| Vascular smooth muscle cells (VSMCs) play a prominent role in the pathogenesis of vascular proliferative disorders such as atherosclerosis, postangioplasty restenosis, bypass vein graft failure, and cardiac allograft vasculopathy. VSMCs can switch between contractile to synthetic/proliferative phenotypes and this phenotype switch is believed to be essential for repair of vascular injure. VSMC migration, proliferation, and hypertrophy triggered by phenotype switch of the vessel wall are considered to be key events in the development of atherosclerosis, postangioplasty restenosis, and venous bypass graft failure. Consequently, anti-proliferative strategies have been demonstrated to successfully prevent the development of vascular proliferative disease. Identification of the key mechanisms involved in VSMC function will help to understand cellular responses to vascular injury.Platelet-derived growth factor (PDGF) is a potent mitogenic agent, which induces the phenotype of VSMCs from differentiated to proliferative status and initiates a multitude of biological effects through the activation of intracellular signal transduction pathways that contribute to VSMC proliferation, migration, and collagen synthesis. The cell cycle is a final common pathway for these mitogenic signaling cascades. The mitogen-activated protein kinase (MAPK) cascade which sequentially involves Ras, Raf, MEK1/2 and the extracellular signal-regulated kinase (ERK)1/2 MAPK, is activated in response to a multitude of mitogenic stimuli, and is essential for cells to exit into a quiescent state (G0) and to pass through the G1–S transition of the cell cycle. ERK1/2 signaling is also necessary for the degradation or down-regulation of CDK inhibitors, such as p21waf1 (p21) and particularly p27kip1 (p27), thereby eliminating their growth-suppressive activities. A previous study has demonstrated that balloon injury rapidly activates the MAP kinases in rat carotid arteries, which is crucial in mediating VSMC proliferation in response to vascular angioplasty. Therefore, any agent that can regulate these processes in VSMCs may have a role in the prevention and treatment of vascular diseases and restenosis after angioplasty. Smooth muscle 22 alpha (SM22α) is one of the proteins serving to regulate the actin cytoskeleton, and is important for inhibiting the phenotypic modulation of VSMCs from contractile to synthetic/proliferative status and plaque growth during atherogenesis. The significant negative correlation between SM22αexpression and VSMC proliferation led us to hypothesize that SM22αmay play a key role in controlling VSMC proliferation. Baicalin is a widely used herb in traditional Chinese medicine with anticancer, antiviral, antibacterial, and anti-inflammatory properties. Baicalin is able to induce apoptosis, and inhibit inflammation. However, regulatory effect of baicalin on VSMC proliferation is unclear. In the present study, we identify the key mechanisms involved in VSMC proliferation and character SM22αand baicalin with these pathways for the prevention of vascular proliferative disease. Our findings not only reveal a fundamental biological function for SM22αand baicalin in VSMC proliferation, but also define a novel genetic pathway for vascular diseases.1 Blockade of Ras-ERK1/2 pathway is involved in SM22αmediated suppression of VSMC proliferation1.1 Overexpression of SM22αinhibits PDGF-BB-induced growth of VSMCsWe first constructed a replication-defective adenovirus, Ad-SM22α, with full-length SM22αcDNA. Infection with Ad-SM22αfor 48 hours increased expression of SM22αprotein in cultured VSMCs, while the levels of PCNA protein which is a marker of cell growth showed inverse correlation with SM22α, as determined by Western blot analysis. Cell count and MTT assays showed that VSMCs infected with Ad-SM22αresulted cell growth arrest in time- and titre-dependent manners. These results suggest that overexpression of SM22αinhibits growth of VSMCs stimulated by PDGF-BB, and this inhibitory effect may be SM22α-dependent.1.2 Overexpression of SM22αarrests cell-cycle progression in VSMCsThe growth suppression effect of SM22αcould be the result of reduced proliferation and/or increased apoptosis of VSMCs. After PDGF-BB stimulation for 24 hours, the G0/G1 phase fraction in cells infected by Ad-SM22αshowed an increase as compared with Ad-null-infected cells. However, we found a slight increase in apoptosis in cells infected with Ad-SM22αunder identical experimental conditions but this was not statistically significant. We also found the activity of caspase-2, 3, 6 and 8 in Ad-SM22α-infected cells had no changes compared with serum-deprived or Ad-null-infected cells. Thus, the reduced proliferation activity may be due mainly to induction of G1/G0 cell cycle arrest by SM22α. Overexpression of SM22αcaused reduction in cyclin D1 levels and an increase in expression of p21 and p27, as compared with VSMCs infected with Ad-null. Taken together, these results suggest that SM22αarrests cell cycle progression via preventing the transition from G1 to S and G2/M phases.1.3 Binding of SM22αto Ras inhibits the Raf–MEK-ERK1/2 signaling pathwayTo delineate the cellular and molecular mechanisms underlying SM22α-mediated VSMC growth arrest, we evaluated the possible role of SM22αin regulating the MAPK signaling cascades. Overexpression of SM22αmarkedly inhibited ERK1/2 activation in VSMCs in response to PDGF-BB stimulation, but there was no change in the activated forms of JNK and p38. Moreover, overexpression of SM22αresulted in a robust reduction in the activated forms of the upstream kinases of ERK1/2, MEK1/2 and Raf-1. SM22α(1-151) mutant with truncation of the actin-binding domain has no actin binding activity. Overexpression of SM22α(1-151) decreased the activity of Raf-1-MEK1/2-ERK1/2 signaling and inhibited cell proliferation, suggesting that the inhibitory effects of SM22αon proliferation signaling are independent of its actin-binding activity. Increasing ERK activation using a constitutively active ERK kinase MEK1 plasmid (pCMV-MEK1) abolished the inhibitory effect of SM22αon cell proliferation. These findings indicate that SM22α-induced cell cycle arrest in the G0/G1 phases may be attributable at least in part to specific inhibition of Raf-MEK1/2-ERK1/2 signaling.Ras is a major upstream signaling protein of Raf-MEK1/2-ERK1/2. Overexpression of SM22αcaused a slight increase in Ras expression. Cross-coimmunoprecipitation analysis and GST pull-down assay showed that SM22αinteracted with Ras both in vivo and in vitro and overexpressed SM22αcompetes with Raf-1 to interact with Ras. Immunofluorescent staining also showed that SM22αco-localized with Ras in VSMCs, which increased in VSMCs infected with Ad-SM22α. To confirm the interaction of SM22αwith Ras is concentration dependent, VSMCs were stimulated with TGF-β1 or all trans-retinoic acid (ATRA) to induce the expression of endogenous SM22α. Cross-coimmunoprecipitation showed an increase in the interaction of SM22αwith Ras in VSMCs stimulated with TGF-β1 or ATRA, associated with increase in SM22αexpression. These data strongly suggest that SM22αcauses a negative regulation of the Raf-1-MEK–ERK1/2 MAPK signaling cascade through segregation of Ras with Raf-1 to prevent the activation of Raf-1 by activated Ras.1.4 Knockdown of SM22αby siRNA increases VSMC proliferation and activates the Raf-1-MEK1/2-ERK1/2 signaling pathwayTo further determine the importance of SM22αin G0/G1 arrest and growth suppression in VSMCs, we used small interfering (si)RNAs to specifically silence SM22αexpression. Western blot analysis revealed efficient knockdown of SM22αexpression using siSM22α, and the expression of Ras was accordingly slightly decreased in VSMCs infected with siSM22α. ATRA treatment led to a significant decrease in the number of cells entering S phase in uninfected or siControl-infected VSMCs. In contrast, transfection of VSMCs with siSM22αaccelerated cell progression into S phase and substantially decreased the population of G0/G1 phase cells under these experiment conditions. In addition, immunofluorescent staining showed that SM22αco-localized with Ras in VSMCs stimulated by ATRA, which decreased in siSM22α-transfected VSMCs. Meanwhile, SM22αknockdown with siSM22αabolished the inhibition of Raf–MEK–ERK1/2 activation by ATRA. These data suggest that high expression of SM22αis not only a marker of contractile VSMCs, but is also required for maintaining differentiation phenotype.2 Overexpression of SM22αinhibits neointimal hyperplasia induced by balloon injury and VSMC migration2.1 Overexpression of SM22αinhibits neointimal hyperplasia induced by balloon injury Currently, almost nothing is known about the effects of SM22αoverexpression on development of neointima hyperplasia. To evaluate this, the carotid arteries of rat were infected with either Ad-SM22αor Ad-null following balloon injury. The intima-to-media (I/M) areas was reduced in Ad-SM22α-infected arteries as compared with Ad-null and uninfected arteries on days 14 and 28 after balloon injury, respectively. Immunohistochemical staining showed that the expression of SM22αmarkedly increased with increase in p27 expression and reduction of PCNA levels in neointimal VSMCs of arteries infected with Ad-SM22α. These data indicate that SM22αoverexpression is closely associated with the suppression of neointimal hyperplasia. 2.2 Overexpression of SM22αsuppresses Raf-1-MEK1/2-ERK1/2 signaling cascade induced by balloon injuryTo determine whether the Raf-1-MEK1/2-ERK1/2 signaling pathway is involved in suppression of neointimal hyperplasia by SM22αoverexpression in vivo, we also evaluated the effect of SM22αon the ERK signaling cascade activated by balloon injury. Western blot analysis showed that the activation of ERK1/2, MEK1/2 and Raf-1 was strongly suppressed in Ad-SM22α-infected samples at day 3 after balloon injury, as compared with uninfected and Ad-null-infected samples. These results further support the concept that SM22αplays an important role in inhibition of the PDGF-BB or injury-activated ERK1/2 signaling cascade and VSMC proliferation in vitro and in vivo.2.3 Overexpression of SM22αinhibits VSMC migrationVSMC migration also contributes to cell growth or intimal thickening. We found that overexpression of SM22αsuppressed the PDGF-BB-induced migration of VSMCs, with a 55% reduction observed in the migration of Ad-SM22α-infected cells compared with Ad-null-infected. Moreover, overexpression of SM22αinhibited the expression of MMP-9 which is a marker of cell migration. Similarly, the MMP-9 level was decreased in the neointima and media of Ad-SM22α-infected arteries as compared with Ad-null and uninfected samples at day 3 and 28 after balloon injury, suggesting that there is a reduction of cell migration in SM22α-overexpressed arteries.3 Baicalin suppresses PDGF-stimulated VSMC proliferation and inhibits neointimal hyperplasia accompanied with reduced ERK signaling and increased p27 accumulation3.1 Baicalin inhibits proliferation of VSMCs induced by PDGF-BBWe first examined the effect of baicalin on proliferation of VSMCs induced with PDGF-BB by MTT assay. The result showed that baicalin inhibited proliferation of VSMCs in a concentration-dependent manner. Higher concentrations of baicalin (40 and 60μM) almost completely blocked the cell proliferation ability. When quiescent cells were treated with baicalin in the absence of PDGF-BB, no significant difference was observed in the cell viability of VSMCs as compared with the control, suggesting that baicalin is not cytotoxic at the concentrations tested. FACS analysis showed baicalin significantly increased the G0-G1 phase cells but decreased the G2-M and S phase cells in VSMCs. Western blot analysis showed that baicalin decreased the protein level of PCNA and increased the expression of p27 protein in a concentration-dependent manner, coinciding with cell cycle arrest. However, the expression of p21 protein was not markedly altered by baicalin. All of these data suggest that baicalin can exert its growth inhibitory effect in VSMCs whose cell cycle progression is arrested likely due to increasing p27 expression.Baicalin pretreatement caused 60% reduction of cell migration, compared with alone PDGF-BB stimulation. Baicalin pretreatment impaired the up-regulation of OPN, ICAM-1, VCAM-1 and MMP-2 proteins by PDGF-BB in a concentration-dependent manner. These results showed that baicalin inhibits VSMC migration induced by PDGF-BB. We found no change in apoptosis cell number and the activity of caspase-3 in VSMCs treated with baicalin compared with untreated cells, which showed baicalin has no effect on apoptosis in VSMCs.3.2 Baicalin inhibits formation of cyclin E-CDK2 complexes and phosphorylation of p27 protein in VSMCsCell cycle progression is controlled by cyclins and CDKs. Western blot analysis showed that treatment with baicalin reduced PDGF-BB-induced cyclin E and CDK2 expression in a concentration-dependent manner. However, the levels of cyclin D1, CDK4 and CDK6 proteins were not affected by baicalin treatment. Cyclin E-CDK2 complex that is active form has been shown to regulate p27 protein level through phosphorylation on Thr187 which is a prerequisite for the proteosome-dependent degradation of p27. Therefore, the observed baicalin-induced increase in p27 protein may be mainly due to a down-regulation of cyclin E-CDK2 activation. Cyclin E-CDK2 complex formation induced by PDGF-BB was concentration-dependently reduced in baicalin-pretreated VSMCs. The reduction of phosphorylated p27 as the result of baicalin treatment was linked to the down-regulated cyclin E-CDK2 complexes, consistent with increase in p27 protein level, suggesting that baicalin treatment may increase p27 protein stability by inhibiting its phosphorylation. To confirm the involvement of p27 in baicalin-induced growth arrest, we examined the effects of p27 knockdown by specific siRNA. The results showed inhibiting of p27 expression impaired the growth suppression induced by baicalin. Taken together, baicalin arrests the cell cycle at G1 phase via inhibiting cyclin E-CDK2 activation and up-regulating p27 protein level. 3.3 Baicalin inhibits PDGFRβ-ERK signaling pathway activated by PDGF-BB in VSMCsTo further delineate the cellular and molecular mechanisms underlying baicalin-induced VSMC growth arrest, we evaluated the effect of baicalin on the MAPK signaling cascade. Baicalin markedly inhibited MEK-ERK1/2 activation in a concentration-dependent manner in VSMCs in response to PDGF-BB stimulation with reduced PDGFRβphosphorylation, but there was no change in the activity of JNK, p38 and Akt. On the other hand, the phosphorylated PDGFRβand ERK1/2 were decreased in a time-dependent manner, phosphorylated PDGFRβstarting 5 min and phosphorylated ERK1/2 starting 10 min of PDGF-BB treatment in baicalin-treated VSMCs, with no change of total PDGFRβand ERK1/2 protein. To elucidate the mechanism by which baicalin inhibits PDGFRβphosphorylation induced by PDGF-BB, cells were treated with simultaneous both PDGF-BB and baicalin or with a mixture of both of them pre-incubated for 30 min, and no changes were observed in PDGFRβ, ERK1/2 and p27 phosphorylation, the interaction of cyclin E and CDK2, and cell proliferation. We also found that treatment of either baicalin or PD98059 resulted in reduced cyclin E-CDK2 complex formation, p27 phosphorylation and degradation, and cell proliferation with inhibiting of ERK1/2 activity, while the opposite change of cyclin E-CDK2 activation, p27 level, and cell proliferation was found upon ERK1/2 activation by transfection of pCMV-MEK1. These results suggest that reduced cyclin E-CDK2 activity and suppression of p27 phosphorylation are the events downstream of blockade of ERK1/2 pathway by baicalin, which is involeved in baicalin-induced proliferation suppression via reduction of p27 phosphorylation and degradation in VSMCs.3.4 Baicalin inhibits neointimal hyperplasiaAfter neointimal formation was induced by balloon injury, an increased I/M ratio of carotid arteries was observed at 14 days. Baicalin significantly reduced I/M ratio by over 70% compared with injured controls. Immunohistochemistry analysis showed that the positive cells of PCNA, ICAM-1 and VCAM-1 were increased in the neointima of balloon-injured arteries, suggesting that proliferative responses were induced by the balloon injury. These results suggest a potent inhibitory effect of baicalin on VSMC proliferation in vivo. Conclusion1 Overexpression of SM22αresulted in inhibition of the Raf-1-MEK1/2-ERK1/2 signaling cascade through segregation of Ras with Raf-1, and resulted in cell cycle arrest in PDGF-stimulated VSMCs.2 High expression of SM22αin vivo inhibited injury-induced neointimal hyperplasia via suppression of VSMC proliferation and inhibition of the Raf-1-MEK1/2-ERK1/2 signaling cascade.3 Growth arrest of VSMCs and suppression of neointimal hyperplasia induced by baicalin are associated with inhibition of cyclin E-CDK2 complex formation and up-regulation of p27 protein via blockade of PDGF receptor-mediated the MEK1/2-ERK1/2 signaling pathway. |
PDF Full Text Request