Manipulating HGF signaling reshapes the cirrhotic liver niche and fills a therapeutic gap in regeneration mediated by transplanted stem cells.

Long-term stem cell survival in the cirrhotic liver niche to maintain therapeutic efficacy has not been achieved. In a well-defined diethylnitrosamine (DEN)-induced liver fibrosis/cirrhosis animal model, we previously showed that liver-resident stem/progenitor cells (MLpvNG2+ cells) or immune cells have improved survival in the fibrotic liver environment but died via apoptosis in the cirrhotic liver environment, and increased levels of hepatocyte growth factor (HGF) mediated this cell death. We tested the hypothesis that inhibiting HGF signaling during the cirrhotic phase could keep the cells alive. We used adeno-associated virus (AAV) vectors designed to silence the c-Met (HGF-only receptor) gene or a neutralizing antibody (anti-cMet-Ab) to block the c-Met protein in the DEN-induced liver cirrhosis mouse model transplanted with MLpvNG2+ cells between weeks 6 and 7 after DEN administration, which is the junction of liver fibrosis and cirrhosis at the site where most intrahepatic stem cells move toward apoptosis. After 4 weeks of treatment, the transplanted MLpvNG2+ cells survived better in c-Met-deficient mice than in wild-type mice, and cell activity was similar to that of the mice that received MLpvNG2+ cells at 5 weeks after DEN administration (liver fibrosis phase when most of these cells proliferated). Mechanistically, a lack of c-Met signaling remodeled the cirrhotic environment, which favored transplanted MLpvNG2+ cell expansion to differentiation into mature hepatocytes and initiate endogenous regeneration by promoting mature host hepatocyte generation and mediating functional improvements. Therapeutically, c-Met-mediated regeneration can be mimicked by anti-cMet-Ab to interfere functions, which is a potential drug for cell-based treatment of liver fibrosis/cirrhosis.

Matrix metalloproteinase-2 inducing COL1A1 synthesis via integrin alpha â ¤ promotes invasion and metastasis of cholangiocarcinoma cells.

INTRODUCTION AND OBJECTIVES: Cholangiocarcinoma (CCA) is characterized by early distant invasion and metastasis, whereas the underlying mechanism is still obscure. Increasing evidence shows that collagen type Ι alpha 1 (COL1A1) is a gene associated with the progression of multiple diseases. Here, we attempted to investigate the role of COL1A1 in CCA. MATERIALS AND METHODS: The expression of COL1A1 between tumor tissues and adjacent normal tissues obtained from CCA patients was detected by Western blot and immunofluorescence, followed by analysis of its clinical significance. Then, the biological effects of COL1A1 overexpression or knockdown on CCA cells were evaluated in vitro and in vivo. Finally, molecular mechanism of COL1A1 in regulating the invasion and metastasis of CCA cells was determined by a series of experiments. RESULTS: COL1A1 expression was significantly higher in CCA pathological tissues than in corresponding adjacent normal tissues. Analysis of 83 CCA patients showed that higher expression of COL1A1 was correlated with poorer patient prognosis. Notably, overexpression or knockdown experiments revealed that COL1A1 contributed to the migration and invasion, as well as epithelial-to-mesenchymal transition (EMT), in CCA cells. Further investigations demonstrated that matrix metalloproteinase-2 (MMP2) promoted COL1A1 upregulation via the integrin alpha Ⅴ pathway, therefore affecting ECM remodelling and inducing EMT in CCA cells. Moreover, COL1A1 expression was positively related to PD-1 and PD-L1 in CCA, and COL1A1 increased PD-L1 expression by activating the NF-κB pathway. CONCLUSIONS: COL1A1 plays an important role in regulating CCA progression and may act as a promising biomarker and therapeutic target for CCA.

New advances of NG2-expressing cell subset in marrow mesenchymal stem cells as novel therapeutic tools for liver fibrosis/cirrhosis.

BACKGROUND: Bone marrow-derived mesenchymal stem cell (BMMSC)-based therapy has become a major focus for treating liver fibrosis/cirrhosis. However, although these cell therapies promote the treatment of this disease, the heterogeneity of BMMSCs, which causes insufficient efficacy during clinical trials, has not been addressed. In this study, we describe a novel Percoll-Plate-Wait procedure (PPWP) for the isolation of an active cell subset from BMMSC cultures that was characterized by the expression of neuroglial antigen 2 (NG2/BMMSCs). METHODS: By using the key method of PPWP and other classical biological techniques we compared NG2/BMMSCs with parental BMMSCs in biological and functional characteristics within a well-defined diethylnitrosamine (DEN)-induced liver fibrosis/cirrhosis injury male C57BL/6 mouse model also in a culture system. Of note, the pathological alterations in the model is quite similar to humans'. RESULTS: The NG2/BMMSCs revealed more advantages compared to parentalBMMSCs. They exhibited greater proliferation potential than parental BMMSCs, as indicated by Ki-67 immunofluorescence (IF) staining. Moreover, higher expression of SSEA-3 (a marker specific for embryonic stem cells) was detected in NG2/BMMSCs than in parental BMMSCs, which suggested that the "stemness" of NG2/BMMSCs was greater than that of parental BMMSCs. In vivo studies revealed that an injection of NG2/BMMSCs into mice with ongoing DEN-induced liver fibrotic/cirrhotic injury enhanced repair and functional recovery to a greater extent than in mice treated with parental BMMSCs. These effects were associated with the ability of NG2/BMMSCs to differentiate into bile duct cells (BDCs). In particular, we discovered for the first time that NG2/BMMSCs exhibit unique characteristics that differ from those of parental BMMSCs in terms of producing liver sinusoidal endothelial cells (LSECs) to reconstruct injured blood vessels and sinusoidal structures in the diseased livers, which are important for initiating hepatocyte regeneration. This unique potential may also suggest that NG2/BMMSCs could be an novel off-liver progenitor of LSECs. Ex vivo studies revealed that the NG2/BMMSCs exhibited a similar trend to that of their in vivo in terms of functional differentiation responding to the DEN-diseased injured liver cues. Additionally, the obvious core role of NG2/BMMSCs in supporting the functions of BMMSCs in bile duct repair and BDC-mediated hepatocyte regeneration might also be a novel finding. CONCLUSIONS: Overall, the PPWP-isolated NG2/BMMSCs could be a novel effective cell subset with increased purity to serve as a new therapeutic tool for enhancing treatment efficacy of BMMSCs and special seed cell source (BDCs, LSECs) also for bioliver engineering.

Mechanism of multidrug resistance to chemotherapy mediated by P­glycoprotein (Review).

Multidrug resistance (MDR) seriously limits the clinical application of chemotherapy. A mechanism underlying MDR is the overexpression of efflux transporters associated with chemotherapeutic drugs. P­glycoprotein (P­gp) is an ATP­binding cassette (ABC) transporter, which promotes MDR by pumping out chemotherapeutic drugs and reducing their intracellular concentration. To date, overexpression of P­gp has been detected in various types of chemoresistant cancer and inhibiting P­gp­related MDR has been suggested. The present review summarizes the mechanisms underlying MDR mediated by P­gp in different tumors and evaluated the related signaling pathways, with the aim of improving understanding of the current status of P­gp­mediated chemotherapeutic resistance. This review focuses on the main mechanisms of inhibiting P­gp­mediated MDR, with the aim of providing a reference for the study of reversing P­gp­mediated MDR. The first mechanism involves decreasing the efflux activity of P­gp by altering its conformation or hindering P­gp­chemotherapeutic drug binding. The second inhibitory mechanism involves inhibiting P­gp expression to reduce efflux. The third inhibitory mechanism involves knocking out the ABCB1 gene. Potential strategies that can inhibit P­gp include certain natural products, synthetic compounds and biological techniques. It is important to screen lead compounds or candidate techniques for P­gp inhibition, and to identify inhibitors by targeting the relevant signaling pathways to overcome P­gp­mediated MDR.

Hepatocyte growth factor-mediated apoptosis mechanisms of cytotoxic CD8+ T cells in normal and cirrhotic livers.

Intrahepatic stem/progenitor cells and cytotoxic CD8+ T cells (CD8+ T cells) in the cirrhotic liver undergo apoptosis, which potentially facilitates progression to cancer. Here, we report that hepatocyte growth factor (HGF) signaling plays an important role in promoting normal and damaged liver CD8+ T cell Fas-mediated apoptosis through its only receptor, c-Met. In addition to binding with HGF, c-Met also binds to Fas to form a complex. Using a diethylnitrosamine (DEN)-induced liver fibrosis/cirrhosis mouse model, immunostaining, and terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) staining, we found that HGF secretion was significantly higher at 10 weeks post-DEN, the liver cirrhotic phase (LCP), than at 3 weeks post-DEN, the liver fibrotic phase (LFP). Correspondingly, differences in CD8+ T cell proliferation and apoptosis were noted between the two phases. Interestingly, staining and TUNEL assays revealed lower smooth muscle actin (α-SMA)+ cell apoptosis, a marker for hepatic stellate cells (HSCs), in the LFP group than in the LCP group, which suggested a beneficial correlation among HGF, CD8+ T cells and HSCs in improving the fibrotic load during damaged liver repair. In cultures, when met different concentrations of recombinant HGF (rHGF), phytohemagglutinin (PHA)-stimulated naive mouse splenic CD8+ T cells (pn-msCD8+ T cells) responded differently; as increases in rHGF increased were associated with decreases in the clonal numbers of pn-msCD8+ T cells, and when the rHGF dose was greater than 200 ng/mL, the clonal numbers significantly decreased. In the presence of 400 ng/mL rHGF, the death-inducing signaling complex (DISC) can be directly activated in both nsCD8+ T cells and healthy human peripheral blood CD8+ T cells (hp-CD8+ T cells), as indicated by recruitment of FADD and caspase-8 because DISC forms via the recruitment of FADD and caspase-8, among others. These findings suggest that Fas-mediated apoptosis, may also indicate a regulatory role of HGF signaling in hepatic homeostasis.

Comparative effects of hepatocyte growth factor and tacrolimus on acute liver allograft early tolerance.

Allostimulated CD8+ T cells (aCD8+ T cells), as the main mediators of acute liver rejection (ARJ), are hyposensitive to apoptosis due to the inactivation of death receptor FAS-mediated pathways and fail to allow tolerance induction, eventually leading to acute graft rejection. Although tacrolimus (FK506), the most commonly used immunosuppressant (IS) in the clinic, allows tolerance induction, its use is limited because its target immune cells are unknown and it is associated with increased incidences of malignancy, infection, and nephrotoxicity, which substantially impact long-term liver transplantation (LTx) outcomes. The dark agouti (DA)-to-Lewis rat LTx model is a well-known ARJ model and was hence chosen for the present study. We show that both hepatocyte growth factor (HGF) (cHGF, containing the main form of promoting HGF production) and recombinant HGF (h-rHGF) exert immunoregulatory effects mainly on allogeneic aCD8+ T cell suppression through FAS-mediated apoptotic pathways by inhibiting cMet to FAS antagonism and Fas trimerization, leading to acute tolerance induction. We also showed that such inhibition can be abrogated by treatment with neutralizing antibodies against cMet (HGF-only receptor). In contrast, we did not observe these effects in rats treated with FK506. However, we observed that the effect of anti-rejection by FK506 was mainly on allostimulated CD4+ T cell (aCD4+ T cell) suppression and regulatory T cell (Treg) promotion, in contrast to the mechanism of HGF. In addition, the protective mechanism of HGF in FK506-mediated nephrotoxicity was addressed. Therefore, HGF as a tolerance inducer, whether used in combination with FK506 or as monotherapy, may have good clinical value. Additional roles of these T-cell subpopulations in other biological systems and studies in these fields will also be meaningful.

Immunotherapeutic Targeting of NG2/CSPG4 in Solid Organ Cancers.

Neuro-glia antigen 2/chondroitin sulfate proteoglycan 4 (NG2/CSPG4, also called MCSP, HMW-MAA, MSK16, MCSPG, MEL-CSPG, or gp240) is a large cell-surface antigen and an unusual cell membrane integral glycoprotein frequently expressed on undifferentiated precursor cells in multiple solid organ cancers, including cancers of the liver, pancreas, lungs, and kidneys. It is a valuable molecule involved in cancer cell adhesion, invasion, spreading, angiogenesis, complement inhibition, and signaling. Although the biological significance underlying NG2/CSPG4 proteoglycan involvement in cancer progression needs to be better defined, based on the current evidence, NG2/CSPG4+ cells, such as pericytes (PCs, NG2+/CD146+/PDGFR-ß+) and cancer stem cells (CSCs), are closely associated with the liver malignancy, hepatocellular carcinoma (HCC), pancreatic malignancy, and pancreatic ductal adenocarcinoma (PDAC) as well as poor prognoses. Importantly, with a unique method, we successfully purified NG2/CSPG4-expressing cells from human HCC and PDAC vasculature tissue blocks (by core needle biopsy). The cells appeared to be spheres that stably expanded in cultures. As such, these cells have the potential to be used as sources of target antigens. Herein, we provide new information on the possibilities of frequently selecting NG2/CSPG4 as a solid organ cancer biomarker or exploiting expressing cells such as CSCs, or the PG/chondroitin sulfate chain of NG2/CSPG4 on the cell membrane as specific antigens for the development of antibody- and vaccine-based immunotherapeutic approaches to treat these cancers.

Comparison of the biological and functional characteristics of mesenchymal stem cells from intrahepatic and identical bone marrow.

In our privious work, our reseach group characterized a population of hepatic-sourced mesenchymal stem cells (MSCs) called MLpvNG2+ cells. In the present study, we compared the biological and functional characteristics of naïve MLpvNG2 cells with identical bone marrow-derived MSCs (niBM-MSCs) using in vitro (conditioned media) and in vivo (a well-set diethylnitrosamine (DEN)-induced liver fibrotic/cirrhotic murine model) procedures. The intrahepatic-sourced mesodermal MLpvNG2+ cells exhibited some biological characteristics (e.g., a set of surface markers) similar to those of extrahepatic niBM-MSCs. In responsed to signals of pathological conditions, such as singals of fibrotic/cirrhotic liver, MLpvNG2+ cells showed higher survival and favored differentiation into ALB(+) and G6Pc(+) hepatocytes, whereas niBM-MSCs predominantly differentiated into CK/KRT19(+) cholangiocytes. We identified C/EBPα/ß expression as a biological characteristic differentiating these two populations of MSCs, wherein MLpvNG2+ cells are likely regulated by C/EBPß transcriptional signaling, whereas niBM-MSCs are likely controlled by C/EBPα transcriptional signaling. Notably, although C/EBPα and C/EBPß transcriptional signaling regulate hepatocyte and cholangiocyte fate, respectively, the expression of these proteins in MLpvNG2+ cells is, to our knowledge, reported for the first time in the present study. We used anti-C/EBP neutralizing antibodies (Abs) both in vitro and in vivo to determine the functional characteristics of these proteins. We conclude that the biological characteristics of these two populations of MSCs depend on their differential C/EBPα/ß expression patterns.

Survival of endogenous hepatic stem/progenitor cells in liver tissues during liver cirrhosis.

AIMS: This study addressed whether endogenous hepatic stem/progenitor (HSP) cells survival were related to the injured signals during liver cirrhosis. MATERIAL AND METHODS: Liver cirrhosis was induced in C57BL/6 mice by administering diethylnitrosamine (DEN) in drinking water. Hematoxylin-eosin staining and Masson's trichrome staining were used to identify infiltrative cells and connective tissues, respectively. The inflammatory activity grade and fibrotic stage, represented as G and S respectively, and evaluated by the International Simplified Grading and Staging System (ISGSS). Endogenous HSP cells (Ng2+HSP) were identified by immunofluorescence staining with an anti-neuro/glial antigen 2 (Ng2) antibody. All data were analyzed using SPSS 22.0 and GraphPad Prism 6 and Student's t-test was performed to determine statistical significance. p-values < 0.05 were considered statistically significant. KEY FINDINGS: All mice administered oral DEN developed liver fibrosis, liver cirrhosis and hepatocellular carcinoma (HCC). During the fibrosis period, observed a positive correlation of survival of endogenous HSP (Ng2+HSP) cells with inflammatory activity. As the disease developed into the cirrhotic stage, when lost correlation of endogenous HSP cells with inflammatory activity, revealed a dramatically reduced survival rate of endogenous HSP (Ng2+HSP) cells. SIGNIFICANCE: The DEN-induced liver cirrhosis could develop into three time zone of fibrosis, cirrhosis and cancer, and the histological patterns in the model are similar to those described in humans. Dramatic survival and less apoptosis of endogenous HSP (Ng2+HSP) cells was within fibrotic state, where inflammation signals is strong, indicating such time zone (1-6 weeks) during liver cirrhosis is important for mobilizing endogenous HSP-based regeneration.

Methylation-mediated LINC00261 suppresses pancreatic cancer progression by epigenetically inhibiting c-Myc transcription.

Background: Due to the limitations of strategies for its early diagnosis and treatment, pancreatic cancer (PC) remains a substantial human health threat. We previously discovered a methylation-mediated lncRNA, LINC00261, which is downregulated in PC tissues. However, the underlying role of LINC00261 in PC remains largely unknown. Methods: Quantitative real-time PCR and in situ hybridization were performed to evaluate the expression levels of LINC00261 in PC, adjacent nontumor and normal pancreas tissues. The clinical significance of LINC00261 was assessed in multicenter PC samples. The functions of LINC00261 in PC were investigated by gain- and loss-of-function assays in vitro and in vivo. Potential downstream pathways and mechanisms were explored via RNA sequencing and bioinformatic analyses. RNA immunoprecipitation and chromatin immunoprecipitation assays were used to validate the underlying mechanisms. Pyrosequencing and targeted demethylation of the LINC00261 promoter were performed to explore the upstream epigenetic mechanisms and therapeutic potential. Results: LINC00261 was significantly downregulated in PC tissues, and its expression was positively associated with the prognosis of PC patients. Phenotypic studies indicated that LINC00261 overexpression significantly suppressed PC cell proliferation, migration and metastasis in vitro and in vivo. c-Myc was identified as a downstream target of LINC00261. LINC00261 repressed c-Myc transcription by physically interacting and binding with the bromo domain of p300/CBP, preventing the recruitment of p300/CBP to the promoter region of c-Myc and decreasing the H3K27Ac level. Moreover, the methylation level of the LINC00261 promoter was high in PC tissues and was correlated with poor prognosis. Targeted demethylation of the LINC00261 promoter inhibited PC progression both in vitro and in vivo. Conclusions: Our findings indicate that methylation-mediated LINC00261 suppresses PC progression by epigenetically repressing c-Myc expression. These findings expand the therapeutic potential of LINC00261, possibly providing evidence to support the development of epigenetic drugs or therapeutic strategies. This research adds further insights into the etiology of PC and indicates that LINC00261 may be a prognostic and therapeutic target in PC.

[Corrigendum] S100A8 facilitates cholangiocarcinoma metastasis via upregulation of VEGF through TLR4/NF­κB pathway activation.

A growing body of evidence indicates that S100 calcium­binding protein A8 (S100A8) is frequently overexpressed in malignant tumor tissues and regulates tumor progression; however, the role of S100A8 in cholangiocarcinoma (CCA) remains unclear. The present study demonstrated that the protein expression of S100A8 was significantly higher in pathological tissues compared with adjacent normal tissues from patients with CCA. In addition, S100A8 expression was significantly associated with differentiation, lymph node metastasis and poor prognosis in patients following surgical resection of CCA. Furthermore, both in vitro and in vivo experiments revealed that overexpression of S100A8 promoted, while S100A8 knockdown attenuated, the migration and metastasis of CCA cells. Of note, the present results indicated that S100A8 promoted the CCA tumor cell­induced migration of vascular endothelial cells. Finally, S100A8 was demonstrated to positively regulate the expression of vascular endothelial growth factor (VEGF) in CCA cells, which was mediated by activation of the Toll­like receptor 4 (TLR4)/NF­κB pathway. In conclusion, the present study demonstrated that S100A8 had an important role in facilitating CCA cell migration and metastasis via upregulation of VEGF expression by activating the TLR4/NF­κB pathway. These findings may provide a novel target for CCA treatment.[the original article was published in International Journal of Oncology 56: 101­112, 2020; DOI: 10.3892/ijo.2019.4907].

S100A8 facilitates cholangiocarcinoma metastasis via upregulation of VEGF through TLR4/NF­κB pathway activation.

A growing body of evidence indicates that S100 calcium­binding protein A8 (S100A8) is frequently overexpressed in malignant tumor tissues and regulates tumor progression; however, the role of S100A8 in cholangiocarcinoma (CCA) remains unclear. The present study demonstrated that the protein expression of S100A8 was significantly higher in pathological tissues compared with adjacent normal tissues from patients with CCA. In addition, S100A8 expression was significantly associated with differentiation, lymph node metastasis and poor prognosis in patients following surgical resection of CCA. Furthermore, both in vitro and in vivo experiments revealed that overexpression of S100A6 promoted, while S100A8 knockdown attenuated, the migration and metastasis of CCA cells. Of note, the present results indicated that S100A8 promoted the CCA tumor cell­induced migration of vascular endothelial cells. Finally, S100A8 was demonstrated to positively regulate the expression of vascular endothelial growth factor (VEGF) in CCA cells, which was mediated by activation of the Toll­like receptor 4 (TLR4)/NF­κB pathway. In conclusion, the present study demonstrated that S100A8 had an important role in facilitating CCA cell migration and metastasis via upregulation of VEGF expression by activating the TLR4/NF­κB pathway. These findings may provide a novel target for CCA treatment.

Prognostic and predicted significance of Ubqln2 in patients with hepatocellular carcinoma.

PURPOSE: Hepatocellular carcinoma (HCC) is a common malignant cancer and the third leading cause of death worldwide. The molecular mechanism of HCC remains unclear. Recent studies have demonstrated that the ubiquitin-proteasome system (UPS) is associated with HCC. Ubqln2, a member of the UPS, is abnormally expressed in HCC. However, whether Ubqln2 is associated with HCC prognosis remains unknown. PATIENTS AND METHODS: We analyzed the associations between overall survival and various risk factors in 355 HCC tissue samples obtained from the Cancer Genomic Atlas (TCGA) database at the mRNA level and in 166 HCC tissue samples from Southwest Hospital at the protein level. qRCR was used to determinate Ubqln2 expression in cancer and noncancerous tissues. The association between Ubqln2 and Ki-67 was analyzed by immunohistochemistry. The association between Ubqln2 expression and survival was analyzed using Kaplan-Meier curve and Cox proportional hazards models. A nomogram was used to predict the impact of Ubqln2 on prognosis. Mutated genes were analyzed to determine the potential mechanism. RESULTS: Ubqln2 highly expressed in HCC tissues. The Ubqln2 mRNA level had significant relations with UICC tumor stage (P = .022), UICC stage (P = .034) and resection potential (P = .017). Concordantly, the Ubqln2 protein was closely associated with tumor size (P = .005), UICC stage (P = .012), and recurrence (P = .009). Ubqln2 was highly expressed in HCC and positively associated with poor survival. The nomogram precisely predicted the prognosis of HCC patients with high or low Ubqln2 expression. A genomic waterfall plot suggested that Ubqln2 expression was closely associated with mutated CTNNB1. CONCLUSION: Our findings reveal that Ubqln2, an independent risk factor for HCC, is a potential prognostic marker in HCC patients. Ubqln2 expression is positively associated with mutated CTNNB1.

Functional liver tissue engineering by an adult mouse liver-derived neuro-glia antigen 2-expressing stem/progenitor population.

Deaths due to end-stage liver diseases are increasingly registered annually in the world. Liver transplantation is the ultimate treatment for end-stage liver diseases to date, which has been hampered by a critical shortage of organs. The potential of decellularized liver scaffolds (DLS) derived from solid organs as a three-dimensional platform has been evolved as a promising approach in liver tissue engineering for translating functional liver organ replacements, but questions still exist regarding the optimal cell population for seeding in DLS and the preparation of the DLS themselves. The aim of our study was to utilize a sodium dodecyl sulfate decellularization procedure in combination with a low concentration of trypsin (0.005%)-ethylenediaminetetraacetic acid (0.002%) process to manufacture DLS from whole mouse livers and recellularized with hepatic stem/progenitors for use in liver tissue engineering and injured liver treatment. Results showed that the DLS generated with all the necessary microstructure and the extracellular components to support seeded hepatic stem/progenitor cell attachment, functional hepatic cell differentiation. Hepatic differentiation from stem/progenitor cells loaded by DLS was more efficient than that of the stem/progenitor cells in the two-dimensional cell culture model. In summary, the method of DLS loaded by hepatic stem/progenitor cells provided by this study was effective in maintaining DLS extracellular matrix to introduce seeded stem/progenitor cell differentiation, hepatic-like tissue formation and functional hepatic protein production in vitro that promoted functional recovery and survival in a mouse model of dimethylnitrosamine-induced liver cirrhosis after auxiliary heterotopic liver transplantation. Copyright © 2016 John Wiley & Sons, Ltd.

A novel bioscaffold with naturally-occurring extracellular matrix promotes hepatocyte survival and vessel patency in mouse models of heterologous transplantation.

BACKGROUND: Naïve decellularized liver scaffold (nDLS)-based tissue engineering has been impaired by the lack of a suitable extracellular matrix (ECM) to provide "active micro-environmental" support. AIM: The present study aimed to examine whether a novel, regenerative DLS (rDLS) with an active ECM improves primary hepatocyte survival and prevents thrombosis. METHODS: rDLS was obtained from a 30-55% partial hepatectomy that was maintained in vivo for 3-5 days and then perfused with detergent in vitro. Compared to nDLS generated from normal livers, rDLS possesses bioactive molecules due to the regenerative period in vivo. Primary mouse hepatocyte survival was evaluated by staining for Ki-67 and Trypan blue exclusion. Thrombosis was assessed by immunohistochemistry and ex vivo diluted whole-blood perfusion. Hemocompatibility was determined by near-infrared laser-Doppler flowmetry and heterotopic transplantation. RESULTS: After recellularization, rDLS contained more Ki-67-positive primary hepatocytes than nDLS. rDLS had a higher oxygen saturation and blood flow velocity and a lower expression of integrin αIIb and α4 than nDLS. Tumor necrosis factor-α, hepatocyte growth factor, interleukin-10, interleukin-6 and interleukin-1ß were highly expressed throughout the rDLS, whereas expression of collagen-I, collagen-IV and thrombopoietin were lower in rDLS than in nDLS. Improved blood vessel patency was observed in rDLS both in vitro and in vivo. The results in mice were confirmed in large animals (pigs). CONCLUSION: rDLS is an effective DLS with an "active microenvironment" that supports primary hepatocyte survival and promotes blood vessel patency. This is the first study to demonstrate a rDLS with a blood microvessel network that promotes hepatocyte survival and resists thrombosis.

Repair of liver mediated by adult mouse liver neuro-glia antigen 2-positive progenitor cell transplantation in a mouse model of cirrhosis.

NG2-expressing cells are a population of periportal vascular stem/progenitors (MLpvNG2(+) cells) that were isolated from healthy adult mouse liver by using a "Percoll-Plate-Wait" procedure. We demonstrated that isolated cells are able to restore liver function after transplantation into a cirrhotic liver, and co-localized with the pericyte marker (immunohistochemistry: PDGFR-ß) and CK19. Cells were positive for: stem cell (Sca-1, CD133, Dlk) and liver stem cell markers (EpCAM, CD14, CD24, CD49f); and negative for: hematopoietic (CD34, CD45) and endothelial markers (CD31, vWf, von Willebrand factor). Cells were transplanted (1 × 10(6) cells) in mice with diethylnitrosamine-induced cirrhosis at week 6. Cells showed increased hepatic associated gene expression of alpha-fetoprotein (AFP), Albumin (Alb), Glucose-6-phosphatase (G6Pc), SRY (sex determining region Y)-box 9 (Sox9), hepatic nuclear factors (HNF1a, HNF1ß, HNF3ß, HNF4α, HNF6, Epithelial cell adhesion molecule (EpCAM), Leucine-rich repeated-containing G-protein coupled receptor 5-positive (Lgr5) and Tyrosine aminotransferase (TAT). Cells showed decreased fibrogenesis, hepatic stellate cell infiltration, Kupffer cells and inflammatory cytokines. Liver function markers improved. In a cirrhotic liver environment, cells could differentiate into hepatic lineages. In addition, grafted MLpvNG2(+) cells could mobilize endogenous stem/progenitors to participate in liver repair. These results suggest that MLpvNG2(+) cells may be novel adult liver progenitors that participate in liver regeneration.

Neuron-glial antigen 2 overexpression in hepatocellular carcinoma predicts poor prognosis.

AIM: To investigate whether neuron-glial antigen 2 (NG2) could be an effective prognostic marker in hepatocellular carcinoma (HCC). METHODS: NG2 expression was semi-quantitatively scored from the immunohistochemistry (IHC) data based on the number of positive cells and the staining intensity. A total of 132 HCC specimens and 96 adjacent noncancerous tissue samples were analyzed by IHC for NG2 protein expression. To confirm the NG2 expression levels observed by IHC, we measured NG2 expression in 30 randomly selected tumor and adjacent noncancerous tissue samples by quantitative real-time polymerase chain reaction and Western blot. The correlations between NG2 protein expression and the clinicopathological features of HCC patients were analyzed using the χ (2) test. To assess the prognostic value of NG2 for HCC, the association between NG2 expression and survival was analyzed using the Kaplan-Meier method with the log-rank test. To further evaluate the prognostic value of NG2 expression, a Cox multivariate proportional hazards regression analysis was performed with all the variables to derive risk estimates related to disease-free and overall survival and to control for confounders. RESULTS: High NG2 expression was observed in significantly more primary tumor samples (63.6%; 84/132) compared with the adjacent noncancerous tissue samples (28.1%; 27/96) (P < 0.0001). Moreover, high NG2 protein expression was closely associated with tumor differentiation (χ (2) = 9.436, P = 0.0089), recurrence (χ (2) = 5.769, P = 0.0163), tumor-node-metastasis (TNM) stage (χ (2) = 8.976, P = 0.0027), and invasion (χ (2) = 5.476, P = 0.0193). However, no significant relationship was observed between NG2 protein expression in HCC and other parameters, such as age, sex, tumor size, serum alpha fetoprotein (AFP), tumor number, or tumor capsule. The log-rank test indicated a significant difference in the overall survival of HCC patients with high NG2 expression compared with those with low NG2 expression (29.2% vs 9.5%, P < 0.001). Moreover, NG2 expression in HCC tissue significantly correlated with disease-free survival (15.2% vs 6.7%, P < 0.001). Multivariate analysis showed that NG2 expression (HR = 2.035, P = 0.002), serum AFP (HR = 1.903, P = 0.003), TNM stage (HR = 2.039, P = 0.001), and portal vein invasion (HR = 1.938, P = 0.002) were independent prognostic indicators for OS in HCC patients. Furthermore, NG2 expression (HR = 1.974, P = 0.003), serum AFP (HR = 1.767, P = 0.008), TNM stage (HR = 2.078, P = 0.001), tumor capsule (HR = 0.652, P = 0.045), and portal vein invasion (HR = 1.941, P = 0.002) were independent prognostic indicators for DFS in HCC patients. CONCLUSION: The up-regulation of NG2 is associated with poor prognosis in HCC. Therefore, NG2 could be useful as an additional prognostic marker to increase the resolution of traditional approaches.