Hexa Histidine–Tagged Recombinant Human Cytoglobin Deactivates Hepatic Stellate Cells and Inhibits Liver Fibrosis by Scavenging Reactive Oxygen Species

BACKGROUND & AIMS: Anti-fibrotic therapy remains an unmet medical need in human chronic liver disease. We report the anti-fibrotic properties of cytoglobin (CYGB), a respiratory protein expressed in hepatic stellate cells (HSCs), the main cell type involved in liver fibrosis. APPROACH & RESULTS: Cygb-deficient mice which had bile duct ligation (BDL)-induced liver cholestasis or choline-deficient L-amino acid-defined (CDAA) diet-induced steatohepatitis significantly exacerbated liver damage, fibrosis and reactive oxygen species (ROS) formation. All these manifestations were attenuated in Cygb-overexpressing mice. We produced 6His-tagged recombinant human CYGB (His-CYGB), traced its bio-distribution and assessed its function in HSCs or in mice with advanced liver cirrhosis using thioacetamide (TAA) or 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). In cultured HSCs, extracellular His-CYGB was endocytosed and accumulated in endosomes via clathrin-mediated pathway. His-CYGB significantly impeded ROS formation spontaneously or in the presence of ROS inducers in HSCs, thus leading to the attenuation of collagen type I alpha 1 production and alpha-smooth muscle actin expression. Replacement the iron centre of the heme group with cobalt nullified the effect of His-CYGB. In addition, His-CYGB induced interferon-β secretion by HSCs which partly contributed to its anti-fibrotic function. Momelotinib incompletely reversed the effect of His-CYGB. Intravenously injected His-CYGB markedly suppressed liver inflammation, fibrosis and oxidative cell damage in TAA- or DDC-administered mice without adverse effects. RNA-seq analysis revealed the downregulation of inflammation and fibrosis-related genes and the upregulation of antioxidant genes in both cell culture and liver tissues. The injected His-CYGB predominantly localised to HSCs but not to macrophages, suggesting specific targeting effects. His-CYGB exhibited no toxicity in humanised liver chimeric PXB mice. CONCLUSIONS: His-CYGB could have anti-fibrotic clinical applications for human chronic liver diseases

Cancer cells produce liver metastasis via gap formation in sinusoidal endothelial cells through proinflammatory paracrine mechanisms

Intracellular gap (iGap) formation in liver sinusoidal endothelial cells (LSECs) is caused by the destruction of fenestrae and appears under pathological conditions; nevertheless, their role in metastasis of cancer cells to the liver remained unexplored. We elucidated that hepatotoxin-damaged and fibrotic livers gave rise to LSECs-iGap formation, which was positively correlated with increased numbers of metastatic liver foci after intrasplenic injection of Hepa1-6 cells. Hepa1-6 cells induced interleukin-23-dependent tumor necrosis factor-α (TNF-α) secretion by LSECs and triggered LSECs-iGap formation, toward which their processes protruded to transmigrate into the liver parenchyma. TNF-α triggered depolymerization of F-actin and induced matrix metalloproteinase 9 (MMP9), intracellular adhesion molecule 1, and CXCL expression in LSECs. Blocking MMP9 activity by doxycycline or an MMP2/9 inhibitor eliminated LSECs-iGap formation and attenuated liver metastasis of Hepa1-6 cells. Overall, this study revealed that cancer cells induced LSEC-iGap formation via proinflammatory paracrine mechanisms and proposed MMP9 as a favorable target for blocking cancer cell metastasis to the liver