[CD137-CD137L signaling promotes angiogenesis in atherosclerosis plaque of mice through activating nuclear factor of activated T cells c1].

ABSTRACT

Objective: To explore whether CD137-CD137L signaling can promote angiogenesis in atherosclerosis plaque via activating nuclear factor of activated T cells c1 (NFATc1). Methods: Apolipoprotein E knock out mice were divided into the following groups control group (n=5), CD137 activated group(n=5)and CD137 inhibited group (n=5). Immunohistochemistry was performed to detect the expression of CD31 in aortic plaque. Endothelial cells (bEnd.3) were purchased from ATCC and divided into the following groups control group, IgG isotype control group, CD137 activated group and CD137 inhibited group. Western blot was used to determine total protein and nucleoprotein expression of NFATc1. The expression level of CD137 protein on the surface of endothelial cells was detected by flow cytometry(FCM) and CD137 protein of lysate of endothelial cells was detected by enzyme-linked immunosorbent assay (ELISA). Transwell assay was used to observe the migration ability of endothelial cells.Matrigel tube formation ability of endothelial cells were tested in the following groups control group, CD137 activated group, silent NFATc1 + CD137 activated group, CD137 inhibited group, and over expressed NFATc1+ CD137 inhibited group. Results: (1) In vivo, the expression level of CD31 was significantly higher in the aortic plaque of CD137 activated group than in control group(1 191±187 vs. 115±30, P<0.05), while which was significantly downregulated in CD137 inhibited group(450±92, P<0.05). (2) The level of nucleoprotein(3.07±0.03 vs. 1.00±0.00, P<0.05) and total protein(2.18±0.30 vs. 1.00±0.00, P<0.05) of NFATc1 were significantly higher in CD137 activated group than in IgG isotype control group. The level of nucleoprotein(0.82±0.04) and total protein(0.84 ± 0.09) of NFATc1 were significantly lower in CD137 inhibited group than in CD137 activated group(both P<0.05). (3) FCM results showed that the fluorescence intensity of CD137 on the cell membrane was significantly higher in endothelial cells stimulated by TNF-α than in normal endothelial cells(5 163±329 vs. 1 660±162, P<0.05). (4) ELISA examination showed that the level of CD137 protein was significantly higher in endothelial cells stimulated by TNF-α than in normal endothelial cells ((573.4±23.7)pg/mg vs.(69.5±16.7)pg/mg, P<0.05). (5) Migration cell number was remarkably higher in CD137 activated group than in IgG isotype control group(1.19±0.13 vs. 1.00±0.00, P<0.05) and significantly lower in CD137 inhibited group(0.82±0.06)than in control group (P<0.05). (6) Values of the formation of the tube length ((5.76±0.18)mm vs. (4.21±0.11)mm, P<0.05) and branch number (29.38±1.28 vs. 21.13±0.96, P<0.05) were both significantly higher in CD137 activated group than in the control group. The formation of the tube length ((1.90±0.11)mm) and branch number(8.91±0.72)were significantly lower in silent NFATc1 + CD137 activated group than in the CD137 activated group (both P<0.05). The formation of the tube length((1.28±0.34)mm) and branch number(5.07±0.35)were also significantly decreased in the CD137 inhibited group compared with the CD137 activated group (both P<0.05). Compared with the CD137 inhibited group, the formation of the tube length((4.82±0.09)mm) and branch number(24.44±1.05) in the over expressed NFATc1+ CD137 inhibited group was increased (both P<0.05). Conclusion: CD137 can promote the angiogenesis in atherosclerosis plaque by activating NFATc1.