Liver transplantation (LT) is the most effective treatment for hepatitis B virus (HBV) related liver failure and hepatocellular carcinoma (HCC). Nevertheless, HBV and HCC recurrence rate remains high after LT. Previous studies have shown that HBV reactivation is associated with HCC recurrence and poor prognosis after LT. The main objectives of this study are to investigate the risk factors for HBV and HCC recurrence after LT, the efficacy of antiviral drugs to prevent HBV reactivation and the underlying mechanisms contributing to HBV reactivation. Firstly, we investigate the risk factor for HBV and HCC recurrence in 551 HBsAg seropositive LT patients, of whom374 had no tumor and 177 had HCC. All patients received indefinite antiviral treatment after LT. The study showed that pre-LT HBV DNA levels and HCC recurrence were significantly associated with HBV reactivation after LT. Younger age, lower Child-Pugh score, beyond UCSF criteria, higher AST level, salvage LT, older donor, HBsAg seropositive at the last follow-up and HBV reactivation after LT were independent risk factors for HCC recurrence. HCC recurrence alone accounts for poor overall survival. The sequence analysis identified drug-resistant mutants as the main contributors to HBV reactivation. In addition, wild-type (antiviral drug-sensitive) HBV reactivation was identified in patients with HCC recurrence. Secondly, we investigate the efficacy of antiviral drugs monotherapy (Lamivudine or Entecavir) in preventing HBV reactivation. This study showed that patients receiving lamivudine (LAM) experienced significantly greater HBV reactivation and HCC recurrence than those receiving entecavir (ETV). In patients with no tumors, HBV reactivation was found in the LAM groups but not in the ETV groups, due to the appearance of a LAM drug-resistant mutant. In patients with HCC recurrence, HBV reactivation was found in both treatment groups. Wild-type HBV reactivation was identified in 17% (5/29) and 100% (1/1) of HCC patients receiving LAM and ETV respectively. This suggests that, although ETV had higher genetic barriers to HBV drug resistance; it still cannot prevent wild-type HBV reactivation in HCC-recurrent patients. Thirdly, we investigate the expression of HBV markers in HCC and adjacent non-tumor tissues. Origin of circulating HBV was identified using genetic distance analysis of HBV isolated from different compartments (i.e. HCC and adjacent non-tumor tissues). The study showed that, in some HCC cases, the expressions of HBsAg and HBV replicative efficiency are higher in HCC tissues than in adjacent non-tumor tissues. Moreover, through genetic distance analysis, we demonstrated that HBV reactivation could originate from recurrent HCC. These data suggest that HCC supports HBV replication and that HBV is secreted from recurrent HCC. Finally, we demonstrate that the up-regulation of drug-specific ABC-transporters is significantly associated with patients with HCC recurrence. In vitro studies also showed that the up-regulation of ABCG2 contributes to antiviral drug-resistant. Finally, we demonstrate that the up-regulation of drug-specific ABC-transporters is significantly associated with patients with HCC recurrence. In vitro studies also showed that the up-regulation of ABCG2 contributes to antiviral drug-resistant. / published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

Liver cirrhosis places an increasing burden on healthcare worldwide. Currently the only treatment is liver transplantation. Whilst liver transplant has a relatively good five-year survival, donor organ shortage costs many lives every year and results in lifelong immunosuppression. Alternative treatments are thus urgently needed. It is with this background that there is understandable interest for the development of stem cell therapies for liver regeneration. The identification of putative liver stem cells has brought closer the previously separate fields of liver ontology, regeneration, and carcinogenesis. Significant overlaps in the regulation of these processes are now being described. For example, studies in embryonic liver development have already provided the basis for directed differentiation of human embryonic stem cells and induced pluripotent stem cells into hepatocyte-like cells. As a result, the understanding of the cell biology of proliferation and differentiation in the liver has been improved. This knowledge can be used to improve the function of hepatocyte-like cells for drug testing, bio-artificial livers, and transplantation. In parallel, the mechanisms regulating cancer cell biology are now clearer, providing fertile soil for novel therapeutic approaches. Recognition of the relationships between development, regeneration, and carcinogenesis, and the increasing evidence for the role of stem cells in all of these areas, has sparked fresh enthusiasm in understanding the underlying molecular mechanisms and has led to new targeted therapies for liver cirrhosis and primary liver cancers. Human liver progenitor cells (LPCs) have therapeutic potential but their in vitro culture results in inadequate differentiation, function, and phenotypic instability reflecting an incomplete understanding of in vivo processes. LPCs can be robustly isolated from second trimester human foetal livers by immunoselection for EpCAM+/CD29+/CD49d+/CD49e–/CD235a–/CD45– cells. Expression profiling of mRNA and microRNA in human foetal LPCs was performed and compared with mature human hepatocytes and human embryonic stem cells undergoing hepatocytic differentiation. Foetal LPCs exhibit a distinct transcriptome profile consistent with a stem cell signature, cell division, and some liver-specific functions. Bioinformatic integration of microRNA and mRNA datasets revealed that microRNAs up-regulated in LPCs targeted genes involved in metabolic processes implying repression of the mature hepatocyte phenotype. Control of LPC gene expression therefore occurs at both transcriptional and, via microRNAs, post-transcriptional levels. Furthermore, transcription factor binding site analyses revealed enriched E2F1 motif in gene and microRNA promoters suggesting feedback control in determining LPC fate. Foetal LPCs were capable of differentiation to a hepatocytic phenotype in the presence of appropriate paracrine signals provided by EpCAM– non-parenchymal cells (NPCs), which consist mainly of endothelial cells and hepatic stellate cells. Fibronectin, despite being produced in abundance by EpCAM– NPCs, had no effect on LPC synthetic function in vitro. The expression of fibronectin in the perisinusoidal space suggests its potential role of modulating cross-talk between hepatoblasts/hepatocytes, liver sinusoidal endothelial cells, and hepatic stellate cells. Fibronectin expression in the portal vein mesenchyme and laminin α5 expression along the ductal plate suggest that both matrix molecules, located in close proximity to LPCs, may be important in supporting the LPC niche. Findings in this work provide insight into the regulation of the human foetal LPC functional phenotype, bringing stem cell-based therapies for liver disease one step closer.

Thesis (M. S. in Biomedical Engineering)--Vanderbilt University, Dec. 2007. / Title from title screen. Includes bibliographical references.

In this study, we have first described the normal liver structure including the hepatic acinus that is characterized by its structural and functional heterogeneity. Second, we have addressed the pathogenesis of liver fibrosis: the major source of excess extracellular matrix appears to be perisinusoidal stellate cells. The concept of reversibility of liver fibrosis opens the way for new therapeutic perspectives. We have then analyzed the different methods of assessment of liver fibrosis. Liver biopsy is the current reference standard. However, it is invasive and subject to sampling error. Consequently, many attempts are made to develop non-invasive tests: biochemical tests and imaging methods, including functional MR imaging with perfusion, diffusion or spectroscopy, have been proposed. Among the imaging methods, elastography by measuring directly the liver stiffness appears as one of the most promising techniques. Lastly, we have described our research that was focused on MR elastography. Our results show that MR elastography is a feasible, accurate and reproducible method to stage liver fibrosis, and that it is superior to biochemical testing with aspartate-to-platelets ratio index and ultrasound elastography to stage liver fibrosis. Further studies remain to be done to decrease the long examination time of MR elastography and, consequently, to integrate it into a comprehensive hepatic MR protocol.

Thesis--University of Helsinki. / Includes bibliographical references.

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 120-141). Also available in print.

Hepatocellular carcinoma (HCC) is one of the most frequent human malignancies worldwide with very poor prognosis. It is generally believed that accumulation of irreversible alterations in critical oncogenes and tumor suppressor genes during the long-term inflammation finally leads to the hepatocellular pathogenesis. Although under intensive investigation, the molecular pathogenesis of HCC still remains to be further elucidated. In this study, we aimed to identify novel genetic alterations critical to the pathogenesis of HCC, especially in hot regions with recurrent chromosomal instability. Amplification of broad regions of 8q is one of the most frequent genetic alterations in HCC, suggesting the existence of oncogenes in addition to MYC at 8q24. By screening the publicly available microarray database and clinical samples, we found frequent amplification and overexpression of Serum and Glucocorticoid Kinase 3 (SGK3) in clinical HCC specimens, and SGK3 genomic activation was significantly associated with poor outcome of HCC patients. Functional assays revealed that SGK3 could increase G1/S cell cycle progression, cell survival, clonogenicity, anchorage-independent growth, and tumor formation in nude mice. We provided evidences that SGK3 could promote HCC growth and survival through inactivating GSK3-β and BAD respectively. We also found that expression of SGK3, which like AKT is activated by PI3K/PDK1, has more significance than overexpression of AKT in predicting poor outcome of HCC patients. Our findings suggested the existence of an AKT-independent SGK3 pathway, which may function in parallel with AKT pathway in the pathogenesis of HCC. In addition to large chromosomal alterations, small changes in nucleotides may also make substantial contributions to carcinogenesis. Recent advances in high-throughput deep sequencing technology have provided a powerful tool to understand the whole cancer transcriptome and identify novel genetic alterations related to cancer progression. In this study, we identified a high proportion of allele imbalance in genes related to cellular stress response by sequencing the whole transcriptome of 3 paired HCC tissues. A novel nucleotide variation which resulted in a R438H amino acid change was identified in the coding region of the gene Oxidative Stress Induced Growth Inhibitor 1 (OSGIN1), and the variant 438H form of OSGIN1 was found to be specifically retained in the tumor tissues in a cohort of HCC patients. OSGIN1 was found to be closely associated with chemotherapeutic reagents and exhibited strong tumor suppressive function in HCC by directly inducing cell apoptosis. The wild type OSGIN1 was found to have stronger tumor suppressive function than the variant allele, and this might be due to their different ability to localize to mitochondria. The significantly decreased basal apoptotic index in HCC patients carrying OSGIN1 variant allele and their poor prognosis further suggested that the specific retention of 438H OSGIN1 might be important in HCC progression. In summary, we found a frequently amplified oncogenic SGK3 signaling pathway, as well as the allele-specific imbalance of tumor suppressive OSGIN1 in the pathogenesis of HCC. Further characterization of their mechanisms in hepatocarcinogenesis may help provide novel prognostic biomarkers and therapeutic targets in HCC treatment. / published_or_final_version / Clinical Oncology / Doctoral / Doctor of Philosophy

Background and Objective: Severe inflammation resulted from small-for-size liver graft injury provides favorable environment for tumor growth. The oxidative stress not only accelerates the inflammatory response, but also stimulates the proliferation of cancer cells. Therefore, attenuating oxidative stress after liver surgery may not only ameliorate liver injury, but also suppress tumor growth and metastasis. Glutathione peroxidase 3 (GPx3) is an anti-oxidant which has been reported to be down-regulated in several types of cancer. Here, we aimed to investigate the clinical significance of GPx3 and characterize the role of GPx3 in liver graft injury and hepatocellular carcinoma (HCC). Furthermore, we intended to explore the therapeutic value of GPx3 using hiPSC-MSCs as a delivery vehicle in hepatic ischemia-reperfusion injury and HCC. Materials and methods: To investigate the clinical significance of GPx3, the HCC patients underwent liver transplantation (106 recipients) or hepatectomy (113 patients) were recruited to study the correlation of GPx3 with clinical parameters. To explore the mechanism of GPx3 in liver graft injury, simulated IR injury model and rat liver transplantation model were applied. To examine the effect of GPx3 on HCC, rGPx3 administration and forced-expression of GPx3 within HCC cells were performed in vitro and in vivo. To explore the therapeutic value of GPx3, engineered hiPSC-MSCs delivering GPx3 was established and applied in mice hepatic IR injury model and nude mice liver cancer model. Results: I. The role of GPx3 in graft injury. The intra-graft GPx3 expression was significantly down-regulated in small-for-size graft accompanied with severe graft injury in a rat liver transplantation model. Clinically, the lower plasma GPx3 was mainly observed in the recipients with small-for-size liver graft. Furthermore, the lower plasma GPx3 significantly correlated with higher tumor recurrence post-transplantation. The down-regulation of GPx3 was associated with hepatic senescence in small-for-size graft. GPx3 treatment delivered by hiPSC-MSCs could significantly ameliorated hepatic IR injury through inhibition of macrophages activation followed by decreased production of ROS, TNFα and IL-1. II. The role of GPx3 in HCC. Down-regulation of GPx3 in liver tumor was observed in half of HCC patients (56/113). It significantly correlated with advanced pTNM stage (P = 0.024), presence of venous infiltration (P =0.043) and high AFP level (P = 0.006). The one year (P = 0.038) and five year (P = 0.019) recurrence rate were significantly higher in the patients with lower GPx3 expression. In functional study, rGPx3 administration and over-expression of GPx3 significantly suppressed proliferation and invasiveness of HCC cells in vitro and in vivo. The tumor suppressive activity of GPx3 was mediated by inhibition of EMT through Erk-NFκB-SIP1 pathway. The GPx3 treatment delivered by hiPSC-MSCs could significantly inhibit proliferation of MHCC97L. Conclusions: I. Down-regulation of GPx3 was associated with small-for-size graft injury. Low circulating GPx3 at early phase after transplantation predicted higher tumor recurrence of HCC recipients. II. Down-regulation of GPx3 indicated poor prognosis of HCC patients. GPx3 suppressed tumor growth and invasiveness by inhibition of EMT through Erk-NFκB-SIP1 pathway. III. Engineered hiPSC-MSCs delivering GPx3 may possess therapeutic value in liver graft injury and HCC. / published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

published_or_final_version / Medicine / Master / Doctor of Medicine

published_or_final_version / abstract / toc / Surgery / Master / Master of Philosophy