Background/Aims: Cardiac interstitial fibrosis is an abnormality of various cardiovascular diseases, including myocardial infarction, hypertrophy, and atrial fibrillation, and it can ultimately lead to heart failure. However, there is a lack of practical therapeutic approaches to treat fibrosis and reverse the damage to the heart. The purpose of this study was to investigate the effect of long-term aspirin administration on pressure overload–induced cardiac fibrosis in mice and reveal the underlying mechanisms of aspirin treatment. Methods: C57BL/6 mice were subjected to transverse aortic constriction (TAC), and treated with 10 mg·kg-1·day-1 of aspirin for 4 weeks. Masson staining and a collagen content assay were used to detect the effects of aspirin on cardiac fibrosis in vivo and in vitro. Western blot and qRT-PCR were applied to examine the impact of aspirin on extracellular signal-regulated kinases (Erks), p-Akt/β-catenin, SerpinE2, collagen I, and collagen III levels in the mice heart. Results: Aspirin significantly suppressed the expression of α-smooth muscle actin (α-SMA; 1.19±0.19-fold) and collagen I (0.95±0.09-fold) in TAC mice. Aspirin, at doses of 100 and 1000 µM, also significantly suppressed angiotensin II-induced α-SMA and collagen I in cultured CFs. The enhanced phosphorylation of Erk1/2 caused by TAC (p-Erk1, 1.49±0.19-fold; p-Erk2, 1.96±0.68-fold) was suppressed by aspirin (p-Erk1, 1.04±0.15-fold; p-Erk2, 0.87±0.06-fold). SerpinE2 levels were suppressed via the Erk1/2 signalling pathway following treatment with aspirin (1.36±0.12-fold for TAC; 1.06±0.07-fold for aspirin+TAC). The p-Akt and β-catenin levels were also significantly inhibited in vivo and in vitro. Conclusions: Our study reveals a novel mechanism by which aspirin alleviates pressure overload-induced cardiac interstitial fibrosis in TAC mice by suppressing the p-Erk1/2 and p-Akt/β-catenin signalling pathways.

Cardiac fibrosis is an important contributor to the development of cardiac dysfunction in diverse pathological conditions. It can be induced by pressure overload, ischemia, and other pathologies [1]. Pressure overload activates a wide variety of signalling pathways in cardiac tissue. For example, increased wall stress leads to the local release of ligands, such as angiotensin II (Ang II) and endothelin-1 [2]. A chronic elevation of Ang II levels is associated with persistent hypertension, myocardial hypertrophy, fibrosis, and adverse remodelling [3]. Fibrosis then exerts an adverse impact on the cardiac electrical properties.

The purpose of this study was to determine whether aspirin could prevent the development of myocardial fibrosis and whether MAPK-extracellular signal-regulated kinase (Erk1/2) and Akt signalling are involved in this process. Acetylsalicylic acid (aspirin), a kind of non-steroidal anti-inflammatory drug (NSAID), is commonly used to treat cardiovascular disease (CVD) because of its efficacy in treating and preventing both primary and secondary cardiac events. Aspirin can inhibit platelet prostaglandin H synthase (also called cyclo-oxygenase, or COX) by decreasing the synthesis of thromboxane A2, a potent platelet stimulant and vasoconstrictor, and consequently, preventing platelet-mediated atherosclerotic plaques from rupturing and causing full vascular occlusion [4]. Our previous study found that aspirin normalized cardiac function and significantly reduced hypertrophic phenotypes induced by transverse aortic constriction (TAC). Aspirin could prevent the development of cardiac hypertrophy via the down-regulation of β-catenin and Akt, which are significantly activated in pressure overload-induced cardiac hypertrophy. These results showed that aspirin possesses significant anti-hypertrophic properties at clinically relevant doses. In this study, we found that low-dose aspirin attenuated cardiac interstitial fibrosis induced by TAC. However, the molecular mechanism remains elusive.

Pressure overload–induced cardiac fibrosis in vivo

Healthy wild-type male mice (C57BL/6) and neonatal Wistar rats were bought from the Experimental Animal Center of Affiliated Second Hospital of Harbin Medical University. Cardiac hypertrophy was induced by TAC in C57BL/6 mice (20-22 g), as described previously [5]. Briefly, the mice were anesthetized using phenobarbital (50 mg·kg-1, intraperitoneally) and were subjected to either TAC or a sham control operation [6]. The TAC procedure consisted of a 7.0 nylon suture being placed around the transverse aorta between the innominate and left carotid arteries. The mice were examined 4 weeks following the surgery. All experimental procedures performed in the studies involving animal participants were in accordance with the Institutional Animal Care and Use Committee of Harbin Medical University, China. All the rats were treated in accordance to the guideline for the Care and Use of Laboratory Animals published by the U.S. National Institute of Health.

In vivo aspirin treatment in mice

Aspirin was purchased from Sigma (Sigma-Aldrich Corp., St. Louis, USA). The mice were then randomly divided into three groups for the subsequent experiments: control group (n = 10), TAC group (n = 10), and TAC treated with aspirin group (TAC+ASA, n = 10). The equivalent animal daily dosage (10 mg·kg-1·day-1) [7] was approximated according to a human dosage of 75 mg·day-1 (equivalent to 1 mg·kg-1·day-1 in humans with an assumed average human weight of 70 kg) [8]. The equivalent animal daily dose (≈ 10 mg·kg-1·day-1), as derived from the body surface area by the Reagan and Nihal equation, was administered to the animal by oral gavage. The controls included non-aspirin-treated TAC and sham control operated mice that received an equivalent volume of vehicle (phosphate buffer solution; PBS). Aspirin was administered both 4 days before the experiments and for 4 consecutive weeks after TAC surgery at daily oral doses of 10 mg·kg-1.

Isolation and culture of cardiac fibroblasts

Cultured cardiac fibroblasts (CFs) were obtained from neonatal Wistar rats, as previously described [9]. Briefly, CFs were separated by the removal of cardiac myocytes via the selective adhesion of non-myocytes at a 1.5-h pre-plating interval. Cardiac fibroblasts were maintained in Dulbecco’s modified Eagle medium (DMEM), supplemented with penicillin, streptomycin, and foetal bovine serum (10% v/v).

In vitro treatment with aspirin

The CFs were randomly divided into seven groups: control; Ang II (50 nM); aspirin (10 µM, 100 µM, 1000 µM) + Ang II; Dickkopf-1 (DKK, 200 nM) + Ang II; and U0126 (10 µM) + Ang II. Dickkopf-1 (DKK-1) is a specific Wnt signalling inhibitor. Dickkopf-1 and Ang II were purchased from Sigma (Sigma Aldrich, St Louis, USA). U0126 has been shown to be a highly selective inhibitor of MEK1/2 (Erk Activator Kinase 1/2). The MEK inhibitor U0126 was purchased from Cell Signaling (Danvers, MA, USA).

Western blot analysis

The total amounts of protein from the fibroblasts or myocardial tissue were resolved using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (Millipore, Bedford, MA) as described in detail previously [10]. Anti-β-catenin, anti-TGF-β1, anti-p-Erk1/2(Phospho-p44/42 Erk1/2), anti-T-Erk1/2(Total p44/42 Erk1/2), and anti-phospho-Akt (Ser473) were obtained from Cell Signaling (Danvers, MA, USA). Anti-col1a, anti-FN1, and anti-α-smooth muscle actin (SMA) were obtained from Santa Cruz (Santa Cruz, CA, USA). Anti-total-Akt (60203-2-Ig) were obtained from PROTEINTECH (Hubei, P.R.C). Membranes were incubated at 4 °C overnight with the specified primary antibody: β-MHC (rabbit, 1: 1000 dilution), β-catenin (rabbit, 1: 1000), p-Akt (rabbit, 1: 200), and GAPDH (mouse, 1: 500). After being washed three times with PBS-Tween 20 (PBST), the membranes were incubated with Alexa Fluor® fluorescence-conjugated goat anti-rabbit or anti-mouse IgG(H+L) (1: 10, 000 dilution, Invitrogen). Membranes were scanned using an Odyssey Imaging System (LI-COR Bioscience, Lincoln, NE). Western blot bands, such as β-catenin, TGF-β1, and col1a, were measured using the Odyssey software v1.2 and normalized using GAPDH as a control. Western blot bands of p-Erk1/2(Phospho-p44/42 Erk1/2) were normalized using the bands of T-Erk1/2 as a control. P-Akt were normalized using the bands of T-Akt as a control.

Masson staining and histological analysis of collagen deposition

Myocardium samples from the normal and TAC model mice were taken from the left ventricle immediately after the mice were killed, and fixed in 4% paraformaldehyde solution. Masson’s trichrome staining was used to evaluate the collagen deposition. The collagen was stained blue. All quantitative evaluations were carried out using the ImagePro Plus software (version 6.0, Media Cybernetics, Bethesda, MD).

Quantitative real-time RT-PCR

Total RNA was extracted using TRIZOL reagent (Invitrogen, Carlsbad, CA, USA) from cultured neonatal cardiac fibroblasts and cardiac tissue, according to the manufacturer’s instructions. Real-time quantitative PCR analysis was performed using an Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA). The expression level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. The primers used in real-time quantitative PCR analysis were as follows: collagen 1 (Col1)a1 (forward 5ʹ-AAGAAGACATCCCTGAAGTCA-3ʹ; reverse 5ʹ-TTGTGGCAGATACAGATCAAG-3ʹ); Col3a1 (forward 5ʹ-TTGGGATGCAGCCACCTTG-3ʹ; reverse 5ʹ-CGCAAAGGACAGATCCTGAG-3ʹ); GAPDH (forward 5ʹ-GGGGCTCTCTGCTCCTCCCTG-3ʹ; reverse 5ʹ-CGGCCAAATCCGTTCACACCG-3ʹ). The relative value of the control sample was obtained using the 2-∆∆CT method.

Measurement of collagen production

Collagen production was detected using a Sircol soluble collagen assay kit (Biocolor Ltd., Northern Ireland, U.K.) according to the manufacturer’s protocol. The Sircol assay is a dye-binding method designed for the analysis of acid and pepsin-soluble collagen. Briefly, the collagen produced by CFs was dissolved in cold acid. Then, the Sircol Dye Reagent was used to stain the dissolved collagen. Absorbance was recorded using a microplate reader (Sunrise, Switzerland) at 555 nm, and the collagen content was calculated.

Immunofluorescence

The intracellular p-Akt, TGF-β1, and α-SMA expression levels were observed via immunofluorescence. In brief, treated and untreated CFs were fixed in 4% paraformaldehyde, blocked with 1% bovine serum albumin, incubated with antibody (1 h at 22 °C, 16 h at 4 °C), and washed in TBST three times (Tris-Buffered Saline containing 0.05% Tween 20; ×3). The cells were then incubated with Alexa Fluor 488-conjugated secondary antibodies (1: 1000, Invitrogen, green fluorescence) or Alexa fluor 594 (1: 1000, Invitrogen, red fluorescence). Nuclei were counterstained with DAPI (4’, 6-diamino-2-phenylindole, 1: 50, Invitrogen, blue fluorescence). Immunofluorescence was visualized under a fluorescence microscope (Nikon 80i, Nikon Corporation).

Enzyme-linked immunosorbent assay (ELISA)

The CFs and cell culture supernatant were collected. The levels of β-catenin and TGF-β1 were measured using ELISA kits according to the manufacturer’s instruction (Elabscience Biotechnology, Wuhan, China).

Statistical analysis

Group data are expressed as the mean ± SEM. Statistical comparisons among the multiple groups were performed using one-way analysis of variance (ANOVA). A two-tailed p<0.05 indicated a statistically significant difference. Statistical values were calculated using the SPSS 10.0 software and illustrated using GraphPad Prism 5.0.

Aspirin reduces pressure overload-induced cardiac interstitial fibrosis in TAC mice

In our previous study, we confirmed that aspirin attenuates cardiac hypertrophy and alleviates cardiac remodelling [6]. This study demonstrated that aspirin reduced cardiac interstitial fibrosis in transverse aortic constriction (TAC) mice. Transverse aortic constriction-induced pressure overload was performed as described previously 10. Extracellular matrix deposition in the left ventricle of TAC mice was assessed using Masson’s trichrome staining. The interstitial fibrotic area of the TAC mice was significantly greater than that of the sham control animals (TAC, 5.56±2.98% versus sham control, 0.55±0.35%, p<0.01) (Fig. 1A, B). After 4 weeks of treatment with aspirin (10 mg·kg-1), the fibrotic area in the TAC mice was reduced (TAC+ASA, 2.05±1.00%, p<0.05) (Fig. 1A, B). The effect of aspirin on the collagen content was detected by a collagen assay of the mouse myocardium, which showed that the collagen content in TAC mice increased approximately 1.34±0.33-fold compared with the control group (Fig. 1C) and that treatment with aspirin led to a reduction in the collagen content (TAC+ASA, 0.99±0.13-fold, p<0.05). Western blotting was used to examine the protein expression levels of α-SMA and collagen I (Col1). The α-SMA and col1 levels significantly increased by 1.69-fold and 1.57-fold in the TAC mice compared with the sham control animals. Aspirin significantly suppressed the expression of α-SMA (1.19±0.19-fold) and col1 (0.95±0.09-fold) (Fig. 1D, n=8). Col1a1 and Col3a1 mRNA were examined using qRT-PCR. Col1a1 and Col3a1 were found to have increased by 2.36-fold and 1.85-fold, respectively, after TAC surgery compared with the sham control group (p<0.05). Treatment with aspirin significantly attenuated the mRNA levels of Col1a1 and Col3a1 (Fig. 1E).

Fig. 1.

Aspirin alleviates pressure overload-induced cardiac interstitial fibrosis in mice with transverse aortic constriction (TAC). A. Representative pictures of Masson trichrome-stained sections. Collagen is stained blue. (upper picture: magnification200×, lower picture: magnification 400×); Scale bar indicates 50 µM. n=6. B. Collagen deposition was quantified via automated image analysis and expressed as a percentage of the fibrotic area. C. Effects of aspirin on collagen content in TAC mice were determined using a Sircol soluble collagen assay. D. Effects of aspirin on the collagen 1 and α-smooth muscle actin (α-SMA) protein levels in the hearts of TAC mice. Western blot analysis of myocardial lysates was from 8 mice, with each lysate from one animal, n=8. E. Effects of aspirin on the COL1A1 and COL3A1 mRNA levels in TAC mice. *p<0.05 vs sham control; + p<0.05 vs TAC.

Fig. 1.

Aspirin alleviates pressure overload-induced cardiac interstitial fibrosis in mice with transverse aortic constriction (TAC). A. Representative pictures of Masson trichrome-stained sections. Collagen is stained blue. (upper picture: magnification200×, lower picture: magnification 400×); Scale bar indicates 50 µM. n=6. B. Collagen deposition was quantified via automated image analysis and expressed as a percentage of the fibrotic area. C. Effects of aspirin on collagen content in TAC mice were determined using a Sircol soluble collagen assay. D. Effects of aspirin on the collagen 1 and α-smooth muscle actin (α-SMA) protein levels in the hearts of TAC mice. Western blot analysis of myocardial lysates was from 8 mice, with each lysate from one animal, n=8. E. Effects of aspirin on the COL1A1 and COL3A1 mRNA levels in TAC mice. *p<0.05 vs sham control; + p<0.05 vs TAC.

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Aspirin attenuates collagen production in cultured cardiac fibroblasts

Markers of myocardial fibrosis, including TGF-β1, FN1 (fibronectin 1, a glycoprotein component of the extracellular matrix), and Col1 were examined using western blot analysis. TGF-β1 is a well-established pro-fibrotic cytokine found in cardiac fibroblasts that mediates collagen production [9]. Aspirin, at doses of 100 and 1000 µM, markedly inhibited the expression of TGF-β1, which had been induced by Ang II (Fig. 2A). Aspirin also significantly suppressed the expression of FN1 at doses of 100 and 1000 µM (Fig. 2A). Ang II (50 nM, 48 h) also caused a significant up-regulation of col1 (approximately 1.51-fold in CFs (p<0.05)), which was attenuated by treatment with aspirin at doses of 100 and 1000 µM, to values of 1.18-fold and 1.09-fold, respectively (Fig. 2A). Immunofluorescence analysis revealed that aspirin suppressed Ang II stimulated α-SMA expression (Fig. 2B).

Fig. 2.

Aspirin attenuates collagen production in cultured CFs. A. Effects of Aspirin on the protein levels of TGF-β1, FN1, and collagen 1 (col1) in cultured CFs. Western blot analysis of CFs lysates was from 3 wells, with each lysate from one animal, n=3. B. Immunofluorescent staining images show α-smooth muscle actin (α-SMA) expression induced by angiotensin II in cultured CFs; scale bar indicates 20 µm. C. Effects of aspirin on the total collagen content in the supernatants of fibroblasts induced by angiotensin II, n=6. D. Effects of aspirin on the total collagen content in the supernatants of fibroblasts induced by TGF-β1, n=6. E. Effects of aspirin on the protein levels of TGF-β1 as detected by an enzyme-linked immunosorbent assay (ELISA), n=10. *p<0.05 vs control; + p<0.05 vs angiotensin II.

Fig. 2.

Aspirin attenuates collagen production in cultured CFs. A. Effects of Aspirin on the protein levels of TGF-β1, FN1, and collagen 1 (col1) in cultured CFs. Western blot analysis of CFs lysates was from 3 wells, with each lysate from one animal, n=3. B. Immunofluorescent staining images show α-smooth muscle actin (α-SMA) expression induced by angiotensin II in cultured CFs; scale bar indicates 20 µm. C. Effects of aspirin on the total collagen content in the supernatants of fibroblasts induced by angiotensin II, n=6. D. Effects of aspirin on the total collagen content in the supernatants of fibroblasts induced by TGF-β1, n=6. E. Effects of aspirin on the protein levels of TGF-β1 as detected by an enzyme-linked immunosorbent assay (ELISA), n=10. *p<0.05 vs control; + p<0.05 vs angiotensin II.

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It is known that collagen is synthesized by fibroblasts; collagen secretion by myocardial fibroblasts directly contributes to cardiac fibrosis [11]. Content of total collagen was measured using a Sircol soluble collagen assay to examine the supernatant of cultured fibroblasts. Aspirin significantly suppressed the Ang II-induced production and secretion of collagen (Fig. 2C) at doses of 100 and 1000 µM, significantly lowering the total collagen content values. We next assessed the effect of aspirin on the collagen synthesis of cardiac fibroblasts and found that 100 and 1000 µM aspirin markedly inhibited the TGF-β1-mediated collagen production (Fig. 2D). Moreover, as a pro-fibrotic cytokine, TGF-β1 can be secreted into the supernatants of fibroblast [12], making it possible to detect TGF-β1 levels using ELISA. Aspirin at a dose of 100 µM markedly lowered the TGF-β1 levels in the supernatant of fibroblasts from 1.97±0.84-fold (Ang II group) to 1.18±0.16 (aspirin+Ang II group) (Fig. 2E, p<0.05).

Aspirin reduces the expression of extracellular signal-regulated kinases 1/2 (Erk1/2) and blocks the MAPK-Erk1/2 pathway

Angiotensin IIcan activate extracellular signal-regulated kinase 1 and 2 (Erk1/2) by discrete G protein-dependent and beta-arrestin2-dependent pathways. Erk1/2 signalling is important in the process of myocardial fibrosis [13]. To explore the underlying molecular mechanism of aspirin’s effect on collagen deposition, the expression of p-Erk1/2 was examined both in vivo and in cultured CFs. As illustrated in Fig. 3A, compared to TAC mice, treatment with 10 mg·kg-1 aspirin resulted in a reduction in the p-Erk1/2 protein levels (p-Erk1, 1.49±0.19-fold for TAC vs. 1.04±0.15-fold for aspirin+TAC, p<0.05; p-Erk2, 1.96±0.68-fold for TAC vs. 0.87±0.06-fold for aspirin+TAC, p<0.05, Fig. 3A). Moreover, Ang II (50 nM) significantly increased the phosphorylation of Erk1/2, and the level of p-Erk increased approximately 1.36±0.21-fold (p-Erk1) and 1.74±0.63-fold (p-Erk2) in CFs compared with the control (p<0.01, Fig. 3B), whereas aspirin at a dose of 100 µM significantly suppressed the expression of p-Erk1/2 (p-Erk1, 1.09±0.11-fold for aspirin+ Ang II, p<0.05; p-Erk2, 0.99±0.11-fold for aspirin+Ang II, p<0.05, Fig. 3B). More importantly, aspirin (100 µM) had a similar effect as U0126 (10 µM), an inhibitor that can inhibit p-Erk1/2. U0126 inhibited the levels of p-Erk1 and Erk2 induced by Ang II by 0.65±0.39- and 0.57±0.33-fold, respectively, compared with the Ang II group (Fig. 3B).

Fig. 3.

Aspirin inhibits the expression of phosphorylated extracellular signal-regulated kinases 1/2 (p-Erk1/2) and blocks the MAPK-Erk1/2 pathway. A. Effects of aspirin on p-Erk1/2 and t-Erk1/2 in mice with transverse aortic constriction (TAC). Western blot analysis of myocardial lysates were from 6 mice, n=6. B. Examples of western blot bands of p-Erk1/2 and t-Erk1/2, which show the effect of aspirin on the protein levels of p-Erk1/2 in CFs. Western blot analyses of CFs lysates were from 6 wells, n=6. *p<0.05 vs sham control; + p<0.05 vs TAC or angiotensin II.

Fig. 3.

Aspirin inhibits the expression of phosphorylated extracellular signal-regulated kinases 1/2 (p-Erk1/2) and blocks the MAPK-Erk1/2 pathway. A. Effects of aspirin on p-Erk1/2 and t-Erk1/2 in mice with transverse aortic constriction (TAC). Western blot analysis of myocardial lysates were from 6 mice, n=6. B. Examples of western blot bands of p-Erk1/2 and t-Erk1/2, which show the effect of aspirin on the protein levels of p-Erk1/2 in CFs. Western blot analyses of CFs lysates were from 6 wells, n=6. *p<0.05 vs sham control; + p<0.05 vs TAC or angiotensin II.

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Aspirin reduces p-Akt and β-catenin levels and blocks the p-Akt/β-catenin pathway

Some signalling pathways significantly participate in cardiac fibrosis pathogenesis. For example, the activity of the PI3K/p-Akt pathway increased the collagen accumulation and fibrosis-related signals [14, 15]. Wnt/β-catenin signalling is involved in heart development and an increase in foetal gene expression occurs during fibrosis [16]. We found that compared to TAC mice, treatment with aspirin (10 mg·kg-1) resulted in a reduction in the p-Akt/t-Akt (TAC 2.11±0.41-fold versus TAC+ASA 0.96±0.33-fold, p<0.01) and β-catenin protein levels (TAC, 1.78±0.45-fold versus TAC+ASA, 1.13±0.13-fold, p<0.05, Fig. 4A). Immunofluorescent staining for p-Akt revealed that aspirin also suppressed the Ang II-stimulated PI3K/AKT levels (Fig. 4B). Activated Akt (p-Akt) positively regulates cell growth and activity [17]. The PI3K/Akt signalling pathway induces cardiac fibroblast proliferation and collagen accumulation, which contributes to fibrosis [18]. As shown in Fig. 4C, the p-Akt (Ser473)/t-Akt levels were significantly upregulated in Ang II-stimulated CFs (1.49±0.31-fold) compared to the control groups. Aspirin (100 µM) efficiently inhibited the up-regulation of p-Akt (0.98±0.19-fold) in Ang II-stimulated CFs. The PI3K/Akt signal pathway can activate β-catenin via AKT/GSK-3β/β-catenin signalling [19].

Fig. 4.

Aspirin reduces p-Akt and β-catenin levels and blocks the p-Akt/ β-catenin pathway. A. Effects of aspirin on the expression of p-Akt and β-catenin in the heart of mice with transverse aortic constriction (TAC). Western blot analysis of myocardial lysates was from 3 mice, n=3. B. Immunofluorescent staining images showing p-Akt expression. Scale bar indicates 20 µm. *p<0.05 vs sham control; + p<0.05 vs TAC or angiotensin II. C. Effects of aspirin on the protein levels of p-Akt. Western blot analysis of CFs lysates were from 3 wells, n=3. D. Effects of aspirin on β-catenin in angiotensin II-treated CFs. Western blot analyses of CFs lysates were from 3 wells, n=3. E. Effects of aspirin and DKK1 on the protein levels of β-catenin as detected using enzyme-linked immunosorbent assay (ELISA), n=12.

Fig. 4.

Aspirin reduces p-Akt and β-catenin levels and blocks the p-Akt/ β-catenin pathway. A. Effects of aspirin on the expression of p-Akt and β-catenin in the heart of mice with transverse aortic constriction (TAC). Western blot analysis of myocardial lysates was from 3 mice, n=3. B. Immunofluorescent staining images showing p-Akt expression. Scale bar indicates 20 µm. *p<0.05 vs sham control; + p<0.05 vs TAC or angiotensin II. C. Effects of aspirin on the protein levels of p-Akt. Western blot analysis of CFs lysates were from 3 wells, n=3. D. Effects of aspirin on β-catenin in angiotensin II-treated CFs. Western blot analyses of CFs lysates were from 3 wells, n=3. E. Effects of aspirin and DKK1 on the protein levels of β-catenin as detected using enzyme-linked immunosorbent assay (ELISA), n=12.

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Effects of aspirin on serpinE2 in vivo and in vitro

SerpinE2, a protein that inhibits serine proteases and is also called protease nexin-1 (PN-1), is encoded by the SERPINE2 gene and can be secreted by fibroblasts [20]. In our previous study, we found that serpinE2 was up-regulated in pathological cardiac fibrosis. The overexpression of serpinE2 leads to the accumulation of extracellular matrix protein and contributes to cardiac fibrosis [10]. In myocardium taken from TAC mice, serpinE2 was increased by 1.36±0.12-fold compared with the sham control group (p<0.01). Treatment with aspirin significantly attenuated this increase of serpinE2 in vivo (1.06±0.07-fold) (Fig. 5A, B). In the presence of Ang II, serpinE2 levels increased approximately 1.50-fold and 1.85-fold in myocardial fibroblasts and in fibroblast supernatants, respectively, compared with the control group (p<0.05). Treatment with aspirin significantly attenuated the serpinE2 levels 0.98-fold in CFs and 1.39-fold in the supernatants of fibroblasts (Fig. 5C, D).

Fig. 5.

Effects of aspirin on the serpinE2 (PN-1) levels in vivo and in vitro. A. Representative Western blot analysis of serpinE2 protein expression (upper panel) and the corresponding statistical analysis (lower panel). Western blot analyses of myocardial lysates from 3 mice, n=3. B. SerpinE2 mRNA levels were measured using qRT-PCR in mice with transverse aortic constrictions (TAC) treated with aspirin, n= 6. C. Effect of aspirin on the serpinE2 levels in the supernatants of fibroblasts induced by angiotensin II (Ang II). The protein levels of serpinE2 were detected using an enzyme-linked immunosorbent assay (ELISA), n=6. D. Effect of aspirin on the serpinE2 levels in myocardial fibroblasts induced by Ang II, n=6; *p<0.05 vs control; + p<0.05 vs TAC or Ang II.

Fig. 5.

Effects of aspirin on the serpinE2 (PN-1) levels in vivo and in vitro. A. Representative Western blot analysis of serpinE2 protein expression (upper panel) and the corresponding statistical analysis (lower panel). Western blot analyses of myocardial lysates from 3 mice, n=3. B. SerpinE2 mRNA levels were measured using qRT-PCR in mice with transverse aortic constrictions (TAC) treated with aspirin, n= 6. C. Effect of aspirin on the serpinE2 levels in the supernatants of fibroblasts induced by angiotensin II (Ang II). The protein levels of serpinE2 were detected using an enzyme-linked immunosorbent assay (ELISA), n=6. D. Effect of aspirin on the serpinE2 levels in myocardial fibroblasts induced by Ang II, n=6; *p<0.05 vs control; + p<0.05 vs TAC or Ang II.

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Recently, many reports have suggested that Wnt-β-catenin signalling is closely associated with tissue fibrosis in several organs 21 and that the activation of Wnt participates in cardiac fibrosis [2, 22, 23]. Ang II (50 nM) increased β-catenin levels approximately 2.18-fold in CFs compared with the control (Fig. 4D, p<0.05), which was significantly reduced by aspirin at doses of 10 µM (1.10-fold), 100 µM (0.96-fold), and 1000 µM (0.90-fold) (Fig. 4D, p<0.05). β-catenin is a key nuclear effector of Wnt signalling and could be antagonized by dickkopf-l(DKK1). Consistent with these findings, Dickkopf-1 (DKK-1) inhibited the Ang II-driven increase of β-catenin (1.08-fold) in CFs, as detected by ELISA (p<0.05). Aspirin produced effects similar to DKK-1 on β-catenin (aspirin+ Ang II,1.23±0.27; Ang II,1.78±0.52 Fig. 4E, p<0.05).

In this study, we provide the first evidence that aspirin is involved in modulating several signalling pathways that reduce fibrosis, including the MAPK-Erk1/2-serpinE2 signalling pathway and the p-Akt/β-catenin signalling pathway (Fig. 6). We also demonstrate that aspirin alleviates cardiac fibrosis by lowering the levels of p-Erk1/2-serpinE2 and p-Akt/β-catenin.

Fig. 6.

Schematic diagram of the effect and mechanisms of aspirin in the pathological process of cardiac fibrosis. Aspirin suppresses p-Erk1/2, which reduces the expression of serpinE2. Aspirin also inhibited the β-catenin and p-Akt levels in vivo and in vitro.

Fig. 6.

Schematic diagram of the effect and mechanisms of aspirin in the pathological process of cardiac fibrosis. Aspirin suppresses p-Erk1/2, which reduces the expression of serpinE2. Aspirin also inhibited the β-catenin and p-Akt levels in vivo and in vitro.

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In our previous study, aspirin was shown to possess significant anti-hypertrophic properties and to down-regulate β-catenin/Akt signalling that had been induced by left ventricular pressure overload in mice [6]. In addition to ventricular hypertrophy, pressure overload results in cardiac remodelling, reactive fibrosis, and apoptosis. In response, there is an activation of the renin-angiotensin-aldosterone system. In particular, the systemic and local production of Ang II [24], a vasoactive peptide that activates mesangial and tubular cells and interstitial fibroblasts, increases the expression and synthesis of extracellular matrix proteins, such as fibronectin, laminin, and collagen [25]. We found that aspirin attenuates the collagen production induced by Ang II in cultured cardiac fibroblasts. This result was supported by the finding that aspirin significantly inhibited CF apoptosis and decreased the levels of apoptosis markers [26]. Ang II also contributes to ongoing inflammation and stimulates the tyrosine phosphorylation of the linker protein SHC, causing Ras activation. Ras/Raf then stimulates Erk1/2, ultimately leading to fibrogenesis. In our results, aspirin repressed p-Erk1/2 expression and blocked the MAPK-Erk1/2 pathway. Our previous study showed that p-Erk1/2 can activate serpinE2 via the Erk-dependent transcription activator Elk1 in myocardial fibroblasts [10]. This current study showed that aspirin can inhibit the expression of serpinE2 by suppressing the MAPK-Erk1/2 signalling pathway. This may explain how aspirin alleviates pressure overload-induced cardiac interstitial fibrosis in vivo.

Aspirin is widely used for its analgesic and antipyretic effects. Because of its inhibitory effects on COX and on platelet aggregation, it is also used in the treatment of cardiovascular diseases. The cardioprotective and antioxidant effects of chronic aspirin administration have previously been reported [27, 28]. Aspirin has many off-targets, one of which is AMPK. Aspirin activates AMPK [29, 30], then activated AMPK can lead to the attenuation of MEK-Erk signalling and inhibits Erk1/2 phosphorylation [31]. AMPK activation in the heart usually improves its function, which will further explain these results. Another study showed that low-dose aspirin influences post-infarct cardiac remodelling by affecting interstitial and perivascular fibrosis after coronary artery ligation in the rat [32], which agrees with our results. A separate report showed that aspirin inhibits the transcription factors NF-κB and AP-1 [33, 34]. Aspirin also inhibits IL-18-induced cardiac fibroblast migration via the induction of IKK/NF-κB and JNK/ AP-1 [35]. Aspirin attenuates Ang II-induced inflammation via the inhibition of Erk1/2 and NF-κB activation in bone marrow mesenchymal stem cells [36]. Our previous study showed that at clinically relevant doses for anti-thrombotic therapy, aspirin possesses significant anti-hypertrophic properties and causes the down-regulation of β-catenin and p-Akt, which may be part of the signalling mechanism necessary for these effects [6]. In the present study, the dosage of aspirin used coincided with the guidelines of the American College of Chest Physicians (ACCP) [37], the American College of Cardiology/American Heart Association, and the European Society of Cardiology [38].

Aspirin suppressed p-Akt and β-catenin expression induced by Ang II in cultured cardiac fibroblasts in our study. Aspirin suppressed TGF-β1 expression and α-SMA induced by Ang II in cultured CFs. Ang II, in contrast, increased TGF-β expression [39]. Some of the Ang II effects are mediated by the release of several factors, including TGF-β [40] and plasminogen activator inhibitor type-1 (PAI-1) [25]. TGF-β1 induces α-SMA expression and collagen production via Erk1/2 activation, and TGF-β1 targets the GSK-3β/β-catenin pathway via Erk activation in the transition of human lung fibroblasts into myofibroblasts [41]. TGF-β induced Wnt secretion and activated the Wnt/β-catenin pathway.

In summary, aspirin attenuated pressure overload-induced interstitial fibrosis in vivo and suppressed the MAPK-Erk1/2 and p-Akt/β-catenin pathways, then inhibited the production of collagen in fibroblasts in vitro.

This study was funded by National Basic Research Program of China (973 program, 2013CB531104); Natural Science Foundation of the HeiLongJiang Province (H201402); Postdoctoral Science Foundation of China (2012T50355), and Postdoctoral Foundation of HeiLongJiang Province (LBH-Q13112, LBH-Z13151).

The authors declare that they have no conflicts of interest.

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X.-L. Li and G.-Y. Wang contributed equally to this work.

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