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

Figure 3

Silencing VEGFR2 impaired CD137 signaling-induced the Akt/eNOS pathway activation, endothelial cell proliferation, migration, and tube formation. (a, b) The protein expression of p-VEGFR2(Tyr1173) after treatment with VEGF165 (20 ng/mL) at 5, 10, 15, and 20 minutes. (c) The silencing efficiency of CD137 VEGFR2-1,-2,-3 (50 nM) after treatment with VEGF165 (20 ng/mL) detected by western blot. (e) After stimulation with VEGF165 (20 ng/mL) for 5 minutes, the effects of different concentrations of XL184 (cabozantinib, an inhibitor of VEGFR2 which was used in tube formation assay) at 0, 0.03, 0.3, 1, 5, and 10 μM on p-VEGFR2(Tyr1173) were examined. (b, d, f) The protein relative expression level to β-actin. (g) Detection of p-VEGFR2(Tyr1173), p-Akt(Ser473), and p-eNOS(Ser1177) proteins after activating CD137 signaling by western blot after the CD137 activation for 10 minutes or/and inhibition of VEGFR2 with siVEGFR2. (h) The protein relative expression level of p-VEGFR2 (Tyr1173), p-Akt (Ser473), and p-eNOS (Ser1177) to total protein. (i) The tube formation of HUVECs after activation of CD137 alone or combined with inhibition of VEGFR2. The number of branch points and total length of tubes were observed under microscope and quantified by Image-Pro Plus 8.0; (l, m). (j) The EdU-555 proliferation assay was used to detect endothelial proliferative ability after activation of CD137 signaling for 3 hours(n); red and blue fluorescence represent the proliferating cells and nuclei, respectively. (k) Endothelial cells were cultured in 24-well transwell plates (pore size: 8 μm) for 12 hours after treatments, and transwell assay was applied to detect endothelial migratory ability (o); the violet color represents migrating cells stained with 0.1% crystal violet. The numbers of proliferative and migratory ECs were analyzed with Image-Pro Plus 8.0 (n, o). , , .

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