Cathepsin â…¤ Participates In HMGB1-promoted Angiogenesis

Objectives:This study is designed to investigate the role of HMGB1in angiogenesis and its signal pathway. Also, the function of Cathepsin V in HMGB1-promoted angiogenesis will be discussed.Methods:Tube formation analysis was used to evaluate the angiogenic ability of ECs in vitro. Western blot was used to detect the content of specific proteins in the cells and cell culture medium. Immunofluorescence staining showed the protein content in the cells and on the membrane. Recombinant HMGB1, Cathepsin V inhibitor SID26681509、HMGB1-TLR4/MD2specific inhibitor Tetramer、TLR4inhibitor Viper、ERK inhibitor U0126and Saquinavir were used to treat the HUVECs. Reagents and siRNA were used to determine the role of proteins:(1) Use SID to inhibit the action of Cathepsin V;(2) Use Tetramer to inhibit the HMGB1-TLR4signaling;(3) Use Viper to inhibit the downstream pathway of TLR4;(4) Use U0126to inhibit ERK phosphorylation;(5) Use siRNA to impair the expression of HMGB1.Results:In hypoxia, HMGB1was released by HUVECs. rHMGB1promotes tube formation. After HMGB1knockdown by siRNA, HUVECs had less ability of tube formation. rHMGB1promotes ERK phosphorylation at both short time point (30min) and longer time point (>3h). ERK inhibitor U0126inhibited tube formation.rHMGB1treated HUVECs had more TLR4expressed on the membrane. TLR4inhibitor Viper inhibited tube formation and ERK phosphorylation. HMGB1-TLR4/MD2specific inhibitor Tetramer had the same effect. Both viper and tetramer inhibits the binding of rHMGB1to HUVECs.Cathepsin V was released into medium in hypoxia, the release was also promoted by rHMGB1. Both Tetramer and U0126inhibited the release of cathepsin V. Cathepsin V inhibitor SID impaired HMGB1-promoted tube formation and ERK phosphorylation but did not affect the binding of rHMGB1.Saquinavir inhibited tube formation. At different time points (6h,12h, and24h), the release of cathepsin V was inhibited by saquinavir. Like SID, saquinavir inhibited ERK phosphorylation but not rHMGB1binding.Conclusions:In hypoxia, HUVECs release HMGB1, which activates ERK phosphorylation through TLR4pathway and results in promotion of angiogenesis. Cathepsin V participates in HMGB1signaling after HMGB1-TLR4interaction. Saquinavir inhibits tube formation in a similar way as cathepsin V inhibitor.