Fig. 4.

Increased NOX-dependent ROS levels in SSRBCs by H/R mediates adhesion to vascular endothelial cellsin vitro. A-C. SSRBCs, AARBCs, SS reticulocytes, and mature SSRBCs were exposed to normoxia, H/R, or hypoxia then incubated during reoxygenation with 50 μM apocynin (A and C), 5 μM MEKI (A), 0.5 (A) or 1 μM (A and C) MnE, or 0.5 or 1 μM MnBuOE (A). D. SSRBCs were incubated with supernatants collected from SSRBC samples exposed to normoxia, H/R, or supernatant collected from SSRBCs exposed to H/R and 1 μM MnE or 5 μM MEKI. Adhesion of RBCs to HMVECs-d was tested in intermittent flow condition assays. Results are presented as % adherent SSRBCs or AARBCs at a shear stress of 2 dynes/cm². Error bars show SEM of five different experiments. A.p < 0.0001 compared to SSRBCs exposed to H/R. C.p < 0.0001 compared to SSRBCs exposed to H/R. D.p < 0.05 compared to SSRBCs; p < 0.01 compared to cells exposed to supernatants collected from SSRBCs exposed to H/R. E. Adhesion of SSRBCs to HMVECs-d exposed to H/R. HMVECs-d were exposed to normoxia, H/R, or hypoxia then treated during reoxygenation with 50 μM apocynin, 1 μM MnE, or 5 μM MEKI. Treated HMVECs-d was tested for their ability to support adhesion of non-treated SSRBCs. Error bars show SEM of three different experiments. p < 0.0001 for SSRBC adhesion to ECs exposed to H/R vs. SSRBC adhesion to ECs exposed to normoxia; p < 0.0001 compared to SSRBC adhesion to ECs exposed to H/R.