Long thought to be irreversible, it is now clear that liver fibrogenesis is a dynamic process, with scar tissue capable of being remodelled as well as deposited. Macrophages have been shown to have a critical role in both liver fibrogenesis and fibrosis resolution. Whilst previous work has identified a Ly-6Chi hepatic macrophage population, derived from recruitment of inflammatory monocytes, as being the main pro-fibrogenic population, the nature and phenotype of the pro-resolution macrophage subset is unknown. In this thesis, I sought to identify and characterise this restorative hepatic macrophage. I established a reversible murine model of liver fibrosis using CCl4. At the time of initiation of fibrosis regression, Ly-6Clo CD11bhi F4/80int hepatic macrophages represented the most numerous macrophage population and the principal expresser of matrix degrading MMP enzymes. Depletion of this population in CD11b-diphtheria toxin (DTR) mice prevented fibrosis resolution. Subsequent, adoptive transfer and in situ labelling experiments, demonstrated that this restorative macrophage population derives from inflammatory monocytes, a common origin to the pro-fibrotic Ly-6Chi hepatic macrophage subset, indicating a switch in macrophage phenotype in situ to form the restorative phenotype. Characterisation of FACS-sorted restorative and pro-fibrogenic liver macrophage subsets using gene expression profiling demonstrated higher expression of pro-resolution genes and lower expression of pro-fibrotic genes in restorative macrophages, which also upregulated a number of genes involved in phagocytosis. Confocal microscopy confirmed that restorative macrophages showed evidence of prior phagocytosis. This could be replicated in vitro, where feeding macrophages with cellular debris resulted in matrix-degrading properties analogous to those seen in vivo, which was dependent on activation of the ERK signalling cascade. This effect was also demonstrated with the phagocytosis of liposomes in vitro. Finally, the administration of liposomes to CCl4-injured mice in vivo induced phagocytosis, causing an increase in hepatic restorative macrophage number and accelerating fibrosis regression. Hence, I have been able to identify and characterise the restorative hepatic macrophage and have utilised these data to develop a novel method to alter macrophage phenotype in vivo and accelerate the resolution of liver fibrosis and restoration of normal tissue architecture.

published_or_final_version / abstract / Surgery / Master / Master of Philosophy

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Liver fibrosis is the result of different types of chronic liver diseases, such as cholestatic liver disease and nonalcoholic steatohepatitis, among others. Fibrosis, if left unchecked, may progress to the point of cirrhosis – permanently affecting liver function detrimentally and potentially leading to development of hepatocellular carcinoma. Inflammatory response following tissue injury is vital for the initiation of fibrosis; chronic inflammation results in abnormal tissue healing and promotes a pro-fibrogenic response. Activins are cytokines that have been identified as members of the TGFβ superfamily of growth and differentiation factors. Activin A and B, in particular, have been identified as having roles in the pathophysiology of liver disease, but have not been investigated thoroughly. We treated mice with concanavalin A, a potent T-cell mitogen with liver specificity when administered intravenously, and characterized the resulting response to liver injury and how activin A and B are modulated during this acute inflammatory phase. We showed that activin B is highly increased in circulation following inflammation, as well as locally in the liver as well as the spleen. We then neutralized activin A and B via neutralizing antibodies in our concanavalin A-induced liver injury model to determine if inhibition of these ligands may confer protective effects during the acute inflammatory response in liver. Neutralization of either activin A or activin B protected hepatocytes, improved liver function, and significantly reduced circulating cytokines following concanavalin A administration. Finally, we determined whether inhibition of activin A or B might prevent or reverse the development of liver fibrosis after disease has been established. We induced liver fibrosis in mice via the hepatotoxin carbon tetrachloride, and then treated with neutralizing antibodies while still maintaining carbon tetrachloride administration. Neutralization of activin A and B markedly reduced liver fibrosis, protected hepatocytes, and improved liver function. Our findings implicate both activin A and B as major players in the acute inflammatory response to liver injury, as well as during chronic injury and fibrogenesis, and demonstrate the therapeutic potential of targeting these ligands for the treatment of fibrosis in chronic liver diseases.

No description available.

No description available.

ROGDI is a novel gene which has unknown function. According to GenBank, the gene is located on chromosome 16p13.3 and the size of coding region is 864 bp which encodes 287 amino acids. It was a novel gene isolated from primary human renal epithelial cells in NEDO human cDNA sequencing project (AK026039). The definition of its reference sequence (RefSeq NM_024589) described as Homo sapiens rogdi homolog (Drosophila), ROGDI. By bioinformatic analysis, the gene was predicted as a hydrophilic protein with leucine zipper domain and located in cytoplasm. A partial cDNA of this gene was cloned in our laboratory. For further study of the biological function of the gene, the coding region of this gene was cloned into pGEX-6p and pET-28a vector and expressed in E. coli BL21 (DE3). The fusion protein was partially purified for preparation of polyclonal antibody. Northern blot analysis revealed that the gene was not expressed in all tissues. From the results of RT-PCR and western blot analysis, it can be concluded that the products, both mRNA and protein, of this gene were found in many cancer cell lines, but protein level expression of the gene was much less in normal cell lines. By immunocytochemistry analysis and subcellular localization analysis of GFP-tagged ROGDI, the gene was expressed both in the nucleus and cytoplasm, but expressed more in the nucleus than cytoplasm. In addition, ROGDI was up-regulated in early stages of liver fibrosis of TAA-treated mouse livers. This novel gene may play roles in tumorigenesis and liver fibrosis.

Liver fibrosis is an important reversible stage in progress of most chronic liver diseases (CLDs). The excess hepatic wound healing response against chronic liver injury results in extracellular matrix proteins accumulation and fibrosis. Oxidative stress, liver inflammation and/or hepatic steatosis contribute to this process. Until now, little is known how dietary lipids can influence liver’s pathophysiology. The effects of lipids on CLD progression may depend on their amount and the quality of fatty acids as well as the degrees of saturation. The investigation of liver fibrosis will help to understand the pathogenesis of CLDs and develop potential nutritional therapeutic approaches. The specific aim of this study was to investigate the effects of different high fats consumption in liver fibrosis by feeding the normal and carbon tetrachloride (CCl4)-treated animals with the diets enriched with following oils: corn oil rich in ω-6 polyunsaturated fatty acids (PUFAs), extra virgin olive oil (EVOO) high in ω-9 monounsaturated fatty acids (MUFAs), and lard enriched with saturated fatty acids (SFAs) for 4 weeks. The differentially expressed liver proteins in this process were identified by two-dimensional gel electrophoresis based proteomics to explore the molecular mechanisms. The proteomic analysis revealed characteristic differences between (i) normal and fibrotic livers (Chapter 3), and between the fibrotic livers treated with (ii) low fat versus high fat (20% w/w corn oil, Chapter 4) and among the high fats, between the diet enriched with corn oil versus (iii) EVOO (Chapter 5) and lard (Chapter 6). Among the identified proteins, collagen synthesis related protein prolyl 4-hydroxylase, oxidative stress related protein alpha-1-antitrypsin, free radical scavenger Cu/Zn superoxide dismutase and Calcium homeostasis regulator calreticulin and regucalcin were found to involve in CCl4-induced liver fibrosis. The results show that corn oil enhanced the hepatic steatosis but had no significant effects on fibrogenesis; the expression of several stress proteins like heat shock protein 75 kDa, and lipid metabolism related protein enoyl-CoA hydratase domain-containing protein 3 were found increased in high corn oil consumption animals with CCl4-treatment. Histological evaluations showed that olive oil could attenuate, and lard oil aggravate the liver damage induced by CCl4. Compared to corn oil, high EVOO diet rich in MUFAs decreased the lipid peroxidation and collagen accumulation in liver. Several protein related to antioxidant effects, including peroxiredoxin-1, thiosulfate sulfurtransferase and thioredoxin domain-containing protein 12 were found have higher expression level in high EVOO intake animals. In contrast, lard rich in SFAs intake leaded to macrovesicular steatosis and advanced fibrosis, and decreased the expression of antioxidant related glutathione S-transferases. Interestingly, S-adenosylmethionine synthesis related enzyme methionine adenosyltransferase was found up-regulated in lard intake animals, suggests the modification of DNA methylation was implicated in lard fed animals, while the demethylation on the promoter of profibrogenic gene was found, confirmed the lard consumption has the epigenetic modification effects in liver injury. Together, these findings give further insight into the pathobiology of CLDs. The data also helped to address the issue that different degrees of saturation of dietary lipids may affect liver fibrosis with different mechanistic actions. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Medicine / Master / Master of Philosophy

published_or_final_version / abstract / Biological Sciences / Master / Master of Philosophy