The Effect Of CD137 Signaling On Vascular Calcification In Mice

ObjectiveTo investigate the effect of CD137 on vascular calcification in vivo using apolipoprotein E–knockout(Apo E-/-) mice, as well as in vitro, vascular smooth muscle cells(VSMCs) isolated from C57BL/6J.MethodsThe present experiment was divided into two parts, one was in vivo, and another was in vitro.(1) In vivo, Apo E-/- mice were fed with high fat diet to induce atherosclerosis and calcification model. After that the mice were intraperitoneally injected with agonist-CD137 antibody or anti-CD137 antibody to activate or inhibit CD137 signaling. Pathological morphological analysis, Masson’s staining and ALP staining were performed; Von kossa staining was used to observe the calcification of the atherosclerotic plaque; Immunohistochemistry was used to observe the expression of Bone morphogenetic protein 2(BMP-2) and runt-related transcription factor 2(Runx2) which are associated with osteogenic differentiation.(2) In vitro, the mouse aortic VSMCs were obtained by Patch-attaching method and VSMCs calcification was induced by β-glycerol phosphate(β-GP). Cells were incubated with agonist-CD137 antibody or anti-CD137 antibody to stimulate or block CD137 signaling. The calcium content was measured by Methylthymol Blue complexone method; Von kossa staining was used to detect the calcification formation; the m RNA levels of BMP-2 and Runx2 were measured by real-time fluorescent quantitative PCR(RT-PCR); the expressions of BMP-2 and Runx2 in VSMCs were determined by Western blot.Result(1) H&E staining revealed that intimal thickening and plaque formation were observed in aortic vascular of all the experimental Apo E-/- mice. In agonist-CD137 group, plaque and calcification area were increased, the number of muscular fibers in vascular wall was decreased, and a large number of round or oval cells which its cytoplasm is basophilous, called osteogenic-cells, were found in plaque. Anti CD137 antibody can reverse the phenomenon.(2) Staining of ALP showed that ALP was located near the areas of calcification,and its activity was significant enhanced in agonist-CD137 group; additional anti-CD137 antibody treatment reduced ALP activity.(3) Immunohistochemical staining showed that expression of BMP-2 and Runx2 are significantly increased after treated with agonist-CD137 antibody; and this effect was partly blocked while treated with added anti-CD137 antibody.(4) Using tissue implant method, we get a number of primary aortic VSMCs of C57BL/6J mice. Third-passage cells were identified by immunofluorescence staining,and the purity of cells reached 95%.(5) VSMCs calcification was more serious in agonist-CD137 group than which in control group, and the anti-CD137 group was lighter than agonist-CD137 group.(6) In VSMCs, compared with control group, calcium content level was increased and ALP activity was enhanced in agonist-CD137 group(P<0.01, P < 0.05respectively); calcium content level was decreased and ALP activity was reduced in anti-CD137 group compared with that in agonist-CD137 group(P<0.01, P < 0.05respectively).(7) The data from RT-PCR and Western blot experiments revealed that the expressions of BMP-2 and Runx2 were all significantly up-regulated after the VSMCs incubated with agonist-CD137 antibody and on this basis down-regulated while VSMCs incubated with additional anti-CD137 antibody.Conclusion(1) Activation of CD137 can promote vascular calcification in vivo and vitro.(2) CD137 can regulate transdifferentiation of VSMCs to osteogenic-cells.