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. STUDY DESIGN AND 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.