Fig. 10

Depletion of GDF11 or TERT impaired angiogenesis and vascular function of young CD133⁺/VEGFR2⁺ cells in vivo. a mRNA expression of Ang-1, bFGF, and VEGF in old MI hearts treated with PBS, old CD133⁺/VEGFR2⁺ cells, young CD133⁺/VEGFR2⁺ cells in the presence/absence of siTERT/siGDF11 evaluating by real-time RT-PCR. b Western blot showed that young CD133⁺/VEGFR2⁺ cells therapy upregulated protein expression of TERT, GDF11, Ang-1, bFGF, and VEGF compared with PBS injection or old CD133⁺/VEGFR2⁺ cells therapy, whereas siGDF11 significantly reduced their expression, and siTERT further abolished theses expression. c Reduction of siTERT or siGDF11 in angiogenesis induced by yEPC therapy in old MI hearts was determined by the number of factor VIII positive-staining vessels per mm² under high-power field view by immunohistochemistry. d, e FACS assessment of the percentages of EGFP-positive cells (EGFP⁺) relative to the whole ventricular cell population (d) and factor VIII (angiogenesis marker), and EGFP double-positive cells (factor VIII⁺EGFP⁺) relative to the whole EGFP⁺ population (e). All graphical data are the mean ± sEM. ^‡p < 0.05 vs. PBS, ^§p < 0.05 vs. oEPC, ^||p < 0.05 vs. yEPC, ^# p < 0.05 vs. yEPC + siTERT, Student’s t-test (n = 5 per group). f, g Representative images of Ang-1 (brown, f) or factor VIII (brown, g) under lower magnification (×10, upper) and higher magnification (×40, lower) by staining cardiomyocytes and vessels in the infarct and peri-infarct area from old MI hearts treated with old CD133⁺/VEGFR2⁺ cells, young CD133⁺/VEGFR2⁺ cells in the presence/absence of siTERT/siGDF11. Staining with anti-β-actin antibody or without primary antibody in the MI hearts treated with PBS served as a positive control and negative control (×40). Arrows indicated the origination of the higher magnification images from the region of interesting in the lower magnification images. h, i Representative phenotypes of gated EGFP⁺ (h) and factor VIII⁺EGFP⁺ (i) cells evaluated by FACS in transplanted CD133⁺/VEGFR2⁺ cells. Although the survival and angiogenesis degree of young CD133⁺/VEGFR2⁺ cells were significantly greater than those of old cells, significantly decreased young cell survival and angiogenesis was seen in young cells pre-treated with siTERT or siGDF11. j Immunofluorescence staining showing that transplanted cells expressed factor VIII. The transplanted cells were prelabeled with EGFP (green); the nuclei were stained with DAPI (blue), and the EC cytoplasm was stained with anti-factor VIII antibody (red). Engrafted EGFP-prelabeled cells expressing factor VIII were the most numerous in yEPC MI hearts, and were lowest in siTERT-transfected young CD133⁺/VEGFR2⁺ cells (arrows). Scale bars = 50 µm