Fig. 6
TERT facilitated GDF11 to activate eNOS signaling pathways in old CD133+/VEGFR2+ cells. a–c Quantitative analysis of NO concentration (a), eNOS protein level (b), and NOS activity (c) in the old CD133+/VEGFR2+ cells transfected with vectors encoding TERT (adTERT) or TERT siRNA (siTERT) in the presence or absence of rhGDF11 under hypoxic conditions. d Representative western blots of the p-eNOS and factor VIII protein levels in old EPCs in the different groups (data represent at least three independent experiments). e, f Quantitative analysis of expression of eNOS phosphorylation (e) and factor VIII (f) determined by western blot in old CD133+/VEGFR2+ cells in the various groups. All data represent means ± s.e.m. p < 0.05: *vs. vehicle, †5 vs. rhGDF11 addition, ‡vs. adTERT addition, §vs. siTERT addition, ||vs. rhGDF11 and adTERT addition (n = 10 per group). g Factor VIII and eNOS expression in cells determined by immunofluorescence with anti-factor VIII (red) and anti-eNOS (green) antibodies, respectively. Also shown are DAPI staining (nuclei; blue) and merged images. Scale bars = 50 μm. h Representative images showing enhanced angiogenesis in the presence of adTERT addition or both adTERT and rhGDF11 addition compared with other groups. Scale bars = 50 μm. i A statistically significant increase in vessel number, length, and branches presented as percentage of vehicle was evident with the cells adding adTERT or adding adTERT and rhGDF11 compared with the vehicle, addition of rhGDF11 alone, or addition of siTERT. j Quantitative analysis of Ang-1, bFGF, and VEGF proteins levels measured by ELISA in supernatant of the old CD133+/VEGFR2+ cells transfected with adTERT or empty vector (WT) post treatments of rhGDF11 or L-NAME (an eNOS inhibitor, 500 µmol/l). Data were shown as means ± s.e.m. p < 0.05: *vs. without any treatment in WT group, †vs. rhGDF11 addition in WT group, ‡vs. L-NAME addition in WT group, §vs. untreated controls in adTERT group, ||vs. rhGDF11 addition in adTERT group (n = 10 per group)
