Activating CD137 Signaling Promotes Sprouting Angiogenesis via Increased VEGFA Secretion and the VEGFR2/Akt/eNOS Pathway

Figure 6

Inhibition of the eNOS activity weakened CD137 signaling-mediated angiogenesis in vitro and ex vivo. (a) The p-eNOS protein level was detected by western blot after treatment with different concentrations of L-NAME (0, 25, 50, 100, 200, and 300 μM) for 24 hours. (b) The p-eNOS protein relative level was standardized by β-actin. (c, e) Treatment with L-NAME before activating CD137 signaling for 3 hours, then the EdU-555 proliferation assay and transwell assay were performed. (d, f) Proliferation and migration cells were detected and counted by red nuclei and the violet cell, respectively. (g) The tube formation of HUVECs was observed after treatment with L-NAME(100 μM) for 30 min under the CD137 signaling activation. (h, i) Total length of tubes and number of branch points, . (j) Aortic rings sprouting after pretreatment with L-NAME(100 μM) for 24 hours before activation of CD137. (k) Number of sprouts per ring(black arrows) was quantified by Image-Pro Plus 8.0, , .

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