Figure 1.
Representive Western blot (A, D) and densitometric quantification of eNOS uncoupling (B, E) and phospho-p66Shc protein (C, F) expression in HAEC treated with oxLDL within sixty minutes (A, B, C) or for twenty four hours (D, E, F).
eNOS uncoupling was presented as ratio of dimer/monomer form of eNOS. The phosphorylation of p66Shc was normalized to total p66Shc protein and total p66Shc was normalized to α-tubulin. Results are presented as means±SEM; n = 6. * p<0.05 vs. cells at 0 minutes or cells under control condition.
Figure 2.
Representive Western blot (A, C) and densitometric quantification of phospho-p66Shc protein expression (B, D) in HAEC after twenty-four hours of incubation with oxLDL in the presence of nitric oxide synthase inhibitor (L-NAME 0.3 mM; A and B) and in the presence of eNOS siRNA (10 nM, C and D).
The phosphorylation of p66Shc was normalized to total p66Shc protein and total p66Shc was normalized to α-tubulin. Results are presented as means±SEM; n = 8. * p<0.05 vs. cells under control conditions. # p<0.05 vs. oxLDL alone. (NFS: nanoparticle formation solution).
Figure 3.
Representative Western blot (A) and densitometric quantification of phospho-p66Shc protein (B) expression in HAEC after twenty-four hours of incubation with oxLDL in the presence of nitric oxide donor (DetaNO 0.1-1 mM or DeaNO 0.1–1 mM).
The phosphorylation of p66Shc was normalized to total p66Shc protein and total p66Shc was normalized to α-tubulin. Results are presented as means±SEM; n = 5. * p<0.05 vs. cells under control conditions. # p<0.05 vs. oxLDL alone.
Figure 4.
Representive Western blot (A) and densitometric quantification of eNOS uncoupling (B) and phospho-p66Shc protein (C) expression in HAEC after twenty-four hours incubation with oxLDL in the presence of tetrahydrobiopterin (BH4 10 µM), apocynin (100 µM), bradykinin (1 µM) or calcium ionophore (A23187 1 µM).
eNOS uncoupling was presented as ratio of dimer/monomer form of eNOS. The phosphorylation of p66Shc was normalized to total p66Shc protein and total p66Shc was normalized to α-tubulin. Results are presented as means±SEM; n = 8. * p<0.05 vs. cells under control conditions. # p<0.05 vs. oxLDL alone.
Figure 5.
Representive Western blot (A) and densitometric quantification of eNOS uncoupling (B) and phospho-p66Shc protein (C) in HAEC after twenty-four hours incubation with oxLDL in the presence of tetrahydrobiopterin [BH4 10 µM; before (B), forty five minutes after (After Early, AE), or sixteen hours after (After Later, AL) oxLDL treatment].
eNOS uncoupling was presented as ratio of dimer/monomer form of eNOS. The phosphorylation of p66Shc was normalized to total p66Shc protein and total p66Shc was normalized to α-tubulin. Results are presented as means±SEM; n = 8. * p<0.05 vs. cells treated with oxLDL alone.
Figure 6.
O2- production after twenty-four hours of incubation with oxLDL in the presence or absence of tetrahydrobiopterin [BH4 10 µM, before (B), forty five minutes after (AE), and sixteen hours after (AL) oxLDL treatment], apocynin (100 µM), bradykinin (1 µM), calcium ionophore (1 µM), L-NAME (0.3 mM) and cGMP (1 mM).
Results are presented as means±SEM; n = 8. * p<0.05 vs. cells under control conditions. # p<0.05 vs. oxLDL alone.
Figure 7.
Putative role of eNOS in oxLDL-induced, p66Shc- mediated oxidative stress in HAEC.
eNOS uncoupling is the primary source of oxLDL-induced oxidative stress in endothelial cells, leading to the p66Shc activation and later surge of ROS production. Supply with nitric oxide or reversal eNOS uncoupling reduces p66Shc activation and ROS production.