Local Oxidative Stress Expansion through Endothelial Cells – A Key Role for Gap Junction Intercellular Communication
Figure 3
De-novo ROS generation in endothelial bystander cells and the effect of ROS scavengers and GJIC inhibition.
bEnd.3 monolayers were incubated with DCFH-DA, rinsed with fresh culture medium and subjected to COI. After illumination the plates were placed in the incubator for 3h and then imaged for PI (pseudo-colored red) and DCF (ROS, pseudo-colored green) fluorescence. A – An overlay of two pseudo-colored captures, the white arches represent distance increments of 100µm from the COI (white rectangle), representative of n = 3. PI+ bystander cells on the top left corner are at 50–100µm from the rim of the COI. Adjacent to them on the first arch (0–100µm) are DCF positive, PI negative bystander cells. B – A plot of DCF fluorescence intensity (mean ± SD of five cells in three separate experiments). LC – light control, cells exposed to laser illumination, without WST11 incubation, imaged for DCF fluorescence 3h later. C – bEnd.3 monolayers were subjected to COI under the following conditions: Control: encompassing standard COI; Vit. C Pre-COI: encompassing incubation with 100µmol/L vitamin C for 1h, rinse and then subjected to COI; Vit. C Post-COI: encompassing the addition of 100µmol/L vitamin C immediately after COI; NAC: incubation with 10mmol/L NAC prior to COI. 3h after COI, cell death was determined by PI fluorescence. Notably, in all cases COI resulted in a complete cell death within the primary illuminated square. Values represent averaged percent of bystander cell death (mean ± SD of at least n = 3 separate experiments in each group) relative to the control. The control values are statistically significant higher than all treated groups (P<0.01). D, E – bEnd.3 monolayers were incubated with GZA (D) or CBX (E), respectively, subjected to COI and probed with DCFH-DA. Dead bystander cells (red) at a distance of 60–80µm from the rim of COI, are accompanied by PI−, DCF+ (representative of n = 3 experiments) in (D) but not in (E). The insert in (E) illustrates the mean DCF fluorescence of CBX compared to GZA treated cells (right and left colum respectively, n = 10 cells) at equal distances from the dead bystander cells. *- P<0.01 between the treatments. # - P<0.05. F – bEnd.3 monolayers were scratched by a surgical scalpel (arrow), underwent COI (near the upper right corner), and placed in the incubator for 10h. Intra-cellular ROS propagation (DCF+) is blocked at the scratch. Insert – right and left columns representing mean DCF fluorescence intensities in the photoactivation side and beyond the scratch, respectively). * P<0.01.