Epigallocatechin inhibits PDGF-BB-induced vascular smooth muscle cells proliferation via DNMT1 regulation of the Nur 77 axis

Epigallocatechin inhibits PDGF-BB-induced vascular smooth muscle cells proliferation via DNMT1 regulation of the Nur 77 axis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Epigallocatechin inhibits PDGF-BB-induced vascular smooth muscle cells proliferation via DNMT1 regulation of the Nur 77 axis Li shen, Juanjuan Tan, Feng Li, Ke Xia, Zhichao Yuan, Zhiqiang Yan This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4260039/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract EGCG inhibits vascular smooth muscle proliferation.Nur77 and DNMT1 have been observd in the plaques of patients with atherosclerosis and are thought to be associated with vascular smooth muscle cell proliferation. This study was designed to investigate the role and mechanism of epigallocatechin gallate (EGCG) on proliferation and migration of vascular smooth muscle cells( VSMCs) and clarified the underlying molecular mechanism of EGCG. We investigated whether EGCG suppressed platelet-derived growth factor(PDGF)-induced vascular smooth muscle cell proliferation, migration and apoptosis in vivo and vitro. The effect of EGCG on smooth muscle cell proliferation and phenotype were evaluated using Cell Counting Kit-8(CCK8), EdU staining, immunohistochemistry and Western blot analysis.The effect of EGCG on smooth muscle cell migration was uising Wound-healing assay and Western blot analysis..The effect of EGCG on smooth muscle cell apoptosis was uising Flow cytometry and Western blot analysis. The current findings demonstrated that EGCG alleviated neointimal hyperplasia in balloon-induced arterial walls in vivo, significantly inhibited PDGF-BB-induced VSMC proliferation, migration, and promoted apoptosis in vitro and identical results were obtained in vivo..Moreover, EGCG attenuated VSMC proliferation by modulating the regulation of the DNMT1-Nur77-signaling axis. Our collective data showed that EGCG inhibited PDGF-BB-induced VSMC proliferation via DNMT1 regulation of the Nur77- signaling axis. In conclusion, These findings suggest that EGCG may be a promising therapeutic agent for the prevention and treatment of CVDs. vascular smooth muscle cell EGCG Nur77 NR4A1 DNMT1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Cardiovascular diseases (CVDs) encompass chronic inflammatory disorders of the arterial vessel walls that eventually result in obstruction of blood flow and are a major cause of ischemic conditions,such as heart and brain infarctions[ 1 ].Vascular smooth muscle cells(VSMCs) are the major cell type in blood vessels, and the proliferation of VSMCs and their phenotypic transformation constitute the pathological basis of CVDs. Modulation of the VSMC phenotype,triggered by harmful microenvironmental stimuli followed by VSMC migration and proliferation, plays an important role in intimal hyperplasia, which is critical for the development of vascular disease[ 2 ]. Platelet-derived growth factor-BB (PDGF-BB) is a major driving factor in VSMC proliferation and migration involved in vascular remodeling and is also a natural ligand of PDGFR-β[ 3 ].PDGF-BB is a potent cellular mitogen and chemoattractant for VSMCs, and by activating its downstream signaling pathways, it contributes to many biological processes and diseases after binding with PDGFR-β[ 3 ]. Nur77 is a transcription factor that is also known as TR3, NR4A1, or NGFI-B, and is an immediate-early gene involved in stress responses in various cell types and organ systems[ 4 , 5 ]. Studies have revealed that Nur77 inhibits the proliferation of both arterial and venous SMCs[ 6 , 7 ] and that Nur77 induces a more differentiated contractile phenotype in SMCs, which is illustrated by the increased expression of SMC-specific marker proteins such as SM-actin and -calponin[ 6 , 8 ]. Inhibition of Nur77 by either small interfering (si)RNAs or overexpression of a dominant-negative variant of Nur77 resulted in enhanced SMC proliferation, and transgenic overexpression of Nur77 in arterial SMCs inhibited flow-induced outward remodeling. Nur77 did not modulate vascular tone but rather reduced macrophage accumulation and concomitantly lowered matrix metalloproteinase levels in remodeled arteries[ 9 ]. Recent studies demonstrated that lipoic acid inhibited neointimal formation in rat carotid arteries after balloon injury and accelerated apoptosis of SMCs through the upregulation of Nur77 [ 10 ] and PDGF-induced migration and proliferation of airway SMCs and increased cellular contractility[ 11 ]. DNA methylation refers to the binding of 5-methylcytosine (5-MC) to S-adenosine methionine in the cytosine ring to form 5-methylcytosine (5MC)[ 12 ]. Therefore,5-MC is an indicator adopted for the evaluation of DNA methylation, with the process occurring at specific dinucleotide sites called CpGs [ 13 ]. DNA methylation is generally acknowledged to be an inhibitory modification that inhibits the binding of the transcription complex to the target gene promoter [ 14 ]; thus, DNA hypermethylation leads to gene silencing [ 13 ]. Furthermore, global hypomethylation was detected in the proliferating intimal SMCs of New Zealand white rabbits that had undergone balloon denudation. Global hypomethylation with a concomitant decrease in DNA methyltransferase (DNMT) activity has also been shown to occur during SMC phenotypic modulation[ 15 ] and proliferation[ 16 ] in culture, and DNMTs are important factors that affect DNA methylation levels. Human-encoded DNMTs included DNMT1, DNMT2, DNMT3A, DNMT3B, and DNMT3L. DNMT1, DNMT3A, and DNMT3B are classical members of the DNMT family that catalyze genomic DNA methylation, whereas DNMT2 and DNMT3L are non-classical members that do not possess catalytic DNMT activity [ 17 ]. Previous studies have suggested that theNur77 is involved in the epigenetic pathway, and recent research has revealed that DNMT1 causes Nur77 DNA hypermethylation and blocks insulin signaling in patients with T2D[ 18 ]. In Dnmt1-KO mice, Nur77 reduced significant losses in methylation, suggesting that theexpressionof pro-apoptotic Nur77 was regulated by CpG methylation levels controlled by DNMT1[ 19 ], which is consistent with earlier findings. (−)-Epigallocatechin-3-gallate (EGCG) is the principal catechin in green tea (50–75%) and has been shown to have numerous potential health effects, including antioxidant, anticarcinogenic, hypocholesterolemic, and cardioprotective epigenetic activities[ 20 ]. EGCG regulates VSMC function through different mechanisms. For example, studies have depicted EGCG suppresses JNK activation, thereby protecting against angiotensin II-induced VSMC hypertrophy [ 21 ]. EGCG can also inhibit PKC- and ERK1/2-signaling pathways, thus improving hyperglycemic VSMC proliferation and injury-induced neointimal formation in the rat common carotid artery[ 21 , 22 ]. Investigators found that EGCG inhibited the proliferation of human aortic VSMCs and improved hypoxia-induced pulmonary artery revascularization by upregulating mitochondrial fusion protein gene-2[ 23 , 24 ]. EGCG also inhibits AT-1R and affects ERK1/2- and p38MAPK-signaling pathways so as to inhibit homocysteine-induced proliferation of VSMCs [ 25 ]. The purpose of this present study, we want to demonstrated that EGCG protects arteries against neointimal hyperplasia in vivo and PDGF-induced VSMC proliferation migrstion in vitro. Examine that EGCG attenuates neointimal hyperplasia through Nur77 and DNMT1, providing a potential natural compound for the treatment of CVD, as well as a theoretical basis for its clinical diagnosis and future treatment. Methods Animal studies Male Sprague–Dawley rats with a mean weight of 250 ± 20 g were purchased from the Shanghai Laboratory Animal Research Center (Shanghai,China) and housed at a constant temperature of 25°C ± 2 and humidity of 60 ± 5% with a 12-h dark/light cycle. This study was conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health and approved by our Ethical Committee (ethics code SCXK, Shanghai 2019-0006). The rats were anesthetized using isoflurane,and a mid line neck incision was made to expose the left common carotid artery. The carotid artery balloon-injury model for rats was then established as previously described[ 31 ], and the rats were randomly assigned to the following experimental groups: control, injured, and EGCG treatment. EGCG was administered once daily at 100 mg/kg for 21 days, whereas normal PBS was administered intragastrically to the control and injured groups. Rats were anesthetized using isoflurane, followed by extraction of the left common carotid artery. H&E staining The carotid arteries were collected, washed with pre-cooled saline to remove excess blood, fixed in 4% paraformaldehyde, and embedded in paraffin. Hematoxylin and eosin (H&E) staining was performed on 4-µm sections, which were imaged using a 09microscope (BX71;OlympusCorporation,Japan). The sizes of the intimal and medial areas were determined using the lumen, internal elastic lamina, and external elastic lamina and were measured using Image J software. Immunohistochemistry (IHC) Immunohistochemical staining was performed to detect PCNA, Nur77, DNMT1, MMP2, COL-I, COL-III, calponin, and α-SMA(Proteintech,Rosemont, IL, USA ).Briefly, 5-µM sections of the rat carotid artery were cut and dewaxed.The slides were then treated with 3% hydrogen peroxide for 30 min at 25°C, incubated with primary antibodies overnight at 4°C, washed three times with PBS, incubated with secondary antibody for 4 h, and stained with DAB (ENZO) after washing with buffer solution. Finally, the sections were counterstained with hematoxylin to visualize cellular nuclei.Image J software was used to analyze the average integrated optical density (IOD) and positively stained regions. Culture of VSMCs We isolated and cultured VSMCs using the explants method as previously described for rat aorta[ 26 ], and their purity was confirmed by staining the cells with α-SMA actin (Proteintech, Rosemont, IL, USA). VSMCs were then seeded in DMEM containing 20% fetal bovine serum(FBS),100 µg/ml streptomycin, and 100 units/ml penicillin and cultured at 37°C in 5% CO2 (Thermo Fisher, Waltham, MA, USA) EGCG was added to VSMCs after starvation for 24 h. Stimulation of VSMCs with PDGF-BB(20ng/ml) for 48 h was performed to examine the effects on cellular proliferation, migration, and apoptosis. All experiments were performed using cells from passages four to eight. Knockdown of Nur77 in VSMCs The pLenti-puro lentiviral vectors containing Nur77-specific small hairpin RNAs (shRNAs) were constructed by Vigene Bioscience Technology (Jinan, China), the pLenti-purolentiviralvectorcontainingnoNur77-specific small hairpin RNAs(siNC) was used as the negative control, and lentivital Nur77 overexpression vectors for Nur77 overexpression were purchased from GenePharma[ 27 ](China). The lentiviral vector and packaging plasmid mix were transfected into HEK293T cells [ 28 ] following the manufacturer’ sinstructions.The medium was replaced after 6h. Fresh medium was collected 72 h post-transfection and passed through a 0.45-µm filter. VSMCs were transduced with filtered lentiviral supernatants supplemented with 8µg/mL polybrene (Sigma-Aldrich, St. Louis, MO,USA)[ 29 ]. VSMC proliferation assay in vitro VSMC proliferation in vitro was determined using the Cell Counting Kit-8(CCK8) and EdU staining. Briefly, 100µL of VSMC suspension (0.5×104cells) was seeded in 96-well plates at 37°C in 5% CO2, and the cells were synchronized in DMEM containing 0.1% double antibiotic for 24h. The cells were then treated with vehicle control, PDGF-BB in DMEM, and /or PDGF-BB in DMEM containing EGCG at different concentrations at the indicated times. Next,10µL of CCK8 solution (MedChem Express,USA) was added to each well for an additional 1h, and absorbance was measured at 450 nm using a microplate reader (BioTek, Winooski, VT, USA) to determine the proliferative rate of the VSMCs. VSMC proliferation was also assessed using a BeyoClickEdU-488 Cell Proliferation Assay Kit(Beyotime,Beijing, China) according to the manufacturer’s instructions. Images were taken using a fluorescence microscope (BX71,Olympus Corporation, Japan), and the signals were counted in three random visual fields per sample. Wound-healing assay Cells were plated in six-well plates at a density of 1.5 × 105 cells/well, and at confluency,the cells were cultured inserum-free medium for 24h and a scratch was made in the cell monolayer with a sterile 200-µL pipette tip. The cells were subsequently treated with vehicle or 20ng/ml PDGF-BB, with or without EGCG(25µM) for the indicated time periods. The scratches were captured at 0 and 48 h using an inverted microscope (BX71; OlympusCorporation,Japan). Cell-cycle analysis VSMCs were seeded in six-well plates at 2 × 105 cells/well and incubated in an atmosphere of 5% CO2 at 37°C. VSMCs in each group were then pretreated with EGCG for 24 h, treated with 20 ng/ml PDGF for 24 h, collected, resuspended in 70% pre-cooled ethanol, and incubated at 4°C for 24 h. The fixed cells were washed twice with phosphate-buffered saline (PBS) and incubated at 37°C in the dark for 30 min using a solution containing DNase-free RNaseA (200µg/ml) and propidium iodide (PI, 50mg/ml). The cells were analyzed by flow cytometry (BD,USA). Western blot analysis Total protein was extracted from VSMCs and lysed with PierceR RIPA buffer (Beyotime Biotechnology, China) containing a cocktail of protease inhibitors, and the protein concentration was measured with a BCA kit (Beyotime Biotechnology,China), followed by immune blotting using dilutions of 1:500 for anti-DNMT1 (Abcam,UK), 1:1000 for Nur77 (Proteintech, Rosemont,IL,USA), and 1:2000 for MMP2, BAX, PCNA, tubulin, and GAPDH antibodies (Proteintech, Rosemont, IL, USA), and separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to nitrocellulose membranes and blocked with5%nonfatmilkinTris-buffered saline (TBS) and Tween (Tween20) (TBST) for 1h. The membranes were incubated with the corresponding primary antibodies at 4°C overnight, washed, and then incubated with the corresponding horseradishperoxidase (HRP)-conjugated goat anti-mouse(1:10,000) or goat anti-rabbit secondary antibody (1:10,000, Proteintech, Rosemont, IL, USA) for 1 h at room temperature. Enhanced chemiluminescence reagent (Absin Bioscience, China) was added to the membranes and immunoblotting bands were visualized using a Tanon Imaging System(Tanon Science, China).Quantitative analysis of band intensity was performed using Image J software (NIH, Bethesda, MD, USA,v1.37). Determination of mRNA expression levels Total RNA was extracted from cells using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA), 1 µg total RNA was reverse transcribed to cDNA, and cDNA was generated using the PrimeScript RT kit (Takara, Japan) according to the manufacturer’s instructions. RT-PCR was performed as previously described[ 30 ] to determine the mRNA expression levels. The primer sequences used in this study were as follows: GAPDH,5'-AGCTTCCCATTCTCAGCCTTGACT-3'(forward) and 5'-ACAAGATGGTGA AGGTCGGTGTGA-3' (reverse); Transgelin primer, (forward:5′-TTCT GCCTCAACATGGCCAAC-3′;reverse:5′-CACCTTCAC TGG CTT GGATC-3′). Dot Blot DNA was prepared from VSMCs, heated at 80°C for 30 min, and blocked with 5% nonfat milk in Tris-buffered saline (TBS) and Tween 20 (TBST) for 1 h. The membranewasincubatedwithmouse5-mC monoclonal antibody (1:1000;EpiGentek,Farmingdale,NY,USA) in TBST containing 5% nonfat milk at 4°C overnight. Membranes were then incubated with peroxidase-conjugated goat anti-mouse antibody in TBST for 1 h at room temperature. The blots were developed using enhanced chemiluminescence (Millipore,Billerica,MA,USA). The signals were quantified using a Tanon image analyzer (TanonScience,Shanghai,China)[ 29 ]. Statistical analysis The data obtained were statistically analyzed using GraphPad Prismsoftware (version 7.0) and expressed as mean ± standard deviation (mean ± SD). We conducted a Student’s t test to compare the means of two independent samples, and one-way ANOVA was applied for multiple groups. Statistical significance was set at P < 0.05. RESULTS EGCG alleviates neointimal hyperplasia of carotid arteries caused by balloon injury To verify the effect of EGCG on neointimal hyperplasia, we established a rat balloon-injured carotid artery model (Fig. 1 ). Our results revealed significant neointimal formation in the rat balloon-injured carotid artery model(injury-group), but EGCG(Drug group) dramatically alleviated neointimal hyperplasia compared to that in the injury group, as assessed by H&E staining and quantification of the intima-media area ratio (I/M area ratio,**P = 0.014 < 0.05, Fig. 1 ). We also measured the expression levels of Nur77, DNMT1, MMP9, α-SMA, calponin, PCNA, COL-Ia, and COL-IIIa in the carotid arteries using IHC. we noted that the expression of PCNA, MMP9, COLIa, and COLIIIa significantly increased in the balloon-injured carotid arteries compared to the normal rats; however, After intragastric EGCG administration, EGCG attenuated these significant changes compared to the injury-only rats (Fig. 2 A–L). In contrast, the expression of SMA and calponin was significantly reduced in the balloon-injured carotid arteries of rats, whereas EGCG reduced the expression levels of SMA and calponin compared to the only balloon-injured rats (Fig. 3 A–F). The expression of Nur77 and DNMT1 was significantly elevated in the balloon-injured carotid arteries of rats, but decreased in the EGCG treatment (100 mg/kg·d) group (Fig. 4 A–F). EGCG inhibits PDGF-induced rat VSMCs proliferation, migration, and apoptosis. We determined the effects of EGCG on PDGF-BB-induced VSMC proliferation using the CCK8 assay, a typical assay used to measure cellular metabolic activity as an indicator of cell viability. VSMCs significantly proliferated in the PDGF-BB (20ng/ml) group, and treatment with EGCG between 10µM and 50µM significantly inhibited this effect(Fig. 5 A). The CCK-8 assay also showed that EGCG suppressed VSMC proliferation for 24h in a dose-dependent manner (Fig. 5 A). These results showed that 25µM EGCG significantly inhibited the proliferation of VSMCs. Consequently, an EGCG concentration of 25 µM was used in subsequent assays. VSMCs significantly migrated in the PDGF-BB (20ng/ml) group, while EGCG inhibited VSMC migration, as revealed by the reduced healing of the VSMC monolayer after EGCG (25 µM) treatment (Fig. 5 C). We next used flow cytometry to determine the influence of EGCG on cell-cycle progression in VSMC, we noted that EGCG (25 µM) treatment caused an obvious drop in the number of cells in the S phase as they were arrested in the G1 phase (Fig. 5 B, D) and underwent significant apoptosis (each experiment was repeated at least three times [* P < 0.01,**P < 0.001 vs. control]). EGCG modulates expression of transgelin (SM22), Bax, MMP2, and PCNA in VSMCs We evaluated the expression of transgelin (SM22), Bax, MMP2, and PCNA in VSMCs. Phenotypic transformation is a common event in the pathophysiology of vascular disease[ 32 ]. To investigate the effects of EGCG on changes in VSMC phenotype, we evaluated transgelin (SM22) mRNA expression levels.Although PDGF-BB induced a significant reduction in transgelin (SM22) expression levels (Fig. 6 D) ,EGCG reversed this effect in a dose-dependent manner (Fig. 6 D). PCNA and MMP2 protein expression was significantly increased in VSMCs (Fig. 6 B,C) after treatment with PDGF-BB, and then decreased after EGCG treatment (Fig. 6 B, C), reflecting an association with cellular proliferative capacity and cellular migration. Bax protein expression was significantly elevated in VSMCs (Fig. 6 A) after treatment with PDGF and decreased after EGCG treatment (Fig. 6 A). EGCG modulates expression and activity of Nur77 and DNMT1, and global DNA methylation inVSMCs We examined the expression of Nur77 and DNMT1 in VSMCs.The expression of Nur77 was significantly increased in VSMCs at the protein (Fig. 7 A) and mRNA (Fig. 7 C) after treatment with PDGF. The protein levels of Nur77 decreased 24h after EGCG treatment (Fig. 7 A).The mRNA expression of Nur77 also decreased 24h after EGCG treatment (Fig. 7 C). EGCG treatment also decreased the protein levels of DNMT1 (Fig. 7 B), indicating that NUR77 and DNMT1 were increased after PDGF-BB treatment, while EGCG inhibited the expression of DNMT1 and Nur77. The expression of 5-mC was evaluated by dot blot analysis (C-E). 5-mc after PDGF-BB treatment, while EGCG inhibited the expression of 5-mc. Nur77 participate in the regulation of EGCG with respect to proliferation, migration, and apoptosis in VSMs lines Nur77 knokdown experiments was conducted to verify whether Nur77 was participate in the regulation of EGCG, We then detected the viability, migration, and apoptosis of VSMCs by Knockdown-Nur77. As shown in Fig. 8 , as determined by EDU assays and Scratch assays, When Si-Nur77, it increased cell proliferation and migration, substantially reduced by EGCG-treated (25µM). Taken together, these results suggest that EGCG suppresses VSMCs proliferation and migration, at least in part, through inducing Nur77 expression. Discussion CVDs constitutes a major health burden worldwide [ 33 ]. Atherosclerosis resulting from hypertension, diabetes, and a high-fat diet plays an important role in the development of CVDs [ 33 ] and is characterized by high morbidity, recurrence, mortality, and disability rates[ 34 ]. Dietary EGCG exhibits anti-inflammatory, anti-oxidative, and anti-lipidemic properties,and has potential for use as an anti-atherosclerotic agent [ 35 , 36 ]. VSMC proliferation has been reported to be a key mechanism in the pathogenesis of CVDs[ 37 ]. In response to vascular injury, VSMCs significantly improve their proliferation, migration, and synthesis capabilities and cause vascular wall thickening, decreased elasticity, and enlargement of the vessel lumen[ 38 ]. Phenotypic switching of VSMCs is a fundamental step that allows proliferation and migration of VSMCs, and increased VSMC proliferation and migration are indispensable in atherosclerosis and restenosis after angioplasty[ 39 ]. Although EGCG regulates the function of VSMCs via various mechanisms, it remains unknown whether abrogated PDGF-BB induces VSMC dedifferentiation. Here, we showed that EGCG pretreatment prevented PDGF-BB-induced VSMCs differentiation, as reflected by increases in VSMC contractile genes,such as α-SMA and Transgelin, and identical results were obtained in vitro. These results indicated that EGCG could serve as a therapeutic agent for the inhibition of VSMC differentiation[ 40 ]. Normal and mature VSMCs exhibit higher levels of contractile proteins, including α-SMA, SM22α, calponin, SMMHC, and myosin light chain kinase (MLCK), and lower levels of synthetic proteins such as OPN,which play a prominent role in blood vessel tone, blood flow, and blood pressure. These changes in molecular concentrations allow the design of novel drugs for the treatment of atherosclerosis, post-angioplasty restenosis, and hypertension via potential underlying mechanisms involved in VSMC phenotypic switching. PCNA is postulated to be a pro-proliferative gene in VSMCs[ 41 ], MMP2 is associated with cellular migration ,and BAX is thought to exert anti-proliferative effects on VSMCs. However, these molecules exert opposite effects on VSMC proliferation. Our results revealed that EGCG reduced the number of EdU-positive and migrating cells in response to PDGF-BB treatment. Furthermore, EGCG mitigated the upregulation of PCNA and MMP2 protein levels, as well as the downregulation of BAX protein expression in VSMCs incubated with PDGF-BB; and we obtained similar results in vito. The cell cycle is a major convergent point in VSMC proliferation, with the process controlled by multiple protein kinases and regulatory cyclins [ 42 ]. Negative regulators of protein kinases and cyclins arrest the cell cycle atthe G0/G1 phase [ 43 ]. we demonstrated that EGCG inhibits DNA synthesis and arrests the cell cycle at the G0/G1 phase. Thus, confirmation of the changes in the protein levels of negative regulators requires further investigation. Nevertheless, these results showed that EGCG diminished PDGF-BB-induced VSMC proliferation and migration, and that the protective effects of EGCG on PDGF-BB-stimulated VSMCs were due to cell cycle arrest. Green tea is one of the most popular beverages, possesses a variety of biological activities, and may be used in the treatment of many diseases, including tumors,CVDs, metabolic disorders, cerebro- vascular diseases, and neurodegenerative diseases[ 44 ]. Several studies have revealed that EGCG inhibits VSMC proliferation[ 45 – 47 ], To further elucidate the mechanism of EGCG inhibiting proliferation of vascular smooth muscle cells, Growing evidence indicates that Nur77 is critical to the pathogenesis of a variety of CVDs, including vascular remodeling, atherosclerosis, cardiac hypertrophy, and cardiac ischemia/reperfusion injury[ 48 – 50 ]. In VSMCs, Nur77 can be induced by multiple stimuli including cytokines, growth factors, oxidized low-densitylipoprotein, and vascular injury[ 51 , 52 ]. We ascertained that Nur77 was highly expressed both in rat PDGF-induced VSMCs in vitro and balloon-injuryed carotid artery in vivo, and that EGCG modulates expression and activity of Nur77,. Therefore, we conclude that EGCG inhibits vascular smooth muscle proliferation by inhibiting NUR77. Our results also showed that Nur77 knockdown promote rat VSMC proliferation, andt inhibited migration, EGCG reversesed these processes. Thus, our data showed that EGCG inhibited PDGF-BB-induced VSMC proliferation via the regulation of Nur77. Further validation test, the results clarified the underlying molecular mechanisms by which EGCG exerts a protective effect onVSMCs. Recent research has shown that inducing genomic DNA hypomethylation can alter SMC phenotypic identity, growth patterns, and the expression levels of contractile genes[ 53 , 54 ], providing preliminary evidence that modulating DNA methylation can shift the phenotypic identity of SMCs. The acquisition and influence of DNA methylation in CVDs and their establishment remain largely unexplored. DNA methylation patterns are established and maintained by three major DNMTs: DNMT1, DNMT3A, and DNMT3B, with DNMT1 being the most abundant and key to the maintenance of methyltransferases in mammalian cells[ 55 ]. It has also been reported that the gene expression profile of Aza-CdR-treated cells was very similar to that of DNMT1-/-cells[ 55 ]. We ascertained that DNMT1 was highly expressed and global DNA methylation at high methylation levels both in rat PDGF-induced VSMCs in vitro and balloon-injuryed carotid artery in vivo, and that EGCG modulates expression and activity of DNMT1and global DNA methylation both in vivo and vitro. Therefore, we hypothesized that EGCG could selectively deplete DNMT1 in a dose-dependent manner in VSMCs, resulting in the in hibition of PDGF-induced VSMC phenotypic modulation. This shows that DNMT1 plays an important role in this modulatory process, implicating DNMT1-mediated DNA methylation in the remodeling of VSMCs. Our investigation provides novel in sights into the mechanism of VSMC remodeling, which may be helpful in developing new treatments for CVDs. Studies have shown that nur77 gene is epigenetically regulated by DNMT1 and is involved in the insulin-signaling pathway[ 56 ]. Nr4a1 is an orphan nuclear. receptor that has been shown to be regulated by DNMT1 and the methylation level of its promoter region[ 19 ]. Consistent with these findings, our results revealed that the protective effects of NUR77 on VSMCs might be regulated by DNMT1. The exact mechanism underlying these actions remains unclear, and further research is required to confirm the exact mechanism. Collectively, our findings suggest that Nur77 and DNMT1 are potential pharmacological targets in CVDs. In summary, EGCG significantly inhibited neointimal hyperplasia induced by balloon injury of rat carotid arteries by attenuating proliferation and migration and promoting apoptosis of rat VSMCs. EGCG also inhibited PDGF-induced rat VSMCs by reducing the expression of Nur77. Mechanistically, si-Nur77 augmented proliferation, whereas EGCG reversed it; the interfering RNA inhibited migration, whereas EGCG exacerbated it. Our results indicate that EGCG significantly ameliorated neointimal hyperplasia by modulating Nur77 and DNMT1signaling. Conclusions Our data revealed disparities inviability, migration, and apoptosis between normal and proliferating VSMCs, and demonstrated the protective role of EGCG inVSMC proliferation in vitro and in vivo. We demonstrated that EGCG inhibited PDGF-BB-induced VSMC proliferation via DNMT1 and Nur77, and this information thus offers insights into future therapeutic. Furthermore, since methylation is involved in the onset and development of CVDs, the application of anti-methylation therapy is expected to provide a new direction in the treatment of such diseases. Abbreviations CVD:cardiovasculardisease; EGCG: EpigallocatechinGallate; VSMCs: Vascular smooth muscle cells; DNMT1:DNA (cytosine-5-)-methyltransferase 1; CCK-8: Cell countingkit-8; EDU: 5-ethynyl-2'-deoxyuridine; SD:Standarddeviation. EGCG=Epigallocatechin-3-gallate;GO=Gene Ontology;KEGG=Kyoto Encyclopedia of Genes and Genomes;PPI=Protein-protein interaction; Declarations Acknowledgments This work was supported in part by Fundamental Research Program of Shanxi Province (Grant Numbers 20210302123019, 202103021224195, 202103021224212, 202103021223189) and Shanxi Scholarship Council of China (Grant numbers 2021-108) Availability of data and materials The data sets used and/or analyzed in this study are available from the corresponding author upon reasonable request. Authors’ contributions S L interpreted the data, and wrote the manuscript. Y ZQ and T JJ designed the study protocol and supervised the project.L F were involved in the interpretation of data, Y ZC and X K revised it critically for important intellectual content. All authors have read and approved the final manuscript. Conflicts of interest The authors declare that they have no competing interests. Ethical approval The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. Disclosure statement The authors have no conflicts of interest to disclose. Funding This study was supported by the Changsha Natural Science Foundation Kq2007043 and the 2022 Key Scientific Research Project of Shaanxi Provincial Education Department (Key Laboratory Project)22JS006. References van Tiel CM, de Vries CJ. NR4All in the vessel wall. J Steroid Biochem Mol Biol. 2012;130(3-5):186-93. 'doi:'10.1016/j.jsbmb.2011.01.010. Yang F, Chen Q, He S, Yang M, Maguire EM, An W et al. miR-22 Is a Novel Mediator of Vascular Smooth Muscle Cell Phenotypic Modulation and Neointima Formation.CIRCULATION.2018;137(17):1824-41. doi:'10.1161/CIRCULATIONAHA.117.027799. Gerthoffer WT. Mechanisms of vascular smooth muscle cell migration. CIRC RES. 2007;100(5):607-21.'doi:'10.1161/01.RES.0000258492.96097.47. Chao LC, Wroblewski K, Ilkayeva OR, Stevens RD, Bain J, Meyer GA et al. Skeletal muscle Nur77 expression enhances oxidative metabolism and substrate utilization. J LIPID RES. 2012;53(12):2610-9.'doi:'10.1194/jlr.M029355. Hanna RN, Carlin LM, Hubbeling HG, Nackiewicz D, Green AM, Punt JA et al. The transcription factor NR4A1 (Nur77) controls bone marrow differentiation and the survival of Ly6C- monocytes. NAT IMMUNOL. 2011;12(8):778-85.'doi:' 10.1038/ni.2063. Arkenbout EK, de Waard V, van Bragt M, van Achterberg TA, Grimbergen JM, Pichon B et al. Protective function of transcription factor TR3 orphan receptor in atherogenesis: decreased lesion formation in carotid artery ligation model in TR3 transgenic mice. CIRCULATION. 2002;106(12):1530-5. 'doi:'10.1161/ 01.cir. 0000028811.03056.bf. de Waard V, Arkenbout EK, Vos M, Mocking AI, Niessen HW, Stooker W et al. TR3 nuclear orphan receptor prevents cyclic stretch-induced proliferation of venous smooth muscle cells. AM J PATHOL. 2006;168(6):2027-35. 'doi:'10.2353/ ajpath.2006.050932. Pires NM, Pols TW, de Vries MR, van Tiel CM, Bonta PI, Vos M et al. Activation of nuclear receptor Nur77 by 6-mercaptopurine protects against neointima formation. CIRCULATION. 2007;115(4):493-500. 'doi:'10.1161/ CIRCULATIONAHA. 106. 626838. Bonta PI, Matlung HL, Vos M, Peters SL, Pannekoek H, Bakker EN et al. Nuclear receptor Nur77 inhibits vascular outward remodelling and reduces macrophage accumulation and matrix metalloproteinase levels. CARDIOVASC RES. 2010;87(3): 561-8. 'doi:'10.1093/cvr/cvq064. Kim HJ, Kim JY, Lee SJ, Kim HJ, Oh CJ, Choi YK et al. alpha-Lipoic acid prevents neointimal hyperplasia via induction of p38 mitogen-activated protein kinase/Nur77-mediated apoptosis of vascular smooth muscle cells and accelerates postinjury reendothelialization. Arterioscler Thromb Vasc Biol. 2010;30(11):2164-72.'doi:'10. 1161/ATVBAHA.110.212308. Ning Y, Huang H, Dong Y, Sun Q, Zhang W, Xu W et al. 5-Aza-2'-deoxycytidine inhibited PDGF-induced rat airway smooth muscle cell phenotypic switching. ARCH TOXICOL. 2013;87(5):871-81. 'doi:'10.1007/s00204-012-1008-y. Miranda TB, Jones PA. DNA methylation: the nuts and bolts of repression. J CELL PHYSIOL. 2007;213(2):384-90. 'doi:'10.1002/jcp.21224. Raftopoulos L, Katsi V, Makris T, Tousoulis D, Stefanadis C, Kallikazaros I. Epigenetics, the missing link in hypertension. LIFE SCI. 2015;129:22-6. 'doi:'10.1016/ j.lfs.2014.08.003. Laird PW. Principles and challenges of genomewide DNA methylation analysis. NAT REV GENET. 2010;11(3):191-203. 'doi:'10.1038/nrg2732. Hiltunen MO, Turunen MP, Hakkinen TP, Rutanen J, Hedman M, Makinen K et al. DNA hypomethylation and methyltransferase expression in atherosclerotic lesions. VASC MED. 2002;7(1):5-11. 'doi:'10.1191/1358863x02vm418oa. Connelly JJ, Cherepanova OA, Doss JF, Karaoli T, Lillard TS, Markunas CA et al. Epigenetic regulation of COL15A1 in smooth muscle cell replicative aging and atherosclerosis. HUM MOL GENET. 2013;22(25):5107-20.'doi:'10.1093/hmg/ddt365. Lyko F. The DNA methyltransferase family: a versatile toolkit for epigenetic regulation. NAT REV GENET. 2018;19(2):81-92. 'doi:'10.1038/nrg.2017.80. Chen YT, Liao JW, Tsai YC, Tsai FJ. Inhibition of DNA methyltransferase 1 increases nuclear receptor subfamily 4 group A member 1 expression and decreases blood glucose in type 2 diabetes. Oncotarget. 2016;7(26):39162-70.'doi:'10.18632/oncotarget.10043. Jorgensen BG, Berent RM, Ha SE, Horiguchi K, Sasse KC, Becker LS et al. DNA methylation, through DNMT1, has an essential role in the development of gastrointestinal smooth muscle cells and disease. CELL DEATH DIS. 2018;9(5):474.'doi:'10.1038/s41419-018-0495-z. Chen YK, Cheung C, Reuhl KR, Liu AB, Lee MJ, Lu YP et al. Effects of green tea polyphenol (-)-epigallocatechin-3-gallate on newly developed high-fat/Western-style diet-induced obesity and metabolic syndrome in mice. J Agric Food Chem. 2011;59(21):11862-71. 'doi:'10.1021/jf2029016. Zheng Y, Song HJ, Kim CH, Kim HS, Kim EG, Sachinidis A et al. Inhibitory effect of epigallocatechin 3-O-gallate on vascular smooth muscle cell hypertrophy induced by angiotensin II. J Cardiovasc Pharmacol. 2004;43(2):200-8. 'doi:'10.1097/00005344-200402000-00006. Orozco-Sevilla V, Naftalovich R, Hoffmann T, London D, Czernizer E, Yang C et al. Epigallocatechin-3-gallate is a potent phytochemical inhibitor of intimal hyperplasia in the wire-injured carotid artery. J VASC SURG. 2013;58(5):1360-5.'doi:'10.1016/j.jvs.2012.11.090. Shu Z, Yu M, Zeng G, Zhang X, Wu L, Tan X. Epigallocatechin-3-gallate inhibits proliferation of human aortic smooth muscle cells via up-regulating expression of mitofusin 2. EUR J CELL BIOL. 2014;93(4):137-44.'doi:'10.1016/j.ejcb.2014.04.001. Zhu TT, Zhang WF, Luo P, He F, Ge XY, Zhang Z et al. Epigallocatechin-3-gallate ameliorates hypoxia-induced pulmonary vascular remodeling by promoting mitofusin-2-mediated mitochondrial fusion. EUR J PHARMACOL. 2017;809:42-51. 'doi:'10.1016/j.ejphar.2017.05.003. Zhan XL, Yang XH, Gu YH, Guo LL, Jin HM. Epigallocatechin gallate protects against homocysteine-induced vascular smooth muscle cell proliferation. MOL CELL BIOCHEM. 2018;439(1-2):131-40. 'doi:'10.1007/s11010-017-3142-6. Tan J, Xie Y, Yao A, Qin Y, Li L, Shen L et al. Long noncoding RNA-dependent regulation of vascular smooth muscle cell proliferation and migration in hypertension. Int J Biochem Cell Biol. 2020;118:105653. 'doi:'10.1016/j.biocel.2019.105653. Tan J, Yang L, Liu C, Yan Z. MicroRNA-26a targets MAPK6 to inhibit smooth muscle cell proliferation and vein graft neointimal hyperplasia. Sci Rep. 2017;7:46602.'doi:'10.1038/srep46602. Stewart SA, Dykxhoorn DM, Palliser D, Mizuno H, Yu EY, An DS et al. Lentivirus-delivered stable gene silencing by RNAi in primary cells. RNA. 2003;9(4):493-501. 'doi:'10.1261/rna.2192803. Yingchun Qin YX, Aihua L, Xiaoqin ZAZ, Yan. Protective effects of astragaloside IV against hypertension-induced vascular remodeling involves the DNMT1 and TET2 signaling pathway. ARCH BIOL SCI. 2021;73(2)(2021Jul.13):301-3 Altman JD, Trendelenburg AU, MacMillan L, Bernstein D, Limbird L, Starke K et al. Abnormal regulation of the sympathetic nervous system in alpha2A-adrenergic receptor knockout mice. MOL PHARMACOL. 1999;56(1):154-61. 'doi:'10.1124/mol.56.1.154. Zhang W, Trebak M. Vascular balloon injury and intraluminal administration in rat carotid artery. J Vis Exp. 2014(94). 'doi:'10.3791/52045. Huhtinen A, Hongisto V, Laiho A, Loyttyniemi E, Pijnenburg D, ScheininM. Gene expression profiles and signaling mechanisms in alpha2B-adrenoceptor-evoked proliferation of vascular smooth muscle cells. BMC SYST BIOL. 2017;11(1):65.'doi:'10.1186/s12918-017-0439-8. Benjamin EJ, Muntner P, Alonso A, Bittencourt MS, Callaway CW, Carson AP et al. Heart Disease and Stroke Statistics-2019 Update: A Report From the American Heart Association. CIRCULATION. 2019;139(10):e56-528.'doi:'10.1161/CIR.0000000000000659. Kimura K, Nagata T, Ohtani M, Nagata M, Kajiki S, Fujino Y et al. Cardiovascular and cerebrovascular diseases risk associated with the incidence of presenteeism and the costs of presenteeism. J OCCUP HEALTH. 2020;62(1):e12167.'doi:'10.1002/1348-9585.12167. Cullen AE, Centner AM, Deitado R, Salazar J. The Impact of Dietary Supplementation of Whole Foods and Polyphenols on Atherosclerosis. NUTRIENTS. 2020;12(7).'doi:'10.3390/nu12072069. Griffiths K, Aggarwal BB, Singh RB, Buttar HS, Wilson D, De Meester F. Food Antioxidants and Their Anti-Inflammatory Properties: A Potential Role in Cardiovascular Diseases and Cancer Prevention. Diseases. 2016;4(3).'doi:'10.3390/diseases4030028. Vazquez-Padron RI, Lasko D, Li S, Louis L, Pestana IA, Pang M et al. Aging exacerbates neointimal formation, and increases proliferation and reduces susceptibility to apoptosis of vascular smooth muscle cells in mice. J VASC SURG. 2004;40(6):1199-207.'doi:'10.1016/j.jvs.2004.08.034. Shi J, Yang Y, Cheng A, Xu G, He F. Metabolism of vascular smooth muscle cells in vascular diseases. Am J Physiol Heart Circ Physiol. 2020;319(3):H613-31. 'doi:'10.1152/ajpheart.00220.2020. Gomez D, Owens GK. Smooth muscle cell phenotypic switching in atherosclerosis. CARDIOVASC RES. 2012;95(2):156-64. 'doi:'10.1093/cvr/cvs115. Eng QY, Thanikachalam PV, Ramamurthy S. Molecular understanding of Epigallocatechin gallate (EGCG) in cardiovascular and metabolic diseases. J ETHNOPHARMACOL. 2018;210:296-310. 'doi:'10.1016/j.jep.2017.08.035. Jiang F, Jiang R, Zhu X, Zhang X, Zhan Z. Genipin inhibits TNF-alpha-induced vascular smooth muscle cell proliferation and migration via induction of HO-1. PLOS ONE. 2013;8(8):e74826. 'doi:'10.1371/journal.pone.0074826. Sherr CJ.Cancer cell cycles. SCIENCE. 1996;274(5293):1672-7. 'doi:' 10. 1126/ science 274.5293.1672. Jiang F, Jiang R, Zhu X, Zhang X, Zhan Z. Genipin inhibits TNF-alpha-induced vascular smooth muscle cell proliferation and migration via induction of HO-1. PLOS ONE. 2013;8(8):e74826. 'doi:'10.1371/journal.pone.0074826. Chowdhury A, Sarkar J, Chakraborti T, Pramanik PK, Chakraborti S. Protective role of epigallocatechin-3-gallate in health and disease: A perspective. BIOMED PHARMACOTHER. 2016;78:50-9. 'doi:'10.1016/j.biopha.2015.12.013. Shu Z, Yu M, Zeng G, Zhang X, Wu L, Tan X. Epigallocatechin-3-gallate inhibits proliferation of human aortic smooth muscle cells via up-regulating expression of mitofusin 2. EUR J CELL BIOL. 2014;93(4):137-44.'doi:'10.1016/j.ejcb.2014.04.001. Liu PL, Liu JT, Kuo HF, Chong IW, Hsieh CC. Epigallocatechin gallate attenuates proliferation and oxidative stress in human vascular smooth muscle cells induced by interleukin-1beta via heme oxygenase-1. Mediators Inflamm. 2014;2014:523684. 'doi:'10.1155/2014/523684. Yang J, Han Y, Sun H, Chen C, He D, Guo J et al. (-)-Epigallocatechin gallate suppresses proliferation of vascular smooth muscle cells induced by high glucose by inhibition of PKC and ERK1/2 signalings. J Agric Food Chem. 2011;59(21):11483-90.'doi:'10.1021/jf2024819. Hamers AA, Vos M, Rassam F, Marinkovic G, Kurakula K, van Gorp PJ et al. Bone marrow-specific deficiency of nuclear receptor Nur77 enhances atherosclerosis. CIRC RES. 2012;110(3):428-38. 'doi:'10.1161/CIRCRESAHA.111.260760. Wang RH, He JP, Su ML, Luo J, Xu M, Du XD et al. The orphan receptor TR3 participates in angiotensin II-induced cardiac hypertrophy by controlling mTOR signalling. EMBO MOL MED. 2013;5(1):137-48. 'doi:'10.1002/emmm.201201369. Cheng Z, Volkers M, Din S, Avitabile D, Khan M, Gude N et al. Mitochondrial translocation of Nur77 mediates cardiomyocyte apoptosis. EUR HEART J. 2011;32(17):2179-88. 'doi:'10.1093/eurheartj/ehq496. Wang L, Gong F, Dong X, Zhou W, Zeng Q. Regulation of vascular smooth muscle cell proliferation by nuclear orphan receptor Nur77. MOL CELL BIOCHEM. 2010;341(1-2):159-66. 'doi:'10.1007/s11010-010-0447-0. Liu Y, Zhang J, Yi B, Chen M, Qi J, Yin Y et al. Nur77 suppresses pulmonary artery smooth muscle cell proliferation through inhibition of the STAT3/Pim-1/NFAT pathway. Am J Respir Cell Mol Biol. 2014;50(2):379-88.'doi:'10.1165/rcmb.2013-0198OC. Hu B, Gharaee-Kermani M, Wu Z, Phan SH. Epigenetic regulation of myofibroblast differentiation by DNA methylation. AM J PATHOL. 2010;177(1):21-8. 'doi:'10.2353/ajpath.2010.090999. Ning Y, Huang H, Dong Y, Sun Q, Zhang W, Xu W et al. 5-Aza-2'-deoxycytidine inhibited PDGF-induced rat airway smooth muscle cell phenotypic switching. ARCH TOXICOL. 2013;87(5):871-81. 'doi:'10.1007/s00204-012-1008-y. Ghoshal K, Majumder S, Datta J, Motiwala T, Bai S, Sharma SM et al. Role of human ribosomal RNA (rRNA) promoter methylation and of methyl-CpG-binding protein MBD2 in the suppression of rRNA gene expression. J BIOL CHEM. 2004;279(8):6783-93. 'doi:'10.1074/jbc.M309393200. Chen YT, Liao JW, Tsai YC, Tsai FJ. Inhibition of DNA methyltransferase 1 increases nuclear receptor subfamily 4 group A member 1 expression and decreases blood glucose in type 2 diabetes. Oncotarget. 2016;7(26):39162-70. 'doi:'10.18632/oncotarget.10043. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4260039","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":290870095,"identity":"21fa043a-83bb-4ae7-9ac2-97596b972c37","order_by":0,"name":"Li shen","email":"","orcid":"","institution":"Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"shen","suffix":""},{"id":290870096,"identity":"1ad31b62-16bd-4393-a023-29a5a3c007ec","order_by":1,"name":"Juanjuan Tan","email":"","orcid":"","institution":"Shaanxi University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Juanjuan","middleName":"","lastName":"Tan","suffix":""},{"id":290870097,"identity":"06c65aa4-7307-4344-aaea-f26c543c48bc","order_by":2,"name":"Feng Li","email":"","orcid":"","institution":"The Third Hospital of Changsha","correspondingAuthor":false,"prefix":"","firstName":"Feng","middleName":"","lastName":"Li","suffix":""},{"id":290870098,"identity":"9cfeb7b4-3b86-41ac-8ce2-0ce2659ad113","order_by":3,"name":"Ke Xia","email":"","orcid":"","institution":"Central South University","correspondingAuthor":false,"prefix":"","firstName":"Ke","middleName":"","lastName":"Xia","suffix":""},{"id":290870099,"identity":"db87a86a-84bf-4696-bf50-e395e5a90796","order_by":4,"name":"Zhichao Yuan","email":"","orcid":"","institution":"The First Affiliated Hospital of Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhichao","middleName":"","lastName":"Yuan","suffix":""},{"id":290870100,"identity":"8d7d693b-b34a-4454-ae6e-cb4c805aa05e","order_by":5,"name":"Zhiqiang Yan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAy0lEQVRIiWNgGAWjYNCCCgZmMM1DvJYzDMw8pGlhbIOqJkqLfHuO4efCeXXs9hIJjA/etjHImxO0oOeNsfTMbYeZeSQSmA3ntjEY7mwgoIVZIsdAmnfbAZAWNmneNoYEgwMEtLBJ5Bj/5p1TB9LC/psoLTwSOWbSvA3MYFuYidIiwfOszJrnGNAvZx42S845J2G4gZAW+fbkzbd5auqS2duTD354U2YjT9AWBoYMAxCZDAy8BpCtBNUDQfoDEGlHjNJRMApGwSgYoQAAIhY0nFL3qDAAAAAASUVORK5CYII=","orcid":"","institution":"Fengxian Hospital Affiliated to the Southern Medical University","correspondingAuthor":true,"prefix":"","firstName":"Zhiqiang","middleName":"","lastName":"Yan","suffix":""}],"badges":[],"createdAt":"2024-04-13 01:59:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4260039/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4260039/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":55059676,"identity":"601ab1fb-70de-449a-ac16-a1dacdd89745","added_by":"auto","created_at":"2024-04-22 02:17:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":744472,"visible":true,"origin":"","legend":"\u003cp\u003eEGCG-attenuated vascular remodeling in the rat balloon-injured carotid artery model. The balloon injury-induced carotid artery model was implemented, and the rats were then administered EGCG (100mg/kg·d,n=4). Neointimal formation in the rat balloon-injured carotid artery model(injury-group), but EGCG(Drug group) dramatically alleviated neointimal hyperplasia compared to that in the injury group, as assessed by H\u0026amp;E staining and quantification of the intima-media area ratio (I/M area ratio,**P = 0.014 \u0026lt; 0.05 vs Control group, **P\u0026lt;0.05 vs. Drug group magnification, x200; scalebar= 100 μm).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4260039/v1/07ec9be1da7907676d8c4ab5.png"},{"id":55059674,"identity":"415dc3a7-1a50-45f1-9eb3-df695442b7f5","added_by":"auto","created_at":"2024-04-22 02:17:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2568999,"visible":true,"origin":"","legend":"\u003cp\u003eImpact of EGCG on damaged carotid arteries of rats in vivo. (A) Representative image of PCNA IHC (B) Quantitative analysis of the percentage of PCNA-positive cells(**P \u0026lt; 0.01 vs.control group;***P \u0026lt; 0.001 vs.Drug(EGCG) group,n=4).(C) Quantitative analysis of PCNA-positive areas (as percentages of the intima-media areas(**P \u0026lt; 0.01 vs.Drug(EGCG) group;***P \u0026lt; 0.001 vs.injury group,n=4). (D) Representative image of MMP9 IHC. (E) Quantitative analysis of the percentage of MMP9-positive cells(***P \u0026lt; 0.001 vs. Drug(EGCG) group;***P \u0026lt; 0.001vs.injury group,n=4). (F) Quantitative analysis of MMP9-positive areas (percentages of the intima-media areas,(**P \u0026lt; 0.01 vs. Drug(EGCG) group;**P \u0026lt; 0.01vs.injury group,n=4). (G) Representative image of Col I IHC. (H) Quantitative analysis of the percentage of Col I-positive cells(**P \u0026lt; 0.01 vs. Control group;***P \u0026lt; 0.01vs.Drug(EGCG) group,n=4). (I) Quantitative analysis of the Col I-positive areas (percentages of the intima-media areas,***P \u0026lt; 0.01vs.control group,***P \u0026lt; 0.01vs.injury group,n=4)). (J) Representative image of Col III IHC. (K) Quantitative analysis of the percentage of Col III-positive cells(**P \u0026lt; 0.01 vs. Control group;***P \u0026lt; 0.01vs.Drug(EGCG) group,n=4). (L) Quantitative analysis of the Col III-positive areas (percentages of the intima-media areas,***P \u0026lt; 0.01vs.Drug(EGCG) group,n=4) (scale bar, 20 μM).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4260039/v1/bf1c995bd29878f359b80008.png"},{"id":55060753,"identity":"2b432158-eeaf-4767-b5ca-08a2c724b799","added_by":"auto","created_at":"2024-04-22 02:33:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1454752,"visible":true,"origin":"","legend":"\u003cp\u003eImpact of EGCG on damaged carotid arteries of rats in vivo. (A) Representative image of calponin IHC. (B) Quantitative analysis of the percentages of calponin-positive cells(***P \u0026lt; 0.01 vs. Control group;***P \u0026lt; 0.01vs.injury group). (C) Quantitative analysis of the calponin-positive areas ( the percentages of the intima-media areas,***P \u0026lt; 0.01 vs. Control group;***P \u0026lt; 0.01vs.injury group). (D) Representative image of α-SMA IHC. (E) Quantitative analysis of the percentages ofα-SMA-positive cells(**P \u0026lt; 0.01 vs. Control group;**P \u0026lt; 0.01vs.injury group). (F) Quantitative analysis of α-SMA-positive areas (the percentages of theintima-media areas,**P \u0026lt; 0.01 vs. Control group;**P \u0026lt; 0.01vs.injury group), (scale bar, 20 μM, n = 4).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4260039/v1/6491cdc03d188fb4d019a437.png"},{"id":55059675,"identity":"367baf1e-cc5e-465a-bd5d-63ab93418650","added_by":"auto","created_at":"2024-04-22 02:17:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1596615,"visible":true,"origin":"","legend":"\u003cp\u003eImpact of EGCG on damaged carotid arteries of rats in vivo. (A) Representative image of Nur77 IHC. (B) Quantitative analysis of the percentages of Nur77-positive cells(**P \u0026lt; 0.01 vs. Control group;***P \u0026lt; 0.001vs.injury group). (C) Quantitative analysis of Nur77-positive areas (the percentages of the intima-media areas,*P \u0026lt; 0.05 vs. Control group;**P \u0026lt; 0.01 vs.injury group). (D) Representative image of DNMT1 IHC. (E) Quantitative analysis of the percentage of DNMT1-positive cells(**P \u0026lt; 0.01 vs. Control group;***P \u0026lt; 0.001vs.Drug(EGCG) group). (F) Quantitative analysis of DNMT1-positive areas ( percentages of intima-media areas,***P \u0026lt; 0.001 vs. Control group;***P \u0026lt; 0.001vs.injury group) (scale bar, 20 μM; *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001; n = 4).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4260039/v1/cbc54a6c25840245cb6af3b7.png"},{"id":55059679,"identity":"bb040100-9e76-44e4-9534-0bed5388e4d0","added_by":"auto","created_at":"2024-04-22 02:17:53","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":730095,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of EGCG on VSMC proliferation, migration, and apoptosis. PDGF-BB stimulated the proliferation of rat VSMCs (A), while EGCG inhibited proliferation (A,*P \u0026lt; 0.05 compared with the control group; \u003csup\u003e##\u003c/sup\u003eP \u0026lt; 0.01, \u003csup\u003e###\u003c/sup\u003eP \u0026lt; 0.001 compared with the PDGF group). PDGF-BB stimulated the migration of rat VSMCs (C,***P \u0026lt; 0.001 compared with the PDGF group ), while EGCG inhibited migration (C,***P \u0026lt; 0.001 compared with the control group). EGCG (25 μM) treatment generated an obvious cell-cycle reduction in the S phase and arrest in the G1 phase (B, D, \u003csup\u003e##\u003c/sup\u003eP \u0026lt; 0.01, compared with the PDGF group) and significantly increased cellular apoptosis(B,D). Data are presented as the mean ± standard error of the mean ).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4260039/v1/c00a19d6edebebf70df3f646.png"},{"id":55059681,"identity":"a7f1f3d6-8fdc-417a-962e-77bd12aea993","added_by":"auto","created_at":"2024-04-22 02:17:53","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":405467,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of EGCG on the expression of transgelin (SM22), PCNA , Bax, and MMP2 in rat VSMCs. The protein expression levels of PCNA, MMP2, and Bax, as determined using western blot analysis (A, B, C,*P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001 compared with the PDGF group). Transgelin mRNA expression, as determined using RT-PCR (D,*P \u0026lt; 0.05,**P \u0026lt; 0.01, ****P \u0026lt; 0.001 compared with the PDGF group,Data are presented as the mean ± standard error of the mean ,n = 5).\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4260039/v1/158ee8765f8271e2b83de9ff.png"},{"id":55059682,"identity":"e1dfdef1-d36e-4676-976f-95933893f483","added_by":"auto","created_at":"2024-04-22 02:17:54","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":357549,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of EGCG on the expression of Nur77 and DNMT1 in VSMCs.VSMCs were treated with various concentrations of EGCG (10,25,50μM) for 24h or for the other indicated time points. Proteins expression for Nur77(A) and DNMT1 (B) were demonstrated by western blotting analysis (*P \u0026lt; 0.05 compared with control group, **P \u0026lt; 0.01 vs. the PDGF group;n=5). Expression of 5-mC ,as evaluated by dot-blot analysis (C,D,Significant differences among groups are indicated by ** P \u0026lt; 0.01 compared with the PDGF group,*P\u0026lt;0.01compared with the control group, *P\u0026lt;0.01compared with the PDGF group, ** P \u0026lt; 0.01 compared with the PDGF group, **** P \u0026lt; 0.01 compared with the PDGF group,Values represent the mean±SD, n=5).\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-4260039/v1/fd1399231b6b5273f83f1efc.png"},{"id":55060553,"identity":"40f35a02-1536-4325-bd53-7611022eee57","added_by":"auto","created_at":"2024-04-22 02:25:53","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1417084,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of knockdown Nur77 on proliferation and migration in VSMCs trated by EGCG. Cells that were transfected to siRNA Nur77 were treated with 25μM EGCG for 24h(A,B,C,D). Cellular proliferation was measured by EDU assay(A,B,*** P \u0026lt; 0.01 compared with the si-NC group,*** P \u0026lt; 0.01 compared with the siNC+PDGF group ),migration was analyzed by (C,D,*** P \u0026lt; 0.01 compared with the si-NC group, *** P \u0026lt; 0.01 compared with the siNur77+PDGF group), (Data are presented as the mean ± SEM, n = 4).\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-4260039/v1/d7412a92416b0bc7e85ba32e.png"},{"id":65898489,"identity":"ed7c6625-2439-40e7-8389-db5077524e4b","added_by":"auto","created_at":"2024-10-04 07:02:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11770891,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4260039/v1/a674c886-d4b8-4a06-a55e-bedda67aca60.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Epigallocatechin inhibits PDGF-BB-induced vascular smooth muscle cells proliferation via DNMT1 regulation of the Nur 77 axis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCardiovascular diseases (CVDs) encompass chronic inflammatory disorders of the arterial vessel walls that eventually result in obstruction of blood flow and are a major cause of ischemic conditions,such as heart and brain infarctions[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].Vascular smooth muscle cells(VSMCs) are the major cell type in blood vessels, and the proliferation of VSMCs and their phenotypic transformation constitute the pathological basis of CVDs. Modulation of the VSMC phenotype,triggered by harmful microenvironmental stimuli followed by VSMC migration and proliferation, plays an important role in intimal hyperplasia, which is critical for the development of vascular disease[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePlatelet-derived growth factor-BB (PDGF-BB) is a major driving factor in VSMC proliferation and migration involved in vascular remodeling and is also a natural ligand of PDGFR-β[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].PDGF-BB is a potent cellular mitogen and chemoattractant for VSMCs, and by activating its downstream signaling pathways, it contributes to many biological processes and diseases after binding with PDGFR-β[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Nur77 is a transcription factor that is also known as TR3, NR4A1, or NGFI-B, and is an immediate-early gene involved in stress responses in various cell types and organ systems[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Studies have revealed that Nur77 inhibits the proliferation of both arterial and venous SMCs[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] and that Nur77 induces a more differentiated contractile phenotype in SMCs, which is illustrated by the increased expression of SMC-specific marker proteins such as SM-actin and -calponin[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Inhibition of Nur77 by either small interfering (si)RNAs or overexpression of a dominant-negative variant of Nur77 resulted in enhanced SMC proliferation, and transgenic overexpression of Nur77 in arterial SMCs inhibited flow-induced outward remodeling. Nur77 did not modulate vascular tone but rather reduced macrophage accumulation and concomitantly lowered matrix metalloproteinase levels in remodeled arteries[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Recent studies demonstrated that lipoic acid inhibited neointimal formation in rat carotid arteries after balloon injury and accelerated apoptosis of SMCs through the upregulation of Nur77 [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] and PDGF-induced migration and proliferation of airway SMCs and increased cellular contractility[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDNA methylation refers to the binding of 5-methylcytosine (5-MC) to S-adenosine methionine in the cytosine ring to form 5-methylcytosine (5MC)[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Therefore,5-MC is an indicator adopted for the evaluation of DNA methylation, with the process occurring at specific dinucleotide sites called CpGs [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. DNA methylation is generally acknowledged to be an inhibitory modification that inhibits the binding of the transcription complex to the target gene promoter [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]; thus, DNA hypermethylation leads to gene silencing [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Furthermore, global hypomethylation was detected in the proliferating intimal SMCs of New Zealand white rabbits that had undergone balloon denudation. Global hypomethylation with a concomitant decrease in DNA methyltransferase (DNMT) activity has also been shown to occur during SMC phenotypic modulation[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] and proliferation[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] in culture, and DNMTs are important factors that affect DNA methylation levels. Human-encoded DNMTs included DNMT1, DNMT2, DNMT3A, DNMT3B, and DNMT3L. DNMT1, DNMT3A, and DNMT3B are classical members of the DNMT family that catalyze genomic DNA methylation, whereas DNMT2 and DNMT3L are non-classical members that do not possess catalytic DNMT activity [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Previous studies have suggested that theNur77 is involved in the epigenetic pathway, and recent research has revealed that DNMT1 causes Nur77 DNA hypermethylation and blocks insulin signaling in patients with T2D[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In Dnmt1-KO mice, Nur77 reduced significant losses in methylation, suggesting that theexpressionof pro-apoptotic Nur77 was regulated by CpG methylation levels controlled by DNMT1[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], which is consistent with earlier findings. (\u0026minus;)-Epigallocatechin-3-gallate (EGCG) is the principal catechin in green tea (50\u0026ndash;75%) and has been shown to have numerous potential health effects, including antioxidant, anticarcinogenic, hypocholesterolemic, and cardioprotective epigenetic activities[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. EGCG regulates VSMC function through different mechanisms. For example, studies have depicted EGCG suppresses JNK activation, thereby protecting against angiotensin II-induced VSMC hypertrophy [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. EGCG can also inhibit PKC- and ERK1/2-signaling pathways, thus improving hyperglycemic VSMC proliferation and injury-induced neointimal formation in the rat common carotid artery[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Investigators found that EGCG inhibited the proliferation of human aortic VSMCs and improved hypoxia-induced pulmonary artery revascularization by upregulating mitochondrial fusion protein gene-2[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. EGCG also inhibits AT-1R and affects ERK1/2- and p38MAPK-signaling pathways so as to inhibit homocysteine-induced proliferation of VSMCs [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe purpose of this present study, we want to demonstrated that EGCG protects arteries against neointimal hyperplasia in vivo and PDGF-induced VSMC proliferation migrstion in vitro. Examine that EGCG attenuates neointimal hyperplasia through Nur77 and DNMT1, providing a potential natural compound for the treatment of CVD, as well as a theoretical basis for its clinical diagnosis and future treatment.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimal studies\u003c/h2\u003e \u003cp\u003eMale Sprague\u0026ndash;Dawley rats with a mean weight of 250\u0026thinsp;\u0026plusmn;\u0026thinsp;20 g were purchased from the Shanghai Laboratory Animal Research Center (Shanghai,China) and housed at a constant temperature of 25\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;2 and humidity of 60\u0026thinsp;\u0026plusmn;\u0026thinsp;5% with a 12-h dark/light cycle. This study was conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health and approved by our Ethical Committee (ethics code SCXK, Shanghai 2019-0006). The rats were anesthetized using isoflurane,and a mid line neck incision was made to expose the left common carotid artery. The carotid artery balloon-injury model for rats was then established as previously described[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], and the rats were randomly assigned to the following experimental groups: control, injured, and EGCG treatment. EGCG was administered once daily at 100 mg/kg for 21 days, whereas normal PBS was administered intragastrically to the control and injured groups. Rats were anesthetized using isoflurane, followed by extraction of the left common carotid artery.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eH\u0026amp;E staining\u003c/h2\u003e \u003cp\u003eThe carotid arteries were collected, washed with pre-cooled saline to remove excess blood, fixed in 4% paraformaldehyde, and embedded in paraffin. Hematoxylin and eosin (H\u0026amp;E) staining was performed on 4-\u0026micro;m sections, which were imaged using a 09microscope (BX71;OlympusCorporation,Japan). The sizes of the intimal and medial areas were determined using the lumen, internal elastic lamina, and external elastic lamina and were measured using Image J software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry (IHC)\u003c/h2\u003e \u003cp\u003eImmunohistochemical staining was performed to detect PCNA, Nur77, DNMT1, MMP2, COL-I, COL-III, calponin, and α-SMA(Proteintech,Rosemont, IL, USA ).Briefly, 5-\u0026micro;M sections of the rat carotid artery were cut and dewaxed.The slides were then treated with 3% hydrogen peroxide for 30 min at 25\u0026deg;C, incubated with primary antibodies overnight at 4\u0026deg;C, washed three times with PBS, incubated with secondary antibody for 4 h, and stained with DAB (ENZO) after washing with buffer solution. Finally, the sections were counterstained with hematoxylin to visualize cellular nuclei.Image J software was used to analyze the average integrated optical density (IOD) and positively stained regions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eCulture of VSMCs\u003c/h2\u003e \u003cp\u003eWe isolated and cultured VSMCs using the explants method as previously described for rat aorta[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], and their purity was confirmed by staining the cells with α-SMA actin (Proteintech, Rosemont, IL, USA). VSMCs were then seeded in DMEM containing 20% fetal bovine serum(FBS),100 \u0026micro;g/ml streptomycin, and 100 units/ml penicillin and cultured at 37\u0026deg;C in 5% CO2 (Thermo Fisher, Waltham, MA, USA) EGCG was added to VSMCs after starvation for 24 h. Stimulation of VSMCs with PDGF-BB(20ng/ml) for 48 h was performed to examine the effects on cellular proliferation, migration, and apoptosis. All experiments were performed using cells from passages four to eight.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eKnockdown of Nur77 in VSMCs\u003c/h2\u003e \u003cp\u003eThe pLenti-puro lentiviral vectors containing Nur77-specific small hairpin RNAs (shRNAs) were constructed by Vigene Bioscience Technology (Jinan, China), the pLenti-purolentiviralvectorcontainingnoNur77-specific small hairpin RNAs(siNC) was used as the negative control, and lentivital Nur77 overexpression vectors for Nur77 overexpression were purchased from GenePharma[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e](China). The lentiviral vector and packaging plasmid mix were transfected into HEK293T cells [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] following the manufacturer\u0026rsquo; sinstructions.The medium was replaced after 6h. Fresh medium was collected 72 h post-transfection and passed through a 0.45-\u0026micro;m filter. VSMCs were transduced with filtered lentiviral supernatants supplemented with 8\u0026micro;g/mL polybrene (Sigma-Aldrich, St. Louis, MO,USA)[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eVSMC proliferation assay in vitro\u003c/h2\u003e \u003cp\u003eVSMC proliferation in vitro was determined using the Cell Counting Kit-8(CCK8) and EdU staining. Briefly, 100\u0026micro;L of VSMC suspension (0.5\u0026times;104cells) was seeded in 96-well plates at 37\u0026deg;C in 5% CO2, and the cells were synchronized in DMEM containing 0.1% double antibiotic for 24h. The cells were then treated with vehicle control, PDGF-BB in DMEM, and /or PDGF-BB in DMEM containing EGCG at different concentrations at the indicated times. Next,10\u0026micro;L of CCK8 solution (MedChem Express,USA) was added to each well for an additional 1h, and absorbance was measured at 450 nm using a microplate reader (BioTek, Winooski, VT, USA) to determine the proliferative rate of the VSMCs. VSMC proliferation was also assessed using a BeyoClickEdU-488 Cell Proliferation Assay Kit(Beyotime,Beijing, China) according to the manufacturer\u0026rsquo;s instructions. Images were taken using a fluorescence microscope (BX71,Olympus Corporation, Japan), and the signals were counted in three random visual fields per sample.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eWound-healing assay\u003c/h2\u003e \u003cp\u003eCells were plated in six-well plates at a density of 1.5 \u0026times; 105 cells/well, and at confluency,the cells were cultured inserum-free medium for 24h and a scratch was made in the cell monolayer with a sterile 200-\u0026micro;L pipette tip. The cells were subsequently treated with vehicle or 20ng/ml PDGF-BB, with or without EGCG(25\u0026micro;M) for the indicated time periods. The scratches were captured at 0 and 48 h using an inverted microscope (BX71; OlympusCorporation,Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eCell-cycle analysis\u003c/h2\u003e \u003cp\u003eVSMCs were seeded in six-well plates at 2 \u0026times; 105 cells/well and incubated in an atmosphere of 5% CO2 at 37\u0026deg;C. VSMCs in each group were then pretreated with EGCG for 24 h, treated with 20 ng/ml PDGF for 24 h, collected, resuspended in 70% pre-cooled ethanol, and incubated at 4\u0026deg;C for 24 h. The fixed cells were washed twice with phosphate-buffered saline (PBS) and incubated at 37\u0026deg;C in the dark for 30 min using a solution containing DNase-free RNaseA (200\u0026micro;g/ml) and propidium iodide (PI, 50mg/ml). The cells were analyzed by flow cytometry (BD,USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003eTotal protein was extracted from VSMCs and lysed with PierceR RIPA buffer (Beyotime Biotechnology, China) containing a cocktail of protease inhibitors, and the protein concentration was measured with a BCA kit (Beyotime Biotechnology,China), followed by immune blotting using dilutions of 1:500 for anti-DNMT1 (Abcam,UK), 1:1000 for Nur77 (Proteintech, Rosemont,IL,USA), and 1:2000 for MMP2, BAX, PCNA, tubulin, and GAPDH antibodies (Proteintech, Rosemont, IL, USA), and separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to nitrocellulose membranes and blocked with5%nonfatmilkinTris-buffered saline (TBS) and Tween (Tween20) (TBST) for 1h. The membranes were incubated with the corresponding primary antibodies at 4\u0026deg;C overnight, washed, and then incubated with the corresponding horseradishperoxidase (HRP)-conjugated goat anti-mouse(1:10,000) or goat anti-rabbit secondary antibody (1:10,000, Proteintech, Rosemont, IL, USA) for 1 h at room temperature. Enhanced chemiluminescence reagent (Absin Bioscience, China) was added to the membranes and immunoblotting bands were visualized using a Tanon Imaging System(Tanon Science, China).Quantitative analysis of band intensity was performed using Image J software (NIH, Bethesda, MD, USA,v1.37).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of mRNA expression levels\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from cells using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA), 1 \u0026micro;g total RNA was reverse transcribed to cDNA, and cDNA was generated using the PrimeScript RT kit (Takara, Japan) according to the manufacturer\u0026rsquo;s instructions. RT-PCR was performed as previously described[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] to determine the mRNA expression levels. The primer sequences used in this study were as follows: GAPDH,5'-AGCTTCCCATTCTCAGCCTTGACT-3'(forward) and 5'-ACAAGATGGTGA AGGTCGGTGTGA-3' (reverse); Transgelin primer, (forward:5\u0026prime;-TTCT GCCTCAACATGGCCAAC-3\u0026prime;;reverse:5\u0026prime;-CACCTTCAC TGG CTT GGATC-3\u0026prime;).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eDot Blot\u003c/h2\u003e \u003cp\u003eDNA was prepared from VSMCs, heated at 80\u0026deg;C for 30 min, and blocked with 5% nonfat milk in Tris-buffered saline (TBS) and Tween 20 (TBST) for 1 h. The membranewasincubatedwithmouse5-mC monoclonal antibody (1:1000;EpiGentek,Farmingdale,NY,USA) in TBST containing 5% nonfat milk at 4\u0026deg;C overnight. Membranes were then incubated with peroxidase-conjugated goat anti-mouse antibody in TBST for 1 h at room temperature. The blots were developed using enhanced chemiluminescence (Millipore,Billerica,MA,USA). The signals were quantified using a Tanon image analyzer (TanonScience,Shanghai,China)[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe data obtained were statistically analyzed using GraphPad Prismsoftware (version 7.0) and expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD). We conducted a Student\u0026rsquo;s t test to compare the means of two independent samples, and one-way ANOVA was applied for multiple groups. Statistical significance was set at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eEGCG alleviates neointimal hyperplasia of carotid arteries caused\u003c/h2\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003eby balloon injury\u003c/h2\u003e \u003cp\u003eTo verify the effect of EGCG on neointimal hyperplasia, we established a rat balloon-injured carotid artery model (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Our results revealed significant neointimal formation in the rat balloon-injured carotid artery model(injury-group), but EGCG(Drug group) dramatically alleviated neointimal hyperplasia compared to that in the injury group, as assessed by H\u0026amp;E staining and quantification of the intima-media area ratio (I/M area ratio,**P\u0026thinsp;=\u0026thinsp;0.014\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe also measured the expression levels of Nur77, DNMT1, MMP9, α-SMA, calponin, PCNA, COL-Ia, and COL-IIIa in the carotid arteries using IHC. we noted that the expression of PCNA, MMP9, COLIa, and COLIIIa significantly increased in the balloon-injured carotid arteries compared to the normal rats; however, After intragastric EGCG administration, EGCG attenuated these significant changes compared to the injury-only rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u0026ndash;L). In contrast, the expression of SMA and calponin was significantly reduced in the balloon-injured carotid arteries of rats, whereas EGCG reduced the expression levels of SMA and calponin compared to the only balloon-injured rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u0026ndash;F). The expression of Nur77 and DNMT1 was significantly elevated in the balloon-injured carotid arteries of rats, but decreased in the EGCG treatment (100 mg/kg\u0026middot;d) group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u0026ndash;F).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEGCG inhibits PDGF-induced rat VSMCs proliferation, migration, and apoptosis.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe determined the effects of EGCG on PDGF-BB-induced VSMC proliferation using the CCK8 assay, a typical assay used to measure cellular metabolic activity as an indicator of cell viability. VSMCs significantly proliferated in the PDGF-BB (20ng/ml) group, and treatment with EGCG between 10\u0026micro;M and 50\u0026micro;M significantly inhibited this effect(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The CCK-8 assay also showed that EGCG suppressed VSMC proliferation for 24h in a dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). These results showed that 25\u0026micro;M EGCG significantly inhibited the proliferation of VSMCs. Consequently, an EGCG concentration of 25 \u0026micro;M was used in subsequent assays.\u003c/p\u003e \u003cp\u003eVSMCs significantly migrated in the PDGF-BB (20ng/ml) group, while EGCG inhibited VSMC migration, as revealed by the reduced healing of the VSMC monolayer after EGCG (25 \u0026micro;M) treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). We next used flow cytometry to determine the influence of EGCG on cell-cycle progression in VSMC, we noted that EGCG (25 \u0026micro;M) treatment caused an obvious drop in the number of cells in the S phase as they were arrested in the G1 phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, D) and underwent significant apoptosis (each experiment was repeated at least three times [* P\u0026thinsp;\u0026lt;\u0026thinsp;0.01,**P\u0026thinsp;\u0026lt;\u0026thinsp;0.001 vs. control]).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eEGCG modulates expression of transgelin (SM22), Bax, MMP2, and PCNA in VSMCs\u003c/h2\u003e \u003cp\u003eWe evaluated the expression of transgelin (SM22), Bax, MMP2, and PCNA in VSMCs. Phenotypic transformation is a common event in the pathophysiology of vascular disease[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. To investigate the effects of EGCG on changes in VSMC phenotype, we evaluated transgelin (SM22) mRNA expression levels.Although PDGF-BB induced a significant reduction in transgelin (SM22) expression levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD) ,EGCG reversed this effect in a dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). PCNA and MMP2 protein expression was significantly increased in VSMCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB,C) after treatment with PDGF-BB, and then decreased after EGCG treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, C), reflecting an association with cellular proliferative capacity and cellular migration. Bax protein expression was significantly elevated in VSMCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA) after treatment with PDGF and decreased after EGCG treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eEGCG modulates expression and activity of Nur77 and DNMT1, and global DNA methylation inVSMCs\u003c/h2\u003e \u003cp\u003eWe examined the expression of Nur77 and DNMT1 in VSMCs.The expression of Nur77 was significantly increased in VSMCs at the protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA) and mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC) after treatment with PDGF. The protein levels of Nur77 decreased 24h after EGCG treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA).The mRNA expression of Nur77 also decreased 24h after EGCG treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). EGCG treatment also decreased the protein levels of DNMT1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB), indicating that NUR77 and DNMT1 were increased after PDGF-BB treatment, while EGCG inhibited the expression of DNMT1 and Nur77. The expression of 5-mC was evaluated by dot blot analysis (C-E). 5-mc after PDGF-BB treatment, while EGCG inhibited the expression of 5-mc.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eNur77 participate in the regulation of EGCG with respect to proliferation, migration, and apoptosis in VSMs lines\u003c/b\u003e \u003c/p\u003e \u003cp\u003eNur77 knokdown experiments was conducted to verify whether Nur77 was participate in the regulation of EGCG, We then detected the viability, migration, and apoptosis of VSMCs by Knockdown-Nur77. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e, as determined by EDU assays and Scratch assays, When Si-Nur77, it increased cell proliferation and migration, substantially reduced by EGCG-treated (25\u0026micro;M). Taken together, these results suggest that EGCG suppresses VSMCs proliferation and migration, at least in part, through inducing Nur77 expression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eCVDs constitutes a major health burden worldwide [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Atherosclerosis resulting from hypertension, diabetes, and a high-fat diet plays an important role in the development of CVDs [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] and is characterized by high morbidity, recurrence, mortality, and disability rates[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Dietary EGCG exhibits anti-inflammatory, anti-oxidative, and anti-lipidemic properties,and has potential for use as an anti-atherosclerotic agent [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. VSMC proliferation has been reported to be a key mechanism in the pathogenesis of CVDs[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In response to vascular injury, VSMCs significantly improve their proliferation, migration, and synthesis capabilities and cause vascular wall thickening, decreased elasticity, and enlargement of the vessel lumen[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Phenotypic switching of VSMCs is a fundamental step that allows proliferation and migration of VSMCs, and increased VSMC proliferation and migration are indispensable in atherosclerosis and restenosis after angioplasty[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAlthough EGCG regulates the function of VSMCs via various mechanisms, it remains unknown whether abrogated PDGF-BB induces VSMC dedifferentiation. Here, we showed that EGCG pretreatment prevented PDGF-BB-induced VSMCs differentiation, as reflected by increases in VSMC contractile genes,such as α-SMA and Transgelin, and identical results were obtained in vitro. These results indicated that EGCG could serve as a therapeutic agent for the inhibition of VSMC differentiation[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Normal and mature VSMCs exhibit higher levels of contractile proteins, including α-SMA, SM22α, calponin, SMMHC, and myosin light chain kinase (MLCK), and lower levels of synthetic proteins such as OPN,which play a prominent role in blood vessel tone, blood flow, and blood pressure. These changes in molecular concentrations allow the design of novel drugs for the treatment of atherosclerosis, post-angioplasty restenosis, and hypertension via potential underlying mechanisms involved in VSMC phenotypic switching. PCNA is postulated to be a pro-proliferative gene in VSMCs[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], MMP2 is associated with cellular migration ,and BAX is thought to exert anti-proliferative effects on VSMCs. However, these molecules exert opposite effects on VSMC proliferation. Our results revealed that EGCG reduced the number of EdU-positive and migrating cells in response to PDGF-BB treatment. Furthermore, EGCG mitigated the upregulation of PCNA and MMP2 protein levels, as well as the downregulation of BAX protein expression in VSMCs incubated with PDGF-BB; and we obtained similar results in vito. The cell cycle is a major convergent point in VSMC proliferation, with the process controlled by multiple protein kinases and regulatory cyclins [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Negative regulators of protein kinases and cyclins arrest the cell cycle atthe G0/G1 phase [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. we demonstrated that EGCG inhibits DNA synthesis and arrests the cell cycle at the G0/G1 phase. Thus, confirmation of the changes in the protein levels of negative regulators requires further investigation. Nevertheless, these results showed that EGCG diminished PDGF-BB-induced VSMC proliferation and migration, and that the protective effects of EGCG on PDGF-BB-stimulated VSMCs were due to cell cycle arrest.\u003c/p\u003e \u003cp\u003eGreen tea is one of the most popular beverages, possesses a variety of biological activities, and may be used in the treatment of many diseases, including tumors,CVDs, metabolic disorders, cerebro- vascular diseases, and neurodegenerative diseases[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Several studies have revealed that EGCG inhibits VSMC proliferation[\u003cspan additionalcitationids=\"CR46\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], To further elucidate the mechanism of EGCG inhibiting proliferation of vascular smooth muscle cells, Growing evidence indicates that Nur77 is critical to the pathogenesis of a variety of CVDs, including vascular remodeling, atherosclerosis, cardiac hypertrophy, and cardiac ischemia/reperfusion injury[\u003cspan additionalcitationids=\"CR49\" citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. In VSMCs, Nur77 can be induced by multiple stimuli including cytokines, growth factors, oxidized low-densitylipoprotein, and vascular injury[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. We ascertained that Nur77 was highly expressed both in rat PDGF-induced VSMCs in vitro and balloon-injuryed carotid artery in vivo, and that EGCG modulates expression and activity of Nur77,. Therefore, we conclude that EGCG inhibits vascular smooth muscle proliferation by inhibiting NUR77. Our results also showed that Nur77 knockdown promote rat VSMC proliferation, andt inhibited migration, EGCG reversesed these processes. Thus, our data showed that EGCG inhibited PDGF-BB-induced VSMC proliferation via the regulation of Nur77. Further validation test, the results clarified the underlying molecular mechanisms by which EGCG exerts a protective effect onVSMCs.\u003c/p\u003e \u003cp\u003eRecent research has shown that inducing genomic DNA hypomethylation can alter SMC phenotypic identity, growth patterns, and the expression levels of contractile genes[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e], providing preliminary evidence that modulating DNA methylation can shift the phenotypic identity of SMCs. The acquisition and influence of DNA methylation in CVDs and their establishment remain largely unexplored. DNA methylation patterns are established and maintained by three major DNMTs: DNMT1, DNMT3A, and DNMT3B, with DNMT1 being the most abundant and key to the maintenance of methyltransferases in mammalian cells[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. It has also been reported that the gene expression profile of Aza-CdR-treated cells was very similar to that of DNMT1-/-cells[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. We ascertained that DNMT1 was highly expressed and global DNA methylation at high methylation levels both in rat PDGF-induced VSMCs in vitro and balloon-injuryed carotid artery in vivo, and that EGCG modulates expression and activity of DNMT1and global DNA methylation both in vivo and vitro. Therefore, we hypothesized that EGCG could selectively deplete DNMT1 in a dose-dependent manner in VSMCs, resulting in the in hibition of PDGF-induced VSMC phenotypic modulation. This shows that DNMT1 plays an important role in this modulatory process, implicating DNMT1-mediated DNA methylation in the remodeling of VSMCs. Our investigation provides novel in sights into the mechanism of VSMC remodeling, which may be helpful in developing new treatments for CVDs.\u003c/p\u003e \u003cp\u003eStudies have shown that nur77 gene is epigenetically regulated by DNMT1 and is involved in the insulin-signaling pathway[\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Nr4a1 is an orphan nuclear. receptor that has been shown to be regulated by DNMT1 and the methylation level of its promoter region[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Consistent with these findings, our results revealed that the protective effects of NUR77 on VSMCs might be regulated by DNMT1. The exact mechanism underlying these actions remains unclear, and further research is required to confirm the exact mechanism. Collectively, our findings suggest that Nur77 and DNMT1 are potential pharmacological targets in CVDs.\u003c/p\u003e \u003cp\u003eIn summary, EGCG significantly inhibited neointimal hyperplasia induced by balloon injury of rat carotid arteries by attenuating proliferation and migration and promoting apoptosis of rat VSMCs. EGCG also inhibited PDGF-induced rat VSMCs by reducing the expression of Nur77. Mechanistically, si-Nur77 augmented proliferation, whereas EGCG reversed it; the interfering RNA inhibited migration, whereas EGCG exacerbated it. Our results indicate that EGCG significantly ameliorated neointimal hyperplasia by modulating Nur77 and DNMT1signaling.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur data revealed disparities inviability, migration, and apoptosis between normal and proliferating VSMCs, and demonstrated the protective role of EGCG inVSMC proliferation in vitro and in vivo. We demonstrated that EGCG inhibited PDGF-BB-induced VSMC proliferation via DNMT1 and Nur77, and this information thus offers insights into future therapeutic. Furthermore, since methylation is involved in the onset and development of CVDs, the application of anti-methylation therapy is expected to provide a new direction in the treatment of such diseases.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCVD:cardiovasculardisease; EGCG: EpigallocatechinGallate; VSMCs: Vascular smooth muscle cells; DNMT1:DNA (cytosine-5-)-methyltransferase 1; CCK-8: Cell countingkit-8; EDU: 5-ethynyl-2\u0026apos;-deoxyuridine; SD:Standarddeviation.\u003c/p\u003e\u003cp\u003eEGCG=Epigallocatechin-3-gallate;GO=Gene Ontology;KEGG=Kyoto Encyclopedia of Genes and Genomes;PPI=Protein-protein interaction;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported in part by Fundamental Research Program of Shanxi Province (Grant Numbers 20210302123019, 202103021224195, 202103021224212, 202103021223189) and Shanxi Scholarship Council of China (Grant numbers 2021-108)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data sets used and/or analyzed in this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS L interpreted the data, and wrote the manuscript. Y ZQ and T JJ designed the study protocol and supervised the project.L F were involved in the interpretation of data, \u0026nbsp;Y ZC and X K revised it critically for important intellectual content. All authors have read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclosure statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no conflicts of interest to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Changsha Natural Science Foundation Kq2007043 and the 2022 Key Scientific Research Project of Shaanxi Provincial Education Department (Key Laboratory Project)22JS006.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003evan Tiel CM, de Vries CJ. NR4All in the vessel wall. J Steroid Biochem Mol Biol. 2012;130(3-5):186-93. \u0026apos;doi:\u0026apos;10.1016/j.jsbmb.2011.01.010.\u003c/li\u003e\n\u003cli\u003eYang F, Chen Q, He S, Yang M, Maguire EM, An W et al. miR-22 Is a Novel Mediator of Vascular Smooth Muscle Cell Phenotypic Modulation and Neointima Formation.CIRCULATION.2018;137(17):1824-41. doi:\u0026apos;10.1161/CIRCULATIONAHA.117.027799.\u003c/li\u003e\n\u003cli\u003eGerthoffer WT. Mechanisms of vascular smooth muscle cell migration. CIRC RES. 2007;100(5):607-21.\u0026apos;doi:\u0026apos;10.1161/01.RES.0000258492.96097.47.\u003c/li\u003e\n\u003cli\u003eChao LC, Wroblewski K, Ilkayeva OR, Stevens RD, Bain J, Meyer GA et al. Skeletal muscle Nur77 expression enhances oxidative metabolism and substrate utilization. J LIPID RES. 2012;53(12):2610-9.\u0026apos;doi:\u0026apos;10.1194/jlr.M029355.\u003c/li\u003e\n\u003cli\u003eHanna RN, Carlin LM, Hubbeling HG, Nackiewicz D, Green AM, Punt JA et al. The transcription factor NR4A1 (Nur77) controls bone marrow differentiation and the survival of Ly6C- monocytes. NAT IMMUNOL. 2011;12(8):778-85.\u0026apos;doi:\u0026apos; 10.1038/ni.2063.\u003c/li\u003e\n\u003cli\u003eArkenbout EK, de Waard V, van Bragt M, van Achterberg TA, Grimbergen JM, Pichon B et al. Protective function of transcription factor TR3 orphan receptor in atherogenesis: decreased lesion formation in carotid artery ligation model in TR3 transgenic mice. CIRCULATION. 2002;106(12):1530-5. \u0026apos;doi:\u0026apos;10.1161/ 01.cir. 0000028811.03056.bf.\u003c/li\u003e\n\u003cli\u003ede Waard V, Arkenbout EK, Vos M, Mocking AI, Niessen HW, Stooker W et al. TR3 nuclear orphan receptor prevents cyclic stretch-induced proliferation of venous smooth muscle cells. AM J PATHOL. 2006;168(6):2027-35. \u0026apos;doi:\u0026apos;10.2353/ ajpath.2006.050932.\u003c/li\u003e\n\u003cli\u003ePires NM, Pols TW, de Vries MR, van Tiel CM, Bonta PI, Vos M et al. Activation of nuclear receptor Nur77 by 6-mercaptopurine protects against neointima formation. CIRCULATION. 2007;115(4):493-500. \u0026apos;doi:\u0026apos;10.1161/ CIRCULATIONAHA. 106. 626838.\u003c/li\u003e\n\u003cli\u003eBonta PI, Matlung HL, Vos M, Peters SL, Pannekoek H, Bakker EN et al. Nuclear receptor Nur77 inhibits vascular outward remodelling and reduces macrophage accumulation and matrix metalloproteinase levels. CARDIOVASC RES. 2010;87(3): 561-8. \u0026apos;doi:\u0026apos;10.1093/cvr/cvq064.\u003c/li\u003e\n\u003cli\u003eKim HJ, Kim JY, Lee SJ, Kim HJ, Oh CJ, Choi YK et al. alpha-Lipoic acid prevents neointimal hyperplasia via induction of p38 mitogen-activated protein kinase/Nur77-mediated apoptosis of vascular smooth muscle cells and accelerates postinjury reendothelialization. Arterioscler Thromb Vasc Biol. 2010;30(11):2164-72.\u0026apos;doi:\u0026apos;10. 1161/ATVBAHA.110.212308.\u003c/li\u003e\n\u003cli\u003eNing Y, Huang H, Dong Y, Sun Q, Zhang W, Xu W et al. 5-Aza-2\u0026apos;-deoxycytidine inhibited PDGF-induced rat airway smooth muscle cell phenotypic switching. ARCH TOXICOL. 2013;87(5):871-81. \u0026apos;doi:\u0026apos;10.1007/s00204-012-1008-y.\u003c/li\u003e\n\u003cli\u003eMiranda TB, Jones PA. DNA methylation: the nuts and bolts of repression. J CELL PHYSIOL. 2007;213(2):384-90. \u0026apos;doi:\u0026apos;10.1002/jcp.21224.\u003c/li\u003e\n\u003cli\u003eRaftopoulos L, Katsi V, Makris T, Tousoulis D, Stefanadis C, Kallikazaros I. Epigenetics, the missing link in hypertension. LIFE SCI. 2015;129:22-6. \u0026apos;doi:\u0026apos;10.1016/ j.lfs.2014.08.003.\u003c/li\u003e\n\u003cli\u003eLaird PW. Principles and challenges of genomewide DNA methylation analysis. NAT REV GENET. 2010;11(3):191-203. \u0026apos;doi:\u0026apos;10.1038/nrg2732.\u003c/li\u003e\n\u003cli\u003eHiltunen MO, Turunen MP, Hakkinen TP, Rutanen J, Hedman M, Makinen K et al. DNA hypomethylation and methyltransferase expression in atherosclerotic lesions. VASC MED. 2002;7(1):5-11. \u0026apos;doi:\u0026apos;10.1191/1358863x02vm418oa.\u003c/li\u003e\n\u003cli\u003eConnelly JJ, Cherepanova OA, Doss JF, Karaoli T, Lillard TS, Markunas CA et al. Epigenetic regulation of COL15A1 in smooth muscle cell replicative aging and atherosclerosis. HUM MOL GENET. 2013;22(25):5107-20.\u0026apos;doi:\u0026apos;10.1093/hmg/ddt365.\u003c/li\u003e\n\u003cli\u003eLyko F. The DNA methyltransferase family: a versatile toolkit for epigenetic regulation. NAT REV GENET. 2018;19(2):81-92. \u0026apos;doi:\u0026apos;10.1038/nrg.2017.80.\u003c/li\u003e\n\u003cli\u003eChen YT, Liao JW, Tsai YC, Tsai FJ. Inhibition of DNA methyltransferase 1 increases nuclear receptor subfamily 4 group A member 1 expression and decreases blood glucose in type 2 diabetes. Oncotarget. 2016;7(26):39162-70.\u0026apos;doi:\u0026apos;10.18632/oncotarget.10043.\u003c/li\u003e\n\u003cli\u003eJorgensen BG, Berent RM, Ha SE, Horiguchi K, Sasse KC, Becker LS et al. DNA methylation, through DNMT1, has an essential role in the development of gastrointestinal smooth muscle cells and disease. CELL DEATH DIS. 2018;9(5):474.\u0026apos;doi:\u0026apos;10.1038/s41419-018-0495-z.\u003c/li\u003e\n\u003cli\u003eChen YK, Cheung C, Reuhl KR, Liu AB, Lee MJ, Lu YP et al. Effects of green tea polyphenol (-)-epigallocatechin-3-gallate on newly developed high-fat/Western-style diet-induced obesity and metabolic syndrome in mice. J Agric Food Chem. 2011;59(21):11862-71. \u0026apos;doi:\u0026apos;10.1021/jf2029016.\u003c/li\u003e\n\u003cli\u003eZheng Y, Song HJ, Kim CH, Kim HS, Kim EG, Sachinidis A et al. Inhibitory effect of epigallocatechin 3-O-gallate on vascular smooth muscle cell hypertrophy induced by angiotensin II. J Cardiovasc Pharmacol. 2004;43(2):200-8. \u0026apos;doi:\u0026apos;10.1097/00005344-200402000-00006.\u003c/li\u003e\n\u003cli\u003eOrozco-Sevilla V, Naftalovich R, Hoffmann T, London D, Czernizer E, Yang C et al. Epigallocatechin-3-gallate is a potent phytochemical inhibitor of intimal hyperplasia in the wire-injured carotid artery. J VASC SURG. 2013;58(5):1360-5.\u0026apos;doi:\u0026apos;10.1016/j.jvs.2012.11.090.\u003c/li\u003e\n\u003cli\u003eShu Z, Yu M, Zeng G, Zhang X, Wu L, Tan X. Epigallocatechin-3-gallate inhibits proliferation of human aortic smooth muscle cells via up-regulating expression of mitofusin 2. EUR J CELL BIOL. 2014;93(4):137-44.\u0026apos;doi:\u0026apos;10.1016/j.ejcb.2014.04.001.\u003c/li\u003e\n\u003cli\u003eZhu TT, Zhang WF, Luo P, He F, Ge XY, Zhang Z et al. Epigallocatechin-3-gallate ameliorates hypoxia-induced pulmonary vascular remodeling by promoting mitofusin-2-mediated mitochondrial fusion. EUR J PHARMACOL. 2017;809:42-51. \u0026apos;doi:\u0026apos;10.1016/j.ejphar.2017.05.003.\u003c/li\u003e\n\u003cli\u003eZhan XL, Yang XH, Gu YH, Guo LL, Jin HM. Epigallocatechin gallate protects against homocysteine-induced vascular smooth muscle cell proliferation. MOL CELL BIOCHEM. 2018;439(1-2):131-40. \u0026apos;doi:\u0026apos;10.1007/s11010-017-3142-6.\u003c/li\u003e\n\u003cli\u003eTan J, Xie Y, Yao A, Qin Y, Li L, Shen L et al. Long noncoding RNA-dependent regulation of vascular smooth muscle cell proliferation and migration in hypertension. Int J Biochem Cell Biol. 2020;118:105653. \u0026apos;doi:\u0026apos;10.1016/j.biocel.2019.105653.\u003c/li\u003e\n\u003cli\u003eTan J, Yang L, Liu C, Yan Z. MicroRNA-26a targets MAPK6 to inhibit smooth muscle cell proliferation and vein graft neointimal hyperplasia. Sci Rep. 2017;7:46602.\u0026apos;doi:\u0026apos;10.1038/srep46602.\u003c/li\u003e\n\u003cli\u003eStewart SA, Dykxhoorn DM, Palliser D, Mizuno H, Yu EY, An DS et al. Lentivirus-delivered stable gene silencing by RNAi in primary cells. RNA. 2003;9(4):493-501. \u0026apos;doi:\u0026apos;10.1261/rna.2192803.\u003c/li\u003e\n\u003cli\u003eYingchun Qin YX, Aihua L, Xiaoqin ZAZ, Yan. Protective effects of astragaloside IV against hypertension-induced vascular remodeling involves the DNMT1 and TET2 signaling pathway. ARCH BIOL SCI. 2021;73(2)(2021Jul.13):301-3\u003c/li\u003e\n\u003cli\u003eAltman JD, Trendelenburg AU, MacMillan L, Bernstein D, Limbird L, Starke K et al. Abnormal regulation of the sympathetic nervous system in alpha2A-adrenergic receptor knockout mice. MOL PHARMACOL. 1999;56(1):154-61. \u0026apos;doi:\u0026apos;10.1124/mol.56.1.154.\u003c/li\u003e\n\u003cli\u003eZhang W, Trebak M. Vascular balloon injury and intraluminal administration in rat carotid artery. J Vis Exp. 2014(94). \u0026apos;doi:\u0026apos;10.3791/52045.\u003c/li\u003e\n\u003cli\u003eHuhtinen A, Hongisto V, Laiho A, Loyttyniemi E, Pijnenburg D, ScheininM. Gene expression profiles and signaling mechanisms in alpha2B-adrenoceptor-evoked proliferation of vascular smooth muscle cells. BMC SYST BIOL. 2017;11(1):65.\u0026apos;doi:\u0026apos;10.1186/s12918-017-0439-8.\u003c/li\u003e\n\u003cli\u003eBenjamin EJ, Muntner P, Alonso A, Bittencourt MS, Callaway CW, Carson AP et al. Heart Disease and Stroke Statistics-2019 Update: A Report From the American Heart Association. CIRCULATION. 2019;139(10):e56-528.\u0026apos;doi:\u0026apos;10.1161/CIR.0000000000000659.\u003c/li\u003e\n\u003cli\u003eKimura K, Nagata T, Ohtani M, Nagata M, Kajiki S, Fujino Y et al. Cardiovascular and cerebrovascular diseases risk associated with the incidence of presenteeism and the costs of presenteeism. J OCCUP HEALTH. 2020;62(1):e12167.\u0026apos;doi:\u0026apos;10.1002/1348-9585.12167.\u003c/li\u003e\n\u003cli\u003eCullen AE, Centner AM, Deitado R, Salazar J. The Impact of Dietary Supplementation of Whole Foods and Polyphenols on Atherosclerosis. NUTRIENTS. 2020;12(7).\u0026apos;doi:\u0026apos;10.3390/nu12072069.\u003c/li\u003e\n\u003cli\u003eGriffiths K, Aggarwal BB, Singh RB, Buttar HS, Wilson D, De Meester F. Food Antioxidants and Their Anti-Inflammatory Properties: A Potential Role in Cardiovascular Diseases and Cancer Prevention. Diseases. 2016;4(3).\u0026apos;doi:\u0026apos;10.3390/diseases4030028.\u003c/li\u003e\n\u003cli\u003eVazquez-Padron RI, Lasko D, Li S, Louis L, Pestana IA, Pang M et al. Aging exacerbates neointimal formation, and increases proliferation and reduces susceptibility to apoptosis of vascular smooth muscle cells in mice. J VASC SURG. 2004;40(6):1199-207.\u0026apos;doi:\u0026apos;10.1016/j.jvs.2004.08.034.\u003c/li\u003e\n\u003cli\u003eShi J, Yang Y, Cheng A, Xu G, He F. Metabolism of vascular smooth muscle cells in vascular diseases. Am J Physiol Heart Circ Physiol. 2020;319(3):H613-31. \u0026apos;doi:\u0026apos;10.1152/ajpheart.00220.2020.\u003c/li\u003e\n\u003cli\u003eGomez D, Owens GK. Smooth muscle cell phenotypic switching in atherosclerosis. CARDIOVASC RES. 2012;95(2):156-64. \u0026apos;doi:\u0026apos;10.1093/cvr/cvs115.\u003c/li\u003e\n\u003cli\u003eEng QY, Thanikachalam PV, Ramamurthy S. Molecular understanding of Epigallocatechin gallate (EGCG) in cardiovascular and metabolic diseases. J ETHNOPHARMACOL. 2018;210:296-310. \u0026apos;doi:\u0026apos;10.1016/j.jep.2017.08.035.\u003c/li\u003e\n\u003cli\u003eJiang F, Jiang R, Zhu X, Zhang X, Zhan Z. Genipin inhibits TNF-alpha-induced vascular smooth muscle cell proliferation and migration via induction of HO-1. PLOS ONE. 2013;8(8):e74826. \u0026apos;doi:\u0026apos;10.1371/journal.pone.0074826.\u003c/li\u003e\n\u003cli\u003eSherr CJ.Cancer cell cycles. SCIENCE. 1996;274(5293):1672-7. \u0026apos;doi:\u0026apos; 10. 1126/ science 274.5293.1672.\u003c/li\u003e\n\u003cli\u003eJiang F, Jiang R, Zhu X, Zhang X, Zhan Z. Genipin inhibits TNF-alpha-induced vascular smooth muscle cell proliferation and migration via induction of HO-1. PLOS ONE. 2013;8(8):e74826. \u0026apos;doi:\u0026apos;10.1371/journal.pone.0074826.\u003c/li\u003e\n\u003cli\u003eChowdhury A, Sarkar J, Chakraborti T, Pramanik PK, Chakraborti S. Protective role of epigallocatechin-3-gallate in health and disease: A perspective. BIOMED PHARMACOTHER. 2016;78:50-9. \u0026apos;doi:\u0026apos;10.1016/j.biopha.2015.12.013.\u003c/li\u003e\n\u003cli\u003eShu Z, Yu M, Zeng G, Zhang X, Wu L, Tan X. Epigallocatechin-3-gallate inhibits proliferation of human aortic smooth muscle cells via up-regulating expression of mitofusin 2. EUR J CELL BIOL. 2014;93(4):137-44.\u0026apos;doi:\u0026apos;10.1016/j.ejcb.2014.04.001.\u003c/li\u003e\n\u003cli\u003eLiu PL, Liu JT, Kuo HF, Chong IW, Hsieh CC. Epigallocatechin gallate attenuates proliferation and oxidative stress in human vascular smooth muscle cells induced by interleukin-1beta via heme oxygenase-1. Mediators Inflamm. 2014;2014:523684. \u0026apos;doi:\u0026apos;10.1155/2014/523684.\u003c/li\u003e\n\u003cli\u003eYang J, Han Y, Sun H, Chen C, He D, Guo J et al. (-)-Epigallocatechin gallate suppresses proliferation of vascular smooth muscle cells induced by high glucose by inhibition of PKC and ERK1/2 signalings. J Agric Food Chem. 2011;59(21):11483-90.\u0026apos;doi:\u0026apos;10.1021/jf2024819.\u003c/li\u003e\n\u003cli\u003eHamers AA, Vos M, Rassam F, Marinkovic G, Kurakula K, van Gorp PJ et al. Bone marrow-specific deficiency of nuclear receptor Nur77 enhances atherosclerosis. CIRC RES. 2012;110(3):428-38. \u0026apos;doi:\u0026apos;10.1161/CIRCRESAHA.111.260760.\u003c/li\u003e\n\u003cli\u003eWang RH, He JP, Su ML, Luo J, Xu M, Du XD et al. The orphan receptor TR3 participates in angiotensin II-induced cardiac hypertrophy by controlling mTOR signalling. EMBO MOL MED. 2013;5(1):137-48. \u0026apos;doi:\u0026apos;10.1002/emmm.201201369.\u003c/li\u003e\n\u003cli\u003eCheng Z, Volkers M, Din S, Avitabile D, Khan M, Gude N et al. Mitochondrial translocation of Nur77 mediates cardiomyocyte apoptosis. EUR HEART J. 2011;32(17):2179-88. \u0026apos;doi:\u0026apos;10.1093/eurheartj/ehq496.\u003c/li\u003e\n\u003cli\u003eWang L, Gong F, Dong X, Zhou W, Zeng Q. Regulation of vascular smooth muscle cell proliferation by nuclear orphan receptor Nur77. MOL CELL BIOCHEM. 2010;341(1-2):159-66. \u0026apos;doi:\u0026apos;10.1007/s11010-010-0447-0.\u003c/li\u003e\n\u003cli\u003eLiu Y, Zhang J, Yi B, Chen M, Qi J, Yin Y et al. Nur77 suppresses pulmonary artery smooth muscle cell proliferation through inhibition of the STAT3/Pim-1/NFAT pathway. Am J Respir Cell Mol Biol. 2014;50(2):379-88.\u0026apos;doi:\u0026apos;10.1165/rcmb.2013-0198OC.\u003c/li\u003e\n\u003cli\u003eHu B, Gharaee-Kermani M, Wu Z, Phan SH. Epigenetic regulation of myofibroblast differentiation by DNA methylation. AM J PATHOL. 2010;177(1):21-8. \u0026apos;doi:\u0026apos;10.2353/ajpath.2010.090999.\u003c/li\u003e\n\u003cli\u003eNing Y, Huang H, Dong Y, Sun Q, Zhang W, Xu W et al. 5-Aza-2\u0026apos;-deoxycytidine inhibited PDGF-induced rat airway smooth muscle cell phenotypic switching. ARCH TOXICOL. 2013;87(5):871-81. \u0026apos;doi:\u0026apos;10.1007/s00204-012-1008-y.\u003c/li\u003e\n\u003cli\u003eGhoshal K, Majumder S, Datta J, Motiwala T, Bai S, Sharma SM et al. Role of human ribosomal RNA (rRNA) promoter methylation and of methyl-CpG-binding protein MBD2 in the suppression of rRNA gene expression. J BIOL CHEM. 2004;279(8):6783-93. \u0026apos;doi:\u0026apos;10.1074/jbc.M309393200.\u003c/li\u003e\n\u003cli\u003eChen YT, Liao JW, Tsai YC, Tsai FJ. Inhibition of DNA methyltransferase 1 increases nuclear receptor subfamily 4 group A member 1 expression and decreases blood glucose in type 2 diabetes. Oncotarget. 2016;7(26):39162-70. \u0026apos;doi:\u0026apos;10.18632/oncotarget.10043. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"vascular smooth muscle cell, EGCG, Nur77, NR4A1, DNMT1","lastPublishedDoi":"10.21203/rs.3.rs-4260039/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4260039/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eEGCG inhibits vascular smooth muscle proliferation.Nur77 and DNMT1 have been observd in the plaques of patients with atherosclerosis and are thought to be associated with vascular smooth muscle cell proliferation. This study was designed to investigate the role and mechanism of epigallocatechin gallate (EGCG) on proliferation and migration of vascular smooth muscle cells( VSMCs) and clarified the underlying molecular mechanism of EGCG. We investigated whether EGCG suppressed platelet-derived growth factor(PDGF)-induced vascular smooth muscle cell proliferation, migration and apoptosis in vivo and vitro. The effect of EGCG on smooth muscle cell proliferation and phenotype were evaluated using Cell Counting Kit-8(CCK8), EdU staining, immunohistochemistry and Western blot analysis.The effect of EGCG on smooth muscle cell migration was uising Wound-healing assay and Western blot analysis..The effect of EGCG on smooth muscle cell apoptosis was uising Flow cytometry and Western blot analysis. The current findings demonstrated that EGCG alleviated neointimal hyperplasia in balloon-induced arterial walls in vivo, significantly inhibited PDGF-BB-induced VSMC proliferation, migration, and promoted apoptosis in vitro and identical results were obtained in vivo..Moreover, EGCG attenuated VSMC proliferation by modulating the regulation of the DNMT1-Nur77-signaling axis. Our collective data showed that EGCG inhibited PDGF-BB-induced VSMC proliferation via DNMT1 regulation of the Nur77- signaling axis. In conclusion, These findings suggest that EGCG may be a promising therapeutic agent for the prevention and treatment of CVDs.\u003c/p\u003e","manuscriptTitle":"Epigallocatechin inhibits PDGF-BB-induced vascular smooth muscle cells proliferation via DNMT1 regulation of the Nur 77 axis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-22 02:17:48","doi":"10.21203/rs.3.rs-4260039/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3824556c-1b58-4f08-9de2-fb5b6860ae5d","owner":[],"postedDate":"April 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-10-04T06:53:39+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-22 02:17:48","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4260039","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4260039","identity":"rs-4260039","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}