Endothelial cell dysfunction during Anoxia-Reoxygenation is associated with a decrease in ATP levels, rearrangement in lipid bilayer phosphatidylserine asymmetry, and an increase in endothelial cell permeability.

Phosphatidylserine (PS) is normally confined in an energy-dependent manner to the inner leaflet of the lipid cell membrane. During cellular stress PS is exteriorized to the outer layer, initiating a cascade of events. Because cellular stress is often accompanied by decreased energy levels and because maintaining PS asymmetry is an energy-dependent process, it would make sense that cellular stress associated with decreased energy levels is also associated with PS exteriorization that ultimately leads to endothelial cell dysfunction. Our hypothesis was that anoxia-reoxygenation (A-R) is associated with decreased ATP levels, increased PS exteriorization on endothelial cell membranes, and increased endothelial cell membrane permeability.The effect on ATP levels during anoxia-reoxygenation was measured via colorimetric assay in cultured cells. To measure the effect of A-R on PS levels, cultured cells underwent A-R and exteriorized PS levels and also total cell PS were measured via biofluorescence assay. Finally, we measured endothelial cell monolayer permeability to albumin after A-R.ATP levels in cell culture decreased 27% from baseline after A-R (p<0.02). There was over a 2-fold increase in exteriorized PS as compared to controls (p<0.01). Interestingly, we found that during A-R, the total amount of cellular PS increased (p<0.01). The finding that total PS changed 2-fold over normal cells suggested that not only is there a change in the distribution of PS across the cell membrane, but there may also be an increase in the amount of PS inside the cell. Finally, A-R increased endothelial cell monolayer permeability (p<0.01).We found that endothelial cell dysfunction during A-R is associated with decreased ATP levels, increased PS exteriorization, and increased in monolayer permeability. This supports the idea that phosphatidylserine exteriorization may a key event during clinical scenarios involving oxygen lack and may one day lead to novel therapies in these situations.Basic Science Paper.