Figure 8
Involvement of oxidative stress in gp-120 mediated PDGF-BB induction in pulmonary endothelial cells. A) Enhanced oxidative stress in pulmonary endothelial cells on Tat and gp120 treatment. Human pulmonary microvascular endothelial cells (HPMVECs) were incubated with carboxy-H2-DCF-DA followed by Tat (25 ng/ml) or gp-120 (100 ng/ml) treatment for 60 min, and assessed for oxidative stress (Mean ± SD., **P ≤ 0.01, ***P < 0.001 vs. control). B) Effect of CCR5 neutralizing antibody on gp-120CM (100 ng/ml) mediated oxidative stress in HPMVECs. Cells were pretreated with CCR5 antibody (10 μg/ml) or equal amount of IgG isotype control for 30 min, followed by DCF assay (Mean ± SD., ***P < 0.001 treatment versus control; #P < 0.05 vs. gp120CM treatment). C) Gp-120CM mediated PDGF-BB expression in the presence of antioxidant cocktail. HPMVECs were pretreated with antioxidant cocktail for 30 min followed by incubation with gp-120CM (100 ng/ml) for 24 hours. Cells were then used for protein extraction followed by sequential immunobloting with antibodies specifically directed to the PDGF-BB and β-actin. Representative western blot images (upper panel) are shown with histograms (lower panel) showing the average densitometric analysis of the PDGF-BB band normalized to corresponding β-actin band from three independent experiments (*** P < = 0.001 versus control; ###P < = 0.001 versus gp120CM treatment).