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Resveratrol (RSVL), a polyphenolic antioxidant present in red wine, has been shown to provide cardiovascular protection by improving endothelial function and reducing myocardial ischemia. However, little is known about how RSVL affects vascular smooth muscle cells (VSMC) differentiation. RSVL blocks VSMC proliferation in vitro and neointimal formation following artery injury in vivo. Thus, one might expect that RSVL will promote VSMC differentiation. Unexpectedly, our results in this study show that RSVL induces VSMCs phenotypic modulation; this is characterized by suppressed transcription of SMC-specific marker genes Tagln, Acta2, Myh11, and Smtn in a dose-dependent and time-dependent manner in cultured VSMCs. Consistent with previous studies, RSVL induces the nuclear translocation of p53 and the expression of p53-responsive genes such as Cdkn1a, Gadd45a, Gadd45, and Fas. In an effort to identify the molecular mechanisms whereby RSVL represses VSMC differentiation, we found that RSVL inhibits the transcription of Myocd and Srf, the key VSMC transcriptional regulators. However, knockingdown and overexpressing p53 did not affect RSVL-induced VSMCs phenotypic modulation: this suggests that RSVL may induce VSMC dedifferentiation via p53-independent mechanisms. This study provides the first evidence showing that RSVL induces VSMC dedifferentiation by regulating Myocd and SRF-mediated VSMC gene transcription.
Phenotypic modulation of vascular smooth muscle cells (VSMCs) plays important roles in normal blood pressure homeostasis and in the pathogenesis of vascular diseases such as atherosclerosis, hypertension, restenosis, as well as aneurysm (Iyemere et al., 2006; Orr et al., 2010). In normal adult arteries, VSMCs maintain the differentiated/contractile phenotype and express high levels of SMC marker genes, such as transgelin (Tagln), smooth muscle-α-actin (Acta2), smooth muscle myosin chain (Myh11) and smoothelin (Smtn). However, in response to pathological stimuli, contractile VSMCs undergo phenotypic modulation, as evident by the presence of a variety of distinct VSMC subtypes such as dedifferentiated, proliferative, migratory and inflammatory phenotypes in the vessel wall (Gerthoffer, 2007; Iyemere et al., 2006; Orr et al., 2010). VSMC phenotypic modulation is characterized by the downregulation of SMC-specific marker genes concomitant with the upregulation of genes regulating cell cycle, apoptosis and migration (Yoshida and Owens, 2005). It is often observed that distinct biological processes such as VSMC differentiation/anti-proliferation and proliferation/dedifferentiation are coupled during SMC phenotypic modulation.
Resveratrol (RSVL, 3,5,4′-trihydroxy-trans-stilbene) is a naturally occurring antioxidant polyphenol found in plants such as grapes, peanuts, and mulberries. It is well recognized for its presence in red wine and the association with the “French paradox”(Das and Das, 2007). Accumulating evidence shows that RSVL has direct beneficial effects on cardiovascular system, including improving endothelial function (Wallerath et al., 2002; Zhang et al., 2009), decreasing myocardial ischemic reperfusion injury (Lekli et al., 2008), inhibiting reactive oxygen species generation (Martinez and Moreno, 2000), anti-inflammation (Manna et al., 2000), inhibition of modification of low-density lipoproteins (Frankel et al., 1993), and anti-platelet aggregation (Olas et al., 2002). RSVL also has anti-cancer (Athar et al., 2009) and extending lifespan effects (Howitz et al., 2003). Thus, RSVL has a plethora of beneficial effects on diverse biological processes.
Several studies have shown that RSVL inhibits the VSMCs proliferation induced by such diverse stimuli as advanced glycation end-products (AGEs) (Mizutani et al., 2000), ox-LDL (Liu and Liu, 2004), TNFα (Lee and Moon, 2005), angiotensin II (Chao et al., 2005), and hydrogen peroxide (El-Mowafy et al., 2008). While RSVL clearly prevents VSMCs proliferation in these situations, little is known about the direct effect of RSVL on VSMCs differentiation. In this study, we used in vitro tissue cultures to test the hypothesis that RSVL promotes VSMCs differentiation, a process that is generally regarded as being associated with anti-proliferation. However, our results show that RSVL blocked VSMC differentiation in cultured VSMCs.
Resveratrol (RSVL, # R5010) and actinomycin D (ActD, #A1410) were purchased from Sigma. The antibodies against p53 (#sc-6243, Santa Cruz), β-actin (#4967, Cell Signaling Technology) and GAPDH (#sc-47724, Santa Cruz) were used in this study.
PAC1 cells (a pulmonary artery-derived SMC line) (Rothman et al., 1992) were maintained in DMEM (Invitrogen) with 10 % FBS at 37 °C with 5 % CO2. PAC1 cells at 80% confluence were treated with vehicle (DMSO) or RSVL (freshly made) at different dosage for different time. 10T1/2 cell line is an embryonic mesenchymal progenitor cell line (#CCL-226, ATCC).
Total RNA from PAC1 cells was extracted and purified using RNeasy Kit (Qiagen). cDNAs were synthesized using the Superscript II reverse transcriptase (Invitrogen). Real-time PCR was performed using the StepOnePlus system (Applied Biosystems) in the presence of SYBR Green. snRNA U6 was used as the internal control, and all PCR primers were designed to cover at least 2 exons (Table 1).
To study the role of mRNA stability in mRNA expression regulation by RSVL, PAC1 cells were treated with either DMSO or RSVL (50 µM) in the presence of the general transcription inhibitor ActD for 1, 3, 6, 12, and 24 hours, respectively. Realtime PCR was used to determine the level of mRNA expression.
The SMC promoter-linked luciferase reporters (50 ng) including Tagln, Acta2, Myh11, 4xTagln CArG and MyoE8 (Myocd enhancer driven) (Creemers et al., 2006; Li et al., 1997; Li et al., 1996; Wang et al., 2001; Yoshida et al., 2003) were transiently transfected with 1 ng CMV-Renilla Luciferase Reporter (Promega) into 10T1/2 or PAC1 cells using the Lipofectamine and Plus transfection reagents (Invitrogen) for 24 hours. The promoter activities were determined by the firefly luciferase activity relative to the internal control renilla luciferase activity using the dual luciferase assay system described by the manufacturer (Promega, Madison, WI).
Whole cell lysate of PAC1 cells was prepared by using M-PER Mammalian Protein Extraction Reagent (Pierce) with Halt Protease Inhibitor Cocktail (Pierce). NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce) plus Halt Protease Inhibitor Cocktail (Pierce) were used to prepare the nuclear extracts. Protein was measured by Qubit fluorometer (Invitrogen).
Equal amounts of proteins were subjected to electrophoresis on 4–12% Bis-Tris NuPAGE Mini-gel (Invitrogen), followed by transfer to PVDF membrane (Millipore). The membrane was blocked with 5% non-fat milk for 1 hour and incubated with primary antibody overnight at 4 °C. After incubation with secondary conjugated antibody for 1 hour, the membrane was subjected to enhanced chemiluminescence detection using SuperSignal West Pico Chemiluminescent Substrate (Pierce). The protein expression levels were quantified by the Adobe Photoshop software.
p53 silencing was achieved by using Dicer-Substrate siRNA duplexes (IDT). PAC1 cells were transfected with p53 siRNA duplex or scrambled siRNA duplex (src, the universal negative control) at 100 nM by using DharmaFECT3 (Dharmacon). After 48 hours, RSVL and vehicle were added for another 24 hours followed by RNA isolation and realtime PCR assay.
At least three independent experiments were performed for all assays. The values presented are means ± standard errors of the means (SEMs). Statistical analysis was performed using one-way ANOVA with post test Dunnett. A value of p<0.05 was considered statistically significant.
To determine the effects of RSVL on VSMC differentiation, we examined the effect on VSMC morphology and the expression of VSMC markers in VSMCs (PAC1 cells, a rat pulmonary arterial derived VSMC line) treated with RSVL at 40 µM and 80 µM for 24 hours (Fig.1). RSVL did not significantly affect the morphology of cultured VSMCs (Fig.1A). Consistent with the antiproliferatory effect of RSVL on VSMCs (Poussier et al., 2005), the density of the VSMCs appeared to be suppressed with increased concentration of RSVL (Fig. 1A). Unexpectedly, RSVL decreased mRNA levels of VSMC marker genes such as Tagln, Acta2, Myh11, and Smtn, in a time- and dose-dependent manner (Fig. 1B and C). Because RSVL at the concentration of 50 µM for 24 hours consistently inhibited VSMC marker gene expression, the experiments described below were performed under this condition.
The transient luciferase assays in PAC1 cells showed that RSVL significantly inhibited promoter activities of SMC marker genes (Fig. 1D). Studies of the transcriptional control of SMC-specific gene expression have highlighted the importance of the CArG box in SMC gene regulation (Miano et al., 2007): each SMC-specific marker gene promoter used in the luciferase assays contains the key CArG box regulatory element. Transient transfection assays also showed that RSVL markedly suppressed the luciferase activities driven by the 4 copies of CArGnear boxes in the Tagln promoter (Fig. 1D), suggesting that RSVL inhibits CArG-mediated VSMC gene transcription.
It is well established that tumor suppressor p53 is involved in RSVL-induced apoptosis on cancer cells (Athar et al., 2009) and anti-proliferation on VSMCs (Mnjoyan and Fujise, 2003; Wang et al., 2006). A recent study demonstrated that p53 down-regulates the expression of myocardin, the master differentiation-inducing transcription factor, and thus inhibits SMC differentiation (Molchadsky et al., 2008). Therefore, it is conceivable that p53 may mediate RSVL-induced VSMCs phenotypic modulation.
As the first step to investigate the mechanism underlying RSVL-induced VSMCs phenotypic modulation, we examined p53-mediated signaling in response to RSVL in PAC1 cells. Consistent with the anti-proliferative effects of RSVL, we found by Western blot assays that RSVL induces p53 expression in both nuclear and whole cell extracts (Fig. 2A). The transcription of several p53-responsive genes such as Cdkn1a, Gadd45a, Gadd45b, and Fas was also upregulated 3–15 folds, although the expression of Tp53 mRNA was not affected (Fig. 2B). This result confirms that p53 signaling is activated in RSVL-treated PAC1 cells.
Extensive studies have been carried out to characterize the molecular mechanisms that regulate SMC differentiation. The serum response factor (SRF)-mediated transcriptional regulatory network plays a central role in regulating SMC gene expression (Miano et al., 2007). SRF recruits myocardin, a potent SMC transcriptional co-activator, and binds to the CArG box to activate the transcription of an array of SMC contractile genes (Wang et al., 2001). Since RSVL downregulated the CArG box-mediated transcription of SMC differentiation marker genes, we next investigated the effects of RSVL on the expression of Myocd and Srf, two key regulators for SMC differentiation: we found that RSVL markedly downregulated the mRNA levels of Myocd and Srf (Fig. 3A, 3B). The expression of Myocd is reduced about 90% 24 hours after RSVL treatment.
The regulation of gene expression involves transcriptional and posttranscriptional mechanisms. It was reported that RSVL decreases Cyp1a1 mRNA stability and hence inhibits Cyp1a1 expression (Lee and Safe, 2001). However, there were studies showing that mRNA stability was not involved in RSVL-induced angiotensin II type 1 receptor (Agtr1a) downregulation in VSMCs (Miyazaki et al., 2008). We examined the effect of RSVL on the mRNA stabilities of Myocd and Srf. ActD is known to inhibit de novo mRNA synthesis. The mRNA degradation rate of Myocd, Srf, and U6 (the internal control) were similar in the presence and absence of RSVL treatment (Fig. 3C), suggesting that RSVL does not affect the stability of Myocd and Srf mRNAs. Therefore, RSVL represses the transcription of Myocd and Srf. Consistent with this result, RSVL significantly decreased Myocd enhancer driven promoter activities (MyoE8-luc) in a dose-dependent manner by luciferase assay in PAC1 cells and 10T1/2 cells (Fig. 3D). This result suggests that RSVL inhibited Myocd and Srf expression at the transcriptional level.
To explore the role of p53 in RSVL-induced VSMCs phenotypic modulation, we knocked down p53 using p53 siRNAs. Comparing with the scramble siRNA (Scr), p53 siRNA efficiently knocked down p53 protein (>70%, Fig. 4A) and mRNA (> more than 80%, Fig. 4B). The nuclear p53 level is significantly lower after p53 silencing in RSVL cells than in the control cells (scr treated in the presence of RSVL) (Fig 4A). p53 siRNA also efficiently knocked down p53 mRNA in the presence of RSVL to the same extend as in the absence of RSVL (Fig.4B). Under this condition, p53 knockdown decreased the mRNA level of its responsive gene Cdkn1a. RSVL-induced upregulation of Cdkn1a was also repressed by p53 knockdown (Fig. 4B), suggesting that RSVL induces the expression of Cdkn1a via p53-mediated signal pathway.
On the contrary, silencing p53 did not prevent RSVL-induced mRNA downregulation of SMC master regulators Myocd and Srf nor SMC-specific marker genes such as Tagln, Acta2, Myh11, and Smtn (Fig. 4B).
Although RSVL did not affect p53 mRNA silencing and protein downregulation induced by p53 siRNA, the p53 protein level after p53 siRNA treatment in the presence of RSVL was higher than that after Scr siRNA treatment in the absence of RSVL (Fig. 4A). Therefore, we cannot exclude the possibility that RSVL-induced expression of p53 might mediate the inhibitory effect of RSVL on the VSMC marker gene expression. To exclude this possibility, we overexpressed p53 in PAC1 cells by transfection. The Western blot assay showed that the nuclear p53 protein level increases more than 4-fold (Fig. 4C); this could mimic the increased p53 translocation induced by RSVL (Fig. 2A). Interestingly, overexpression of p53 did not significantly change the basal Myocd-enhancer driving promoter activity nor RSVL-induced downregulation of this promoter activity (Fig. 4D). Taken together, these results indicate that RSVL induces p53 activation in VSMCs and promotes VSMCs phenotypic modulation through p53-independent mechanisms.
This study demonstrated that RSVL downregulates the transcription of SMC key regulators Myocd and Srf, and hence decreases SMC maker gene expression. VSMCs phenotypic switch from contractile to synthetic phenotype play important roles in the pathogenesis of vascular diseases such as atherosclerosis and aneurysm.
RSVL is known for its beneficial cardiovascular and cancer preventive effects. However, RSVL may also have adverse pathological effect such as renal toxicity in rat (Crowell et al., 2004). The present study shows that RSVL induces VSMC dedifferentiation in vitro. Since SMC dedifferentiation is usually regarded as a pathological process participating in the pathogenesis of cardiovascular diseases, such as hypertension, atherosclerosis, and aneurysm, it is possible that RSVL may have the potential of inducing VSMCs phenotypic modulation in vivo, thus aggravating the pathogenesis of cardiovascular diseases.
In this study the cell models used to carry out the VSMC phenotypic modulation induced by RSVL were cultured PAC1 cells (a rat pulmonary artery-derived SMC line), and 10T1/2 cells (a mouse embryonic mesenchymal progenitor cell line). The PAC1 cells has been shown to retain the VSMC phenotype even after extensive passages as evident by the spindle shaped morphology, the expression of functional surface receptors for angiotensin II, norepinephrine, and alpha-thrombin as well as the expression of smooth muscle markers such as isoforms of alpha-actin, myosin heavy chain, myosin regulatory light chain, and alpha-tropomyosin (Rothman et al., 1992). Nevertheless, it should be noted that cultured VSMC lines, VSMC progenitor cell line, as well as primary VSMCs could behave differently from VSMC and their progenitor cells in vivo. In view of this, in future studies the potential adverse effect of RSVL on the vascular system should be examined using animal model of vascular diseases.
VSMC dedifferentiation is characterized by the down regulation of VSMC contractile gene expression, which has been associated with the pathogenesis of vascular diseases such as atherosclerosis and aneurysms (Ailawadi et al., 2009; Lenk et al., 2007; Regan et al., 2000; Shanahan et al., 1994). However, the functional roles of VSMC dedifferentiation in VSMC phenotypic modulation remain unknown. Studies of SM22 knockout mice showed that SM22 deficiency promotes VSMC inflammation in response to high fat diet and carotid artery injury in vivo (Feil et al., 2004; Shen et al., 2010). Therefore, downregulation of cytoskeleton proteins may play an active role in promoting VSMC phenotypic modulation in response to injury. Consistent with this notion, the down regulation of smooth muscle cell marker genes occurs early before aneurysm formation (Ailawadi et al., 2009). More evidence suggests that VSMC dedifferentiation may be a phenotype precondition for VSMC remodeling into other subtypes in response to environmental stimuli.
The phenotypic modulation of different VSMC subtypes is controlled by complex regulatory mechanisms involving the regulatory circuits of transcription factors and the crosstalks of intracellular pathways (Yoshida and Owens, 2005). Myocd, and SRF are the key regulators in coordinating the transcriptional regulation of VSMC marker genes. The expression of Myocd, and Srf has been associated with VSMC phenotypic modulation in response to a variety of intracellular signal pathways and extracellular stimuli such as treatment of PDGF-BB, cyclosporine and loss of tensile stress (Deaton et al., 2009; Garvey et al., 2010; Liu et al., 2005; Wang and Olson, 2004; Yoshida and Owens, 2005; Zheng et al., 2010). To explore the molecular mechanisms mediating RSVL-induced VSMC phenotypic modulation, we examined the expression of Myocd and Srf in response to RSVL. We found that RSVL suppresses the transcription of Myocd,and Srf without affecting their mRNA stabilities (Fig. 3).
Although the direct molecular target of RSVL has not yet been identified, RSVL affects the activation of a variety of intracellular signal pathways. The present data showed that RSVL induces p53 activation in VSMCs. Unlike the recent study showing that p53 decreased myocardin expression and hence inhibited differentiation from mesenchymal cells to SMC (Molchadsky et al., 2008), p53 activation does not appear to participate directly in the RSVL-induced VSMC phenotypic modulation (Fig. 4). The effect of p53 on Myocd-mediated SMC differentiation may depend on the cell models.
To determine the molecular mechanisms underlying RSVL-induced VSMCs phenotypic modulation, we also explored the potential participation of several RSVL regulated targets such as Sirt1 and β-catenin in SMC phenotypic modulation (Gracia-Sancho et al., 2010) (Zhou et al., 2009). We found that knockingdown Sirt1 or β-catenin is not sufficient to block RSVL-induced VSMC dedifferentiation by the siRNA knockdown approach (data not shown). Further studies are needed to understand the molecular mechanisms underlying RSVL-induced VSMCs phenotypic modulation.
It is worth noting that microRNAs (miRNAs) have emerged as central players in governing the transcriptional regulatory programs essential for cardiovascular development and diseases (Liu and Olson, 2010). It is possible that RSVL may regulate the expression of miRNAs to inhibit VSMC marker genes expression. It is likely that genome-wide gene profile analyses using microarray and microRNA arrays may reveal the molecular mechanisms mediating RSVL-induced VSMC phenotypic modulation.
In summary, this study provides the first evidence demonstrating that RSVL induces VSMCs dedifferentiation via p53-independent mechanisms using both gain-of-function and loss-of-function approaches in vitro. Since VSMC phenotypic modulation is prevalent in the pathogenesis of vascular wall remodeling in cardiovascular diseases, these results point to the possibility of RSVL having potential adverse effects on the vascular system under certain conditions in spite of its cardiovascular protective activities.
This work was supported by the grant from the National Heart, Lung, and Blood Institute (HL087014 to L.L). We appreciate the constructive comments and valuable discussion with Dr. Hui J Li and Zhonghui Xu. We are grateful to Dr. Eric Olson for critical reagents and Dr. Alex Gow for sharing lab equipments.
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Conflicts of interest
The authors had no conflicts of interest to declare in relation to this article.