CD137 Signaling Induce Mouse Atherosclerotic Calcification Via P38 Signaling

ObjectiveTo explore whether CD137-CD137L signaling regulates calcification via P38 signaling pathway in mouse smooth muscle cell.MethodsThis experiment includes zoopery and cell culture experiments:1.In cell culture experiments,mouse vascular smooth muscle cells were cultured by using method of tissue piece inoculation.The experiment was divided into three groups:control group,agonist-CD137 group（control group+recombinant CD137L protein）,anti-P38 group（agonist-CD137 group+P38 inhibitor）.The medium of all the calcification groups would be added a mixture of 10mmol/lβ-glycerophosphate+10-8mol/l dexamethasone+10^-7mol/l insulin.RT-PCR was used to observe the expression of p-P38,OPN and RUNX-2 mRNA.Western Blot was obtained to observe the expression of p-P38,OPN and RUNX-2 protein.The degeree of local calcium deposition and calcium-regulating markers,that includes Von Kossa staining,Alizarin red staining,alkaline activity,calcium ion concentration were tested to reflect the osteogenic transformation.2.In zoopery,15 ApoE^-/-mices at 8 weeks were divided into three groups randomly as follows:control group,agonist-CD137 group（control group+recombinant CD137L protein）,anti-P38 group（agonist-CD137 group+P38 inhibitors）.For pathological morphological analysis,Masson’s staining were performed.Von Kossa staining was applied to observe the calcification of the atherosclerotic plaque in each group.Results1.Western Blot showed that the expression of p-P38 protein was significantly upregulated at 24h after treated with recombinant CD137L protein in VSMC（0.26±0.03 vs 0.02±0.01,P<0.05）.Compared with the control group,the protein level of p-P38（0.51±0.01 vs 0.32±0.01,P<0.05）,OPN（0.5±0.01 vs 0.09±0.01,P<0.05）,RUNX-2（0.34±0.02 vs 0.18±0.03,P<0.05）in agonist-CD137 group was significantly increased;the effects above were blocked by adding specific P38 inhibitor SB203580（0.41±0.03 vs 0.51±0.01;0.38±0.01 vs 0.5±0.01 and 0.12±0.01 vs 0.18±0.03,P<0.05）.2.Compared with the control group,the mRNA level of OPN（1.63±0.4 vs 1,P<0.05）and RUNX-2（2.64±0.16 vs 1,P<0.05）in agonist-CD137 group was significantly increased;the effects above were blocked by adding specific P38 inhibitor SB203580（0.38±0.02 vs 1 and 0.47±0.03 vs 1,P<0.05）.3.Compared with the control group,the calcification was more serious in agonist-CD137 group,which could be significantly attenuated by cotreatment with SB203580.4.The ALP activity（2.4±0.25 vs 1.39±0.22,P<0.05）and calcium ion concentration （5.52±0.33 vs 3.15±0.32,P<0.05）was increased in agonist-CD137 group while the effects could be bloacked by adding specific P38 inhibitor SB203580（1.98±0.07 vs 1.39±0.22;3.74±0.19 vs 3.15±0.32,P<0.05）.5.In zoopery,compared with the control group,the calcification in ApoE^-/-mouse aortic plaque was more serious in agonist-CD137 group,which could be significantly reduced in anti-P38 group.Conclusions CD137-CD137L signaling may regulate the formation of atherosclerotic calcification through P38 pathway in mice.