Liver-specific β-catenin knockout (β-Catenin-LKO) mice have revealed an essential role of β-catenin in metabolic zonation where it regulates pericentral gene expression and in initiating liver regeneration (LR) after partial hepatectomy (PH), by regulating expression of Cyclin-D1. However what regulates β-catenin activity in these events remains an enigma. Here, we investigate to what extent β-catenin activation is Wnt-signaling dependent and the potential cell source of Wnts. We studied liver-specific Lrp5/6 KO (Lrp-LKO) mice where Wnt-signaling was abolished in hepatocytes while the β-catenin gene remained intact. Intriguingly, like β-catenin-LKO mice, Lrp-LKO exhibited a defect in metabolic zonation observed as lack of glutamine synthetase (GS), Cyp1a2 and Cyp2e1. Lrp-LKO also displayed a significant delay in initiation of LR due to absence of β-catenin-TCF4 association and lack of Cyclin-D1. To address the source of Wnt proteins in liver, we investigated conditional Wntless (Wls) KO mice, which lacked ability to secrete Wnts from either liver epithelial cells (Wls-LKO), or macrophages including Kupffer cells (Wls-MKO), or endothelial cells (Wls-EKO). While Wls-EKO was embryonic lethal precluding further analysis in adult hepatic homeostasis and growth, Wls-LKO and Wls-MKO were viable but did not show any defect in hepatic zonation. Wls-LKO showed normal initiation of LR, however Wls-MKO showed a significant but temporal deficit in LR that was associated with decreased β-catenin-TCF4 association and diminished Cyclin-D1 expression. Conclusion Wnt-signaling is the major upstream effector of β-catenin activity in pericentral hepatocytes and during LR. Hepatocytes, cholangiocytes or macrophages are not the source of Wnts in regulating hepatic zonation. However, Kupffer cells are a major contributing source of Wnt secretion necessary for β-catenin activation during LR.

Many studies have shown that tumor‐associated macrophages (TAMs) contribute to tumor development and poor prognosis in various cancers. In this study, we investigated the macrophage populations and phenotypes, and their correlation to angiogenesis, immunosuppression, and clinical prognosis in intrahepatic cholangiocarcinoma (ICC). CD68 (+) and CD163 (+) macrophage infiltration was analyzed in paraffin‐embedded tissue samples from 39 patients. CD163 is used as a marker of M2 macrophages. Neovascularization and infiltration of forkhead box P3 (FOXP3) (+) regulatory T cells were also evaluated. The number of CD68 (+) and CD163 (+) macrophages was positively correlated with the numbers of vessels and regulatory T cells. The number of CD163 (+) cells was more closely associated with them. Intrahepatic cholangiocarcinoma (ICC) patients with high counts of CD163 (+) macrophages showed poor disease‐free survival (P = 0.0426). The macrophage density was not correlated with overall survival. In an in vitro study using ICC cell lines (HuCCT1, RBE, and MEC) and human macrophages, tumor cell supernatant (TCS) from cell lines induced an activation of signal transducers and activators of transcription‐3 (Stat3) and macrophage polarization toward the M2 phenotype. Tumor cell supernatant (TCS) from HuCCT1 most strongly induced Stat3 activation and production of cytokines and other bioactive molecules such as interleukin (IL)‐10, vascular endothelial growth factor (VEGF)‐A, transforming growth factor (TGF)‐β, and matrix metalloproteinase (MMP)‐2. Down‐regulation of Stat3 by siRNA significantly suppressed the production of IL‐10 and VEGF‐A. These results provide suggestive evidence that TAMs contribute to cancer progression via Stat3 activation, and CD163 is useful for evaluating M2 TAMs and predicting the clinical prognosis of ICC patients. (Cancer Sci)

The prognosis for individuals diagnosed with hepatocellular carcinoma (HCC) remains poor because of the high frequency of invasive tumor growth, intrahepatic spread, and extrahepatic metastasis. Here, we investigated the role of the standard isoform of CD44 (CD44s), a major adhesion molecule of the extracellular matrix and a cancer stem cell marker, in the TGF-b-mediated mesenchymal phenotype of HCC. We found that CD44s was the dominant form of CD44 mRNA expressed in HCC cells. Overexpression of CD44s promoted tumor invasiveness and increased the expression of vimentin, a mesenchymal marker, in HCC cells. Loss of CD44s abrogated these changes. Also in the setting of CD44s overexpression, treatment with TGF-b1 induced the mesenchymal phenotype of HCC cells, which was characterized by low E-cadherin and high vimentin expression. Loss of CD44s inhibited TGF-b-mediated vimentin expression, mesenchymal spindle-like morphology, and tumor invasiveness. Clinically, overexpression of CD44s was associated with low expression of E-cadherin, high expression of vimentin, a high percentage of phospho-Smad2-positive nuclei, and poor prognosis in HCC patients, including reduced disease-free and overall survival. Together, our findings suggest that CD44s plays a critical role in the TGF-b-mediated mesenchymal phenotype and therefore represents a potential therapeutic target for HCC. Cancer Res; 72(13); 3414-23. Ó2012 AACR.

β‐Catenin in hepatocytes, under the control of Wnts, regulates pericentral gene expression. It also contributes to liver regeneration (LR) after partial hepatectomy (PH) by regulating cyclin‐D1 gene expression as shown in the β‐catenin and Wnt coreceptors low‐density lipoprotein receptor‐related protein 5/6 conditional knockouts (KO). However, conditional deletion of Wntless (Wls), required for Wnt secretion, in hepatocytes, cholangiocytes, or macrophages lacked any impact on zonation, while Wls deletion in macrophages only marginally affected LR. Here, we address the contribution of hepatic endothelial cells (ECs) in zonation and LR by characterizing EC‐Wls‐KO generated by interbreeding Wls‐floxed and lymphatic vessel endothelial hyaluronan receptor (Lyve1)‐cre mice. These mice were also used to study LR after PH. While Lyve1 expression in adult liver is limited to sinusoidal ECs only, Lyve1‐cre mice bred to ROSA26‐Stopflox/flox‐enhanced yellow fluorescent protein (EYFP) mice showed EYFP labeling in sinusoidal and central vein ECs. EC‐Wls‐KO mice showed decreased liver weights; lacked glutamine synthetase, cytochrome P450 2e1, and cytochrome P450 1a2; and were resistant to acetaminophen‐induced liver injury. After PH, EC‐Wls‐KO showed quantitative and qualitative differences in cyclin‐D1 expression at 24‐72 hours, which led to a lower hepatocyte proliferation at 40 hours but a rebound increase by 72 hours. ECs and macrophages isolated from regenerating livers at 12 hours showed significant up‐regulation of Wnt2 and Wnt9b messenger RNA; these are the same two Wnts involved in baseline β‐catenin activity in pericentral hepatocytes. Conclusion: At baseline, ECs secrete Wnt proteins essential for β‐catenin activation in pericentral hepatocytes. During LR, sinusoidal and central vein ECs and secondarily macrophages secrete Wnt2, while predominantly central vein ECs and secondarily macrophages are the likely source of Wnt9b. This process spatiotemporally regulates β‐catenin activation in hepatocytes to induce cell proliferation. (Hepatology Communications 2018;2:845‐860)

β-catenin is important for liver regeneration after CDE diet-induced liver injury, and BEC-derived hepatocytes can permanently incorporate into the liver parenchyma to mediate liver regeneration. This article is protected by copyright. All rights reserved.

Our data showed that tumour infiltrating inflammatory and immune cells may play a pivotal role in ECC progression and a high-risk signature predicted poor prognosis in ECC patients.

Hepatic repair is directed chiefly by the proliferation of resident mature epithelial cells. Further if predominant injury is to cholangiocytes, the hepatocytes can transdifferentiate to cholangiocytes to assist in the repair and vice versa as shown by various fate-tracing studies. However, the molecular bases of reprograming remain elusive. Using two models of biliary injury where repair occurs via cholangiocyte proliferation and hepatocyte transdifferentiation to cholangiocytes, we identify an important role of Wnt signaling. First we identify upregulation of specific Wnt proteins in the cholangiocytes. Next, using conditional knockouts of Wntless and Wnt co-receptors LRP5/6, transgenic mice expressing stable β-catenin, and in vitro studies, we show a role of Wnt signaling through β-catenin in hepatocyte to biliary transdifferentiation. Lastly, we show that specific Wnts regulate cholangiocyte proliferation but in a β-catenin-independent manner. Conclusion: Wnt signaling regulates hepatobiliary repair after cholestatic injury in both β-catenin dependent and independent manners.

For biomedical application of nanoparticles, the surface chemical functionality is very important to impart additional functions, such as solubility and stability in a physiological environment, and targeting specificity as an imaging probe and a drug carrier. Although polyethylene glycol (PEG) has been used extensively, here, it is proposed that hyperbranched polyglycerol (PG) is a good or even better alternative to PEG. Superparamagnetic iron oxide nanoparticles (SPIONs) prepared using a polyol method are directly functionalized with PG through ring‐opening polymerization of glycidol. The resulting SPION‐PG is highly soluble in pure water (>40 mg mL−1) and in a phosphate buffer solution (>25 mg mL−1). Such high solubility enables separation of SPION‐PG according to size using size exclusion chromatography (SEC). The size‐separated SPION‐PG shows a gradual increase in transverse relaxivity (r2) with increasing particle size. For biological application, SPION‐PG is functionalized through multistep organic transformations (–OH → –OTs (tosylate) → –N3 → –RGD) including click chemistry as a key step to impart targeting specificity by immobilization of cyclic RGD peptide (Arg‐Gly‐Asp‐D‐Tyr‐Lys) on the surface. The targeting effect is demonstrated by the cell experiments; SPION‐PG‐RGD is taken up by the cells overexpressing αvβ3‐integrin such as U87MG and A549.