Antioxidant cocktail attenuates Tat and cocaine-mediated augmentation of endothelial dysfunction.

<p>Effect of antioxidants (A) and catalase or SOD (B) on cocaine (1 µM) and Tat (25 ng/ml) mediated barrier dysfunction of HPMECs. Confluent monolayers were grown on collagen-coated Transwell inserts and treated with antioxidant cocktail followed by Tat and cocaine exposure for 6 or 24 hours. Monolayers were then treated with FITC-dextran and after 15 min the fluorescence in the lower compartment was measured and expressed as percentage of basal fluorescence. (C) Effect of antioxidants on Tat and/or cocaine mediated down-regulation of tight junction protein expression in pulmonary arterial endothelial cells. Cells grown on coverslip were immunostained for TJP-1. (D) Quantification of ZO-1 immunofluorescence using ImageJ software. (E) Western blot analysis of ZO-1 in various cellular compartments. Blot is representative of at least three independent experiments with histogram showing the average densitometry analysis normalized to β-integrin for membrane fraction, β-actin for cytosolic fraction and PCNA for nuclear compartment. The values shown are means (±S.E.M.). *P≤0.001 compared to control; ^@P≤0.001, compared to cocaine and Tat treatment.</p

Blocking Tat and cocaine-mediated ERK activation reverses ZO-1 disruption in HPMECs.

<p>Phosphorylated ERK was detected by western blot analysis of HPMECs treated with (A) Tat and cocaine for different time intervals as indicated and (B) with cocaine and/or Tat for 1.5 hours. (C) ZO-1 expression analysis in HPMECs pre-treated with U0126 for 30 min followed by Tat and cocaine treatment for 24 hours. Membrane fraction was isolated using compartment protein fractionation kit and ZO-1 was detected by western blot analysis. Lower panels show the average densitometry analysis normalized to β-actin for total cellular extract (A and B) and β-integrin in case of membrane fraction (C) of at least three independent experiments. Mean (±S.E.M.). *P≤0.05, **P≤0.01, ***P≤0.001 compared to control; ^#P≤0.001 compared to cocaine treatment. ^$p<0.001 compared to Tat treatment, ^@P≤0.01, compared to combined cocaine and Tat treatment.</p

Enhanced oxidative stress on treatment of pulmonary endothelial cells with Tat and cocaine.

<p>(A) Generation of ROS in HPMECs treated with Tat and/or cocaine was quantified by DCF assay at indicated time-points. (B) ROS production in cells treated with Tat and cocaine in the presence or absence of SU5416 (antagonist of VEGFR-2) or BD1047 (antagonist of sigma receptor) for 1 hour as analyzed by DCF assay. (C) Generation of H₂O₂ was quantified using Amplex red assay kit. HPMECs were treated with Tat and cocaine in the presence or absence of catalase (10U/ml) or SOD (100U/ml) for 1 hour. (D) Reduction of H₂O₂ formation on pre-treatment of Tat and cocaine exposed HPMECs with SU5416 or BD1047. (E) Changes in Tat and cocaine-mediated superoxide generation on SU5416 or BD1047 pre-treatment. Formation of superoxide (O₂⁻) was quantified by SOD-inducible cytochrome c reductase assay. The values shown are means (±SD) of at least three independent experiments. *P≤0.05, **P≤0.01, ***P≤0.001 compared to control; ^#P≤0.05, ^##P≤0.01,^###P≤0.001 compared to cocaine treatment; ^$P≤0.001 compared to Tat treatment;^@ P≤0.05, ^@@ P≤0.001, compared to Tat and cocaine combinational treatment; ^&P≤0.01, ^&&P≤0.001 compared to catalase-treated control; ^%‘P≤0.001, compared to SOD-treated control.</p

Activation of Ras/Raf/Erk pathway in Tat and cocaine exposed HPMECs.

<p>(A) Ras activation was assessed by pull-down assay in cells treated with Tat and cocaine for 30 or 60 min. (B) HPMECs were pre-treated with antioxidant cocktail, SU5416 or BD1047 for 5 min followed by Tat and cocaine treatment for 30 min. Representative western blot images are shown with histogram showing the average densitometry analysis of at least three independent experiments. Mean (±S.E.M.), *P≤0.001, compared to control; ^@P≤0.001, compared to cocaine and Tat treatment.</p

Attenuation of Tat and cocaine mediated endothelial dysfunction in the presence of VEGFR-2 or sigma receptor antagonists.

<p>(A) Expression of sigma and dopamine receptors in HPMECs as analyzed by Western blot of total cellular extract. (B) HPMECs were treated with Tat (25 ng/ml) and cocaine (1 µM) for 6 or 24 hours in the presence or absence of SU5416 (antagonist of VEGFR-2) or BD1047 (antagonist of sigma receptor). FITC-Dextran permeability was assessed by using <i>in-vitro</i> vascular permeability assay kit. The values shown are means (±SD) of at least three independent experiments. (C) Membrane fraction was isolated and analyzed for ZO-1 by western blot analysis. Blot is representative of at least three independent experiments with histogram showing (lower panel) the average densitometry analysis normalized to β-integrin (mean ± S.E.M). (D) Representative images showing immunocyto-fluorescence staining of ZO-1. (E) Quantification of ZO-1 immunofluorescence using ImageJ software. The values are represented as fold change compared to untreated control. *P≤0.01, **P≤0.001 compared to untreated control; ^@P≤0.05, ^@@P≤0.01, ^@@@P≤0.001 compared to Tat and cocaine treatment.</p