The Role Of Histone Methylation In Vascular Restenosis And Its Mechanisms

Vascular restenosis is an abnormal neointimal formation disease complicated after surgery on narrowing blood vessels in vascular diseases,e.g.atherosclerosis.It compromises the prognosis of coronary heart disease,stroke,and other vascular narrowing diseases.Restenosis is elicited by endothelial denudation during surgery which exposes medial vascular smooth muscle cells（VSMCs）to complicated cues in blood as well as shear stress developed by bloodstream.As a result,static VSMCs,which maintain low proliferation,migration,and secretion activity under physiological condition,are switched to de-differentiated VSMCs with robust proliferation,migration,and secretion activity whereas low contractility.These alterations induce proliferation of local VSMCs,secretion of various MMPs as well as ECMs followed by VSMCs migration and vascular remodeling.To date,various drug-eluting stents are applied to treat vascular restenosis and have been the top priority of this condition due to the long-term drug release and inhibitory activity on restenosis.Although drug-eluting stent improves prognosis of restenosis remarkably,defects of this recipe still exist,such as partially murky mechanisms,side effects,stents replacement,and incomplete repressive activity.Recently,spotlight is shedding on the involvement of histone methylation in cardiovascular diseases.Overall,its involvement in vascular disease remains to be manifested.Here,we aim to unveil novel histone methylation sites and to decipher their respective mechanisms,and thus obtaining potential targets for restenosis treatment.Here in our study,we applied histone methylation-targeting HPLC/MS to profile histone methylations in human VSMCs.It manifested that VSMCs treated with PDGF-BB showed significantly increase in H3K27me3 which indicated its close association with phenotypic switch of VSMCs.Further,immunoblotting showed that H3K27me3 increased remarkably after both PDGF-BB and FBS challenge.Immunofluorescence and immunoblotting using blood vessels obtained from rats with wire injury on left common carotids showed similar phenomena with studies in vitro.Subsequently,alteration of EZH2,the enzyme introducing H3K27me3,was determined.In accordance with H3K27me3,EZH2 was elevated significantly in phenotypically switched VSMCs and injuried vessels.In contrast,its homologue EZH1 was not altered by PDGF-BB challenge.Underlying mechanism investigation showed that PDGF-BB stabilized EZH2 after CHX challenge.UNC1999,the specific inhibitor of EZH1/2 with oral bioactivity,was capable of decreasing H3K27me3induced by PDGF-BB.Cell number counting,MTS,Ki67 immunostaing,Q-PCR and immunoblotting of PCNA analysis showed that UNC1999 inhibited VSMCs proliferation.Cell cycle flow cytometry indicated that UNC1999 blocked cell cycle entry.Moreover,scratch-healing and transwell showed that UNC1999 inhibited migration of VSMCs significantly.Mechanistically,PDGF-BB promoted VSMCs proliferation through elevating the enrichment of H3K27me3 on region neighboring transcriptional start site of p16^INK4A which attenuated p16^INK4A transcription.As a result,entry of VSMCs into S phase was increased.Consistently,studies in vivo indicated that systemic and perivascular administration of UNC1999 mitigated vascular neointimal formation significantly.In sum,we discovered a novel histone methylation site H3K27me3 was implicated in vascular restenosis with histone methylation-targeting HPLC/MS.Inhibition of the methylation of this site was capable of inhibiting proliferation and migration of VSMCs.Mechanistically,phenotypic switch induced H3K27me3 enrichment on region neighboring transcriptional start site of p16^INK4A and thus decreased its expression which promoted cell cycle entry and proliferation of VSMCs.Reversing the process using EZH1/2 inhibitor UNC1999 was capable of increasing p16^INK4ANK4A expression and inhibiting VSMCs proliferation in vitro and neointimal formation in vivo.Therefore,EZH2-mediated H3K27me3 could be a potential target for clinical treatment of restenosis.