IL-1?-induced Senescence Promotes Osteoblastic Transition Of Vascular Smooth Muscle Cells

Objective IL-1?,as a key biomarker and mediator of vascular calcification in the patients with end-stage renal disease(ESRD),may be involved in the process of premature senescence of vascular smooth muscle cells(VSMCs).We tried to investigate whether IL-1?-induced premature senescence contributes to the process of osteoblastic transition and vascular calcification in VSMCs in this study.Methods 1.Histological analysis Both an aging-related protein(P21)and an osteoblastic marker(bone morphogenetic protein-2,BMP2)was detected by immunohistochemistry(IHC)to compare its expression in vascular tissue between the health and the patients with ESRD.Vascular calcification was detected with alizarin red staining.Clinical data and image data were collected from hospital to investigate vascular smooth cell senescence,osteoblastic transition and vascular calcification.2.Cytological studies Obtaining VSMCs as object of study by primary culture,then treating VCMCs in different ways and times to survey the change of cellular morphology.To find the relationship between cellular aging and calcification by using SA-?-Gal staining analysis,calcification staining and quantification of calcium deposition.3.Molecular mechanism analysis(1)To investigate IL-1?-induced premature senescence of VSMCs via activation of NF-?B/P53/P21 pathway,western blot(WB)was performed to detect expression of P65, P53,p21 and on other molecules.(2)To study whether IL-1?-induced premature senescence contributes to osteoblastic transition in VSMCs.WB was used to detect the VSMCs marker of alpha smooth muscle-actin(?-SMA)and osteoblastic markers of bone morphogenetic protein-2(BMP2)and Runt-related transcription factor-2(RUNX2)expression.Results 1.There are 11 cases of patients(73.3%)in 15 patients which exhibited mineral deposition in both BMP2 and p21 positive VSMCs of the distal radial arteries.It means that senescence was consistent with osteoblastic transition and calcification of VSMCs in the patients with ESRD.2.IL-1? could induce premature senescence of VSMCs.(1)Comparing with control group,human VSMCs exhibited flat and enlarged morphology when exposed to phosphate and IL-1? which stated VSMCs were aging.(2)Comparing with the control group,the number of human VSMCs exposed to phosphate and IL-1? were increased with the passage of time by using SA-?-Gal staining analysis.(3)The expression of aging-related protein P21 was increased when human VSMCs were treated with phosphate and IL-1? comparing with control group.3.Treating with IL-1?,p-P65,Ac-P65 and Ac-P53 were activated and the expression of P53 and P21 were increased,which stated phosphate and IL-1?-induced premature senescence through NF-?B/P53/P21 pathway.4.Calcification was dramatically enhanced in VSMCs by exposure to IL-1?.However,resveratrol treatment effectively repressed hyperphosphate-induced and IL-1?-induced calcification in VSMCs.Then calcium content in VSMCs was determined with the calcium assay kit.Compared with 3-day vehicle cells,VSMCs showed a higher level of intracellular calcium induced by both phosphate and IL-1?.5.The increased expression of BMP2 and RUNX2 was markedly observed in VSMCs treated by IL-1?,coincident with an increased p21 expression.But significant alterations of RUNX2 and BMP2 expression were not observed in VSMCs by exposure to both IL-1? and resveratrol compared with those of the vehicle cells.Taken together,IL-1?-induced calcification was required for senescence-associated osteoblastic transition in VSMCs,and the senescence-associated osteoblastic transition was markedly inhibited by resveratrol.Conclusion 1.IL-1? may be useful as a biomarker and predictor of calcification risk in the patients with ESRD.2.The higher levels of plasma IL-1? induces senescent VSMCs via the activation of NF-?B/p53/p21 pathway and contributes to the vascular calcification owing osteoblastic transition in the senescent VSMCs.3.Resveratrol would be helpful to prevent the senescence-associated medial calcification owing to the osteoblastic transition of VSMCs during the senescence.