Effect Of Cd137-cd137l Signal On The Formation Of Vascular Calcification In Mice By Regulating Mmp-9 Of Macrophages

Objective To investigate whether CD137-CD137 L signal can affect the formation of vascular calcification by mediating the expression of matrix metalloproteinase 9(MMP-9)in macrophages.Methods 1.In vitro experiment: peritoneal macrophages(PM)of C57 BL / 6J mice were extracted by peritoneal lavage with thioglycolate.The primary culture of VSMC was carried out by the method of tissue block attachment.Construct PM-VSMC co-culture system for further experiments.The experiment was separated into five groups: control group,co-culture group,control+CD137L activation group,coculture+CD137L activation group and MMP-9 inhibition group.The activity of MMP-9 was detected by gelatinase.The m RNA and protein expression of OPN and Runx-2 were detected by real-time quantitative polymerase chain reaction(RT-PCR)and Western blot.The calcium concentration and alkaline phosphatase(ALP)activity were measured by microplate method.The degree of calcification was observed by Von Kossa and alizarin red staining.2.In vivo experiment: 30 Apo E-/-mice of 6-8 weeks old were separated into 5 groups: control group,macrophage activation group,control group+CD137L activation group,macrophage activation+CD137L activation group,MMP-9 inhibition group.HE staining was used to detect the size and shape of aortic plaque,and Runx-2 immunohistochemical staining was used to detect the degree of calcification.Results 1.After stimulation of PM by recombinant CD137 L protein for 0,6,12 and 24 hours,the expression of MMP-9 was the highest at 12 hours(P<0.05),and the activity of MMP-9 was the highest(P<0.01).2.The expression of calcification related proteins in VSMC of each group: RT-PCR showed that compared with the control group,co-culture group and CD137 L excited control group,the m RNA expression level of Runx-2 and OPN in CD137 L excited co-culture group was significantly higher(P<0.05).No significant difference was observed in m RNA expression of Runx-2 and OPN between the first three groups(P>0.05).Compared with CD137 L excited co-culture group,MMP-9 inhibited the m RNA expression of Runx-2 and OPN in VSMC significantly(P<0.05).Western blot showed that the expression level of Runx-2 and OPN in VSMC of CD137 L excited co-culture group was significantly higher(P<0.01).Compared to the control group,the level of Runx-2 and OPN expression in VSMC of co-culture group and CD137 L excited control group was not statistically significant(P>0.05).Compared with CD137 L excited co-culture group,the expression of Runx-2 and OPN in VSMC of MMP-9 inhibition group was significantly lower(P<0.01).3.The Ca2+ concentration and ALP activity of VSMC in each group showed that: the Ca2+ concentration and ALP activity of VSMC in CD137 L excited co-culture group were significantly higher(P<0.01).Compared with the control group,the Ca2+ concentration and ALP activity of VSMC in co-culture group and CD137 L excited co-culture group were not statistically significant(P>0.05).Compared with CD137 L excited co-culture group,the Ca2+ concentration and ALP activity of VSMC in MMP-9 inhibition group decreased significantly(P<0.05).4.The results of Von Kossa and alizarin red staining showed that the calcium deposition of VSMC in CD137 L co-culture group was obviously higher than that in MMP-9 inhibition group.5.In vivo experiments exhibited that HE staining showed that the plaque area and necrotic core area of mouse aorta increased in macrophage activation+CD137L stimulation group,while the plaque area and necrotic core area decreased in MMP-9 inhibition group.Immunohistochemical staining showed that the expression of Runx-2 calcification index increased significantly in macrophage activated+CD137L activated group,but decreased in MMP-9 inhibited group.Conclusion CD137-CD137 L signal may affect the formation of vascular smooth muscle cells and vascular calcification by inducing the expression of MMP-9 in macrophages.