Roles of high mobility group box 1 protein released from endothelial cells with hypoxic injury on neuronal amyloidogenesis

Abstract

Hypoxia-reperfusion injury is one of the major risk factors for neurodegeneration. However, it is unclear whether ischaemic damage in brain microvascular endothelial cells plays roles in neurodegeneration, particularly in the amyloidogenic changes contributing to the development of Alzheimer's disease (AD) pathologies. Therefore, we investigated the roles of hypoxia-reoxygenation (H/R)-induced release of high mobility group box protein 1 (HMGB1), a risk molecule for AD pathogenesis in the ischaemic damaged brain, from human brain microvascular endothelial cells (HBMVECs) in neuronal amyloid beta (Aβ) production. H/R experiments were carried out in a purpose-built hypoxia glove-box chamber with continuous monitoring of O2 concentration. HBMVECs were treated with hypoxia (0.5% O2 ) and glucose deprivation for 1 h, and then the cells were restored to 37 °C with fresh M199 medium in a humidified atmosphere of 95% air and 5% CO2 . Endothelial permeability was assessed by measuring FITC-tagged dextran flux across monolayers of cultured HBMVECs. H4swe or SH-SY5Y cells were exposed to CM from HBMVECs with or without H/R for 24 h. Aβ40 and Aβ42 levels in the CM of H4swe cells were determined with commercial ELISA kits. Transfections of HMGB1 or Sirt1 siRNAs were performed using RNAi Max. Plasmids encoding Flag-tagged WT or dominant negative (DN) mutant H363Y SIRT1 were transfected using Lipofectamine. H/R increased nuclear-cytosolic translocation and secretion of HMGB1 in HBMVECs, along with increased permeability and HMGB1-dependent p-c-Jun activation. In addition, H/R increased the expression of Sirtuin 1 (Sirt1), coincident with an increase of intracellular Sirt1-HMGB1 binding in HBMVECs. H/R increased the acetylation of HMGB1 and extracellular secretion, which was significantly inhibited by Sirt1 overexpression. Furthermore, Sirt1 contributed to autophagy-mediated endogenous HMGB1 degradation. More importantly, treatment of neuronal cells with conditioned medium from H/R-stimulated HBMVECs (H/R-CM) activated their amyloidogenic pathways. The neuronal amyloidogenic changes (i.e., increased levels of extracellular Aβ40 and Aβ42) by H/R-CM from HBMVECs were further increased by Sirt1 inhibition, which was significantly suppressed by neutralization of the HMGB1 in H/R-CM. Our results suggest that HMGB1 derived from H/R-stimulated HBMVECs contributes to amyloidogenic pathways in neurons playing roles in the pathogenesis of AD, which are regulated by endothelial Sirt1.