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.


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 increases 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.
Collard
C D
CD
Department of Anesthesia, Brigham and Women's Hospital, Harvard Medical School, Boston, Mass. 02115, USA.
Väkevä
A
A
Büküsoglu
C
C
Zünd
G
G
Sperati
C J
CJ
Colgan
S P
SP
Stahl
G L
GL
D015703:Antigens, CD / Q000032:analysis; D015153:Blotting, Western; D018958:CD55 Antigens / Q000032:analysis; D003167:Complement Activation / Q000502:physiology*; D003176:Complement C3 / Q000032:analysis*; D003169:Complement Inactivator Proteins / Q000032:analysis; D004730:Endothelium, Vascular / Q000502:physiology*; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006801:Humans; D000860:Hypoxia / Q000503:physiopathology*; D051916:Membrane Cofactor Protein; D008562:Membrane Glycoproteins / Q000032:analysis; D015427:Reperfusion Injury / Q000503:physiopathology*; D013499:Surface Properties; D014471:Umbilical Veins / Q000502:physiology*
D015703:Antigens, CD; C497857:CD46 protein, human; D018958:CD55 Antigens; D003176:Complement C3; D003169:Complement Inactivator Proteins; D051916:Membrane Cofactor Protein; D008562:Membrane Glycoproteins