found that upregulated expression of tenascin-C in Ang II-induced hypertensive mice was blocked by an aldosterone blocker, eplerenone, without affecting the blood pressure level, which suggests that Ang II may induce tenascin-C expression in myocardium through an aldosterone-dependent pathway but independent of blood pressure. Based on our in situ hybridization analysis demonstrating that the source of tenascin-C was cardiac fibroblasts in the perivascular region, we speculated that aldosterone might stimulate interstitial fibroblasts to synthesize tenascin-C. However, aldosterone did not induce tenascin-C synthesis in cardiac fibroblasts in culture, although Ang II enhanced tenascin-C expression as reported for other types of cells.29,30 Although it remains controversial whether cardiac fibroblasts express mineral corticoid receptors,31-33 several reports have suggested synergism between Ang II and aldosterone34,35 because of induction of Ang II receptor levels36 or receptor binding37 by the latter. However, neither addition of aldosterone nor blocking with eplerenone exerted any influence on tenascin-C expression induced by Ang II in the present study. Therefore, it seems likely that aldosterone facilitates tenascin-C gene expression in vivo not through a direct action on cardiac fibroblasts but by actions on other factors secreted by other cells.
There is a growing body of evidence that Ang II/aldosterone treatment induces inflammation and accumulation of macrophages in perivascular regions in the myocardium.5,6 Generally, macrophages are important regulators in inflammation in various tissue and the main source of fibrogenic mediators such as IL-1, TGF-β, and PDGF (reviewed in reference 38). In the present study, Ang II infusion caused accumulation of macrophages and upregulation of PDGF-A, -B, PDGF receptor α, and TGF-β1 in mouse hearts. These changes were inhibited by eplerenone, and their extent correlated with the expression level of tenascin-C. In culture, TGF-β1 and PDGF upregulated tenascin-C expression by cardiac fibroblasts. Taken together, it seems likely that aldosterone elicits inflammatory reaction in perivascular regions in Ang II-induced hypertensive mouse hearts, which might, in turn, induce tenascin-C synthesis of fibroblasts partly through 2 signaling pathways mediated by TGFβ and PDGF-A-B/PDGF-receptor α.
Although further studies are necessary to elucidate the complex multistep molecular pathways involved, induction of tenascin-C by aldosterone in the hypertensive heart might be a key step in perivascular fibrosis and thus a prime target for therapy.
The present results suggest involvement of tenascin-C in hypertensive cardiac fibrosis and that blockade of mineralocorticoid receptor with eplerenone reduces expression of tenascin-C by reducing inflammatory reaction, subsequently resulting in attenuation of perivascular fibrosis.
This study was supported by a grant-in-aid for scientific research (No. 17590725) from the Ministry of Education, Culture Sports, Science and Technology of Japan to K.I.-Y and by a grant for Intractable Disease from the Ministry of Health, Labor and Welfare of Japan to K.I.-Y and M. H. Part of the study was also supported from a grant by Pfizer, Inc.
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