Background and Aims Antifibrotic therapy remains an unmet medical need in human chronic liver disease. We report the antifibrotic properties of cytoglobin (CYGB), a respiratory protein expressed in hepatic stellate cells (HSCs), the main cell type involved in liver fibrosis. Approach and Results Cygb‐deficient mice that had bile duct ligation–induced liver cholestasis or choline‐deficient amino acid–defined diet–induced steatohepatitis significantly exacerbated liver damage, fibrosis, and reactive oxygen species (ROS) formation. All of these manifestations were attenuated in Cygb‐overexpressing mice. We produced hexa histidine–tagged recombinant human CYGB (His‐CYGB), traced its biodistribution, 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 through a 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 1 alpha 1 production and α‐smooth muscle actin expression. Replacement the iron center of the heme group with cobalt nullified the effect of His‐CYGB. In addition, His‐CYGB induced interferon‐β secretion by HSCs that partly contributed to its antifibrotic function. Momelotinib incompletely reversed the effect of His‐CYGB. Intravenously injected His‐CYGB markedly suppressed liver inflammation, fibrosis, and oxidative cell damage in mice administered TAA or DDC mice without adverse effects. RNA‐sequencing analysis revealed the down‐regulation of inflammation‐ and fibrosis‐related genes and the up‐regulation of antioxidant genes in both cell culture and liver tissues. The injected His‐CYGB predominantly localized to HSCs but not to macrophages, suggesting specific targeting effects. His‐CYGB exhibited no toxicity in chimeric mice with humanized livers. Conclusions His‐CYGB could have antifibrotic clinical applications for human chronic liver diseases.

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.

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Pancreatic cancer is a highly challenging malignancy with extremely poor prognosis. Cytoglobin (CYGB), a hemeprotein involved in liver fibrosis and cancer development, is expressed in pericytes of all organs. Here, we examined the role of CYGB in the development of pancreatic cancer. CYGB expression appeared predominately in the area surrounding adenocarcinoma and negatively correlated with tumor size in patients with pancreatic cancer. Directly injecting 7, 12-dimethylbenz[a]anthracene into the pancreatic tail in wild-type mice resulted in time-dependent induction of severe pancreatitis, fibrosis, and oxidative damage, which was rescued by Cygb overexpression in transgenic mice. Pancreatic cancer incidence was 93% in wild-type mice but only 55% in transgenic mice. Enhanced CYGB expression in human pancreatic stellate cells in vitro reduced cellular collagen synthesis, inhibited cell activation, increased expression of antioxidant-related genes, and increased CYGB secretion into the medium. Cygb-overexpressing or recombinant human CYGB (rhCYGB) -treated MIA PaCa-2 cancer cells exhibited dose-dependent cell cycle arrest at the G1 phase, diminished cell migration, and reduction in colony formation. RNA sequencing in rhCYGB-treated MIA PaCa-2 cells revealed downregulation of cell cycle and oxidative phosphorylation pathways. An increase in MIA PaCa-2 cell proliferation and reactive oxygen species production by H2O2 challenge was blocked by rhCYGB treatment or Cygb overexpression. PANC-1, OCUP-A2, and BxPC-3 cancer cells showed similar responses to rhCYGB. Known antioxidants N-acetyl cysteine and glutathione also inhibited cancer cell growth. These results demonstrate that CYGB suppresses pancreatic stellate cell activation, pancreatic fibrosis, and tumor growth, suggesting its potential therapeutic application against pancreatic cancer.