Given the important role of angiogenesis in liver pathology, the current study investigated the role of Runt-related transcription factor 1 (RUNX1), a regulator of developmental angiogenesis, in the pathogenesis of non-alcoholic steatohepatitis (NASH). Quantitative RT-PCRs and a transcription factor analysis of angiogenesis-associated differentially expressed genes in liver tissues of healthy controls, patients with steatosis and NASH, indicated a potential role of RUNX1 in NASH. The gene expression of RUNX1 was correlated with histopathological attributes of patients. The protein expression of RUNX1 in liver was studied by immunohistochemistry. To explore the underlying mechanisms, in vitro studies using RUNX1 siRNA and overexpression plasmids were performed in endothelial cells (ECs). RUNX1 expression was significantly correlated with inflammation, fibrosis and NASH activity score in NASH patients. Its expression was conspicuous in liver non-parenchymal cells. In vitro, factors from steatotic hepatocytes and/or VEGF or TGF-β significantly induced the expression of RUNX1 in ECs. RUNX1 regulated the expression of angiogenic and adhesion molecules in ECs, including CCL2, PECAM1 and VCAM1, which was shown by silencing or over-expression of RUNX1. Furthermore, RUNX1 increased the angiogenic activity of ECs. This study reports that steatosis-induced RUNX1 augmented the expression of adhesion and angiogenic molecules and properties in ECs and may be involved in enhancing inflammation and disease severity in NASH.

Liver injury caused by drugs, viruses, and toxins that impede the proliferation of mature hepatocytes results in the activation of hepatic progenitor cells (HPCs), which then participate in the restoration of the damaged liver tissue. HPCs are known to be bipotential cells, capable of forming both hepatocytes and cholangiocytes when regeneration by mature hepatocytes is plagued or impaired. Both clinical studies of liver disease and certain experimental animal models of liver injury conspicuously show the presence of activated HPC response and proliferation. However, in addition to regeneration, the proliferation of HPCs also determines the appearance of a ductular reaction that has been correlated with progressive portal fibrosis, suggesting intricate links between activation of HPCs and fibrogenesis. The current review highlights the role of activated HPCs in both hepatic regeneration and fibrosis during liver injury.

Background: Hepatitis B-X Protein (HBx) encoded in Hepatitis B virus (HBV) is known to play a critical role in development and progression of HBV induced hepatocellular carcinoma (HCC). HBx interacts with and activates various cells in HCC microenvironment to promote tumor initiation, progression and invasion. In this study, we investigated how surrounding stromal cells interact with HBx-infected hepatoma cells by a series of in vitro co-culture studies. Methods: Huh7 hepatoma cells were cultured and transfected with the mammalian expression vector pGFP-HBx. Co-culture assays were performed between HBx-transfected Huh7 cells and conditioned media (CM) from stromal cells [endothelial cell lines (HUVECs) and hepatic stellate cell lines (LX2 cells)]. The effect of these interactions was studied by a series of functional assays like chemotaxis, invasion, and wound healing scratch assays. Also, quantitative real time (RT)-PCRs of the mesenchymal genes was performed in the hepatoma cells with and without the co-cultures. Hep3B cells with an integrated HBV genome were taken as positive controls. Results: HBx-transfected Huh7 cells cultured in presence of CM from HUVECs illustrated enhanced migration and tube formation as compared to HBx-transfected cells cultured alone or co-cultured with LX2 cells. HBx-transfected hepatoma cells incubated with CM from HUVECs also expressed mesenchymal genes including Thy1, CDH2, TGFβR1, VIM, and CD133. ELISAs revealed increased levels of TGF-β in CM from HUVECs. In comparison to unstimulated HBx-transfected Huh7 cells, TGF-β stimulated cells displayed increased invasive properties and mesenchymal gene expression. RT-PCR and flow cytometry analysis further demonstrated that incubation with either CM from HUVECs or TGF-β significantly increased the expression of a stemness marker, CD133 in HBx-infected hepatoma cells. Gene inhibition experiments with CD133 siRNA showed a downregulation of mesenchymal gene expression and properties in TGF-β induced HBx-infected hepatoma cells as compared to that observed in control siRNA treated cells, indicating CD133 as one of the key molecules affecting epithelial to mesenchymal transition (EMT) in HBx-infected cells. Conclusion: The study indicates that secretory factors like TGF-β from neighboring endothelial cells may enhance expression of CD133 and impart an aggressive EMT phenotype to HBx-infected hepatoma cells in HBV induced HCC.

Use of tumor-associated antigens for cancer immunotherapy is limited due to their poor in vivo stability and low cellular uptake. Delivery of antigenic peptides using synthetic polymer-based nanostructures has been actively pursued but with limited success. Peptide-based nanostructures hold much promise as delivery vehicles due to their easy design and synthesis and inherent biocompatibility. Here, we report self-assembly of a dipeptide containing a non-natural amino acid, α,β-dehydrophenylalanine (ΔF), into nanotubes, which efficiently entrapped a MAGE-3-derived peptide (M3). M3 entrapped in F-ΔF nanotubes was more stable to a nonspecific protease treatment and both F-ΔF and F-ΔF-M3 showed no cellular toxicity for four cancerous and noncancerous cell lines used. F-ΔF-M3 showed significantly higher cellular uptake in RAW 267.4 macrophage cells compared to M3 alone and also induced in vitro maturation of dendritic cells (DCs). Immunization of mice with F-ΔF-M3 selected a higher number of IFN-γ secreting CD8+ T cells and CD4+ T compared to M3 alone. On day 21, a tumor growth inhibition ratio (TGI, %) of 41% was observed in a murine melanoma model. These results indicate that F-ΔF nanotubes are highly biocompatible, efficiently delivered M3 to generate cytotoxic T lymphocytes responses, and able to protect M3 from degradation under in vivo conditions. The F-ΔF dipeptide-based nanotubes may be considered as a good platform for further development as delivery agents.