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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. 3 — (a) Western blotting showing protein expression of VEGF-A, phospho-Akt, total Akt, phospho-eNOS, and total eNOS. GAPDH was used as loading control. HUVEC were infected with Ad.Null or Ad.p75^NTR (100, 250, and 500 M.O.I.) and then lysated for western blotting. Bands are representative of three independent experiments. (b) HUVEC were infected with Ad.Null or Ad.p75^NTR (250 M.O.I.). The release of NO was analyzed using the fluorescent probe DAF-2DA in the presence or absence of the eNOS preferential inhibitor L-NIO. FL1 fluorescence, emitted by DAF-2DA after reaction with NO, was determined by FACS. The bar graph shows the NOS-mediated FL1 mean fluorescence intensity after subtraction of eNOS-independent fluorescence emitted in presence of L-NIO. Representative histographs are shown on the right. Full curves are representative of NO release in the absence of L-NIO. Empty curves show NO release in the presence of L-NIO. Values in bar graph are means±SEM **P<0.01 vs. Ad.Null; (c) Left Panel: Protein expression of phospho-FAK, phospho-Akt, total Akt, phospo-eNOS, and total eNOS, in HUVEC infected with Ad.Null or Ad.p75^NTR (each at 250 M.O.I.) plus Ad.VEGF-A or its control Ad.Null (each at 10 M.O.I.). GAPDH is used as a loading control. Bands are representative of three independent experiments. Right Panel: Protein expression of VEGF-A, phospho-FAK, total Akt, phospho-Akt, total eNOS, and phospo-eNOS in HUVEC infected with Ad.Null or Ad.p75^NTR (each at 250 M.O.I.) plus Ad.Myr-Akt or its control Ad.Null (each at 100 M.O.I.). GAPDH is used as a loading control. Bands are representative of three independent experiments. (d) Both Ad.VEGF-A and Ad.Myr-Akt rescued tube formation capacity of p75^NTR-transduced HUVEC. Cells were infected as described in (c) at 12h before being seeded in matrigel. Images were taken at 24h from seeding. Values in the quantification graph are means±SEM.* P<0.05 vs. Ad.Null plus Ad.Null, ⁺P<0.05 vs. Ad.p75^NTR plus Ad.Null. (e) Relative mRNA expression (determined by real-time RT-PCR, using RNA 18S as reference) of BIRC5 (survivin) and PTTG1 (securin) in p75^NTR-HUVEC and Null-HUVEC. Co-infection with Myr-Akt enhanced BIRC5 and PTTG1 content of p75^NTR-HUVEC. Values are means±SEM.* P<0.05 vs. Ad.Null, ⁺P<0.05 vs. Ad.p75^NTR. (f) Relative mRNA expression of VEGF-A, ITGB1, and VEZF1 in the adductor muscles of normoglycemic mice following its transduction with Null (Null) or p75^NTR (p75^NTR) and in Null-transduced diabetic limb muscles (diabetes). Values are means±SEM.*P<0.05 and **P<0.01 vs. Ad.Null in normoglycemic muscles. (g) Relative mRNA expression of VEGF-A, ITGB1, and VEZF1 in p75^NTR-HUVEC and Null-HUVEC. Co-infection with VEGF-A rescued the mRNA level of ITGB1 but not of VEZF1 in p75^NTR-HUVEC. Values are means±SEM.** P<0.01 vs. Ad.Null, ⁺P<0.05 vs. Ad.p75^NTR. (h) Schematic representation of the molecular signalling emanating from p75^NTR in EC.

Fig. 3 — (a) Western blotting showing protein expression of VEGF-A, phospho-Akt, total Akt, phospho-eNOS, and total eNOS. GAPDH was used as loading control. HUVEC were infected with Ad.Null or Ad.p75^NTR (100, 250, and 500 M.O.I.) and then lysated for western blotting. Bands are representative of three independent experiments. (b) HUVEC were infected with Ad.Null or Ad.p75^NTR (250 M.O.I.). The release of NO was analyzed using the fluorescent probe DAF-2DA in the presence or absence of the eNOS preferential inhibitor L-NIO. FL1 fluorescence, emitted by DAF-2DA after reaction with NO, was determined by FACS. The bar graph shows the NOS-mediated FL1 mean fluorescence intensity after subtraction of eNOS-independent fluorescence emitted in presence of L-NIO. Representative histographs are shown on the right. Full curves are representative of NO release in the absence of L-NIO. Empty curves show NO release in the presence of L-NIO. Values in bar graph are means±SEM **P<0.01 vs. Ad.Null; (c) Left Panel: Protein expression of phospho-FAK, phospho-Akt, total Akt, phospo-eNOS, and total eNOS, in HUVEC infected with Ad.Null or Ad.p75^NTR (each at 250 M.O.I.) plus Ad.VEGF-A or its control Ad.Null (each at 10 M.O.I.). GAPDH is used as a loading control. Bands are representative of three independent experiments. Right Panel: Protein expression of VEGF-A, phospho-FAK, total Akt, phospho-Akt, total eNOS, and phospo-eNOS in HUVEC infected with Ad.Null or Ad.p75^NTR (each at 250 M.O.I.) plus Ad.Myr-Akt or its control Ad.Null (each at 100 M.O.I.). GAPDH is used as a loading control. Bands are representative of three independent experiments. (d) Both Ad.VEGF-A and Ad.Myr-Akt rescued tube formation capacity of p75^NTR-transduced HUVEC. Cells were infected as described in (c) at 12h before being seeded in matrigel. Images were taken at 24h from seeding. Values in the quantification graph are means±SEM.* P<0.05 vs. Ad.Null plus Ad.Null, ⁺P<0.05 vs. Ad.p75^NTR plus Ad.Null. (e) Relative mRNA expression (determined by real-time RT-PCR, using RNA 18S as reference) of BIRC5 (survivin) and PTTG1 (securin) in p75^NTR-HUVEC and Null-HUVEC. Co-infection with Myr-Akt enhanced BIRC5 and PTTG1 content of p75^NTR-HUVEC. Values are means±SEM.* P<0.05 vs. Ad.Null, ⁺P<0.05 vs. Ad.p75^NTR. (f) Relative mRNA expression of VEGF-A, ITGB1, and VEZF1 in the adductor muscles of normoglycemic mice following its transduction with Null (Null) or p75^NTR (p75^NTR) and in Null-transduced diabetic limb muscles (diabetes). Values are means±SEM.*P<0.05 and **P<0.01 vs. Ad.Null in normoglycemic muscles. (g) Relative mRNA expression of VEGF-A, ITGB1, and VEZF1 in p75^NTR-HUVEC and Null-HUVEC. Co-infection with VEGF-A rescued the mRNA level of ITGB1 but not of VEZF1 in p75^NTR-HUVEC. Values are means±SEM.** P<0.01 vs. Ad.Null, ⁺P<0.05 vs. Ad.p75^NTR. (h) Schematic representation of the molecular signalling emanating from p75^NTR in EC.