HMGB1/RAGE pro-inflammatory axis promotes vascular endothelial cell apoptosis in limb ischemia/reperfusion injury

OBJECTIVE: High-Mobility Group Box 1 (HMGB1) promotes vascular injuries induced by limb Ischemia and Reperfusion (IR), but the molecular mechanisms are not well understood. This study aimed to investigate the role of Receptor for Advanced-Glycation End products (RAGE) in HMGB1-regulated inflammatory response and vascular injury in limb IR using the rat IR and cellular Hypoxia and Reoxygenation (HR) models. METHODS: We analyzed the vascular structure and elastic fiber deposition in rat femoral arteries by histological staining. We determined gene expression in vascular tissues and cells by quantitative RT-PCR, Western blotting and immunofluorescence; analyzed the protein levels in rat serum or cell supernatant by ELISA; and assessed protein interaction by co-immunoprecipitation. We used CCK-8 for analyzing cell viability, and assessed apoptosis by Hoechst staining and flow cytometry. RESULTS: RAGE inhibition by FPS-ZM1 significantly repressed rat vascular injury that was induced by limb IR treatment. HMGB1 and RAGE expression, P38, ERK1/2, P65 and IKBa phosphorylation, as well as HIF-1a, NLRP3, Caspase-1, TNF-a and IL-6 expression and P65 in nucleus, increased in femoral arteries of a rat IR model and HUVEC undergoing HR treatment, whereas all the factors except HMGB1 and RAGE were inhibited by FPS-ZM1 treatment. In addition, we found that HMGB1 binds with RAGE in HUVEC undergoing HR treatment, which was suppressed by FPS-ZM1. Finally, the apoptosis of HUVEC also increased by HR treatment, but repressed under FPS-ZM1 treatment. CONCLUSION: HMGB1 binds with RAGE to promote vascular inflammation and endothelial cell apoptosis, which mediates vascular injury during acute limb IR.