Extracellular histones induce inflammation and senescence of vascular smooth muscle cells by activating the AMPK/FOXO4 signaling pathway.

ABSTRACT

BACKGROUND: Sepsis is an abnormal immune-inflammatory response that is mainly caused by infection. It can lead to life-threatening organ dysfunction and death. Severely damaged tissue cells will release intracellular histones into the circulation as damage-related molecular patterns (DAMPs) to accelerate the systemic immune response. Although various histone-related cytotoxicity mechanisms have been explored, those that affect extracellular histones involved in vascular smooth muscle cell (VSMC) dysfunction are yet to be determined. METHODS: Mouse aortic vascular smooth muscle cells (VSMCs) were stimulated with different concentrations of histones, and cell viability was detected by CCK-8 assay. Cellular senescence was assessed by SA ß-gal staining. C57BL/6 mice were treated with histones with or without BML-275 treatment. RT-qPCR was performed to determine the expression of inflammatory cytokines. Western blotting was used to analyze the expression of NLRP3, ASC and caspase-1 inflammasome proteins. The interaction of NLRP3 and ASC was detected by CoIP and immunofluorescence staining. RESULTS: In this study, we found that extracellular histones induced senescence and inflammatory response in a dose-dependent manner in cultured VSMCs. Histone treatment significantly promoted apoptosis-associated speck-like protein containing CARD (ASC) as well as NACHT, LRR and PYD domains-containing protein 3 (NLRP3) interaction of inflammasomes in VSMCs. Forkhead box protein O4 (FOXO4), which is a downstream effector molecule of extracellular histones, was found to be involved in histone-regulated VSMC inflammatory response and senescence. Furthermore, the 5'-AMP-activated protein kinase (AMPK) signaling pathway was confirmed to mediate extracellular histone-induced FOXO4 expression, and blocking this signaling pathway with an inhibitor can suppress vascular inflammation induced by extracellular histones in vivo and in vitro. CONCLUSION: Extracellular histones induce inflammation and senescence in VSMCs, and blocking the AMPK/FOXO4 pathway is a potential target for the treatment of histonemediated organ injury.