Resveratrol attenuates hyperproliferation of vascular smooth muscle cells from spontaneously hypertensive rats: Role of ROS and ROS-mediated cell signaling
A B S T R A C T
Resveratrol, a natural polyphenolic compound has been reported to attenuate angiotensin II -induced vascular smooth muscle cell (VSMC) hypertrophy; however, whether resveratrol could also inhibit hyperproliferation of VSMC from spontaneously hypertensive rats (SHR) is unexplored. The present study investigates the effect of resveratrol on hyperproliferation of VSMC from SHR and the underlying molecular mechanisms responsible for this response. For these studies, aortic VSMC from SHR and Wistar-Kyoto (WKY) rats were used. The pro- liferation of VSMC was determined by [3H] thymidine incorporation and the levels of proteins were determined by Western blotting. The enhanced proliferation exhibited by VSMC from SHR was attenuated by resveratrol. In addition, resveratrol attenuated the overexpression of cyclin D1, cyclin E, cyclin dependent kinase 4 (Cdk4),Cdk2, phosphorylated retinoblastoma protein (pRb), Giα proteins and enhanced phosphorylation of ERK1/2 and AKT in VSMC from SHR. Furthermore, the overproduction of superoxide anion, increased NADPH oxidase ac- tivity, overexpression of Nox2, Nox4 and p47phox proteins, increased phosphorylation of EGFR, IGF-IR, and c-Src were all abrogated by resveratrol. These results suggest that resveratrol attenuates the hyperproliferation of VSMC from SHR through the inhibition of ROS, c-Src, growth factor receptor activation, MAPK/PI3K, Giα and cell cycle proteins that are implicated in VSMC hyperproliferation.
1.Introduction
Hypertension is a chronic systemic disease, affects approximately 25% of the adult population [1]. Uncontrolled high blood pressure can lead to serious complications including heart attack, heart failure, and atherosclerosis [2]. Despite hypertension has been focus of intense re- search, 20 to 30% of patients with hypertension are resistant to cur- rently available antihypertensive treatment [3]. One of the pathophy- siological mechanisms involved in both development and complications of hypertension is vascular remodeling including abnormal VSMC growth, proliferation, migration, etc. [4,5]. Hyperproliferation of VSMCs not only plays pivotal role in the development of essential hy- pertension, but also atherosclerosis and restenosis [6]. Small mesenteric arteries of spontaneously hypertensive rats (SHR) (animal model for genetic hypertension) exhibited typical hallmarks of vascular re- modeling including smaller lumen, a greater media thickness, and augmented media-to-lumen ratios than Wistar–Kyoto (WKY) rat [7].VSMC cultured from the aorta of SHR have been reported to exhibit enhanced proliferation compared with normotensive WKY [8,9]. The cellular signaling pathways mediating hyperproliferation in VSMCs are complex. Several distinct signal transduction pathways in- cluding reactive oxygen species (ROS) and ROS mediated signaling pathway are implicated in vascular remodeling by promoting VSMC proliferation [10].
One of the most important sources of ROS in the VSMC are membrane bound NADPH oxidases which are responsible for the formation of superoxide anion (O2−) [11] [12]. The levels of ROS, have been shown to be augmented in VSMC from SHR due to the in- creased levels of superoxide anion (O −), NADPH oxidase activity and increased expression of the NADPH oxidase subunits Nox1/Nox2/Nox4 and p47phox [13].The enhanced oxidative stress in VSMC from SHR was attributed toincreased level of endogenous vasoactive peptides such as angiotensin II (Ang II), augmented expression of Giα proteins, and the decreased levels of cAMP [14]. It was reported that endogenous vasoactive pep- tides, through increased oxidative stress and resultant activation of c- Src, transactivate EGFR, which through mitogen-activated protein (MAP) kinase signaling and resultant overexpression of Giα proteins,contributes to the hyperproliferation of VSMC from SHR [9] [14,15]. Hyperproliferation of VSMC from SHR is associated with accel- erating entry of cells from G0/G1 phase of cell cycle to the synthetic phase [6]. In addition, the cell cycle proteins from G1-phase were re- ported to be over expressed in VSMC from SHR and implicated in the hyper-proliferation [16,17].
We previously demonstrated that the overexpression of Giα proteins and enhanced MAP kinase/PI3 kinase acti-vation contribute to the enhanced proliferation and expression of cell cycle proteins in VSMC from SHR, because PD98059, wortmannin and pertussis toxin, the inhibitors of MAP kinase, PI3 kinase and Giα pro- teins respectively, attenuated the hyperproliferation of VSMC from SHR and overexpression of cell cycle proteins to control levels [17].Resveratrol (RV) is polyphenolic molecule found in many natural sources including skin of grapes, berries and peanut [18]. Polyphenols including RV have been proposed to contribute to the “French paradox”phenomenon which consists of lower incidence of coronary heart dis-ease (CHD) in the French population [19]. Therefore, RV is receiving recently huge attention for its beneficial effects against different car- diovascular diseases like hypertension [20]. In addition, RV has also been shown to exert antimitogenic effect on hyperproliferation of VSMC induced by hyperglycemia [21] serum [22], and tumor necrosis factor alpha (TNF-α) [23]. However, whether RV exerts antiproliferative ef-fect in VSMC from SHR is not completely understood. Therefore, thepresent study was undertaken to examine the effect of RV on the pro- liferation of VSMC from SHR and to explore the underlying molecular mechanisms mediating this effect. We showed that RV inhibits hyper- proliferation of VSMC from SHR that involves the attenuation of en- hanced expression of cell cycle proteins, Giα proteins, enhanced acti-vation of MAP kinase/PI3 kinase, transactivation and over expression ofgrowth factor receptors, enhanced c-Src activation and oxidative stress.
2.Materials and methods
Resveratrol (3, 4′, 5-Trihydroxy-trans-stilbene) and β-Actin (AC-15) antibody were purchased from Sigma (St-Louis, MO, USA). Antibodiesagainst cyclin D1 (sc-20,044), Cdk4 (sc-23,896), cyclin E (sc-481), Cdk2(sc-6248), phospho-specific-Ser249/Thr252 Rb (sc-377528), Rb (sc-102), Giα-2 (sc-13534), Giα-3 (sc-262), ERK1/2 (sc-135900), phospho-spe- cific-Tyr204 ERK1/2 (sc-7383), phospho-specific-Ser473 AKT (sc-7985- R), AKT (sc-8312), phosphospecific-Tyr530 c-Src (sc-16,846), c-Src (sc- 19), phospho-specific-Tyr1173 EGFR (sc-12,351), EGFR (sc-03), phos- phor-specific-Tyr1165/1166 IGF-IR (sc-101704), IGF-IRβ (sc-713), dyneinIC1/2 (sc-13524), and goat anti-mouse IgG-HRP (sc-2005) from SantaCruz Biotechnology Inc. (Santa Cruz, DA, USA). Nox2/gp91 antibody was purchased from Abcam Inc. (Toronto, ON, Canada). Polyclonal Nox4 antibody was from protein tech. (Manchester, United Kingdom). Polyclonal p47phox antibody was purchased from Bioss Inc. (Massachusetts, USA). Western blotting reagents were from St. Cruz Biotech (Santa Cruz, CA). The L-[4,5-3H]-Thymidine was from PerkinElmer Inc. (Waltham, Massachusetts, USA).VSMC from 14-week-old SHR and their age-matched WKY rats were cultured from aortas, as described previously [24]. The purity of the cells was checked by immunofluorescence technique using α-actin, asdescribed previously [25]. These cells were found to contain high levelsof smooth-muscle-specific actin [26].
The cells were plated in 75 cm2 flasks and incubated at 37 °C in 95% air and 5% CO2 humidified at- mosphere in Dulbecco’s modified Eagle’s medium (DMEM) (with glu- cose, L-glutamine, and sodium bicarbonate) containing 1% antibiotics and 10% heat-inactivated fetal bovine serum (FBS). The cells were passaged upon reaching confluence with 0.5% trypsin containing 0.2% EDTA and utilized between passages 3 and 10. Confluent cells were then starved by incubation for 4 h in DMEM without FBS at 37 °C to reduce the interference by growth factors present in the serum. The cells were then incubated in the absence or presence of various con- centrations of RV (10 to 100 μM) or as otherwise indicated for 16 h. After incubation, the cells were washed three times with PBS and lysedin 200 μl of buffer containing 25 mM Tris·HCl (pH 7.5), 25 mM NaCl, 1 mM sodium orthovanadate, 10 mM sodium fluoride, 10 mM sodiumpyrophosphate, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 1%Triton X-100, 0.1% sodium dodecyl sulfate, (SDS), and 0.5 μg/ml leupeptin on ice. The cell lysates were centrifuged at 12,000 rpm for 10 min at 4 °C. Protein concentration was measuredby Bradford assay [27]. All the animal procedures used in the present study were approved by the Comité de Déontologie de l’Expérimenta- tion sur les Animaux (CDEA) of the University of Montreal (protocol #99050). The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (Guide, NRC 2011).The levels of proteins were determined by Western blotting using specific antibodies as described previously [28]. Equal amounts of protein (30 μg) were subjected to 10% SDS-polyacrylamide gel elec-trophoresis (SDS-PAGE), transferred to nitrocellulose membranes andincubated with the primary antibodies against cyclin D1, Cdk4, cyclin E, Cdk2, p-Rb, Rb, Giα-2, Giα-3, ERK1/2, p-ERK1/2, p-AKT, AKT, p-c- Src, c-Src, p-EGFR, EGFR, p-IGF-IR, IGF-IRβ, Nox2/gp91, Nox4,p47phox, β-actin and dynein.
The antigen-antibody complexes weredetected by incubating the blots with respective secondary antibodies conjugated with horseradish peroxidase for 1 h at room temperature and then the blots were then washed three times with PBS before re- acting with enhanced-chemiluminescence Western-blotting detection reagents. Quantitative analysis of specific bands was performed by densitometric scanning of the autoradiographs with the enhanced laser densitometer LKB Ultroscan XL and quantified using the gel-scan XL evaluation software (version 2.1) from Pharmacia (Baie d’Urfé, QC, Canada).Cell proliferation was quantified by DNA synthesis which was evaluated by incorporation of [3H] thymidine into cells as described previously [29]. Subconfluent VSMC from SHR and WKY rats were plated in 6-well plates for 24 h and were serum deprived for 4 h toinduce cell quiescence. The cells were then incubated in the absence or presence of different concentration of RV (10–100 μM) for 16 h. [3H] thymidine (1 μCi) was added and further incubated for 4 h before the cells were harvested. The cells were rinsed twice with ice-cold PBS andincubated with 5% trichloroacetic acid for 1 h at 4 °C. After being washed twice with ice-cold water, the cells were incubated with 0.4 N sodium hydroxide solution for 30 min at room temperature, and radioactivity was determined by liquid scintillation counter.Basal superoxide anion production in VSMC was measured using the lucigenin-enhanced chemiluminescence method with low concentra- tion (5 μM) of lucigenin as described previously [30]. The cells aftertreatment with RV (50 μM), were washed in oxygenated Kreb–Hepesbuffer, scraped and placed in scintillation vials containing lucigenin solution, and the emitted luminescence was measured with a liquid scintillation counter (Wallac 1409; Perkin Elmer Life Science, St Laurent, Quebec, Canada) for 5 min. The average luminescence value was estimated, the background value subtracted and the result was divided by the total weight of proteins in each sample. After the emitted luminescence for basal superoxide anion produc- tion was measured, 0.1 mM β-Nicotinamide adenine dinucleotide 2′- phosphate reduced tetrasodium salt hydrate (NADH, Sigma-Adrich) was added in the vials and the luminescence was measured con- tinuously for 5 min in a liquid scintillation counter (Wallac 1409;PerkinElmer Life Science). NADPH oxidase activity was calculated by subtracting the basal superoxide-induced luminescence from the lumi- nescence value induced by NADH.Results are expressed as means ± SEM. Comparisons between groups were made with one-way analysis of variance (ANOVA) in conjunction with Newman-Keuls test using GraphPad Prism5 (GraphPad Software Inc., La Jolla, California, USA). A difference be- tween groups was considered statistically significant at p < 0.05. 3.Results We earlier reported that RV attenuated Ang II-induced hypertrophy of VSMC [31]. To investigate if RV could also attenuate the hyperpro- liferation of VSMC from SHR, the effect of various concentrations of RV on DNA synthesis (a marker of proliferation) was examined on VSMC from SHR and WKY rats and the results are shown in Fig. 1. As reported earlier [14], the level of DNA synthesis in VSMCs from SHR as de- termined by [3H] thymidine incorporation was enhanced by about 120% as compared to WKY rats and RV attenuated this enhancedproliferation in a concentration-dependent manner. At 100 μM, RV al-most completely attenuated the augmented proliferation of VSMC from SHR to WKY level. In addition, RV also attenuated the proliferation of VSMC from WKY rats and at 100 μM, it inhibited the proliferation by about 70%.The enhanced expression of cyclin D1, Cdk4, cyclin E and Cdk2 was shown to contribute to the hyperproliferation of VSMC from SHR [17]. To investigate if RV-induced attenuation of hyperproliferation of VSMC from SHR is due to the inhibition of the enhanced expression of cell cycle proteins, we examined the effect of RV on the levels of different cell cycle proteins. As shown in Fig. 2, the expression of cyclin D1 (A) and Cdk4 (B) was significantly enhanced by about 90% and 35% re- spectively in VSMC from SHR as compared to WKY and RV completely abolished the enhanced expression of cyclin D1 and Cdk4. In addition; the levels of cyclin E (C) and Cdk2 (D) were significantly enhanced by 35% and 125% respectively in VSMC from SHR as compared to WKY and RV completely attenuated the augmented expression of cyclin E to control levels (C) whereas the enhanced expression of Cdk2 was atte- nuated by about 30% (D). On the other hand, RV treatment did not have any significant effect on the levels of these proteins in WKY rats except that Cdk4 level was decreased by about 35%.Furthermore, the expression of phosphorylated retinoblastomaprotein (pRb) (Fig. 3A) that was enhanced by about 85% in VSMC from SHR was also decreased by RV treatment by about 65%, however, RV did not affect the levels of pRb in WKY rats. In addition, the levels of Rb were not different in VSMC from SHR from WKY rats and RV did not have any effect on the expression of Rb proteins in VSMC from both SHR and WKY rats (Fig. 3B).The overexpression of Giα proteins is implicated in the increased proliferation of VSMC from SHR [9,17]. To determine if RV-induced attenuation of hyperproliferation of VSMC from SHR is also attributedto its ability to inhibit the enhanced level of Giα proteins, the effect of RV on the expression of Giα proteins was examined in VSMC from SHR and WKY and the results are shown in Fig. 4. As reported earlier [9], the levels of Giα-2 (A) and Giα-3 (B) were enhanced by about 85% and 35% respectively in VSMC from SHR as compared to WKY rats and RV at- tenuated the overexpression of Giα proteins to control levels. On the other hand, RV treatment did not affect the expression of Giα proteins in VSMC from WKY rats.We have earlier shown that VSMC from SHR exhibited increased activity of ERK1/2 and AKT as compared with VSMC from WKY rats [9,17] that contributes to enhanced expression of Giα proteins andresultant increase in DNA synthesis in VSMC from SHR [9]. To examineif RV-induced decreased expression of Giα and cell cycle proteins and resultant decreased proliferation is due to its ability to attenuate the enhanced activation of MAP kinase/PI3 kinase, we investigated theeffect of RV on the phosphorylation of ERK1/2 and AKT in VSMC from SHR and the results are shown in Fig. 5. As reported earlier [17], the phosphorylation of ERK1/2 (5A) and AKT (5B) was significantly aug- mented by about 40% and 35% respectively in VSMC from SHR as compared to WKY and RV completely abolished the augmented phos- phorylation or ERK 1/2 and AKT. On the other hand, this treatment did not affect the basal phosphorylation of AKT and ERK1/2 in VSMC from WKY rats.Earlier studies have shown that growth factor receptor transacti- vation contributes to the enhanced expression of Giα proteins [26] and proliferation of VSMC from SHR [14], Therefore, it was desirable toinvestigate if RV could also attenuate the growth factor receptor acti- vation that results in the attenuation of Giα protein expression and decreased DNA synthesis. To test this, the effect of RV on the expression and phosphorylation of EGFR and IGFR was examined in VSMC from SHR and WKY and the results are shown in Fig. 6. As previously re- ported [32] the phosphorylation of EGFR (A) and IGF-IR (C) was in- creased by about 120% and 55% respectively in VSMCs from SHR compared to WKY rats and RV treatment attenuated the enhanced phosphorylation of EGFR and IGF-IR by about 80% and 30% respec- tively. In addition, the expression of EGFR (B) and IGF-IR (D) was also increased in VSMCs from SHR as compared with WKY rats by about 60% and 130% respectively and RV treatment restored overexpression of EGFR (B) and IGF-IR (C) in VSMC from SHR to WKY control levels. On the other hand, RV treatment did not significantly affect the basal phosphorylation and expression of these growth factor receptors in VSMC from WKY rats. The role of enhanced phosphorylation of non-receptor tyrosine ki- nase c-Src in transactivation of EGFR and resultant hyperproliferation of VSMC from SHR has been demonstrated [14]. To investigate if the inhibition of enhanced phosphorylation of c-Src by RV contributes to the decreased activation of EGFR and resultant attenuated DNA synthesis, we examined the effect of RV on the activation of c-Src in VSMC from SHR and WKY rats. Results shown in Fig. 7 indicate that the phosphorylation of tyrosine419 on c-Src was significantly increased by about 45% in VSMC from SHR compared with WKY rats and RV atte- nuated the enhanced phosphorylation of c-Src by about 60%. On the other hand, this treatment did not have any significant effect on the phosphorylation of c-Src in WKY rats.Earlier studies have shown that enhanced oxidative stress exhibited by VSMC from SHR contribute to the enhanced expression of Giα proteins [28] and hyperproliferation of VSMC from SHR [14]. There- fore, it was of interest to investigate if RV-induced attenuation of DNA synthesis is due to its ability to inhibit oxidative stress, the effect of RVon production of O2– and NADPH oxidase activity was examined in VSMC from SHR and WKY rats. As shown in Fig. 8, both production of O2– (A) and NADPH oxidase activity (B) were augmented in VSMC from SHR compared with WKY by about 90% and RV treatment completely abolished the enhanced O – production and NADPH oxidase activity. On the other hand, this treatment did not have any effect on the O –production and NADPH oxidase activity in VSMC from WKY rats.In order to investigate if RV-evoked decreased production of O – and NADPH oxidase activity was associated with decreased expression of different subunits of NADPH oxidase, the effect of RV on the ex-pression of different subunits of NADPH oxidase was examined in VSMC from SHR and WKY rats. Results shown in Fig. 9 indicate that the ex- pression of p47phox (A), Nox4 (B), and Nox2 (C) was elevated by about 90%, 110% and 240% respectively in SHR as compared to WKY and RV treatment attenuated the augmented levels of p47phox, Nox4, and Nox2 by about 65%, 75%, and 60% respectively without affecting the levels of these proteins in WKY rats. 4.Discussion Vascular remodeling including hypertrophy and hyperplasia of VSMC plays important role in the development of essential hyperten- sion and atherosclerosis [33]. Resveratrol (RV) has been shown to lower the remodeling process by partially reversing lumen narrowing and media thickening in SHR arteries [18]. Although, RV was reported to inhibit VSMC proliferation induced by serum, endothelin and PDGF [34,35], we showed for the first time that RV attenuates the hyper- proliferation of VSMC from SHR and provide a new insight into the molecular mechanisms underlying the antiproliferative effect of RV.The overexpression of cell cycle proteins from G1 to S phase in VSMC from SHR and their role in hyperproliferation has been reported [16,17]. RV has been shown to decrease the enhanced level of cyclin A, B, D, and E, and Cdk 2, 4, and 6 in human lymphocytes and epidermoid carcinoma cells [36,37]. However, our data show for the first time that RV attenuates the enhanced expression of cyclin D1, cyclin E, Cdk2 and Cdk4 in VSMC from SHR, In addition, the fact that RV decreases the phosphorylation of retinoblastoma protein, a repressor of progression towards S-phase, further supports that RV-evoked attenuation of hy- perproliferation of VSMC from SHR may be attributed to its ability to decrease the expressions of cyclin D1/Cdk4 and cyclin E/Cdk2 com- plexes.The enhanced levels of Giα proteins have been implicated in over-expression of cell cycle proteins and hyper-proliferation in VSMC from SHR because the treatment of VSMC from SHR with pertussis toxin that inactivates Giα proteins was shown to attenuate the overexpression of cell cycle proteins (Cdk2, Cdk4, cyclin D1, and pRb) and hyperproli-feration [9,17]. Furthermore, the knocking down of Giα proteins by siRNA or antisense treatment result in attenuation of hyperproliferation of VSMC from SHRs [9,38]. We report for the first time that RV at- tenuates the enhanced expression of Giα proteins in VSMC from SHR to control levels. Taken together, it may be suggested that the RV-induced inhibition of augmented levels of cell cycle proteins from G1-phase andthe resultant attenuation of the hyperproliferation of VSMCs from SHR may be attributed to its ability to decrease the enhanced expression of Giα proteins levels.The implication of MAP kinase (MAPK) and PI3-kinase (PI3K) in theenhanced expression of Giα proteins and hyper proliferation of VSMC induced by vasoactive peptides and in VSMC from SHR is well docu- mented [9,29]. We show that RV attenuates the enhanced phosphor-ylation of ERK1/2 and AKT in VSMC from SHR and suggest that the antiproliferative effect of RV in VSMC from SHR may also be mediated through the inhibition of MAP kinase/PI3 kinase signaling pathways. This notion is supported by the studies of other investigators who have also shown that the inhibition of MAP kinase/PI3 kinase pathways by RV was involved in blocking high glucose -induced cell proliferation in rat VSMC and endothelin-1 (ET-1)-induced cell proliferation in human coronary artery smooth muscle cells [21,39].Earlier studies have shown the involvement of growth factor re- ceptor IGFR and EGFR transactivation in vasoactive peptide-induced enhanced activation of MAP kinase and enhanced DNA synthesis in A10 and aortic VSMC [40,41]. Furthermore, epidermal growth factor re- ceptor transactivation by endogenous Ang II and ET-1 has been shown to contribute to the enhanced proliferation of VSMC from SHR through augmenting the activity of MAP kinase [14]. Wang and his group have shown that RV inhibits EGFR phosphorylation with consequent de- creased AKT phosphorylation in prostate cancer cell lines [42]. In ad-dition, the inhibition of transforming growth factor-α (TGF- α) ex-pression and insulin-like growth factor I receptor (IGF-IR) mRNA was reported by RV in human breast cancer cells [43]. Our results showing that RV inhibits the enhanced phosphorylation and expression of EGFR and IGF-IR suggest that the antiproliferative effect of RV in VSMCs from SHR may also be mediated through the inhibition of the enhanced ex- pression and activation of growth factor receptors.We earlier showed the involvement of c-Src in the enhanced pro- liferation of VSMC from SHR and the transactivation of growth factor receptors [14,32]. RV has also been shown to interfere with Src tyrosine kinase activity [44,45]. Recent study has also demonstrated the in- hibition of Ang II- induced activation of c-Src by RV in VSMC. However, we demonstrate for the first time that RV treatment attenuated the enhanced activation of c-Src in VSMC from SHR. Taken together, it may be suggested that the antiproliferative effect of RV may also be medi- ated through the inhibition of c-Src activation and subsequent inhibi- tion of growth factor receptor activation.The role of oxidative stress generated by ROS in the pathogenesis of many vascular diseases, including hypertension and vascular re- modeling is well established [46,47]. Oxidative stress has also been shown to contribute in the hyperproliferation of VSMCs from SHR through c-Src activation and transactivation of growth factor receptors [14,48]. The cardio and vasoprotective effects of RV are shown to be related to its antioxidant activity [49]. Schreiner et al. reported that RV reduces intracellular ROS and extracellular H2O2 release from rat VSMC [50]. We also show that the treatment of VSMCs from SHR with RVattenuates the enhanced levels of O2– production, NADPH oxidase ac- tivity as well as NADPH oxidase subunits p47phox, Nox2 and Nox4.Taken together, these results suggest that RV induced-inhibition of VSMCs proliferation in SHR may be mediated by its ability to suppress ROS generation and NADPH oxidase activity. 5.Conclusions In summary, we demonstrate that RV attenuates the enhanced ex- pression of Giα proteins, cell cycle proteins, oxidative stress, c-Src Fig. 9. Effect of RV on the levels of p47phox, Nox4, and Nox2 proteins in VSMC from SHR and WKY rats. Serum- starved, quiescent aortic VSMC from SHR and WKY rats were incubated in the absence or presence of RV (50 μM) for 16 h. Cell lysates were prepared and subjected to Western blot analysis using specific antibodies against p47phox (A), Nox4 (B), and Nox2 (C) as described in Materials and methods. Dynein was used as the loading control. Protein bands were quantified by densitometric scanning. Values are means ± SE of 4 separate experi- ments. Results are expressed as percentage of WKY CTL, taken as 100%. ⁎⁎p < 0.01, ⁎⁎⁎p < 0.001 versus WKY CTL; p < 0.05, p < 0.01, p < 0.001 versus SHR CTL activation, activation of growth factor receptors such as EGF-R and IGF- R, and MAP kinase, all the signaling pathways that were shown to be implicated in hyperproliferation of VSMC from SHR. Thus, it may be suggested that RV inhibits hyperproliferation of VSMC from SHR and improves vascular remodeling through its ability to attenuate enhanced oxidative stress and downstream signaling cascade including c-Src and growth factor receptor activation, MAPK signaling and overexpression of Giα proteins and cell cycle proteins and that RV could be used as a therapeutic agent in the treatment of vascular complications associated with hypertension and MTX-211 hypertrophy.