Endothelial cells injury and pro-inflammation cytokines release are the initial steps of hyperhomocysteinemia (HHcy)-associated vascular inflammation. Pyroptosis is a newly identified proinflammation form of programmed cell death, causing cell lysis and IL-1 beta release, and characterized by the caspases-induced cleavage of its effector molecule gasdermins (GSDMs). However, the effect of homocysteine (Hcy) on endothelial cells pyroptosis and the underlying mechanisms have not been fully defined. We have previously reported that Hcy induces vascular endothelial inflammation accompanied by the increase of high mobility group box-1 protein (HMGB1) and lysosomal cysteine protease cathepsin V in endothelial cells, and other studies have shown that HMGB1 or cathepsins are involved in activation of NLRP3 inflammasome and caspase-1. Here, we investigated the role of HMGB1 and cathepsin V in the process of Hcy-induced pyroptosis. We observed an increase in plasma IL-1 beta levels in HHcy patients and mice models, cathepsin V inhibitor reduced the plasma IL-1 beta levels and cleavage of GSDMD full-length into GSDMD N-terminal in the thoracic aorta of hyperhomocysteinemia mice. Using cultured HUVECs, we observed that Hcy promoted GSDMD N-terminal expression, silencing GSDMD or HMGB1 rescued Hcy-induced pyroptosis. HMGB1 also increased GSDMD N-terminal expression, and silencing cathepsin V reversed HMGB1-induced pyroptosis. HMGB1 could increase lysosome permeability, and silencing cathepsin V attenuated HMGB1-induced activation of caspase-1. In conclusion, this study has delineated a novel mechanism that HMGB1 mediated Hcy-induced endothelial cells pyroptosis partly via cathepsin V-dependent pathway. (C) 2020 Elsevier Inc. All rights reserved.