Secreted Protein Acidic And Rich In Cysteine Modulates Molecular Arterial Homeostasis Of Human Arterial Smooth Muscle Cells In Vitro

Objective:Intracranial aneurysm(IA)is a serious life-threatening disease and the primary cause of spontaneous subarachnoid hemorrhage(SAH),with a high mortality and disability rate.The exact pathogenesis of IAs remains unclear and it also lacks specific biological markers to screen the formation of IAs or assess the risk of its rupture.Our research group proposed the concept of "Arterial Homeostasis",which represent a dynamic equilibrium between damage and repair of arterial wall.To counteract the damage caused by blood flow stress,arterial wall itself is capable of repairing this damage.The arterial homeostasis plays a vital role in maintain the stability of the arterial wall,while imbalance of homeostasis may lead to the formation of IA.Apart from the hemodynamic factors,several congenital factors are also associated with the formation and progression of IA.Secreted protein acidic and rich in cysteine(SPARC)has been proved significantly overexpression in the vascular smooth muscle cells(VSMCs)of IAs,but its effect on VSMCs is still unknown.This paper is aimed at probing the underlying mechanism of SPARC on VSMCs during the formation and progression of lAs.Methods:Human umbilical arterial smooth muscle cells(HUASMCs)were used as a model of tunica media of the artery,which mainly consisted of VSMCs.After exposure to a gradient concentrations(0,0.125,0.25,0.5,1,2,and 4ug/ml)of SPARC in vitro for 24 h,cell counting kit-8(CCK-8)assay was used to detect the cell viability.With the exposure of(0,2ug/ml)SPARC for(0,2,6,12,24,48 h),flow cytometry were used to investigate cell cycle distribution,cell apoptosis ratio.Meanwhile,western bolt was used to measure the expression of P21,P53,CyclinD1,Bax,Bcl2,Caspase-3,Cleaved Caspase-3,MMP2,MMP9,TIMP1,TIMP2.Results:After exposure to 2 and 4 ?g/ml SPARC,cell viability of HUASMCs were 89.3 ± 2.00%and 87.57 ± 2.17%(P<0.05 vs.control),respectively.Induced by 2?g/ml SPARC,the proportion of cells in G0/G1 phase was 74.77 ± 1.33%(P<0.05 vs.control),and the early and late apoptosis ratio were 7.38 ± 1.25%and 4.86 ± 0.81%(P<0.01 vs.control),respectively.After exposure to 2 ?g/ml SPARC for 2,6,12,24,and 48 h,Western blot analysis showed that the protein level of p21 was upregulated significantly at 2-12 h(P<0.05 vs.control),while the expression of p53 remained stable within 48 h.The expression of Bax protein increased markedly and peaked at 24(P<0.01 vs.control),while Bcl2 protein decreased significantly at 48 h(P<0.01 vs.control).Cleaved caspase3 was also upregulated dramatically and peaked at 24 h(P<0.05 vs.control).The protein level of MMP2 increased significantly and peaked at 24 h(P<0.01 vs.control),while TIMP2 remained stable.Taken together,SPARC could arrest HUASMCs in G0/G1 phase by overexpression of p21 and induce mitochondria-mediated apoptosis in vitro,which could result in the decreased cell viability.Besides,SPARC might also lead to the activation of MMP2 instead of MMP9.Conclusion:These results indicated SPARC could reduce the self-repair capability and increase injury of media layer and internal elastic lamina of intracranial artery,which would disrupt the normal homeostatic mechanism controlling vascular repair,thus promoting the formation and progression of IA.