Reoxygenation of Hypoxic Human Umbilical Vein Endothelial Cells Activates the Classic Complement Pathway

Background Ischemia-reperfusion injury leads to the activation and endothelial deposition of complement. We investigated whether exposure of human umbilical vein endothelial cells (HUVECs) to hypoxia and/or reoxygenation activates complement and decreases HUVEC-surface expression of the C3 regulatory proteins CD46 and CD55. Methods and Results HUVECs were subjected to 0, 12, or 24 hours of hypoxia (O2=1%) and then reoxygenated for 3 hours (O2=21%) in the presence of 30% human serum. C3 deposition and HUVEC-surface expression of CD46 and CD55 were evaluated by ELISA and flow cytometry. C3 deposition on HUVECs subjected to 12 or 24 hours of hypoxia followed by 3 hours of reoxygenation was significantly greater than normoxic HUVECs. Inhibition of the classic but not the alternative complement pathway during reoxygenation attenuated C3 deposition. Western blot analysis of HUVEC lysates under reducing conditions demonstrated significantly increased iC3b deposition in hypoxic/reoxygenated HUVECs compared with n...