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SIRT1 activation attenuates cardiac fibrosis by endothelial-to-mesenchymal transition
Biomedicine & Pharmacotherapy 118 (2019) 109227
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SIRT1 activation attenuates cardiac fibrosis by endothelial-to-mesenchymal transition
T
Zhen-Hua Liua, Yanhong Zhanga, Xue Wangb, Xiao-Fang Fana, Yuqing Zhangc, Xu Lid, ⁎ Yong-sheng Gonga, Li-Ping Hana,d, a
Institute of Hypoxia Medicine, School of Basic Medical Sciences, Wenzhou Medical University, Wenzhou, China Teaching Center of Medical Functional Experiment, School of Basic Medical Sciences, Wenzhou Medical University, Wenzhou, China c Department of Biochemistry, School of Basic Medical Sciences, Wenzhou Medical University, Wenzhou, China d Department of Physiology, School of Basic Medical Sciences, Wenzhou Medical University, Wenzhou, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: SIRT1 EndMT Cardiac fibrosis TGF-β/Smad2/3 VE-cadherin
Endothelial-to-mesenchymal transition (EndMT) is closely related to the pathogenesis of various diseases, including cardiac fibrosis. Transforming growth factor (TGF)-β1 strongly induces EndMT, and sirtuin 1 (SIRT1) may play vital roles in TGF-β/Smad pathway inhibition. This study aimed to determine whether SIRT1 activation inhibits EndMT, thereby attenuating cardiac fibrosis. Cardiac fibrosis was induced in C57BL/6 mice by subcutaneously injecting isoproterenol. SIRT1 was activated and then suppressed by intraperitoneally injecting resveratrol (RSV) and EX527, respectively. EndMT was induced by adding TGF-β1 to H5V cells and measured by immunofluorescence and western blot. The role of SIRT1 in EndMT was determined by lentivirus-mediated overexpression of SIRT1. Interactions between SIRT1 and Smad2/3 in the TGF-β/Smad2/3 pathway were examined by immunoprecipitation. SIRT1 activation upregulated CD31 and vascular endothelial-cadherin, and downregulated α-smooth muscle actin, fibroblast-specific protein 1, and vimentin. SIRT1 upregulated and EX527 inhibited TGF-β receptor 1 (TGF-βR1) and P-Smad2/3 expression, respectively. SIRT1 activation and overexpression by RSV/SRT2104 and lentivirus transfection, respectively, reduced TGF-β1-induced EndMT. SIRT1 and Smad2/3 interaction was shown by immunoprecipitation in vivo and in vitro. TGF-βR1 and P-Smad2/3 expression was downregulated and Smad2/3 nuclear translocation was inhibited. In conclusion, SIRT1 activated by RSV attenuated isoproterenol-induced cardiac fibrosis by regulating EndMT via the TGF-β/Smad2/3 pathway.
1. Introduction Despite great clinical advances and strategies during the past decades, heart failure continues to pose a major health concern worldwide [1]. Cardiac fibrosis, in which normal myocardium is substituted by non-functional fibrotic tissue, leads to progressive heart failure [2]. Cardiac fibrosis is strongly associated with multiple cardiovascular diseases characterized by the deposition of extracellular matrix (ECM) components [3]. The process of fibrosis consists of the proliferation of fibroblasts and their subsequent differentiation into myofibroblasts, the latter resulting in an increased expression of α-smooth muscle actin (αSMA) compared to that induced by fibroblasts. The production of ECM proteins, including collagen I, III, VI, and V, is also increased in myofibroblasts [4,5]. Other sources of myofibroblasts include CD34+ progenitor cells, which originate from the bone marrow [6]. Recently, epithelial-to-mesenchymal transition (EndMT) has been reported as a
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novel source of fibroblasts [7] and endothelial cells (ECs), giving rise to the myofibroblasts associated with fibrotic diseases [8]. EndMT is the process by which endothelial cells obtain characteristics of mesenchymal cells and lose the characteristics of ECs, and commonly occurs in the intima of cardiovascular tissue. During this transition, the expression of endothelial-specific markers, such as CD31 and vascular endothelial (VE)-cadherin, is decreased, while that of mesenchymal-specific markers, such as α-SMA, fibroblast-specific protein 1 (FSP1), and vimentin, is increased [9]. This transition promotes the migration of myofibroblasts to interstitial tissues, which leads to fibrosis [10]. EndMT is reportedly closely related to the pathogenesis of numerous diseases, including cardiac fibrosis [5], systemic sclerosis [11], atherosclerosis [12], cancer, pulmonary hypertension, myocardial infarction, and other fibrotic diseases [8,13,14]. Therefore, the regulation of EndMT could be a potential therapeutic strategy against cardiac fibrosis [15]. Transforming growth factor (TGF)-β, an inducer
Corresponding author. E-mail address: [email protected] (L.-P. Han).
https://doi.org/10.1016/j.biopha.2019.109227 Received 14 March 2019; Received in revised form 6 July 2019; Accepted 15 July 2019 0753-3322/ © 2019 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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of EndMT, promotes the Smad3-mediated synthesis of α-SMA, which plays a vital role in the differentiation of myofibroblasts [16,17]. It has been reported that the TGF-β/Smad signaling pathway is a potential stimulator and major regulator of EndMT during the progression of myocardial fibrosis [18,19]. In addition, TGF-β-induced extracellular signal-regulated kinase (Erk) activation was observed to potentiate or interfere with Smad signaling [19,20], and TGF-β can activate phosphoinositide 3-kinase/protein kinase B (Akt) pathways [21]. These pathways in myofibroblasts can lead to novel and effective therapies against fibrosis and other diseases. Sirtuin 1 (SIRT1), also known as nicotine adenine dinucleotide-dependent deacetylase, has great therapeutic potential because of its protective role in cardiovascular diseases. The pharmacological activation of SIRT1 could be applied as a novel treatment strategy in the prevention of cardiac fibrosis [22]. However, the exact mechanism of its activation has not yet been elucidated. SIRT1 is reportedly involved in attenuating cardiac fibrosis by inhibiting oxidative stress [23]. There is reason to believe that EndMT participates in the above process because of evidence showing that TGF-β/Smad2/3 is a specific target of SIRT1 [22,24]. SIRT1 might also be associated with the inhibition of Erk/Akt phosphorylation in cardiac hypertrophy [25]. In turn, it is hypothesized that SIRT1 attenuates fibrosis by regulating EndMT. In this study, both resveratrol (RSV) and SRT2104 were used to activate SIRT1. Some experimental studies have verified that RSV, a polyphenol extracted from plants [26], is able to delay the processes involved in diverse cardiovascular diseases by upregulating SIRT1 [27,28]. A previous report has also shown that SIRT1 was increased by SRT2104, a synthetic small molecule activator, to extend the lifespan of mice [29]. The aim of the present study was to investigate whether the activation of SIRT1 could attenuate cardiac fibrosis induced by isoproterenol (ISO) in vivo, and whether the TGF-β/Smad2/3 pathway is involved in this process. The role of SIRT1 in TGF-β-induced EndMT in vitro was evaluated in H5V cells, a type of mouse heart microvascular endothelial cells, and the relevant molecular mechanisms were determined. This study provides significant experimental evidence suggesting SIRT1 to be a potential therapeutic target for cardiac fibrosis and other fibrotic diseases.
staining. The ISO-treated and ISO + RSV + EX527 groups showed greater myofibril disarray and collagen deposition than did those in the RSV + ISO group (Fig. 2A). ISO treatment also significantly increased the level of immunostaining for collagen I and collagen III, while ISO + RSV significantly attenuated the extent of interstitial fibrosis compared to that in both the ISO and ISO + RSV + EX527 groups (Fig. 2A). Quantitative analysis of mRNA expression and western blot confirmed these results (Fig. 2B). These findings suggest that SIRT1 activated by RSV inhibited ISO-induced interstitial collagen deposition.
2.3. SIRT1 expression was downregulated by ISO Quantitative reverse transcription polymerase chain reaction (qRTPCR) and western blot were performed to determine the changes in SIRT1 expression during cardiac fibrosis. The results showed that ISO suppressed the expression of SIRT1 at the mRNA and protein levels, while RSV significantly upregulated SIRT1. EX527 eliminated the effects of RSV in terms of SIRT1 expression (Fig. 3A).
2.4. SIRT1 activated by RSV attenuated EndMT in ISO-induced cardiac fibrosis As EndMT was previously indicated as a significant contributor to cardiac fibrosis, endothelial markers (VE-cadherin and CD31) and mesenchymal markers (α-SMA, vimentin, and FSP1) were examined to assess the extent of EndMT. As shown in Fig. 3B and C, the expression of VE-cadherin and CD31 was downregulated, while that of α-SMA, vimentin, and FSP1 was upregulated in the hearts of ISO-treated mice. Although RSV reversed these changes, EX527 eliminated the effect of RSV on EndMT (Fig. 3B and C). These results suggest that SIRT1 activated by RSV attenuated EndMT in ISO-induced cardiac fibrosis.
2.5. TGF-β/Smad2/3 was involved in the mechanism of SIRT1-regulated EndMT in vivo
2. Results
The role of SIRT1 in the inhibition of the TGF-β/Smad pathway has been confirmed in previous studies. In this study, the expression of TGFβR1 and phosphorylated Smad2/3 (P-Smad2/3) was increased in the ISO and ISO + RSV + EX527 groups, but was decreased in the ISO + RSV group (Fig. 4A). Immunoprecipitation (IP) revealed an interaction between SIRT1 and Smad2/3 (Fig. 4B, C), which suggests that the TGF-β/Smad2/3 pathway is involved in the regulation of EndMT by SIRT1.
2.1. SIRT1 activated by RSV attenuated ISO-induced myocardial hypertrophy After two weeks of RSV treatment, myocardial function was evaluated by echocardiographic measurements. No significant difference was observed in the heart rate during anesthesia among the different groups. EX527 is an effective, selective SIRT1 inhibitor and EX527 could effectively inhibit the activity of SIRT1 deacetylase. The corresponding echocardiographic images indicated that the thickness of the left ventricular posterior wall and interventricular septal was increased in the ISO and ISO + RSV + EX527 groups, whereas no thickness was observed in the ISO + RSV group (Fig. 1A). RSV treatment also prevented the ISO-mediated increase in the left ventricular mass, fractional shortening (FS), and ejection fraction (EF), and decrease in the left ventricular internal dimension at diastole (LVIDd), left ventricular internal dimension at systole (LVIDs), left ventricular end-diastolic volume (LVEDV), and left ventricular end-systolic volume (LVESV) (Fig. 1B). These results suggest that SIRT1 activated by RSV attenuated compensatory myocardial hypertrophy in the ISO-induced mouse model.
2.6. Activation of SIRT1 inhibited TGF-β1-induced EndMT in H5V cells To examine the effect of SIRT1 on EndMT in vitro, EndMT was induced by TGF-β1 in H5V cells. SIRT1, endothelial markers (VE-cadherin and CD31), and fibroblast markers (α-SMA and vimentin) were evaluated. TGF-β1 changed the shape of the cells from oval to spindleshaped (Fig. 5A), but it exerted no significant cellular cytotoxicity (Fig. 5B). In addition, TGF-β1 downregulated VE-cadherin and CD31 while upregulating vimentin and α-SMA, indicating that the progression of EndMT was promoted. TGF-β1 also inhibited SIRT1 expression (Fig. 5D), whereas both RSV and SRT2014 upregulated SIRT1 expression in TGF-β1-treated cells while inhibiting EndMT development (Fig. 5D). These changes were further verified by double immunofluorescence staining (Fig. 5C), which showed that TGF-β1 greatly reduced the number of VE-cadherin-positive cells and increased that of αSMA-positive cells. SIRT1 activated by either RSV or SRT2104 reversed the TGF-β1-induced changes. These findings demonstrate that the activation of SIRT1 attenuated TGF-β1-induced EndMT in H5V cells.
2.2. SIRT1 activated by RSV prevented cardiac interstitial collagen deposition To measure the degree of cardiac fibrosis, histological analysis was performed using hematoxylin and eosin (H&E) and Masson’s trichrome 2
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Fig. 1. SIRT1 activated by resveratrol (RSV) attenuated cardiac hypertrophy induced by isoproterenol (ISO). (A) Representative echocardiographic photos from M-mode. (B) Echocardiographic parameters including interventricular septal thickness at diastole (IVSd), interventricular septal thickness at systole (IVSs), left ventricular internal dimension at diastole (LVIDd), left ventricular internal dimension at systole (LVIDs), left ventricular posterior wall thickness at diastole (LVPWd), left ventricular posterior wall thickness at systole (LVPWs), left ventricular enddiastolic volume (LVEDV), left ventricular endsystolic volume (LVESV), ejection fraction (EF), fractional shortening (FS), and left ventricular mass. *p < 0.05, **p < 0.01. Data are represented as the mean ± standard error of the mean (SEM), n = 6.
2.7. Overexpression of SIRT1 suppressed TGF-β1-induced EndMT in H5V cells
2.9. TGF-β/Smad2/3 was involved in the regulation of EndMT by SIRT1 in H5V cells
To further demonstrate that SIRT1 plays a key role in EndMT, lentiviruses were used to overexpress SIRT1 in H5V cells. Lentiviral transfection was performed for 48 h followed by treatment with TGF-β1 for 48 h. The transfection efficiency was evaluated using green fluorescent protein (Fig. 6A), and positive cells were selected using 2 μg/mL puromycin before TGF-β1 treatment. Western blot revealed that SIRT1 overexpression inhibited TGF-β1-induced EndMT (Fig. 6B).
The mechanism by which SIRT1 inhibited TGF-β1-induced EndMT in vitro is consistent with the in vivo mechanism. TGF-βR1 and PSmad2/3 were upregulated in TGF-β1-induced EndMT and the activation of SIRT1 attenuated the expression of relevant proteins (Fig. 8A). EX527 increased the expression of the abovementioned proteins compared to that in the RSV group. The overexpression of SIRT1 also reduced the expression of TGF-βR1 and P-Smad2/3 in TGF-β1-induced EndMT (Fig. 8B). Moreover, IP revealed an interaction between SIRT1 and Smad2/3 (Fig. 8C, D). These findings indicate that SIRT1 regulated TGF-β1-induced EndMT via the TGF-β/Smad pathway.
2.8. SIRT1 inhibited nuclear translocation of Smad2/3 in TGF-β1-treated H5V cells
3. Discussion SIRT1 and Smad2/3 were expressed in the cytoplasm of normal H5V cells. When the cells were treated with TGF-β, SIRT1 was downregulated and Smad2/3 activation was inhibited. Then, the nuclear translocation of P-Smad2/3 in TGF-β1-treated H5V cells was inhibited by SIRT1 overexpression (Fig. 7).
In the current study, we revealed that SIRT1 activated by RSV attenuated ISO-induced cardiac dysfunction and fibrosis by regulating EndMT in vivo. Additionally, SIRT1 overexpression suppressed the development of TGF-β1-induced EndMT in vitro. To our knowledge, our 3
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Fig. 2. SIRT1 activated by RSV attenuates ISO-induced cardiac fibrosis. (A) H&E staining of the left ventricular myocardium. Masson’s trichrome staining and immunohistochemistry of collagen I and collagen III. Scale bar =50 μm. (B) mRNA and protein expression of collagen I and collagen III. *p < 0.05, **p < 0.01. Data are represented as the mean ± SEM, n = 6.
study is the first to prove that SIRT1 attenuated cardiac fibrosis by regulating EndMT both in vivo and in vitro, although numerous studies have shown that SIRT1 attenuated cardiac fibrosis by inhibiting oxidative stress [23]. The activation of SIRT1 suppressed that of TGF-βR1 and P-Smad2/3 both in vivo (Fig. 4) and in vitro (Fig. 8). The interaction between SIRT1 and Smad2/3 was demonstrated by IP, and SIRT1 was found to inhibit the nuclear translocation of Smad2/3 in TGF-β1treated H5V cells. Sirtuins have been shown to dictate “replicative lifespan” and control stress resistance and longevity [30]. Numerous studies have shown that overexpression of sirtuins in fruit flies resulted in a renewable increase in longevity [31,32]. RSV is the most potent sirtuin-activating compound (STAC) discovered for SIRT1 [33]. SRT2104, a STAC, mimics aspects of calorie restriction and extends male mouse lifespan [34]. The present study showed a reduction of SIRT1 expression and activity in cardiac fibrosis. By activating SIRT1, RSV attenuated compensatory myocardial hypertrophy and reduced interstitial collagen deposition in an ISO-induced mouse model. When SIRT1 was inhibited by EX527, the protective effects of RSV was eliminated. Cardiac fibrosis is a serious global health concern involving the excess deposition of ECM in cardiac muscles and is associated with multiple forms of cardiovascular disease [3]. Recent studies have shown that EndMT is a significant contributor to the development of various types of organic fibrosis, including cardiac fibrosis [8]. Cardiac myofibroblasts could also originate from ECs through EndMT during the development of tissue fibrosis [8]. Myofibroblasts originate from the proliferation of resident fibroblasts and are activated in response to
TGF-β stimulation [8]. Since EndMT may be a source of mesenchymal cells, this transition could activate myofibroblasts and contribute to the development of myocardial fibrosis [8]. TGF-β is the main factor driving fibrosis in most forms of fibrotic diseases. Additionally, it contributes to the development of fibrotic diseases by enhancing the expression of collagen and inducing the differentiation of cells into myofibroblasts [35]. TGF-β also induces EndMT to further contribute to the development of fibrosis [36,37]. The present study showed that the expression of TGF-βR1, α-SMA, and FSP1 (mesenchymal markers) was upregulated while that of VE-cadherin and CD31 (endothelial markers) was downregulated in ISO-induced cardiac fibrosis. Moreover, the activation of SIRT1 by RSV suppressed the activation of TGF-βR1, VE-cadherin, and CD31 while increasing the activation of α-SMA and FSP1. Similar results were observed in H5V cells treated with TGF-β1-induced EndMT. These findings suggest that SIRT1 attenuated cardiac fibrosis via the regulation of EndMT both in vivo and in vitro. Numerous fibrotic diseases, including cardiac fibrosis, are attenuated in Smad3-deficient animals [16]. One study showed that Smad2/3 may be a specific target of SIRT1 and that SIRT1 could therefore be used as a novel therapeutic strategy to prevent heart failure [22]. Both Smad2 and Smad3 were found to be key mediators of TGF-β-induced tissue fibrosis and ECM production, maintaining the activity of activated fibroblasts. Moreover, Smad3 was required when TGF-β induced the synthesis of α-SMA [16]. In fibroblasts, the TGF-β type I receptor could phosphorylate Smad2/3. SIRT1 activation by geniposide attenuated ISO-induced cardiac fibrosis via the TGF-β/Smad 4
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Fig. 3. SIRT1 activated by RSV inhibited cardiac EndMT in mice. (A) mRNA and protein expression of SIRT1 in cardiac tissue. (B) Double immunofluorescence staining of VE-cadherin (green) and α-SMA (red) was performed to evaluate EndMT in fibrotic hearts at ×400 magnification. Scale bar =50 μm. (C) Representative protein expression of endothelial markers, CD31 and VE-cadherin, and mesenchymal markers, α-SMA, vimentin, and fibroblast-specific protein-1 (FSP1), in cardiac tissue assayed by western blot. Western blot results were normalized to β-actin. *p < 0.05, **p < 0.01. Data are represented as the mean ± SEM, n = 6.
experiments. A previous study has shown that SIRT1 repressed TGF-βinduced EndMT in human umbilical cord ECs through direct Smad4 deacetylation [38]. P‑Smad2/3 bound to Smad4 is transferred to the nucleus and activates gene transcription [39]. In the current study, TGF-β was also used to induce EndMT. As a result, we elucidated a new mechanism whereby SIRT1 binding to Smad2/3 inhibited the nuclear translocation of Smad2/3. These findings indicate that overexpression of SIRT1 suppressed TGF-βR1 and P-Smad2/3 levels. The fact that P-
signaling pathway [23]. Thus, we speculated that SIRT1 regulates EndMT via the TGF-β/Smad signaling pathway. In the current study, P-Smad2/3 expression was increased in ISOinduced cardiac fibrosis, but was attenuated by RSV-activated SIRT1. To further elucidate the underlying mechanisms, we used IP to identify whether an interaction exists between SIRT1 and Smad2/3. We chose to use H5V cells, a type of mouse heart microvascular ECs, in the in vitro study as they are from the same species as that in the in vivo
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Fig. 4. Western blot analysis of TGF-β/Smad and immunoprecipitation (IP) of the interaction between SIRT1 and Smad2/3 in the ISOinduced mouse model. (A) The expression levels of TGF-β receptor 1 (TGF-βR1) and its downstream proteins P-Smad2/3 were examined. (B, C) The interaction between SIRT1 and Smad2/3 was examined by IP. *p < 0.05, **p < 0.01. Data are represented as the mean ± SEM, n = 6.
obtained from Peprotech (Rocky Hill, NJ, USA). Antibodies against the following proteins were purchased from Abcam(Cambridge, MA, USA): TGF-βR1 (ab31013), CD31 (ab28364), VE-cadherin (ab33168), vimentin (ab92547), α-SMA (ab32575), and FSP1 (ab197896). The following antibodies were purchased from Cell Signaling Technology (Boston, MA, USA): P-Smad2/3 (8828), T-Smad2/3 (8685). The following antibodies were purchased from Proteintech (Chicago, IL, USA): SIRT1, collagen I, collagen III, and β-actin. Cell counting kit 8 was purchased from Invitrogen (Carlsbad, CA, USA).
Smad2/3 is found downstream of TGF-β suggests that SIRT1 suppressed the progress of TGF-β-induced EndMT by binding to Smad2/3 (Fig. 9). In the TGF-β/Smad pathway, TGF-β can activate both Ras/Erk mitogen-activated protein kinase and phosphoinositide 3-kinase/Akt signaling, which also play important roles in EndMT in a TGF-β-dependent manner [40]. SIRT1 reportedly inhibited both the Erk1/2 and Akt signaling pathways in cardiac fibrosis [41,42]. SIRT1 could act on Akt and Erk1/2 directly or via other pathways, but further research is required in order to elucidate this mechanism in detail. Although TGF-β has been identified as one of the most important growth factors in the induction of EndMT, other signaling pathways, such as the Notch and Wnt/β-catenin pathways, can also regulate EndMT in the cardiovascular system [43]. Therefore, further investigation is required to clarify whether other signaling pathways also participate in the regulation of EndMT via SIRT1.
5.2. Animals and treatment Adult male C57BL/6 mice, aged 8–10 weeks, were purchased from the Wenzhou Medical University Laboratory Animal Centre (Wenzhou, China). All animal procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). The animals were housed individually in cages under hygienic conditions in a 12/12-h light/dark cycle at 22 ± 3 °C and 45 ± 10% humidity for seven days before the experiments. The study was evaluated and approved by the Wenzhou Medical University Animal Care and Use Ethics Committee (permission number wydw2013-0054). The animals were allowed free access to a standard commercial diet and tap water. The mice were then randomly divided into four groups (10 per group) for treatment: Control, ISO, ISO + RSV, and ISO + RSV + EX527. In the ISO model mice, ISO was injected subcutaneously at 10 mg/kg/day for three days and then at 5 mg/kg/day for 11 days [46]. In the treatment groups, RSV (20 mg/kg/day) was intraperitoneally injected with or without EX527 (5 mg/kg/day) for 14 days [45,47,48]. The control group was treated with the corresponding solvent.
4. Conclusions Our study demonstrated that SIRT1 activated by RSV attenuated cardiac fibrosis induced by ISO via the regulation of EndMT. SIRT1 binding to Smad2/3 inhibited Smad2/3 nuclear translocation, thereby regulating EndMT via the TGF-β/Smad2/3 pathway in vivo and in vitro. The present study provides significant experimental evidence supporting the use of SIRT1 as a potential therapeutic target for cardiac fibrosis and suggests a theoretical basis for the treatment of other fibrotic diseases. 5. Materials and methods 5.1. Reagents ISO was purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in phosphate-buffered saline (PBS). RSV (HY-16561, 98.90% purity) was obtained from MedChem Express (Monmouth Junction, NJ, USA) and dissolved in 5% dimethyl sulfoxide (DMSO), 5% Tween 80, and 90% saline [44]. EX527, an inhibitor of SIRT1, was purchased from MedChem Express and dissolved in a special solvent composed of 1% DMSO, 30% PEG-400, and 1% Tween 80 [45]. SRT2104 (HY-15262), a synthetic small molecule activator of SIRT1, was purchased from MedChem Express and dissolved in 10% DMSO. TGF-β1 (100-21) was
5.3. Echocardiographic analysis of cardiac hypertrophy The mice were anesthetized with 1.5% isoflurane before being subjected to echocardiography using a Vevo1100 system (VisualSonics System, Toronto, Ontario, Canada). The following parameters were recorded: interventricular septal thickness at diastole (IVSd), interventricular septal thickness at systole (IVSs), left ventricular internal dimension at diastole (LVIDd), left ventricular internal dimension at systole (LVIDs), left ventricular posterior wall thickness at diastole 6
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Fig. 5. Activation of SIRT1 inhibited TGF-β1-induced EndMT in H5V cells. (A) Changes in endothelial morphological phenotype induced by TGF-β1 in H5V cells. Scale bar =50 μm. The cells gradually changed from an oval to a spindle shape. (B) The cytotoxicity of different treatments in H5V cells was assessed using a cell counting kit 8 assay. (C) Confocal microscopic images of VE-cadherin and α-SMA representing EndMT. Scale bar =20 μm. (D) Protein expression of SIRT1, CD31, VEcadherin, α-SMA, and vimentin was assessed by western blot. *p < 0.05, **p < 0.01. Data are represented as the mean ± SEM, n = 6.
5.4. Histological quantification of cardiac fibrosis
(LVPWd), left ventricular posterior wall thickness at systole (LVPWs), left ventricular end-diastolic volume (LVEDV), left ventricular endsystolic volume (LVESV), ejection fraction (EF), fractional shortening (FS), and left ventricular mass.
After the 14-day treatment described above, all mice were sacrificed and the hearts were quickly excised, arrested in diastole with 1.34 M KCl, weighed, placed in 4% paraformaldehyde, and embedded in
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Fig. 6. Overexpression of SIRT1 suppressed TGF-β1-induced EndMT in H5V cells. (A) H5V cells were transfected with lentiviral (LV)SIRT1 or LV-Con for 48 h. Green fluorescent protein was used to evaluate the transfection efficiency of LV-SIRT1 and empty vectors. Scale bar =50 μm. (B) The protein expression of SIRT1, CD31, VE-cadherin, α-SMA, and vimentin was assessed by western blot after transfection. *p < 0.05, **p < 0.01. Data are represented as the mean ± SEM, n = 6.
were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 U/mL streptomycin (Sigma, Milan, Italy) at 37 °C in a humidified atmosphere containing 5% CO2. After overnight serum starvation, H5V cells were randomly treated with TGF-β (20 ng/mL) and RSV or SRT2104 (20 μM) for 24 h [49]. SIRT1 expression was inhibited by EX527 (10 μM).
paraffin. The hearts were cut transversely close to the apex. Sections of the heart (4–5 μm) were mounted onto slides and stained with hematoxylin and eosin (H&E, Solarbio, Beijing, China) for histopathological analysis. To determine collagen deposition, tissue sections were subjected to Masson’s trichrome staining (Solarbio, Beijing, China) and visualized by light microscopy (ECLIPSE 80i, Nikon Corporation). The photographs were analyzed using digital analysis software (ImageJ) in a blinded manner. For immunohistochemical analysis, cardiac sections were deparaffinized in xylene and rehydrated in a graded ethanol series. Endogenous peroxidase activity was quenched in 3% methanolH2O2 for 10 min. Antigen retrieval was performed by heating the sections in 10 mM citrate buffer (pH 6.0) in a microwave oven. Non-specific binding was blocked in 1% bovine serum albumin. Next, the sections were incubated with primary antibodies against collagen I and collagen III for 2 h, followed by incubation with horseradish peroxidase goat anti-rabbit IgG for 1 h. Labeling was visualized using chromogen diaminobenzidine staining (Boster, Wuhan, China) and the slides were counterstained with hematoxylin.
5.7. Overexpression of SIRT1 via lentiviral transfection Empty vectors (LV-Con) and LV with mouse SIRT1 (LV-SIRT1) were constructed by Genechem (Shanghai, China). To distinguish the transfected cells and measure transfection efficiency, enhanced green fluorescent protein cDNA was inserted into the plasmid sequence. LVs (LV-Con and LV-SIRT1) at a multiplicity of infection of 50 were added to the H5V cells. Twelve hours after transfection, the lentiviruses were removed. Forty-eight hours after transfection, cells were selected with 2 μg/mL puromycin in the culture medium. The transfection efficiency of LV-Con and LV-SIRT1 was evaluated by quantifying the green fluorescent protein in the cells.
5.5. qRT-PCR 5.8. Histological analysis of EndMT The mRNA levels of SIRT1, collagen I, collagen III, TGF-β1, and GAPDH were analyzed by qRT-PCR. Total RNA was extracted from frozen, pulverized left ventricular cardiac tissue using TRIzol reagent following the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). The RNA purity was evaluated based on the OD260/OD280 ratios detected with NanoDrop One (Thermo Scientific, USA). The mRNA was reverse-transcribed into cDNA and synthesized from 2 μg of total RNA using oligo (dT) primers and the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, USA). qRT-PCR was performed according to the instructions using a SuperReal PreMix Plus (SYBR Green) (Tiangen, Beijing, China) by StepOnePlus (Applied Biosystems, Carlsbad, CA, USA). GAPDH and 18S were used as internal controls. The primers used in the study are shown in Table 1.
In vivo, tissue sections were co-incubated with the antibodies against VE-cadherin (1:100) and α-SMA (1:100) at 4 °C overnight and washed three with PBS for 5 min per wash. The sections were then incubated with Alexa Fluor 488/Alexa Fluor 546 antibodies (1:1000; Invitrogen) for 1 h before incubating with 4′,6-diamidino-2-phenylindole (Sigma, Milan, Italy) for nuclear staining. The results were analyzed by fluorescence microscopy (ECLIPSE Ti-S; Nikon Corporation). In vitro, H5V cells subjected to various treatments were fixed with 4% paraformaldehyde in 0.1 M PBS for 15 min. Then, the cells were washed three times with PBS, followed by permeabilization with 0.3% Triton X-100 for 15 min at 25℃. After culturing with 5% bovine serum albumin for 1 h, the cells were stained for VE-cadherin (1:100) and αSMA (1:100) at 4 °C overnight. Then, the cells were stained with Alexa Fluor 488/Alexa Fluor 546 antibodies (1:1000; Invitrogen) for 1 h and washed three times with PBS before nuclear staining. The results were analyzed by fluorescence microscopy (LSM800; Zeiss Corporation) and
5.6. Cell culture and treatments H5V cells, a type of mouse heart microvascular endothelial cell, 8
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Fig. 7. SIRT1 inhibited the nuclear translocation of Smad2/3 in TGF-β1-treated H5V cells. (A&B) SIRT1 overexpression inhibited the activation of Smad2/3, then inhibited P-Smad2/3 translocation into the nuclei of TGF-β1-treated H5V cells. Scale bar =50 μm. (C) SIRT1 was inhibited in TGF-induced EndMT and expressed in the cytoplasm. Scale bar =50 μm.
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Fig. 8. SIRT1 suppressed the expression of TGF-βR1 and P-Smad2/3 and interacted with Smad2/3 in TGF-β1-treated H5V cells. (A, B) Western blot of TGF-β/Smad and other signaling pathways in TGF-β1-induced EndMT. (A) SIRT1 was activated by RSV and SRT2014. (B) SIRT1 was overexpressed via lentivirus (LV) transfection. (C, D) The interaction between SIRT1 and Smad2/3 was revealed by IP. *p < 0.05, **p < 0.01. Data are represented as the mean ± SEM, n = 6.
Fig. 9. SIRT1 regulates EndMT by inhibiting the nuclear translocation of Smad2/3 in TGF-β1-treated H5V cells.
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Table 1 Gene-specific primers used in qRT-PCR. Species
Genes
Sequences
Mouse Mouse Mouse Mouse
SIRT1 Collagen I Collagen III GAPDH
Forward: Forward: Forward: Forward:
5'-GCTGACGACTTCGACGACG-3' Reverse: 5'-TCGGTCAACAGGAGGTTGTCT-3' 5'-GCTCCTCTTAGGGGCCACT-3' Reverse: 5'-CCACGTCTCACCATTGGGG-3' 5'-CTGTAACATGGAAACTGGGGAAA-3' Reverse: 5'-CCATAGCTGAACTGAAAACCACC-3' 5'-AGGTCGGTGTGAACGGATTTG-3' Reverse: 5'-TGTAGACCATGTAGTTGAGGTCA-3'
all images acquired were analyzed in a blinded way using ImageJ.
© 2019 by the authors. Submitted for possible open access publication under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
5.9. Western blot
Appendix A. Supplementary data
Heart tissues and H5V cells were lysed in radioimmunoprecipitation assay buffer (Beyotime, Shanghai, China) and centrifuged at 4 °C at 13,000 rpm for 10 min. The supernatant was collected and protein quantification was carried out using a bicinchoninic acid protein assay (Beyotime). The extracted protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride membranes. The membranes were blocked and incubated with primary antibodies and the expression levels of the target proteins were normalized to that of β-actin. After incubation at 4 °C overnight, the membranes were washed three times with Trisbuffered saline/Tween, incubated with horseradish peroxidase goat anti-rabbit IgG or goat anti-mouse IgG for 1 h at room temperature (1:5000, Invitrogen), and scanned using the Molecular Imager ChemiDoc XRS Imaging System (Bio-Rad, Hercules, CA, USA).
Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.biopha.2019.109227. References [1] A.L. Bui, T.B. Horwich, G.C. Fonarow, Epidemiology and risk profile of heart failure, Nat. Rev. Cardiol. 8 (2011) 30–41. [2] A. Gulati, A. Jabbour, T.F. Ismail, K. Guha, J. Khwaja, S. Raza, K. Morarji, T.D. Brown, N.A. Ismail, M.R. Dweck, E. Di Pietro, M. Roughton, R. Wage, Y. Daryani, R. O’Hanlon, M.N. Sheppard, F. Alpendurada, A.R. Lyon, S.A. Cook, M.R. Cowie, R.G. Assomull, D.J. Pennell, S.K. Prasad, Association of fibrosis with mortality and sudden cardiac death in patients with nonischemic dilated cardiomyopathy, Jama 309 (2013) 896–908. [3] D. Al Hattab, M.P. Czubryt, A primer on current progress in cardiac fibrosis, Can. J. Physiol. Pharmacol. 95 (2017) 1091–1099. [4] Y.Y. Ho, D. Lagares, A.M. Tager, M. Kapoor, Fibrosis-a lethal component of systemic sclerosis, Nat. Rev. Rheumatol. 10 (2014) 390–402. [5] B. Hinz, S.H. Phan, V.J. Thannickal, M. Prunotto, A. Desmouliere, J. Varga, O. De Wever, M. Mareel, G. Gabbiani, Recent developments in myofibroblast biology: paradigms for connective tissue remodeling, Am. J. Pathol. 180 (2012) 1340–1355. [6] E.L. Herzog, R. Bucala, Fibrocytes in health and disease, Exp. Hematol. 38 (2010) 548–556. [7] S.N. Flier, H. Tanjore, E.G. Kokkotou, H. Sugimoto, M. Zeisberg, R. Kalluri, Identification of epithelial to mesenchymal transition as a novel source of fibroblasts in intestinal fibrosis, J. Biol. Chem. 285 (2010) 20202–20212. [8] E.M. Zeisberg, O. Tarnavski, M. Zeisberg, A.L. Dorfman, J.R. McMullen, E. Gustafsson, A. Chandraker, X.L. Yuan, W.T. Pu, A.B. Roberts, E.G. Neilson, M.H. Sayegh, S. Izumo, R. Kalluri, Endothelial-to-mesenchymal transition contributes to cardiac fibrosis, Nat. Med. 13 (2007) 952–961. [9] L. Xiao, A.C. Dudley, Fine-tuning vascular fate during endothelial-mesenchymal transition, J. Pathol. 241 (2017) 25–35. [10] S. Lamouille, J. Xu, R. Derynck, Molecular mechanisms of epithelial-mesenchymal transition, Nat Rev Mol Cell Bio 15 (2014) 178–196. [11] D.T.B. Thuan, H. Zayed, A.H. Eid, H. Abou-Saleh, G.K. Nasrallah, A.A. Mangoni, G. Pintus, A potential link between oxidative stress and endothelial-toMesenchymal transition in systemic sclerosis, Front. Immunol. 9 (2018). [12] Q.Q. Ling, J. Zhao, C.G. Zheng, J. Chun, Roles of notch signaling pathway and endothelial-mesenchymal transition in vascular endothelial dysfunction and atherosclerosis, Eur. Rev. Med. Pharmacol. Sci. 22 (2018) 6485–6491. [13] L. Maddaluno, N. Rudini, R. Cuttano, L. Bravi, C. Giampietro, M. Corada, L. Ferrarini, F. Orsenigo, E. Papa, G. Boulday, E. Tournier-Lasserve, F. Chapon, C. Richichi, S.F. Retta, M.G. Lampugnani, E. Dejana, EndMT contributes to the onset and progression of cerebral cavernous malformations, Nature 498 (2013) 492-+. [14] S. Piera-Velazquez, Z.D. Li, S.A. Jimenez, Role of endothelial-mesenchymal transition (EndoMT) in the pathogenesis of fibrotic disorders, Am. J. Pathol. 179 (2011) 1074–1080. [15] P. Kong, P. Christia, N.G. Frangogiannis, The pathogenesis of cardiac fibrosis, Cell. Mol. Life Sci. 71 (2014) 549–574. [16] M. Dobaczewski, M. Bujak, N. Li, C. Gonzalez-Quesada, L.H. Mendoza, X.F. Wang, N.G. Frangogiannis, Smad3 signaling critically regulates fibroblast phenotype and function in healing myocardial infarction, Circ. Res. 107 (2010) 418-U176. [17] I. Cucoranu, R. Clempus, A. Dikalova, P.J. Phelan, S. Ariyan, S. Dikalov, D. Sorescu, NAD(P)H oxidase 4 mediates transforming growth factor-beta 1-induced differentiation of cardiac fibroblasts into myofibroblasts, Circ. Res. 97 (2005) 900–907. [18] M. Wu, Z.Y. Peng, C.H. Zu, J. Ma, S.J. Lu, J.H. Zhong, S.D. Zhang, Losartan attenuates myocardial endothelial-to-Mesenchymal transition in spontaneous hypertensive rats via inhibiting TGF-beta/Smad signaling, PLoS One 11 (2016). [19] E. Pardali, G. Sanchez-Duffhues, M.C. Gomez-Puerto, P. Ten Dijke, TGF-betaInduced endothelial-mesenchymal transition in fibrotic diseases, Int. J. Mol. Sci. 18 (2017). [20] M. Funaba, C.M. Zimmerman, L.S. Mathews, Modulation of Smad2-mediated signaling by extracellular signal-regulated kinase, J. Biol. Chem. 277 (2002) 41361–41368. [21] R. Derynck, Y.E. Zhang, Smad-dependent and Smad-independent pathways in TGF-
5.10. Immunoprecipitation For immunoprecipitation, 500 μg of total protein extract was blocked with 20 μL of Protein A–Agarose (Sigma-Aldrich, St. Louis, MO, USA). Then, 2 μL of the appropriate antibody was added to the supernatant and incubated overnight at 4 °C. The antibodies were also purified using Protein A–Agarose. In all cases, the samples were analyzed by SDS-PAGE, followed by western blot with the appropriate antibodies. 5.11. Statistical analysis The results are expressed as mean ± standard error of the mean and analyzed by one-way analysis of variance and Tukey's post-hoc test (*p < 0.05 and **p < 0.01) with GraphPad Prism 5.0. Supplementary Materials: Table S1. Echocardiographic parameters in RSV control and EX527 control group; Figure S1. Neither RSV nor EX527 caused significant histological alterations. Declaration of competing interest The authors declare that they have no conflict of interest. Author contributions Methodology and investigation, Z.H.L. and X.W; formal analysis and writing—review and editing, Y.H.Z., Y.Q.Z. and X.F.F; supervision and writing—original draft preparation, Y.S.H.G. and L.P.H. Funding This research was funded by the Natural Science Foundation of Zhejiang Province, grant number LY17H020010.
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SIRT1 activation attenuates cardiac fibrosis by endothelial-to-mesenchymal transition
T
Zhen-Hua Liua, Yanhong Zhanga, Xue Wangb, Xiao-Fang Fana, Yuqing Zhangc, Xu Lid, ⁎ Yong-sheng Gonga, Li-Ping Hana,d, a
Institute of Hypoxia Medicine, School of Basic Medical Sciences, Wenzhou Medical University, Wenzhou, China Teaching Center of Medical Functional Experiment, School of Basic Medical Sciences, Wenzhou Medical University, Wenzhou, China c Department of Biochemistry, School of Basic Medical Sciences, Wenzhou Medical University, Wenzhou, China d Department of Physiology, School of Basic Medical Sciences, Wenzhou Medical University, Wenzhou, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: SIRT1 EndMT Cardiac fibrosis TGF-β/Smad2/3 VE-cadherin
Endothelial-to-mesenchymal transition (EndMT) is closely related to the pathogenesis of various diseases, including cardiac fibrosis. Transforming growth factor (TGF)-β1 strongly induces EndMT, and sirtuin 1 (SIRT1) may play vital roles in TGF-β/Smad pathway inhibition. This study aimed to determine whether SIRT1 activation inhibits EndMT, thereby attenuating cardiac fibrosis. Cardiac fibrosis was induced in C57BL/6 mice by subcutaneously injecting isoproterenol. SIRT1 was activated and then suppressed by intraperitoneally injecting resveratrol (RSV) and EX527, respectively. EndMT was induced by adding TGF-β1 to H5V cells and measured by immunofluorescence and western blot. The role of SIRT1 in EndMT was determined by lentivirus-mediated overexpression of SIRT1. Interactions between SIRT1 and Smad2/3 in the TGF-β/Smad2/3 pathway were examined by immunoprecipitation. SIRT1 activation upregulated CD31 and vascular endothelial-cadherin, and downregulated α-smooth muscle actin, fibroblast-specific protein 1, and vimentin. SIRT1 upregulated and EX527 inhibited TGF-β receptor 1 (TGF-βR1) and P-Smad2/3 expression, respectively. SIRT1 activation and overexpression by RSV/SRT2104 and lentivirus transfection, respectively, reduced TGF-β1-induced EndMT. SIRT1 and Smad2/3 interaction was shown by immunoprecipitation in vivo and in vitro. TGF-βR1 and P-Smad2/3 expression was downregulated and Smad2/3 nuclear translocation was inhibited. In conclusion, SIRT1 activated by RSV attenuated isoproterenol-induced cardiac fibrosis by regulating EndMT via the TGF-β/Smad2/3 pathway.
1. Introduction Despite great clinical advances and strategies during the past decades, heart failure continues to pose a major health concern worldwide [1]. Cardiac fibrosis, in which normal myocardium is substituted by non-functional fibrotic tissue, leads to progressive heart failure [2]. Cardiac fibrosis is strongly associated with multiple cardiovascular diseases characterized by the deposition of extracellular matrix (ECM) components [3]. The process of fibrosis consists of the proliferation of fibroblasts and their subsequent differentiation into myofibroblasts, the latter resulting in an increased expression of α-smooth muscle actin (αSMA) compared to that induced by fibroblasts. The production of ECM proteins, including collagen I, III, VI, and V, is also increased in myofibroblasts [4,5]. Other sources of myofibroblasts include CD34+ progenitor cells, which originate from the bone marrow [6]. Recently, epithelial-to-mesenchymal transition (EndMT) has been reported as a
⁎
novel source of fibroblasts [7] and endothelial cells (ECs), giving rise to the myofibroblasts associated with fibrotic diseases [8]. EndMT is the process by which endothelial cells obtain characteristics of mesenchymal cells and lose the characteristics of ECs, and commonly occurs in the intima of cardiovascular tissue. During this transition, the expression of endothelial-specific markers, such as CD31 and vascular endothelial (VE)-cadherin, is decreased, while that of mesenchymal-specific markers, such as α-SMA, fibroblast-specific protein 1 (FSP1), and vimentin, is increased [9]. This transition promotes the migration of myofibroblasts to interstitial tissues, which leads to fibrosis [10]. EndMT is reportedly closely related to the pathogenesis of numerous diseases, including cardiac fibrosis [5], systemic sclerosis [11], atherosclerosis [12], cancer, pulmonary hypertension, myocardial infarction, and other fibrotic diseases [8,13,14]. Therefore, the regulation of EndMT could be a potential therapeutic strategy against cardiac fibrosis [15]. Transforming growth factor (TGF)-β, an inducer
Corresponding author. E-mail address: [email protected] (L.-P. Han).
https://doi.org/10.1016/j.biopha.2019.109227 Received 14 March 2019; Received in revised form 6 July 2019; Accepted 15 July 2019 0753-3322/ © 2019 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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of EndMT, promotes the Smad3-mediated synthesis of α-SMA, which plays a vital role in the differentiation of myofibroblasts [16,17]. It has been reported that the TGF-β/Smad signaling pathway is a potential stimulator and major regulator of EndMT during the progression of myocardial fibrosis [18,19]. In addition, TGF-β-induced extracellular signal-regulated kinase (Erk) activation was observed to potentiate or interfere with Smad signaling [19,20], and TGF-β can activate phosphoinositide 3-kinase/protein kinase B (Akt) pathways [21]. These pathways in myofibroblasts can lead to novel and effective therapies against fibrosis and other diseases. Sirtuin 1 (SIRT1), also known as nicotine adenine dinucleotide-dependent deacetylase, has great therapeutic potential because of its protective role in cardiovascular diseases. The pharmacological activation of SIRT1 could be applied as a novel treatment strategy in the prevention of cardiac fibrosis [22]. However, the exact mechanism of its activation has not yet been elucidated. SIRT1 is reportedly involved in attenuating cardiac fibrosis by inhibiting oxidative stress [23]. There is reason to believe that EndMT participates in the above process because of evidence showing that TGF-β/Smad2/3 is a specific target of SIRT1 [22,24]. SIRT1 might also be associated with the inhibition of Erk/Akt phosphorylation in cardiac hypertrophy [25]. In turn, it is hypothesized that SIRT1 attenuates fibrosis by regulating EndMT. In this study, both resveratrol (RSV) and SRT2104 were used to activate SIRT1. Some experimental studies have verified that RSV, a polyphenol extracted from plants [26], is able to delay the processes involved in diverse cardiovascular diseases by upregulating SIRT1 [27,28]. A previous report has also shown that SIRT1 was increased by SRT2104, a synthetic small molecule activator, to extend the lifespan of mice [29]. The aim of the present study was to investigate whether the activation of SIRT1 could attenuate cardiac fibrosis induced by isoproterenol (ISO) in vivo, and whether the TGF-β/Smad2/3 pathway is involved in this process. The role of SIRT1 in TGF-β-induced EndMT in vitro was evaluated in H5V cells, a type of mouse heart microvascular endothelial cells, and the relevant molecular mechanisms were determined. This study provides significant experimental evidence suggesting SIRT1 to be a potential therapeutic target for cardiac fibrosis and other fibrotic diseases.
staining. The ISO-treated and ISO + RSV + EX527 groups showed greater myofibril disarray and collagen deposition than did those in the RSV + ISO group (Fig. 2A). ISO treatment also significantly increased the level of immunostaining for collagen I and collagen III, while ISO + RSV significantly attenuated the extent of interstitial fibrosis compared to that in both the ISO and ISO + RSV + EX527 groups (Fig. 2A). Quantitative analysis of mRNA expression and western blot confirmed these results (Fig. 2B). These findings suggest that SIRT1 activated by RSV inhibited ISO-induced interstitial collagen deposition.
2.3. SIRT1 expression was downregulated by ISO Quantitative reverse transcription polymerase chain reaction (qRTPCR) and western blot were performed to determine the changes in SIRT1 expression during cardiac fibrosis. The results showed that ISO suppressed the expression of SIRT1 at the mRNA and protein levels, while RSV significantly upregulated SIRT1. EX527 eliminated the effects of RSV in terms of SIRT1 expression (Fig. 3A).
2.4. SIRT1 activated by RSV attenuated EndMT in ISO-induced cardiac fibrosis As EndMT was previously indicated as a significant contributor to cardiac fibrosis, endothelial markers (VE-cadherin and CD31) and mesenchymal markers (α-SMA, vimentin, and FSP1) were examined to assess the extent of EndMT. As shown in Fig. 3B and C, the expression of VE-cadherin and CD31 was downregulated, while that of α-SMA, vimentin, and FSP1 was upregulated in the hearts of ISO-treated mice. Although RSV reversed these changes, EX527 eliminated the effect of RSV on EndMT (Fig. 3B and C). These results suggest that SIRT1 activated by RSV attenuated EndMT in ISO-induced cardiac fibrosis.
2.5. TGF-β/Smad2/3 was involved in the mechanism of SIRT1-regulated EndMT in vivo
2. Results
The role of SIRT1 in the inhibition of the TGF-β/Smad pathway has been confirmed in previous studies. In this study, the expression of TGFβR1 and phosphorylated Smad2/3 (P-Smad2/3) was increased in the ISO and ISO + RSV + EX527 groups, but was decreased in the ISO + RSV group (Fig. 4A). Immunoprecipitation (IP) revealed an interaction between SIRT1 and Smad2/3 (Fig. 4B, C), which suggests that the TGF-β/Smad2/3 pathway is involved in the regulation of EndMT by SIRT1.
2.1. SIRT1 activated by RSV attenuated ISO-induced myocardial hypertrophy After two weeks of RSV treatment, myocardial function was evaluated by echocardiographic measurements. No significant difference was observed in the heart rate during anesthesia among the different groups. EX527 is an effective, selective SIRT1 inhibitor and EX527 could effectively inhibit the activity of SIRT1 deacetylase. The corresponding echocardiographic images indicated that the thickness of the left ventricular posterior wall and interventricular septal was increased in the ISO and ISO + RSV + EX527 groups, whereas no thickness was observed in the ISO + RSV group (Fig. 1A). RSV treatment also prevented the ISO-mediated increase in the left ventricular mass, fractional shortening (FS), and ejection fraction (EF), and decrease in the left ventricular internal dimension at diastole (LVIDd), left ventricular internal dimension at systole (LVIDs), left ventricular end-diastolic volume (LVEDV), and left ventricular end-systolic volume (LVESV) (Fig. 1B). These results suggest that SIRT1 activated by RSV attenuated compensatory myocardial hypertrophy in the ISO-induced mouse model.
2.6. Activation of SIRT1 inhibited TGF-β1-induced EndMT in H5V cells To examine the effect of SIRT1 on EndMT in vitro, EndMT was induced by TGF-β1 in H5V cells. SIRT1, endothelial markers (VE-cadherin and CD31), and fibroblast markers (α-SMA and vimentin) were evaluated. TGF-β1 changed the shape of the cells from oval to spindleshaped (Fig. 5A), but it exerted no significant cellular cytotoxicity (Fig. 5B). In addition, TGF-β1 downregulated VE-cadherin and CD31 while upregulating vimentin and α-SMA, indicating that the progression of EndMT was promoted. TGF-β1 also inhibited SIRT1 expression (Fig. 5D), whereas both RSV and SRT2014 upregulated SIRT1 expression in TGF-β1-treated cells while inhibiting EndMT development (Fig. 5D). These changes were further verified by double immunofluorescence staining (Fig. 5C), which showed that TGF-β1 greatly reduced the number of VE-cadherin-positive cells and increased that of αSMA-positive cells. SIRT1 activated by either RSV or SRT2104 reversed the TGF-β1-induced changes. These findings demonstrate that the activation of SIRT1 attenuated TGF-β1-induced EndMT in H5V cells.
2.2. SIRT1 activated by RSV prevented cardiac interstitial collagen deposition To measure the degree of cardiac fibrosis, histological analysis was performed using hematoxylin and eosin (H&E) and Masson’s trichrome 2
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Fig. 1. SIRT1 activated by resveratrol (RSV) attenuated cardiac hypertrophy induced by isoproterenol (ISO). (A) Representative echocardiographic photos from M-mode. (B) Echocardiographic parameters including interventricular septal thickness at diastole (IVSd), interventricular septal thickness at systole (IVSs), left ventricular internal dimension at diastole (LVIDd), left ventricular internal dimension at systole (LVIDs), left ventricular posterior wall thickness at diastole (LVPWd), left ventricular posterior wall thickness at systole (LVPWs), left ventricular enddiastolic volume (LVEDV), left ventricular endsystolic volume (LVESV), ejection fraction (EF), fractional shortening (FS), and left ventricular mass. *p < 0.05, **p < 0.01. Data are represented as the mean ± standard error of the mean (SEM), n = 6.
2.7. Overexpression of SIRT1 suppressed TGF-β1-induced EndMT in H5V cells
2.9. TGF-β/Smad2/3 was involved in the regulation of EndMT by SIRT1 in H5V cells
To further demonstrate that SIRT1 plays a key role in EndMT, lentiviruses were used to overexpress SIRT1 in H5V cells. Lentiviral transfection was performed for 48 h followed by treatment with TGF-β1 for 48 h. The transfection efficiency was evaluated using green fluorescent protein (Fig. 6A), and positive cells were selected using 2 μg/mL puromycin before TGF-β1 treatment. Western blot revealed that SIRT1 overexpression inhibited TGF-β1-induced EndMT (Fig. 6B).
The mechanism by which SIRT1 inhibited TGF-β1-induced EndMT in vitro is consistent with the in vivo mechanism. TGF-βR1 and PSmad2/3 were upregulated in TGF-β1-induced EndMT and the activation of SIRT1 attenuated the expression of relevant proteins (Fig. 8A). EX527 increased the expression of the abovementioned proteins compared to that in the RSV group. The overexpression of SIRT1 also reduced the expression of TGF-βR1 and P-Smad2/3 in TGF-β1-induced EndMT (Fig. 8B). Moreover, IP revealed an interaction between SIRT1 and Smad2/3 (Fig. 8C, D). These findings indicate that SIRT1 regulated TGF-β1-induced EndMT via the TGF-β/Smad pathway.
2.8. SIRT1 inhibited nuclear translocation of Smad2/3 in TGF-β1-treated H5V cells
3. Discussion SIRT1 and Smad2/3 were expressed in the cytoplasm of normal H5V cells. When the cells were treated with TGF-β, SIRT1 was downregulated and Smad2/3 activation was inhibited. Then, the nuclear translocation of P-Smad2/3 in TGF-β1-treated H5V cells was inhibited by SIRT1 overexpression (Fig. 7).
In the current study, we revealed that SIRT1 activated by RSV attenuated ISO-induced cardiac dysfunction and fibrosis by regulating EndMT in vivo. Additionally, SIRT1 overexpression suppressed the development of TGF-β1-induced EndMT in vitro. To our knowledge, our 3
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Fig. 2. SIRT1 activated by RSV attenuates ISO-induced cardiac fibrosis. (A) H&E staining of the left ventricular myocardium. Masson’s trichrome staining and immunohistochemistry of collagen I and collagen III. Scale bar =50 μm. (B) mRNA and protein expression of collagen I and collagen III. *p < 0.05, **p < 0.01. Data are represented as the mean ± SEM, n = 6.
study is the first to prove that SIRT1 attenuated cardiac fibrosis by regulating EndMT both in vivo and in vitro, although numerous studies have shown that SIRT1 attenuated cardiac fibrosis by inhibiting oxidative stress [23]. The activation of SIRT1 suppressed that of TGF-βR1 and P-Smad2/3 both in vivo (Fig. 4) and in vitro (Fig. 8). The interaction between SIRT1 and Smad2/3 was demonstrated by IP, and SIRT1 was found to inhibit the nuclear translocation of Smad2/3 in TGF-β1treated H5V cells. Sirtuins have been shown to dictate “replicative lifespan” and control stress resistance and longevity [30]. Numerous studies have shown that overexpression of sirtuins in fruit flies resulted in a renewable increase in longevity [31,32]. RSV is the most potent sirtuin-activating compound (STAC) discovered for SIRT1 [33]. SRT2104, a STAC, mimics aspects of calorie restriction and extends male mouse lifespan [34]. The present study showed a reduction of SIRT1 expression and activity in cardiac fibrosis. By activating SIRT1, RSV attenuated compensatory myocardial hypertrophy and reduced interstitial collagen deposition in an ISO-induced mouse model. When SIRT1 was inhibited by EX527, the protective effects of RSV was eliminated. Cardiac fibrosis is a serious global health concern involving the excess deposition of ECM in cardiac muscles and is associated with multiple forms of cardiovascular disease [3]. Recent studies have shown that EndMT is a significant contributor to the development of various types of organic fibrosis, including cardiac fibrosis [8]. Cardiac myofibroblasts could also originate from ECs through EndMT during the development of tissue fibrosis [8]. Myofibroblasts originate from the proliferation of resident fibroblasts and are activated in response to
TGF-β stimulation [8]. Since EndMT may be a source of mesenchymal cells, this transition could activate myofibroblasts and contribute to the development of myocardial fibrosis [8]. TGF-β is the main factor driving fibrosis in most forms of fibrotic diseases. Additionally, it contributes to the development of fibrotic diseases by enhancing the expression of collagen and inducing the differentiation of cells into myofibroblasts [35]. TGF-β also induces EndMT to further contribute to the development of fibrosis [36,37]. The present study showed that the expression of TGF-βR1, α-SMA, and FSP1 (mesenchymal markers) was upregulated while that of VE-cadherin and CD31 (endothelial markers) was downregulated in ISO-induced cardiac fibrosis. Moreover, the activation of SIRT1 by RSV suppressed the activation of TGF-βR1, VE-cadherin, and CD31 while increasing the activation of α-SMA and FSP1. Similar results were observed in H5V cells treated with TGF-β1-induced EndMT. These findings suggest that SIRT1 attenuated cardiac fibrosis via the regulation of EndMT both in vivo and in vitro. Numerous fibrotic diseases, including cardiac fibrosis, are attenuated in Smad3-deficient animals [16]. One study showed that Smad2/3 may be a specific target of SIRT1 and that SIRT1 could therefore be used as a novel therapeutic strategy to prevent heart failure [22]. Both Smad2 and Smad3 were found to be key mediators of TGF-β-induced tissue fibrosis and ECM production, maintaining the activity of activated fibroblasts. Moreover, Smad3 was required when TGF-β induced the synthesis of α-SMA [16]. In fibroblasts, the TGF-β type I receptor could phosphorylate Smad2/3. SIRT1 activation by geniposide attenuated ISO-induced cardiac fibrosis via the TGF-β/Smad 4
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Fig. 3. SIRT1 activated by RSV inhibited cardiac EndMT in mice. (A) mRNA and protein expression of SIRT1 in cardiac tissue. (B) Double immunofluorescence staining of VE-cadherin (green) and α-SMA (red) was performed to evaluate EndMT in fibrotic hearts at ×400 magnification. Scale bar =50 μm. (C) Representative protein expression of endothelial markers, CD31 and VE-cadherin, and mesenchymal markers, α-SMA, vimentin, and fibroblast-specific protein-1 (FSP1), in cardiac tissue assayed by western blot. Western blot results were normalized to β-actin. *p < 0.05, **p < 0.01. Data are represented as the mean ± SEM, n = 6.
experiments. A previous study has shown that SIRT1 repressed TGF-βinduced EndMT in human umbilical cord ECs through direct Smad4 deacetylation [38]. P‑Smad2/3 bound to Smad4 is transferred to the nucleus and activates gene transcription [39]. In the current study, TGF-β was also used to induce EndMT. As a result, we elucidated a new mechanism whereby SIRT1 binding to Smad2/3 inhibited the nuclear translocation of Smad2/3. These findings indicate that overexpression of SIRT1 suppressed TGF-βR1 and P-Smad2/3 levels. The fact that P-
signaling pathway [23]. Thus, we speculated that SIRT1 regulates EndMT via the TGF-β/Smad signaling pathway. In the current study, P-Smad2/3 expression was increased in ISOinduced cardiac fibrosis, but was attenuated by RSV-activated SIRT1. To further elucidate the underlying mechanisms, we used IP to identify whether an interaction exists between SIRT1 and Smad2/3. We chose to use H5V cells, a type of mouse heart microvascular ECs, in the in vitro study as they are from the same species as that in the in vivo
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Fig. 4. Western blot analysis of TGF-β/Smad and immunoprecipitation (IP) of the interaction between SIRT1 and Smad2/3 in the ISOinduced mouse model. (A) The expression levels of TGF-β receptor 1 (TGF-βR1) and its downstream proteins P-Smad2/3 were examined. (B, C) The interaction between SIRT1 and Smad2/3 was examined by IP. *p < 0.05, **p < 0.01. Data are represented as the mean ± SEM, n = 6.
obtained from Peprotech (Rocky Hill, NJ, USA). Antibodies against the following proteins were purchased from Abcam(Cambridge, MA, USA): TGF-βR1 (ab31013), CD31 (ab28364), VE-cadherin (ab33168), vimentin (ab92547), α-SMA (ab32575), and FSP1 (ab197896). The following antibodies were purchased from Cell Signaling Technology (Boston, MA, USA): P-Smad2/3 (8828), T-Smad2/3 (8685). The following antibodies were purchased from Proteintech (Chicago, IL, USA): SIRT1, collagen I, collagen III, and β-actin. Cell counting kit 8 was purchased from Invitrogen (Carlsbad, CA, USA).
Smad2/3 is found downstream of TGF-β suggests that SIRT1 suppressed the progress of TGF-β-induced EndMT by binding to Smad2/3 (Fig. 9). In the TGF-β/Smad pathway, TGF-β can activate both Ras/Erk mitogen-activated protein kinase and phosphoinositide 3-kinase/Akt signaling, which also play important roles in EndMT in a TGF-β-dependent manner [40]. SIRT1 reportedly inhibited both the Erk1/2 and Akt signaling pathways in cardiac fibrosis [41,42]. SIRT1 could act on Akt and Erk1/2 directly or via other pathways, but further research is required in order to elucidate this mechanism in detail. Although TGF-β has been identified as one of the most important growth factors in the induction of EndMT, other signaling pathways, such as the Notch and Wnt/β-catenin pathways, can also regulate EndMT in the cardiovascular system [43]. Therefore, further investigation is required to clarify whether other signaling pathways also participate in the regulation of EndMT via SIRT1.
5.2. Animals and treatment Adult male C57BL/6 mice, aged 8–10 weeks, were purchased from the Wenzhou Medical University Laboratory Animal Centre (Wenzhou, China). All animal procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). The animals were housed individually in cages under hygienic conditions in a 12/12-h light/dark cycle at 22 ± 3 °C and 45 ± 10% humidity for seven days before the experiments. The study was evaluated and approved by the Wenzhou Medical University Animal Care and Use Ethics Committee (permission number wydw2013-0054). The animals were allowed free access to a standard commercial diet and tap water. The mice were then randomly divided into four groups (10 per group) for treatment: Control, ISO, ISO + RSV, and ISO + RSV + EX527. In the ISO model mice, ISO was injected subcutaneously at 10 mg/kg/day for three days and then at 5 mg/kg/day for 11 days [46]. In the treatment groups, RSV (20 mg/kg/day) was intraperitoneally injected with or without EX527 (5 mg/kg/day) for 14 days [45,47,48]. The control group was treated with the corresponding solvent.
4. Conclusions Our study demonstrated that SIRT1 activated by RSV attenuated cardiac fibrosis induced by ISO via the regulation of EndMT. SIRT1 binding to Smad2/3 inhibited Smad2/3 nuclear translocation, thereby regulating EndMT via the TGF-β/Smad2/3 pathway in vivo and in vitro. The present study provides significant experimental evidence supporting the use of SIRT1 as a potential therapeutic target for cardiac fibrosis and suggests a theoretical basis for the treatment of other fibrotic diseases. 5. Materials and methods 5.1. Reagents ISO was purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in phosphate-buffered saline (PBS). RSV (HY-16561, 98.90% purity) was obtained from MedChem Express (Monmouth Junction, NJ, USA) and dissolved in 5% dimethyl sulfoxide (DMSO), 5% Tween 80, and 90% saline [44]. EX527, an inhibitor of SIRT1, was purchased from MedChem Express and dissolved in a special solvent composed of 1% DMSO, 30% PEG-400, and 1% Tween 80 [45]. SRT2104 (HY-15262), a synthetic small molecule activator of SIRT1, was purchased from MedChem Express and dissolved in 10% DMSO. TGF-β1 (100-21) was
5.3. Echocardiographic analysis of cardiac hypertrophy The mice were anesthetized with 1.5% isoflurane before being subjected to echocardiography using a Vevo1100 system (VisualSonics System, Toronto, Ontario, Canada). The following parameters were recorded: interventricular septal thickness at diastole (IVSd), interventricular septal thickness at systole (IVSs), left ventricular internal dimension at diastole (LVIDd), left ventricular internal dimension at systole (LVIDs), left ventricular posterior wall thickness at diastole 6
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Fig. 5. Activation of SIRT1 inhibited TGF-β1-induced EndMT in H5V cells. (A) Changes in endothelial morphological phenotype induced by TGF-β1 in H5V cells. Scale bar =50 μm. The cells gradually changed from an oval to a spindle shape. (B) The cytotoxicity of different treatments in H5V cells was assessed using a cell counting kit 8 assay. (C) Confocal microscopic images of VE-cadherin and α-SMA representing EndMT. Scale bar =20 μm. (D) Protein expression of SIRT1, CD31, VEcadherin, α-SMA, and vimentin was assessed by western blot. *p < 0.05, **p < 0.01. Data are represented as the mean ± SEM, n = 6.
5.4. Histological quantification of cardiac fibrosis
(LVPWd), left ventricular posterior wall thickness at systole (LVPWs), left ventricular end-diastolic volume (LVEDV), left ventricular endsystolic volume (LVESV), ejection fraction (EF), fractional shortening (FS), and left ventricular mass.
After the 14-day treatment described above, all mice were sacrificed and the hearts were quickly excised, arrested in diastole with 1.34 M KCl, weighed, placed in 4% paraformaldehyde, and embedded in
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Fig. 6. Overexpression of SIRT1 suppressed TGF-β1-induced EndMT in H5V cells. (A) H5V cells were transfected with lentiviral (LV)SIRT1 or LV-Con for 48 h. Green fluorescent protein was used to evaluate the transfection efficiency of LV-SIRT1 and empty vectors. Scale bar =50 μm. (B) The protein expression of SIRT1, CD31, VE-cadherin, α-SMA, and vimentin was assessed by western blot after transfection. *p < 0.05, **p < 0.01. Data are represented as the mean ± SEM, n = 6.
were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 U/mL streptomycin (Sigma, Milan, Italy) at 37 °C in a humidified atmosphere containing 5% CO2. After overnight serum starvation, H5V cells were randomly treated with TGF-β (20 ng/mL) and RSV or SRT2104 (20 μM) for 24 h [49]. SIRT1 expression was inhibited by EX527 (10 μM).
paraffin. The hearts were cut transversely close to the apex. Sections of the heart (4–5 μm) were mounted onto slides and stained with hematoxylin and eosin (H&E, Solarbio, Beijing, China) for histopathological analysis. To determine collagen deposition, tissue sections were subjected to Masson’s trichrome staining (Solarbio, Beijing, China) and visualized by light microscopy (ECLIPSE 80i, Nikon Corporation). The photographs were analyzed using digital analysis software (ImageJ) in a blinded manner. For immunohistochemical analysis, cardiac sections were deparaffinized in xylene and rehydrated in a graded ethanol series. Endogenous peroxidase activity was quenched in 3% methanolH2O2 for 10 min. Antigen retrieval was performed by heating the sections in 10 mM citrate buffer (pH 6.0) in a microwave oven. Non-specific binding was blocked in 1% bovine serum albumin. Next, the sections were incubated with primary antibodies against collagen I and collagen III for 2 h, followed by incubation with horseradish peroxidase goat anti-rabbit IgG for 1 h. Labeling was visualized using chromogen diaminobenzidine staining (Boster, Wuhan, China) and the slides were counterstained with hematoxylin.
5.7. Overexpression of SIRT1 via lentiviral transfection Empty vectors (LV-Con) and LV with mouse SIRT1 (LV-SIRT1) were constructed by Genechem (Shanghai, China). To distinguish the transfected cells and measure transfection efficiency, enhanced green fluorescent protein cDNA was inserted into the plasmid sequence. LVs (LV-Con and LV-SIRT1) at a multiplicity of infection of 50 were added to the H5V cells. Twelve hours after transfection, the lentiviruses were removed. Forty-eight hours after transfection, cells were selected with 2 μg/mL puromycin in the culture medium. The transfection efficiency of LV-Con and LV-SIRT1 was evaluated by quantifying the green fluorescent protein in the cells.
5.5. qRT-PCR 5.8. Histological analysis of EndMT The mRNA levels of SIRT1, collagen I, collagen III, TGF-β1, and GAPDH were analyzed by qRT-PCR. Total RNA was extracted from frozen, pulverized left ventricular cardiac tissue using TRIzol reagent following the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). The RNA purity was evaluated based on the OD260/OD280 ratios detected with NanoDrop One (Thermo Scientific, USA). The mRNA was reverse-transcribed into cDNA and synthesized from 2 μg of total RNA using oligo (dT) primers and the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, USA). qRT-PCR was performed according to the instructions using a SuperReal PreMix Plus (SYBR Green) (Tiangen, Beijing, China) by StepOnePlus (Applied Biosystems, Carlsbad, CA, USA). GAPDH and 18S were used as internal controls. The primers used in the study are shown in Table 1.
In vivo, tissue sections were co-incubated with the antibodies against VE-cadherin (1:100) and α-SMA (1:100) at 4 °C overnight and washed three with PBS for 5 min per wash. The sections were then incubated with Alexa Fluor 488/Alexa Fluor 546 antibodies (1:1000; Invitrogen) for 1 h before incubating with 4′,6-diamidino-2-phenylindole (Sigma, Milan, Italy) for nuclear staining. The results were analyzed by fluorescence microscopy (ECLIPSE Ti-S; Nikon Corporation). In vitro, H5V cells subjected to various treatments were fixed with 4% paraformaldehyde in 0.1 M PBS for 15 min. Then, the cells were washed three times with PBS, followed by permeabilization with 0.3% Triton X-100 for 15 min at 25℃. After culturing with 5% bovine serum albumin for 1 h, the cells were stained for VE-cadherin (1:100) and αSMA (1:100) at 4 °C overnight. Then, the cells were stained with Alexa Fluor 488/Alexa Fluor 546 antibodies (1:1000; Invitrogen) for 1 h and washed three times with PBS before nuclear staining. The results were analyzed by fluorescence microscopy (LSM800; Zeiss Corporation) and
5.6. Cell culture and treatments H5V cells, a type of mouse heart microvascular endothelial cell, 8
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Fig. 7. SIRT1 inhibited the nuclear translocation of Smad2/3 in TGF-β1-treated H5V cells. (A&B) SIRT1 overexpression inhibited the activation of Smad2/3, then inhibited P-Smad2/3 translocation into the nuclei of TGF-β1-treated H5V cells. Scale bar =50 μm. (C) SIRT1 was inhibited in TGF-induced EndMT and expressed in the cytoplasm. Scale bar =50 μm.
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Fig. 8. SIRT1 suppressed the expression of TGF-βR1 and P-Smad2/3 and interacted with Smad2/3 in TGF-β1-treated H5V cells. (A, B) Western blot of TGF-β/Smad and other signaling pathways in TGF-β1-induced EndMT. (A) SIRT1 was activated by RSV and SRT2014. (B) SIRT1 was overexpressed via lentivirus (LV) transfection. (C, D) The interaction between SIRT1 and Smad2/3 was revealed by IP. *p < 0.05, **p < 0.01. Data are represented as the mean ± SEM, n = 6.
Fig. 9. SIRT1 regulates EndMT by inhibiting the nuclear translocation of Smad2/3 in TGF-β1-treated H5V cells.
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Table 1 Gene-specific primers used in qRT-PCR. Species
Genes
Sequences
Mouse Mouse Mouse Mouse
SIRT1 Collagen I Collagen III GAPDH
Forward: Forward: Forward: Forward:
5'-GCTGACGACTTCGACGACG-3' Reverse: 5'-TCGGTCAACAGGAGGTTGTCT-3' 5'-GCTCCTCTTAGGGGCCACT-3' Reverse: 5'-CCACGTCTCACCATTGGGG-3' 5'-CTGTAACATGGAAACTGGGGAAA-3' Reverse: 5'-CCATAGCTGAACTGAAAACCACC-3' 5'-AGGTCGGTGTGAACGGATTTG-3' Reverse: 5'-TGTAGACCATGTAGTTGAGGTCA-3'
all images acquired were analyzed in a blinded way using ImageJ.
© 2019 by the authors. Submitted for possible open access publication under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
5.9. Western blot
Appendix A. Supplementary data
Heart tissues and H5V cells were lysed in radioimmunoprecipitation assay buffer (Beyotime, Shanghai, China) and centrifuged at 4 °C at 13,000 rpm for 10 min. The supernatant was collected and protein quantification was carried out using a bicinchoninic acid protein assay (Beyotime). The extracted protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride membranes. The membranes were blocked and incubated with primary antibodies and the expression levels of the target proteins were normalized to that of β-actin. After incubation at 4 °C overnight, the membranes were washed three times with Trisbuffered saline/Tween, incubated with horseradish peroxidase goat anti-rabbit IgG or goat anti-mouse IgG for 1 h at room temperature (1:5000, Invitrogen), and scanned using the Molecular Imager ChemiDoc XRS Imaging System (Bio-Rad, Hercules, CA, USA).
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5.10. Immunoprecipitation For immunoprecipitation, 500 μg of total protein extract was blocked with 20 μL of Protein A–Agarose (Sigma-Aldrich, St. Louis, MO, USA). Then, 2 μL of the appropriate antibody was added to the supernatant and incubated overnight at 4 °C. The antibodies were also purified using Protein A–Agarose. In all cases, the samples were analyzed by SDS-PAGE, followed by western blot with the appropriate antibodies. 5.11. Statistical analysis The results are expressed as mean ± standard error of the mean and analyzed by one-way analysis of variance and Tukey's post-hoc test (*p < 0.05 and **p < 0.01) with GraphPad Prism 5.0. Supplementary Materials: Table S1. Echocardiographic parameters in RSV control and EX527 control group; Figure S1. Neither RSV nor EX527 caused significant histological alterations. Declaration of competing interest The authors declare that they have no conflict of interest. Author contributions Methodology and investigation, Z.H.L. and X.W; formal analysis and writing—review and editing, Y.H.Z., Y.Q.Z. and X.F.F; supervision and writing—original draft preparation, Y.S.H.G. and L.P.H. Funding This research was funded by the Natural Science Foundation of Zhejiang Province, grant number LY17H020010.
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