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EGCG inhibits CTGF expression via blocking NF-KB activation in cardiac fibroblast.

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Keywords: Epigallocatechin-3-gallate Cardiac fibrosis Connective tissue growth factor Reactive oxygen species NF-KB

ABSTRACT

Connective tissue growth factor (CTGF) has been reported to play an important role in tissue fibrosis and presents a promising therapeutic target for fibrotic diseases. In heart, inappropriate increase in level of CTGF promotes fibroblast proliferation and extracellular matrix (ECM) accumulation, thereby exacerbating cardiac hypertrophy and subsequent failure. Epigallocatechin-3-gallate (EGCG), the major polyphenol found in green tea, possesses multiple protective effects on the cardiovascular system including cardiac fibrosis. However, the molecular mechanism by which EGCG exerts its anti-fibrotic effects has not been well investigated. In this study, we found that EGCG could significantly reduce collagen synthesis, fibronectin (FN) expression and cell proliferation in rat cardiac fibroblasts stimulated with angiotensinll (Ang11). It also ameliorated cardiac fibrosis in rats submitted to abdominal aortic constriction (AAC). Moreover, EGCG attenuated the excessive expression of CTGF induced by AAC or Angll, and reduced the nuclear translocation of NF-KB p65 subunit and degradation of IKB-et. Subsequently, we demonstrated that in cardiac fibroblasts NF-KB inhibition could suppress Angll-induced CTGF expression. Taken together, these findings provide the first evidence that the effect of EGCG against cardiac fibrosis may be attributed to its inhibition on NF-KB activation and subsequent CTGF overexpression, suggesting the therapeutic potential of EGCG on the prevention of cardiac remodeling in patients with pressure overload hypertrophy.

[c] 2012 Elsevier GmbH. All rights reserved.

Introduction

Cardiac fibrosis is a pathological feature of many heart diseases, which is characterized by interstitial fibroblast proliferation and excessive production and deposition of myocardial extracellular matrix (ECM) including collagens and fibronectin (FN) (Weber et al. 1994). Although much progress has been made in recent years, the pathogenesis of cardiac fibrosis remains to be clarified. Connective tissue growth factor (CTGF) is a matricellular protein (Perbal 2004), which is expressed in multiple tissues and cell types (Chen et al. 2000). Over the last decade it has become increasingly clear that CTGF is an important mediator and marker of tissue fibrosis (Daniels et al. 2009: Ruperez et al. 2003). Enhanced CTGF protein levels were reported in cardiac tissue from patients with heart failure, and the CTGF-stained area was found to correlate with the degree of myocardial fibrosis (Koitabashi et al. 2007). It is suggested that CTGF may be an attractive target for the treatment or prevention of cardiac fibrosis involved in cardiac hypertrophy and heart failure.

Tea is the most popular and widely consumed beverage in the world. Epidemiological studies have proved that tea consumption is associated with a reduced risk of cardiovascular disease (Higdon and Frei 2003; Zheng et al. 2004). Epigallocatechin-3-gallate (EGCG) is a major polyphenol found in green tea (dried fresh leaves of the plant, Camellia sinensis, Theaceae). Recently, it has attracted considerable attention for the potential effect on prevention of oxidative stress-related diseases such as ischemic heart injury and hypertension (Huo et al. 2008). To date, the antioxidant properties of EGCG have been well addressed (Lambert and Elias 2010), and accumulating evidences indicate that the EGCG-induced cardioprotection may be mediated through free radical scavenging (Higdon and Frei 2003; Wolfram 2007). Besides these findings, researchers also revealed that EGCG blocked cardiac hypertrophy and fibrosis both in vivo and in vitro (Li et al. 2006; Sheng et al. 2009). However, the molecular mechanisms underlying the anti-hypertrophic effect of EGCG remain to be clarified, and little is known about the potential target of EGCG on cardiac fibrosis relating to cardiac hypertrophy. The aim of this study is to determine whether EGCG protects against pressure overload-induced cardiac fibrosis by suppressing CTGF expression, and to further investigate the potential molecular mechanism underlying.

Materials and methods

Abdominal aortic constriction surgery

Sprague-Dawley (SD) rats (male, weighing 180-200g. SPF grade, certification no. 0067950) were supplied by the Experimental Animal Center of Sun Yat-Sen University (Guangzhou, China). Pressure overload was induced in rats by abdominal aortic constriction (AAC) according to procedure reported by others (Phrommintikul et al. 2008). The investigation conformed to 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 animal experiments were approved by the Institutional Ethics Review Board of Sun Yat-Sen University.

SD rats were randomly assigned into the following five groups and the intragastrically treatment was performed as below:

Sham groups:

Group (1.): saline group, sham-operated, treated for 4 weeks with saline, i.g., daily, from the 1st day after surgery (n=10).

Group (2.): EGCG group, sham-operated, treated for 4 weeks with 50 mg/kg EGCG, i.g., daily, from the 1st day after surgery (n = 10).

AAC groups:

Group (3.): saline group, submitted to AAC, treated for 4 weeks with saline, i.g., daily, from the 1st day after surgery (n=10).

Group (4.): EGCG low-dose group (EGCG-L), submitted to MC, treated for 4 weeks with 25 mg/kg EGCG, i.g., daily, from the 1st day after surgery (n= 10).

Group (5.): EGCG high-dose group (EGCG-H), submitted to MC, treated for 4 weeks with 50 mg/kg EGCG, i.g., daily, from the 1st day after surgery (n=10).

EGCG ([greater than or equal to]98%, purity by HPLC) was purchased from Aladdin Reagent Co., Ltd. (Shanghai, China), which was separated from green tea. After 4 weeks, all animals were sacrificed and the hearts were removed.

Cell culture

Adult rat cardiac fibroblasts were prepared and cultured as previously described (Zhang et al. 2007). The purity of cardiac fibroblasts was greater than 95% as determined by positive staining for vimentin and negative staining for a-actin and von Willebrand factor. Third to fifth passage cardiac fibroblasts were used for all experiments, and the cells were starved for 24 h in Dulbecco's modified Eagle's medium (DMEM) supplemented with 0.5% fetal bovine serum.

Histological analysis

Left ventricles were fixed with 4% paraformaldehyde, dehydrated, and embedded in paraffin. Sections of heart (4-5 p.m thick) were prepared and stained with picrosirius red (PSR) for collagen deposition. To determine the degree of cardiac fibrosis, collagen area fraction was calculated as percent surface area occupied by collagen. The quantification was performed with Image Pro-Plus software (Media-cybernetics) by two independent investigators blinded to the experiment procedure.

Hydroxyproline and malondialdehyde measurement

The content of hydroxyproline and malondialdehyde (MDA) in myocardial tissue was assayed by two commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's instructions.

Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA from tissues or cultured cells was extracted using Trizol reagent (Invitrogen). One microgram of total RNA was reverse transcripted using One-step RT Kit (Takara Biotechnology) and the resulting cDNA was used as a PCR template. The mRNA expression levels were determined using SYBR-Green Quantitative PCR Kit (Takara Biotechnology) by iCycler iQ system (Bio-Rad). All PCRs were done in triplicate. Rat-specific primers (Supplementary Table S1) for collagen-I, collagen-III and CfGF were synthesized by lnvitrogen. GAPDH was used as an endogenous control.

EdU incorporation assay

Cell proliferation was measured by 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay as described previously (Guo et al. 2011). In brief, cardiac fibroblasts were seeded into 96-well plates (5 x 103 cells per well) and exposed to 50pM EdU (Ribobio, Guangzhou, China) for 4h at 37 C. The cells were fixed with 4% formaldehyde for 15 min and treated with 0.5% Triton X-100 for 20 min at room temperature. After washing with phosphate buffered saline for three times, the cells were reacted with 100 ti..l of 1 x Apollo[R] reaction cocktail for 30 min and subsequetly stained with 100 p..1 Hoechst 33342 (5 [micro],g/m1) for 30 min. The EdU incorporation rate was expressed as the ratio of EdU positive cells to total Hoechst 33342 positive cells.

Western blot analysis

Rabbit anti-FN monoclonal antibody was purchased from Boster (Wuhan, China). Rabbit anti-CTGF polyclonal antibody and mouse anti-a-tubulin monoclonal antibody were purchased from Sigma. Mouse anti-NF-KB p65 subunit polyclonal antibody and rabbit anti-IkBot monoclonal antibody were purchased from Santa Cruz. Western blot analyses were performed as previously described (Zhou et al. 2006), and a-tubulin was used as a loading control.

Quantification of intracellular reactive oxygen species (ROS)

ROS was detected by fluorescent probe dye, 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA, Sigma) and dihythoethidium (DHE) (lnvitrogen) following the manufacturer's instructions.

Luciferase reporter gene assay

The pRL-TK and NFKB reporter plasmids pGL4.32, and dual-luciferase reporter assay system were all purchased from Promega. Adult rat cardiac fibroblasts were seeded at 5 x 104 cells/well into 96-well plates, and cotransfected with NF-KB reporter plasmid (100 ng/well) and pRL-TK (10 ng/well) as an internal control. Total amounts of transfected DNA were equalized by the addition of empty vector. After 8 h of incubation, the cells were serum-deprived for 12h and subjected to Angll stimulation with or without EGCG. The cell lysates were prepared with passive lysis buffer, and the luciferase activity was measured by Luciferase Reporter Assay System (Promega) and normalized by the transfection efficiency calculated by the Renilla luciferase activity from pRL-TK.

RNA interference

Three different duplex siRNAs for p65 (si01, si02, siO3) and negative control siRNA were purchased from lnvitrogen. Briefly, rat card iomyocytes were transfected with 05 siRNAs and negative control siRNA respectively using Lipofectamine 2000 (lnvitrogen) according to the manufacturer's instructions. At 48 h after transfection, Western blot were performed to determine the silencing efficiency. Among these siRNAs, si02 exhibited the best efficiency for 1365 knockdown and thereby was used in the following experiments (Supplementary Fig. S1).

Statistical analysis

Data are presented as mean [+ or -] SE. Statistical analyses between two groups were performed by unpaired Student's t-test. Differences among groups were tested by one-way analysis of variance (ANOVA) with Tukey's post hoc test. In all cases, differences were considered statistically significant with p <0.05.

Results

EGCG attenuates cardiac fibrosis from pressure overload

Sirius red staining was used to detect collagen distribution in left ventricular cross sections. The results showed that at 4 weeks after AAC surgery the collagen deposition was increased 3.6-fold, which was attenuated by treatment with EGCG. No significant difference was observed between the sham animals treated with saline and EGCG (Supplementary Fig. S2). In addition, the increase in hydroxyproline content in left ventricle of AAC rats was significantly reduced after EGCG treatment (Fig. 1A). Consistently, ciRT-PCR analysis revealed that the mRNA expression of myocardial collagen-I and III markedly increased after AAC, which could be inhibited by EGCG (Fig. 1B). Furthermore, Western blot analysis showed that EGCG suppressed the upregulation of FN protein level induced by AAC, while EGCG itself did not affect the expression of FN (Fig. 1C).

EGCG reduces rat cardiac fibroblasts proliferation and ECM deposition

The cultured rat cardiac fibroblasts were preincubated with 0.1, 1, and 10 nM EGCG for 1 h followed by treatment with 100 nM AngII for 24 h. The cell proliferation was determined by EdU incorporation assay, and collagen synthesis was detected by hydroxyproline and qRT-PCR assay. As shown in Fig. 2A, Ang11-induced cardiac fibroblasts proliferation was inhibited by 10 [micro]M EGCG. Moreover, the increase in hydroxyproline content induced by Ang11 was significantly reduced after EGCG treatment (Fig. 2B). Consistently, collagen-1 and collagen-III mRNA levels were both significantly upregulated by AngII which could be attenuated by EGCG pretreatment (Fig. 2C). Angll led to obvious upregulation of FN protein expression in cardiac fibroblasts, but it was blocked by 1 or 10 [micro]M EGCG (Fig. 2D).

EGCG inhibits CTGF expression induced by AAC or Angll

The effects of EGCG on CTGF expression were detected both in vivo and in vitro. As shown in Fig. 3A and B, the mRNA and protein levels of CTGF in myocardial tissues from AAC rats were significantly upregulated. EGCG itself showed no affect on CTGF expression, but it could reduce the excessive CTGF expression induced by AAC. Similar effects of EGCG were observed in cardiac fibroblasts treated with Angll (Fig. 3C and D).

EGCG blocks NF-k13 activation and transcriptional activity induced by Ang II

Translocation of NF-KB from the cytoplasm to the nucleus and degradation of IKB-a are known to be involved in the process of the activation of NF-KB (Ghosh et al. 1998; Wu et al. 2010). To determine the effect of EGCG on the NF-KB pathway, the relative nuclear level of NF-KB p65 subunit and the cytoplasmic level of 1KB-a were examined by Western blot analysis. As shown in Fig. 4A, the nucleic p65 level was detected to be significantly elevated and the degradation of IKB-a in cytoplasma was observed in myocardial tissues from AAC rats. Treatment with 25 mg/kg EGCG (EGCG-L) or 50 mg/kg EGCG (EGCG-H) could markedly block the increase in p65 expression and 1KB-a degradation following AAC. Moreover, preincubation of cardiac fibroblasts with EGCG for 1 h also suppressed NF-KB activation induced by Angll (Fig. 4B). Subsequently, the results of luciferase reporter gene assay revealed that EGCG dose-dependently inhibited Ang1I-induced NF-KB-Luc reporter gene activity in cultured cardiac fibroblasts submitted to Angll treatment (Fig. 4C).

NF-KB inhibition decreases the expression of CTGF induced by Angll in cardiac fibroblast

To further investigate the role of NF-KB pathway in the regulation of CTGF expression during cardiac fibrosis, the cultured rat cardiac fibroblasts were treated with pyrrolidine dithiocarbamate (PDTC), an inhibitor for NF-KB (Snyder et al. 2002), or transfected with siRNA directed against p65 (sip65) followed by Angll stimulation for 24h. The mRNA and protein levels of CTGF were detected respectively by ciRT-PCR and Western blot respectively. As shown in Fig. 5A and B, the expression of CTGF was significantly upregulated after Angll treatment, which could be reversed by PDTC or sip65. It indicated that the elevated CTGF expression in response to fibrotic stimuli required the participation of NF-KB.

Discussion

EGCG, the most abundant polyphenol in green tea, has attracted considerable attention for its protective effects against cardiovascular diseases through ameliorating inflammatory reaction (Jeong et al. 2004), lowering serum cholesterol levels, increasing high-density lipoprotein, and reducing evolving atherosclerosis (Chyu et al. 2004). Although Sheng et al. (2009) have recently shown that EGCG inhibited proliferation of cardiac fibroblasts in rats with cardiac hypertrophy, the role of EGCG in cardiac fibrosis is still not fully evaluated and its potential molecular target remains to be elucidated. In the present work, we revealed that EGCG attenuated cardiac fibrosis induced by Angll treatment or pressure overload induced by AAC, which might be mediated by suppressing CTGF expression. Furthermore, our results indicated that the inhibition effect of EGCG on CTGF could be at least partially attributed to the suppression of redox-sensitive NF-KB activation. These findings shed new light on the mechanism underlying anti-fibrosis effect of EGCG and support the potential use of EGCG as a preventive and therapeutic candidate against cardiac fibrosis and heart failure.

The pathogenesis of cardiac fibrosis is complex and far from well-understood. During the last decade, emerging evidence has suggested that TGF plays a crucial role in fibrogenic processes in multiple tissues including the heart (Daniels et al. 2009; Ruperez et al. 2003). It has been proved that CTGF is characteristically overexpressed in cardiac fibrosis in a fashion correlating with severity of fibrosis (Dziadzio etal. 2005). Moreover, CTGF can stimulate collagen synthesis in both cardiac fibroblasts and cardiomyocytes, and contribute to fibrosis by inducing the expression of tissue inhibitor of matrix metalloproteinases (TIMPs) to inhibit ECM degradation (Wang et al. 2010). Therefore, CTGF may be an important biomarker for the cardiovascular diseases where ECM accumulation is a key feature, such as ischemic cardiomyopathy (Chen et al. 2000) and myocardial infarction (Dean et al. 2005). In this study, we reported for the first time the inhibition effect of EGCG on CTGF expression under the circumstance of cardiac fibrosis. EGCG treatment could significantly reduce the upregulation of CTGF expression in heart of rats submitted to MC or cultured rat cardiac fibroblasts stimulated with Angll (Fig. 3). These results support the growing body of findings suggesting TGF as a potential candidate for therapeutic intervention to mitigate fibrosis in heart.

Recently, it has been recognized that Angll upregulates CTGF via a process mediated by production of transforming growth factor-3 (TGF-13) and ROS (Ruperez et al. 2003). TGF-f3 is an important regulator of cell proliferation and ECM (Huang and Huang 2005), and it can strongly induce CTGF expression in many cell types including cardiomyocytes and fibroblasts (Chen et al. 2000). Thus, the effect of EGCG on TGF-13 level in Ang11-incluced cardiac fibrosis was evaluated. Our result showed that the elevated TGF-13 level induced by Angll was not affected by EGCG treatment (Supplementary Fig. S3), indicating the anti-fibrotic effects of EGCG may not be dependent on the suppression of TGF-13 and subsequent CTGF expression. On the other hand, mounting evidences have suggested that oxidative stress, especially a chronic increase in ROS, stimulates and exacerbate fibrotic response (Liu et al. 2010). Not surprisingly, we observed that EGCG, as an effective antioxidant, could reduce ROS generation in both AAC and AngII-induced cardiac fibrosis (Supplementary Fig. S4). Therefore, the anti-fibrotic effect of EGCG may be partially related to the scavenging of ROS and the inhibition of subsequent CI'GF expression.

It has been well established that KOS generation leads to the activation of the transcription factor NF-KB associating with excessive accumulation of ECM including collagen and fibronectin, which participates in the progression of cardiac fibrosis (Kumar et al. 2011). Here, we found that that EGCG significantly decreased 1KB- cx and p65 translocation into the nucleus induced by Angll in cardiac fibroblasts. Similar results were observed in AAC rats after EGCG treatment (Fig. 4A and B). Moreover, EGCG could dose-dependently suppress the transcriptional activity of NF-KB as revealed by luciferase reporter gene assay (Fig. 4C). These findings are consistent with previous studies (Li et al. 2006), indicating that inhibition of NF-KB pathway is involved in the effect of EGCG against cardiac fibrosis. It has been reported that there are several consensus sequence of NF-KB on CTGF promoter (Chen and Zheng 2008). Thus we further examined the effect of PDTC, an inhibitor of NF-KB, and siRNA for p65 on CTGF levels in cultured rat cardiac fibroblasts. The results showed that NF-KB inhibition led to significant downregulation of CTGF mRNA and protein expression (Fig. 5). Convergently, it suggests that EGCG may inhibit CTGF expression via blocking NF-KB activation in cardiac fibroblast and thereby ameliorate cardiac fibrosis. Certainly, further studies are warranted to elucidate in detail the mechanism underlying EGCG-induced suppression of CTGF.

Conclusions

In summary, the present study demonstrates that EGCG prevents myocardial fibrosis induced by Angll and pressure overload, which may be attributed to inhibition on NF-KB signaling pathway and subsequent CTGF expression (Fig. 6). Since cardiac fibrosis is not only the pathological phenomenon during cardiac hypertrophy and remodeling but also a main risk factor for cardiac dysfunction, our findings suggest the potential application of EGCG in clinic to prevent the transition from cardiac hypertrophy to heart failure.

Conflict of interest

No conflict of disclose.

Acknowledgements

This work was supported by research grants from the National Natural Science Foundation of China (No. 81072641); NSFC-CIHR China-Canada Joint Health Research Initiative Proposal (No. 330811120434); Major Project of Guangdong Province (No. 2008A030201013); Major Project of Guangzhou City (No. 200821E571)

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at http://dx.cloi.org/10.1016/j.phymed.2012.10.002.

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* Corresponding authors at: Department of Pharmacology and Toxicology, School of Pharmaceutical Sciences, Sun Yat-sen University (Higher Education Mega Center), 132 East Wai-huan Rd., Guangzhou 510006, PR China. Tel.: +86 2039943116: fax: +86 20 39943026.

E-mail addresses: yejt@mail.sysu.edu.cn (J.-T. Ye), liupq@mail.systheclu.cn (P.-Q. Liu).

1 These authors contributed equally to this work.

0944-71131$-see front matter C 2012 Elsevier GmbH. All rights reserved. http://dx.doLorg/10.1016/j.phymed.2012.10.002

Yi Cai (a), (b), (c), Shan-Shan Yu (a), (c), (1), Ting-Ting Chen (a), Si Gao (a), Biao Geng (a), Yang Yu (a), Jian-Tao Ye (a), *, Pei-Qing Liu (a), *

(a.) Department of Pharmacology and Toxicology, School of Pharmaceutical Sciences, Sun Yat-sen University. Higher Education Mega Center, Guangzhou 510006, Guangdong, PR China

(b.) Guangzhou Research 1nsititute of Snake Venom,Guangzhou Medical College, Guangzhou 510182, Guangdong, PR China

(c.) Department of Pharmaceutical Science, Zhujiang Hospital. Southern Medical University, Guangzhou 510280,Guangdong, PR China
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Title Annotation:epigallocatechin-3-gallate; connective tissue growth factor; nuclear factor kappa-light-chain-enhancer of activated B cells
Author:Caia, Yi; Yu, Shan-Shan; Chen, Ting-Ting; Gao, Si; Geng, Biao; Yu, Yang; Yea, Pan-Tao; Liu, Pei-Qing
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9CHIN
Date:Jan 15, 2013
Words:4340
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