Chemokines and chemokine receptors contribute to the migration of hepatic stellate cells (HSCs) and Kupffer cells, two key cell types in fibrogenesis. Here, we investigate the role of CCR2, the receptor for monocyte chemoattractant protein (MCP)-1, MCP-2, and MCP-3, in hepatic fibrosis. Hepatic CCR2, MCP-1, MCP-2, and MCP-3 messenger RNA expression was increased after bile duct ligation (BDL). Both Kupffer cells and HSCs, but not hepatocytes, expressed CCR2. BDL- and CCl₄-induced fibrosis was markedly reduced in CCR2^−/− mice as assessed through collagen deposition, α-smooth muscle actin expression, and hepatic hydroxyproline content. We generated CCR2 chimeric mice by the combination of clodronate, irradiation, and bone marrow (BM) transplantation allowing full reconstitution of Kupffer cells, but not HSCs, with BM cells. Chimeric mice containing wild-type BM displayed increased macrophage recruitment, whereas chimeric mice containing CCR2^−/− BM showed less macrophage recruitment at 5 days after BDL. Although CCR2 expressed in the BM enhanced macrophage recruitment in early phases of injury, CCR2 expression on resident liver cells including HSCs, but not on the BM, was required for fibrogenic responses in chronic fibrosis models. In vitro experiments demonstrated that HSCs deficient in CCR2^−/− or its downstream mediator p47phox^−/− did not display extracellular signal-regulated kinase and AKT phosphorylation, chemotaxis, or reactive oxygen species production in response to MCP-1, MCP-2, and MCP-3. Conclusion: Our results indicate that CCR2 promotes HSC chemotaxis and the development of hepatic fibrosis. (Hepatology 2009.)

The origin of fibrogenic cells in liver fibrosis remains controversial. We assessed the emerging concept that hepatocytes contribute to production of extracellular matrix (ECM) in liver fibrosis through epithelial-mesenchymal transition (EMT). We bred triple transgenic mice expressing ROSA26 stop β-galactosidase (β-gal), albumin Cre, and collagen α1(I) green fluorescent protein (GFP), in which hepatocyte-derived cells are permanently labeled by β-gal and type I collagen-expressing cells are labeled by GFP. We induced liver fibrosis by repetitive carbon tetrachloride (CCl4) injections. Liver sections and isolated cells were evaluated for GFP and β-gal as well as expression of α-smooth muscle actin (α-SMA) and fibroblast-specific protein 1 (FSP-1). Upon stimulation with transforming growth factor β-1, cultured hepatocytes isolated from untreated liver expressed both GFP and β-gal with a fibroblast-like morphological change but lacked expression of other mesenchymal markers. Cells from CCl4-treated livers never showed double-positivity for GFP and β-gal. All β-gal-positive cells exhibited abundant cytoplasm, a typical morphology of hepatocytes, and expressed none of the mesenchymal markers including α-SMA, FSP-1, desmin, and vimentin. In liver sections of CCl4-treated mice, GFP-positive areas were coincident with fibrotic septa and never overlapped X-gal-positive areas. Conclusion: Type I collagen-producing cells do not originate from hepatocytes. Hepatocytes in vivo neither acquire mesenchymal marker expression nor exhibit a morphological change clearly distinguishable from normal hepatocytes. Our results strongly challenge the concept that hepatocytes in vivo acquire a mesenchymal phenotype through EMT to produce the ECM in liver fibrosis. (Hepatology 2009.)

Cholestatic liver fibrosis is caused by obstruction of the biliary tract and is associated with early activation of portal fibroblasts (PFs) that express Thy-1, fibulin 2, and the recently identified marker mesothelin (MSLN). Here, we have demonstrated that activated PFs (aPFs) and myofibroblasts play a critical role in the pathogenesis of liver fibrosis induced by bile duct ligation (BDL). Conditional ablation of MSLN⁺ aPFs in BDL-injured mice attenuated liver fibrosis by approximately 50%. Similar results were observed in MSLN-deficient mice (Msln^–/– mice) or mice deficient in the MSLN ligand mucin 16 (Muc16^–/– mice). In vitro analysis revealed that MSLN regulates TGF-β1–inducible activation of WT PFs by disrupting the formation of an inhibitory Thy-1–TGFβRI complex. MSLN also facilitated the FGF-mediated proliferation of WT aPFs. Therapeutic administration of anti-MSLN–blocking Abs attenuated BDL-induced fibrosis in WT mice. Liver specimens from patients with cholestatic liver fibrosis had increased numbers of MSLN⁺ aPFs/myofibroblasts, suggesting that MSLN may be a potential target for antifibrotic therapy.

Background: Combination therapy with anti-programmed death-ligand 1 monoclonal antibody atezolizumab plus anti-vascular endothelial growth factor agent bevacizumab (Atezo/Bev) was approved in 2020 as a first-line treatment for unresectable hepatocellular carcinoma (HCC). Atezo/Bev therapy is relatively well tolerated; however, factors that can predict its response have not yet been reported. Thus, we aimed to investigate whether the pretreatment neutrophil-to-lymphocyte ratio (NLR) could predict the therapeutic response in patients with HCC treated with Atezo/Bev therapy. Methods: We analyzed the course of 40 patients with HCC who received Atezo/Bev therapy at our hospital and attempted to identify pretreatment factors that could predict response by comparing those who achieved disease control with those who did not. Results: The pretreatment NLR value in patients who achieved disease control was significantly lower than that in patients with disease progression (2.47 vs. 4.48, p = 0.013). Using the optimal NLR cut-off value for predicting response (3.21) determined by receiver operating characteristic curve analysis, patients with NLR ≤ 3.21 had significantly better progression-free survival than those with NLR > 3.21 (p < 0.0001), although there were no significant differences in liver function or tumor-related background factors between the two groups. Conclusions: The pretreatment NLR value may be a useful predictor of response to Atezo/Bev therapy for HCC.