(Circulation. 1997;96:326-333.)
© 1997 American Heart Association, Inc.
Articles |
Reoxygenation of Hypoxic Human Umbilical Vein Endothelial Cells Activates the Classic Complement Pathway
From the Center for Experimental Therapeutics and Reperfusion Injury, Department of Anesthesia, Brigham and Women's Hospital, Harvard Medical School, Boston, Mass.
Correspondence to Gregory L. Stahl, PhD, Center for Experimental Therapeutics and Reperfusion Injury, Department of Anesthesia, Brigham and Women's Hospital, Harvard Medical School, 75 Francis St, Boston, MA 02115. E-mail gstahl{at}zeus.bwh.harvard.edu
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 normoxic HUVECs. FACS analysis confirmed iC3b deposition. HUVEC-surface expression of CD46 and CD55 increased after hypoxia and/or reoxygenation.
Conclusions We conclude that (1) hypoxia and reoxygenation of HUVECs significantly increases iC3b deposition on HUVECs, (2) C3 deposition after hypoxia and reoxygenation is largely mediated by the classic complement pathway, and (3) HUVEC-surface expression of CD46 and CD55 increases after hypoxia and reoxygenation. These data demonstrate that hypoxia and reoxygenation of human endothelial cells activates the classic complement pathway despite an increase in complement C3 regulatory proteins.
Key Words: endothelium • hypoxia • ischemia • reperfusion
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