SM22α Accumulation Is Involved In VSMC Senescence And Vascular Aging

Senescence of vascular smooth muscle cells(VSMCs) contribute to the pathogenesis of cardiovascular disease, such as atherosclerosis and hypertension. Senescent cell is usually characterized by enlarged and flattened shape, accompanied by alteration of cell function and gene expression, such as elevated p53 and p21, negative regulators of the cell cycle, and the increase of SA-β-gal activity, a biomarker of cellular senescence. These changes have been implicated in aging and age-related vascular disorder. Although the function of cellular senescence has been extensively examined, its underlying molecular mechanism remains elusive.Smooth muscle(SM) 22α, known as a specific marker for smooth muscle cell, is abundantly expressed in mature smooth muscle cells. The recent studies show that SM22α is involved in various cell behaviors via affecting different signaling pathway, and plays a key role in pathogenesis of atherosclerosis and tumors. SM22α is overexpressed in senescent cells. Furthermore, SM22α-mediated actin stabilization may trigger cell death and dysfunction. We have also found that the overexpression of SM22α upregulates the levels of CDK-inhibitors p21 and p27 proteins. However, the causal relationship between the increased SM22α and senescence is unclear. Methods:Rat VSMCs were treated with Ang II(10-7 mol/L) for 5 days to induce stress-induced premature senescence(SIPS), and replicative senescence(RS) of VSMCs was induced by serial passage. In addition, Ldlr-/-/Sm22α+/+ and Ldlr-/-/Sm22α-/- mice were infused with Ang II(1 μg/kg/min) for 4 weeks to reproduce hypertension model. We tested the effect of SM22α on VSMC senescence and senescence-related signaling pathway by knockdown and overexpression of SM22α. SA-β-gal staining was used to detect senescent VSMCs. Expression of m RNA and protein were examined by Real-time PCR, Western blot, immunofluorescence and immunohistochemistry, respectively. Protein interactions were determined using co-immunoprecipitation assay. Results: 1 SM22α promotes Ang II-induced VSMC senescence 1.1 Ang II induces VSMC premature senescence, accompanied with accumulation of SM22α.To prepare the model of VSMC senescence, VSMCs were induced by Ang II. We showed that Ang II chronic treatment increased the expression of p53 and p21 in a time-dependent manner. However, the level of proliferating cell nuclear antigen protein(PCNA, a marker of cell growth) gradually decreased under the same conditions. The expression of γ-H2 AX, a marker of DNA damage, significantly increased after treatment with Ang II for 5 days, compared with control group, supporting the notion that chronic stimulation of Ang II induces VSMC senescence. We showed that expression of SM22α was increased in parallel with VSMC senescence, speculating that SM22α may be involved in Ang II-induced VSMC senescence. 1.2 SM22α promotes Ang II-induced premature senescence in VSMCs.To determine the significance of SM22α upregulationin in VSMC senescence, the expression of SM22α in VSMCs was knocked down and upregulated, respectively. Knockdown of SM22α significantly decreased the number of SA-β-gal-positive cells and reduced expression of p53 and p21 in Ang II-treated VSMCs. Conversely, the overexpression of SM22α markedly increased SA-β-gal-positive cells and the expression of p53 and p21 under the same conditions, suggesting that SM22α exerts stimulatory effect on Ang II-induced premature senescence. 1.3 Deletion of SM22α improves Ang II-induced hypertension and vascular aging in mice.To investigate whether SM22α has the same effect on VSMC senescence in vivo, we treated Ldlr-/-/Sm22α+/+ and Ldlr-/-/Sm22α-/- mice with Ang II(1.44mg/kg per day) infusion for 4 weeks. We showed that both SBP and DBP in Sm22α-/- mice were lower than that in the Sm22α+/+ group. SA- β-gal-positive cells and the expression of p53 and p21 in the aortic media of Sm22α-/- mice were less than that in Sm22α+/+mice. These data suggest a casual relationship between high expression of SM22α and VSMC senescence. 2 SM22α promotes Ang II-induced VSMC senescence via stabilizing of p53. 2.1 SM22α inhibits ubiquitination and degradation of p53.To define the causal relationship between SM22α and p53 during VSMC senescence, we examined the effect of SM22α knockdown and overexpression on p53 expression. We found that both knockdown and overexpression of SM22α did not change the m RNA level of p53. However, the level of p53 ubiquitination was increased following knockdown of SM22α, accompanied with decreased p53 protein. Moreover, a reduced p53 ubiquitination and degradation was found in VSMCs with overexpressing SM22α. The data demonstrate that SM22α may have an inhibitory effect on p53 ubiquitination and degradation. 2.2 SM22α inhibits interaction of Mdm-2 with p53 in senescent VSMCs.Mdm-2, as E3 ubiquitin ligase, mediates p53 ubiquitination and degradation. To confirm the mechanism underlying SM22α inhibiting p53 ubiquitination, the interaction of Mdm-2 and p53 in senescent VSMCs was detected using Co-immunoprecipitation assay. The results showed that knockdown of SM22α led to an enhanced interaction of Mdm-2 with p53, which was abolished by overexpression of SM22α. Pretreatment with nutlin-3, an inhibitor of Mdm-2/p53 interaction, markedly decreased the interaction between p53 and Mdm-2 in chronic Ang II-induced VSMCs with SM22α knockdown, accompanied by decreased p53 ubiquitination and increased level of p53 expression. The results suggest that SM22α suppresses the interaction of Mdm-2 with p53, reduces p53 ubiquitination and degradation. 2.3 SM22α inhibits activation of Akt-Mdm-2 pathway.To determine the upstream signaling pathway of p53 ubiquitination and 8 degradation regulated by SM22α, we examined the effect of SM22α on the activation of Akt-Mdm-2 pathway. We showed that knockdown of SM22α increased phosphorylation levels of Akt and Mdm-2 in chronic Ang II-induced VSMCs. Conversely, overexpression of SM22α decreased it. Inhibition of Akt activity with PI3K/Akt inhibitor LY294002 abolished the phosphorylation of Mdm-2 induced by SM22α knockdown. Similarly, phosphorylation of Akt and Mdm-2 were higher in Sm22α-/- mice than that in Sm22α+/+ mice. Our findings indicate that SM22α inhibits the activation of Akt-Mdm-2 pathway, and excessive expression of SM22α improves the p53 stability. 3 Preliminary study of vascular calcification triggered by VSMC senescence 3.1 Replicative senescence of VSMCs enhances the calcification.To explore the relationship between VSMC senescence and vascular calcification, replicative senescence of VSMCs was induced by serial passage, and then VSMCs were cultured in calcification medium. SA-β-gal activity, expression of p53 and p21 were increased in P13 VSMCs. Alizarin red staining and quantitation of calcium deposition showed that calcium deposition was markedly increased in senescent VSMCs. Expression of BMP-2, OPN and activity of ALP that are calcification-related regulatory factors, were significantly upregulated in senescent VSMCs, suggesting their osteoblastic transition during replicative senescence of VSMCs. Similarly, SIPS of VSMCs can also enhance their calcification. 3.2 Downregulation of SM22α promotes the senescence-associated calcification.Based on SM22α knockdown inhibiting VSMC senescence, we determined whether SM22α is involved in senescence–associated calcification. We found that calcium deposition was reduced in senescent VSMCs with disruption of SM22α, and the expression of BMP-2 and activity of ALP were also declined. These results suggest a link between VSMC senescence and calcification.Conclusions:1 The expression of SM22α is upregulated in VSMC senescence. 2 Excessive expression of SM22α promotes VSMC senescence andvascular aging. 3 Accumulation of SM22α inhibits ubiquitination and degradation of p53via blockade of Akt-Mdm-2 activation, and improves p53 stability. 4 Senescence accelerates the osteoblastic transition in VSMCs.