Advanced glycation end products on stored red blood cells increase endothelial reactive oxygen species generation through interaction with receptor for advanced glycation end products.
BACKGROUND
Recent evidence suggests that storage-induced alterations of the red blood cell (RBC) are associated with adverse consequences in susceptible hosts. As RBCs have been shown to form advanced glycation end products (AGEs) after increased oxidative stress and under pathologic conditions, we examined whether stored RBCs undergo modification with the specific AGE N-(carboxymethyl)lysine (N(ε) -CML) during standard blood banking conditions.


METHODS
Purified, fresh RBCs from volunteers were compared to stored RBCs (35-42 days old) obtained from the blood bank. N(ε) -CML formation was quantified using a competitive enzyme-linked immunosorbent assay. The receptor for advanced glycation end products (RAGE) was detected in human pulmonary microvascular endothelial cells (HMVEC-L) by real-time polymerase chain reaction, Western blotting, and flow cytometry. Intracellular reactive oxygen species (ROS) generation was measured by the use of 5-(and 6-)chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester-based assays.


RESULTS
Stored RBCs showed increased surface N(ε) -CML formation when compared with fresh RBCs. HMVEC-L showed detectable surface RAGE expression constitutively. When compared to fresh RBCs, stored RBCs triggered increased intracellular ROS generation in both human umbilical vein endothelial cells and HMVEC-L. RBC-induced endothelial ROS generation was attenuated in the presence of soluble RAGE or RAGE blocking antibody.


CONCLUSIONS
The formation of the AGE N(ε) -CML on the surface of stored RBCs is one functional consequence of the storage lesion. AGE-RAGE interactions may be one mechanism by which transfused RBCs cause endothelial cell damage.
Mangalmurti
Nilam S
NS
Department of Medicine, Division of Pulmonary, Allergy, and Critical Care, University of Pennsylvania, Philadelphia 19104, USA. nspatel@mail.med.upenn.edu
Chatterjee
Shampa
S
Cheng
Guanjun
G
Andersen
Emily
E
Mohammed
Aishat
A
Siegel
Donald L
DL
Schmidt
Ann Marie
AM
Albelda
Steven M
SM
Lee
Janet S
JS
D000293:Adolescent; D000328:Adult; D000368:Aged; D001793:Blood Preservation / Q000009:adverse effects*; D002450:Cell Communication; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D017707:Erythrocyte Transfusion / Q000009:adverse effects*; D004912:Erythrocytes / Q000166:cytology / Q000378:metabolism*; D005434:Flow Cytometry; D017127:Glycation End Products, Advanced / Q000378:metabolism*; D006801:Humans; D008239:Lysine / Q000031:analogs & derivatives / Q000378:metabolism; D008875:Middle Aged; D018384:Oxidative Stress / Q000502:physiology; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic / Q000378:metabolism*; D013997:Time Factors; D014471:Umbilical Veins / Q000166:cytology; D055815:Young Adult
D017127:Glycation End Products, Advanced; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic; C048496:N(6)-carboxymethyllysine; D008239:Lysine