Fig 2: Upregulation of SDF1 in NPCs increases the activation of phosphatidylinositol-3-kinase/AKT signaling in VECs. (A) Primary NPCs were identified by immunofluorescence. The cells simultaneously expressed green (aggrecan) and red (type II collagen) fluorescence, demonstrated a chondrocyte-like phenotype and were identified as NPCs. (B and C) After transfection of OE-SDF1 adenovirus, the mRNA and protein expression of SDF1 in NPCs was detected by reverse transcription-quantitative PCR and western blotting. (D) Immunofluorescence of NPCs revealed visible SDF1 expression. (E) VECs were stimulated with the conditioned media from NPCs with OE-SDF1 or control, and a western blot was used to assess the protein expression levels of p-AKT, AKT and PTEN in VECs. n=3, *P<0.05; ***P<0.001. Magnification, ×200. SDF1, stromal cell-derived factor 1; NPCs, nucleus pulposus cells; OE-, overexpression; p-, phosphorylated; AKT, AKT serine/threonine kinase 1; PTEN, phosphatase and tensin homolog; NC, negative control; VECs, vascular endothelial cells.

Fig 3: Influence of AKT inhibition on the angiogenesis of VECs. VECs with MK-2206 (10 µM, 30 min) treatment or non-treated were cultured in the cultural supernatant derived from NPCs with OE-SDF1 or controls, and then the following assays were performed. (A) Western blotting was performed to detect the expression levels of p-AKT and AKT in VECs. (B) VEC viability was detected by Cell Counting Kit-8. (C) Tube formation ability in VECs was analyzed by Matrigel tube formation assay, and the total branch length was calculated by ImageJ (magnification, ×100). (D and E) Migration and invasion abilities of VECs were analyzed by Transwell assay, with SDF1 overexpressed or non-treated NPCs as a chemokine (magnification, ×100). n=3, **P<0.01, ***P<0.001. SDF1, stromal cell-derived factor 1; NPCs, nucleus pulposus cells; OE-, overexpression; p-, phosphorylated; AKT, AKT serine/threonine kinase 1; VECs, vascular endothelial cells.

Fig 4: Influence of the SDF1/C-X-C receptor 4 axis on the proliferation, migration, invasion and angiogenesis of VECs. VECs were first co-treated with AMD3100 (10 µM, 30 min) and the conditioned media from NPCs with OE-SDF1 or controls, and then the following assays were carried out. (A) Western blot analysis was performed to detect the expression levels of p-AKT and AKT in VECs. (B) VEC viability was detected by Cell Counting Kit-8 assay. (C) The tube formation ability of VECs was analyzed by Matrigel tube formation assay, and the total branch length was calculated by ImageJ (magnification, ×100). (D and E) The migration and invasion of VECs was analyzed by Transwell assay with SDF1 overexpressed or non-treated NPCs as a chemokine (magnification, ×100). n=3, *P<0.05, **P<0.01, ***P<0.001. SDF1, stromal cell-derived factor 1; NPCs, nucleus pulposus cells; OE-, overexpression; p-, phosphorylated; AKT, AKT serine/threonine kinase 1; VECs, vascular endothelial cells.

Fig 5: Influence of SDF1 on the migration, invasion and angiogenesis of VECs. (A) Expression levels of SDF1 in the cultured supernatants of NPCs were detected by ELISA following 24 h of treatment with various concentrations (0, 10, 50, 100, 200 and 500 µg/ml) of anti-SDF1 antibody. (B) Expression levels of SDF1 in the cultured supernatants of NPCs were detected by ELISA following 0, 12, 24 and 48 h treatment with 100 µg/ml of anti-SDF1 antibody. (C) Tube formation ability of VECs was analyzed by Matrigel tube formation assay, and the total branch length was calculated with ImageJ (magnification, ×100) after VECs were treated with the conditioned media from OE-SDF1 or the control group transfected-NPCs along with anti-SDF1 antibody (100 µg/ml, 24 h) or not. (D and E) Cell migration and invasion abilities were analyzed by Transwell assay with SDF1 overexpressed or non-treated NPCs as a chemokine (magnification, ×100). n=3, *P<0.05, **P<0.01, ***P<0.001. SDF1, stromal cell-derived factor 1; NPCs, nucleus pulposus cells; OE-, overexpression; VECs, vascular endothelial cells.