Mechanism Study On Wnt/β-catenin Signaling Pathway In Vasa Vasorum Neovascularization In Atherosclerosis

Objective Vasa vasorum neovascularization is a key feature of atherosclerosis(AS).Anti-angiogenesis is a hopeful strategy in the prevention of atherosclerosis.Previously,recombinant human endostatin(endostar)were proven ant-angiogenic agents both in vivo and in vitro.And cryptotanshinone(CPT)exerts anti-atherosclerosic effect in a series of studies.However,the mechanism of two drugs remain further studied.Here we investigate the effects of Endostar on vasa vasorum neovascularization in the experimental porcine model of early AS,and explore the role of Wnt/β-catenin signaling pathway in the process of anti-angiogenic effect of Endostar and CPT in human umbilical vein endothelial cells(HUVEC)to provide theoretical basis for prevention and treatment of AS.Methods Eighteen adult male Ba-Ma mini pigs were randomly divided into three groups,with six animals in each group.The pigs in the normal(N)group were fed a normal diet for 18 weeks without balloon injury surgery.The animals in the AS control group and the AS+Endostar group were fed a hypercholesterolemic diet for 12 weeks after balloon injury surgery,and received either saline or Endostar treatment for additional six weeks,while remaining on the hypercholesterolemic diet.After 12 weeks’ hypercholesterolemic diet,atherosclerosis plaque of early stage was observed in the abdominal aortas of swine.At the end of the18th experimental week,animals were killed.The blood lipid(TC,LDL-C,HDL-C,TG)and inflammatory fectors(IL-6,hs-CRP)were measured.The abdominal aortas where injured by balloon catheter were taken for HE staining and fluorescence staining.β-catenin mRNA and VEGF mRNA were detected by qRT-PCR and protein levels by Western blot.HUVECs were treated with different concentrations Endostar(0,50ug/ml,100ug/ml,150ug/ml,200 ug/ml)or CPT(0,2.5p,mol/1,5μmol/1,10μmol/1,20μmol/1),then cytotoxicity was detected by the MTT method and Annexin V/PI staining.To gain insight into cellular signaling,the effects on TCF/LEF transcription factors were detected by the Dual-luciferase reporter assay.Next,the nuclear expression of β-catenin was evaluated using Western blot and Immunochemistry.Finally,vascular endothelial growth factor(VEGF)and cyclin D1,downstream proteins of the Wnt/β-catenin pathway were examined with Western blot.Results The AS group had significantly higher body weight and serum lipid concentration levels than the N group(p<0.05),attesting to the success of the hypercholesterolemic diet.However,no statistical differences were noted between the AS and AS+Endostar groups(p>0.05).Histopathology results revealed that foam cell accumulation,vasa vasorum density and intima-media thickness(IMT)also increased in the AS group,as compared with the N group(p<0.05).Endostar treatment significantly alleviated atherosclerosis with decreased vasa vasorum density and IMT(AS vs.AS+Endostar,p<0.05).The results from qRT-PCR and Western blot analysis indicated that expression of angiogenesis markers VEGF and p-catenin in the atherosclerotic abdominal aorta was considerably reduced by the Endostar treatment.In addition,immunohistochemistry results showed that VEGF and β-catenin were predominately localized in endothelial cells of adventitial vasa vasorum.The levels of serum inflammatory markers hs-CRP and IL-6 were significantly higher in the AS group compared with the N group(p<0.05),but showed no significant difference during the Endostar treatment,suggesting that local inhibition of angiogenesis was not accompanied by a change in serum inflammatory markers.Endostar or CPT dose-dependently suppressed endothelial cell viability without no cytotoxicity under experimental doses.In particular,Endostar or CPT blocked β-catenin-dependent transcription in HUVEC in a dose-dependent manner.In Western blot,Endostar or CPT decreased expression of β-catenin in nucleus of HUVEC(P<0.01).In Immunohistochemistry,β-catenin signals were weaker in the nucleus of the Endostar or CPT intervene group,compared to the positive control.Also,VEGF and cyclin D1 were both eliminated by both agents(P<0.05).Conclusion Our results support the role of vasa vasorum neovascularization in the development of atherosclerosis and the therapeutic potential of anti-angiogenesis intervention in atherosclerosis.Endostar and CPT are potential angiogenic inhibitors,which may impact on the Wnt/β-catenin signaling pathway via down-regulating endogenous β-catenin,TCF/LEF transcription,and pro-angiogenic factors such as VEGF and cyclin D1.