Figure 4.

Oxidized hemoglobin variants cause rapid disruption of pulmonary endothelial monolayer and generate oxidative stress. (A) HPAEC monolayer were grown on Transwell inserts and then exposed to either ferric or ferryl forms of HbA, HbS, or HbE at equimolar concentration (100 μM) for 6 h in the presence of FITC conjugated dextran, 40 KD. Endothelial monolayer integrity was assessed by measuring the passage of FITC-dextran as mentioned in the methods section. Values are presented as average fold difference over control (N = 3). Differences between groups were compared using Mann-Whitney U-test. ^*P < 0.05 and ^#P < 0.001 vs. respective controls. (B) HPAEC were exposed to ferryl Hb proteins at equimolar concentration (100 μM) for 6 h. Following incubation cells were washed thoroughly and probed with spin trapping agent DMPO. DMPO-nitrone adducts formed with oxidatively modified proteins within HPAEC were visualized by immunofluorescence method using anti-DMPO-nitrone antibody. Images shown are from one representative set of experiments repeated three times.