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. Author manuscript; available in PMC: 2009 Feb 12.

Published in final edited form as: Circ Res. 2008 Jun 19;103(2):e15–e26. doi: 10.1161/CIRCRESAHA.108.177386

Fig. 1 — (a) HUVEC were infected with 100, 250 and 500 M.O.I. of Ad.Null or Ad.p75^NTR, as indicated. After 48h, cell lysates were collected and subjected to western blotting with antibodies to p75^NTR, cleaved caspase-3, and GAPDH (used as loading control). (b) FACS analyses for p75^NTR show abundant receptor expression (95.8±3%) at 12h from gene transfer with Ad.p75^NTR (250 M.O.I.), while at 12h from Null gene transfer, p75^NTR-expressing HUVEC are only 6.7±0.7%. (c) p75^NTR-expressing HUVEC were gated and studied at 12h, 24h, and 48h for co-expression with Annexin-V and propidium iodide (PI) to detect apoptosis. This analysis revealed that at 12h from Ad.p75^NTR, less than 9% of p75^NTR-carrying cells presented Annexin-V on the external plasma membrane. Early apoptosis (cells positive for Annexin-V and negative for PI, lower right squares) peaked at 24h (39.1±3% of p75^NTR-expressing HUVEC), followed by late apoptosis (cells positive for both Annexin-V and PI, upper right squares) at 48h from Ad.p75^NTR (17.05±2% of p75^NTR-expressing HUVEC). (d) HUVEC were treated as described in (a) and apoptotic nuclei were detected by TUNEL assay. Fluorescent images are representative of apoptosis rate in Null-HUVEC and p75^NTR-HUVEC. Bar graphs quantify apoptosis, which is expressed as percentage of TUNEL-positive nuclei (green fluorescence) to total nuclei (stained in blue fluorescence by DAPI). Data are presented as means±SEM. *P<0.05 and **P<0.001 vs. Ad.Null. (e,f) Caspase-3 activity assay was performed on HUVEC (e) or MVEC (f). Cells (5000 cells/well) were infected with 100, 250 or 500 M.O.I. of Ad.Null or Ad.p75^NTR or left uninfected (PBS). After 48h, Caspase-Glo 3/7 was incubated for 1h before recording luminescence. The apoptosis inducer staurosporin (stauro, 1μM) was used as reference in (e). Values are means±SEM. *P<0.05 and **P<0.001 vs. Ad.Null. (g) Upper panel: Concentration of endothelial apoptotic microparticles (EMP) released by Null-HUVEC or p75^NTR-HUVEC under no other stimulation or following incubation with the apoptosis inducer staurosporin (100 nM, 16h). Values are means±SEM. **P<0.01 vs. Ad.Null. Lower panel: p75^NTR is present in EMP released by HUVEC. The shadow peak corresponds to fluorescence background obtained with the isotypic control of p75^NTR antibody. The second peak represents the specific labelling of EMPs with fluorescent p75^NTR antibody. (h) Caspase-3 activity assay performed on HUVEC infected with Ad.Null or Ad. p75^NTR (each at 100 and 250 M.O.I.) before being treated with PBS, proNGF (5ng/mL), NGF (100ng/mL), or BDNF (100ng/mL) for 24h. Values are means±SEM. *P<0.01 vs. PBS.

Fig. 1 — (a) HUVEC were infected with 100, 250 and 500 M.O.I. of Ad.Null or Ad.p75^NTR, as indicated. After 48h, cell lysates were collected and subjected to western blotting with antibodies to p75^NTR, cleaved caspase-3, and GAPDH (used as loading control). (b) FACS analyses for p75^NTR show abundant receptor expression (95.8±3%) at 12h from gene transfer with Ad.p75^NTR (250 M.O.I.), while at 12h from Null gene transfer, p75^NTR-expressing HUVEC are only 6.7±0.7%. (c) p75^NTR-expressing HUVEC were gated and studied at 12h, 24h, and 48h for co-expression with Annexin-V and propidium iodide (PI) to detect apoptosis. This analysis revealed that at 12h from Ad.p75^NTR, less than 9% of p75^NTR-carrying cells presented Annexin-V on the external plasma membrane. Early apoptosis (cells positive for Annexin-V and negative for PI, lower right squares) peaked at 24h (39.1±3% of p75^NTR-expressing HUVEC), followed by late apoptosis (cells positive for both Annexin-V and PI, upper right squares) at 48h from Ad.p75^NTR (17.05±2% of p75^NTR-expressing HUVEC). (d) HUVEC were treated as described in (a) and apoptotic nuclei were detected by TUNEL assay. Fluorescent images are representative of apoptosis rate in Null-HUVEC and p75^NTR-HUVEC. Bar graphs quantify apoptosis, which is expressed as percentage of TUNEL-positive nuclei (green fluorescence) to total nuclei (stained in blue fluorescence by DAPI). Data are presented as means±SEM. *P<0.05 and **P<0.001 vs. Ad.Null. (e,f) Caspase-3 activity assay was performed on HUVEC (e) or MVEC (f). Cells (5000 cells/well) were infected with 100, 250 or 500 M.O.I. of Ad.Null or Ad.p75^NTR or left uninfected (PBS). After 48h, Caspase-Glo 3/7 was incubated for 1h before recording luminescence. The apoptosis inducer staurosporin (stauro, 1μM) was used as reference in (e). Values are means±SEM. *P<0.05 and **P<0.001 vs. Ad.Null. (g) Upper panel: Concentration of endothelial apoptotic microparticles (EMP) released by Null-HUVEC or p75^NTR-HUVEC under no other stimulation or following incubation with the apoptosis inducer staurosporin (100 nM, 16h). Values are means±SEM. **P<0.01 vs. Ad.Null. Lower panel: p75^NTR is present in EMP released by HUVEC. The shadow peak corresponds to fluorescence background obtained with the isotypic control of p75^NTR antibody. The second peak represents the specific labelling of EMPs with fluorescent p75^NTR antibody. (h) Caspase-3 activity assay performed on HUVEC infected with Ad.Null or Ad. p75^NTR (each at 100 and 250 M.O.I.) before being treated with PBS, proNGF (5ng/mL), NGF (100ng/mL), or BDNF (100ng/mL) for 24h. Values are means±SEM. *P<0.01 vs. PBS.