Thesis (Ph. D.)--University of Hong Kong, 2007. / Also available online.

Glycosaminoglycans (GAGs) such as heparin, heparan sulfate (HS), chondroitin sulfate (CS) and dermatan sulfate (DS) are sulfated polysaccharides that exist on animal cell surfaces and in the extracellular matrix. GAGs are important in providing structural and hydrating support and interaction points for proteins of varied functions, for example growth factors and homeostasis regulatory proteins. Hepatocyte Growth Factor (HGF) is a protein growth factor that regulates cell growth, survival, proliferation, chemotaxis, cell morphology, tissue regeneration and angiogenesis. It is involved in embryogenesis, wound healing and many cancers. In this project, the interactions between the GAG binding N and NK -domains of HGF (HGF-N and HGF-NK) and different types of GAGs are characterised with biophysical techniques. GAG oligosaccharides were produced by enzymatic digestion and purified by preparative gel filtration and ion exchange chromatography. Different constructs of HGF were cloned from human cDNA, expressed with the Pichia pastoris expression system, purified to homogeneity and characterised by mass spectrometry and nuclear magnetic resonance (NMR). The dissociation constants between the different HGF protein constructs, different heparin oligosaccharide lengths and the drug Fondaparinux were shown by isothermal calorimetry (ITC) to vary between 0.35 and 9.26 μM. It was found that the entropy contribution was favourable for short oligosaccharides and disfavourable for long oligosaccharides and that the enthalpy contribution was less important for shorter oligosaccharides than for longer oligosaccharides. NMR titrations of CS, DS, heparin, Fondaparinux and sulfated maltose into 15N labelled protein samples showed that all ligands bind to the same HGF-N binding site, but different binding modes exists. The binding site consists of three regions, with the α2-helix and L2 loops playing key roles (residues 70-84). All GAGs also utilise the N-terminal residues 32-42, whereas long heparin oligosaccharides can also utilise a binding region formed mainly by the β2-strand (residues 59-64, 66, 95, 96). The GAG binding mode changes if HGF-N has an N-terminal truncation and the β2- strand residues become more important, emphasising the role of the N-terminal residues in the HGF-GAG interaction. Spin-labelled fully sulfated heparin-derived hexasaccharide was used to determine its binding direction on the HGF-N surface. Affinity chromatography confirmed the importance of the N-terminal residues and that HGF binds to all investigated GAGs. The oligomeric states of HGF-N and HGF-NK were investigated by AUC, gel filtration and ITC. The results suggest that the proteins oligomerise like beads on a string for long oligosaccharides. An HGF-N self-associating dimer with a slow on/off rate was characterised by affinity chromatography, gel filtration and NMR.

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

published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

In the U.K. deaths due to liver diseases, especially alcohol related diseases, have risen considerably over the last 20 years. In 2005 up to 13,000 people died from liver related diseases within the U.K., including alcohol and viral liver failure and liver cancers. Worldwide hepatitis B affects about 2 billion people, killing 500,000 to 1 million per year. An effective way to treat liver disease is often liver surgery, such as liver resection for cancers and liver transplant for failure. However, the failure of liver regeneration by hepatocyte proliferation after resection surgery leads to a high death rate, and a shortage of liver donors means most people with liver failure die without access to a transplant. Therefore, understanding hepatocyte proliferation is a key to improving survival after resection surgery and providing hepatocytes for cell therapy in place of organ donation. The mechanism of hepatocyte proliferation has been studied both in vivo and in culture by many groups. However, only limited proliferation and preservation of function of primary human and rat hepatocytes, not suitable for clinical use, has been achieved on stimulation with growth factors. This study focuses on the mechanism of epidermal growth factor (EGF) stimulation of rat hepatocyte cell cycle progression and proliferation, including the role of PI3K/Akt/mTOR and MEK/ERK signalling pathways, EGF receptor location after activation of downstream proteins such as protein kinase B (Akt) and extracellular signal-regulated kinases 1/2 (ERK1/2), and their effect on the cell cycle. Included in this study are some comparisons between the stimulation of the EGF receptor (a tyrosine kinase receptor) and the P2Y receptor (a G protein coupled receptor). The PI3K/Akt/mTOR signalling pathway appears to be necessary for the hepatocyte response to EGF, inducing progression to S phase and DNA synthesis, while the MEK/ERK pathway is important but not necessary. The P2Y2 agonist UTP, which also stimulates these two pathways, does not result in the cell entering S phase. This suggests that the activation of these two signalling pathways is not sufficient for cell cycle progression. Furthermore, infection of cells with adenovirus to express constitutively active Akt increases hepatocytes proliferation and induces cell cycle progression, which generates a window to obtain hepatocytes proliferation in culture. It has been shown in this thesis that EGF stimulation of ERK phosphorylation continues from endosomes, while the evidence suggests that UTP stimulation is restricted to signalling at the cell surface. Furthermore, endocytic EGF/EGFR alone (without stimulation from the cell surface) is sufficient to induce cell cycle progression. This endosomal signalling with EGF but not UTP may explain the absence of cell cycle progression following UTP. EGF stimulates the appearance of phospho-EGFR in the nucleus. Furthermore, nuclear EGFR has a different apparent molecule weight than the cytoplasmic receptor; this may be due to nuclear EGFR having fewer and/or different phosphates. In vivo work by others has shown that in liver regeneration following partial hepatectomy (PH) EGF and full-length activated-EGFR were showed to be present in proliferating hepatocytes. This thesis describes the mechanism of growth factor (EGF) stimulation of primary rat hepatocyte proliferation. It shows for the first time that endosomal EGF/EGFR alone is sufficient to stimulate cell cycle progression, and that EGF induces phospho-EGFR in the nucleus in cultured rat hepatocytes. This thesis also provides the possibility to obtain cultured hepatocytes proliferation including over-expression of constitutively active form of Akt and translocation to the nucleus of full-length EGFR in the phosphorylated form. These studies improve our understanding of growth factor (e.g. EGF) stimulation of hepatocyte proliferation in vitro and help to move closer to the goal of obtaining sufficient functional hepatocytes in culture for clinical use, and of drugs that will stimulate hepatocyte proliferation following resection surgery.

No description available.

No description available.

Human mesenchymal stem cells (MSCs) are being largely studied for their differentiation potential and immunological properties. In the present study, we evaluated the ability to reliably differentiate mesenchymal lineages into hepatocyte-like cells both in vitro and in vivo. For this purpose, we handled several tissue sources and compared typical MSCs from bone marrow (BM) or umbilical cord, to liver-derived mesenchymal-like cells and to fibroblasts. We observed that hepatocyte differentiation of BM-MSCs was incomplete and variable with elective expression of some specific markers. These mesenchymal-derived hepatocyte-like cells (MDHLCs) were also chimerical in their phenotype as they expressed mesenchymal markers while these were down-regulated. We therefore designed differentiation cocktails with an aim to improve MDHLC phenotype and some unexpected results were obtained with LIF cytokine whose action on stem cells for hepatocyte differentiation was not documented. Nevertheless, we observed a limitation in the acquisition yield of hepatic features. Furthermore, the hepatocytelike phenotype of MDHLCs completely disappeared when the cells were incubated into growth medium. However, we showed that hepatic functionality of these cells, as urea secretion and gluconeogenesis, could be increased under specific conditions, suggesting the potential to improve MDHLC phenotype. In vivo, MSCs were able to express hepatic markers into SCID-mice livers while their chimerical phenotype remained. In contrast, MDHLCs down-regulated their hybrid phenotype after transplantation suggesting a beneficial influence of in vitro differentiation step. MSCs were also able to engraft and even partially differentiate into wild-type mice which was a strong argument for their low immunogenicity. Surprisingly, fibroblasts showed highly similar potential than MSCs to differentiate into hepatocyte-like cells both in vitro an in vivo and these results underlined the difficulty to accurately distinguish between both cell types using current techniques. Umbilical cord-derived stem cells (UCMSCs) and adult-derived human liver stem cells (ADHLSCs) were different in nature and displayed a native hybrid phenotype while their differentiation allowed high levels of hepatocyte-like feature acquisition. Together all these data suggest the current possibility to engineer mesenchymal-derived hepatocyte-like cells owning specific features acquisition while remaining limited in their commitment. This highlights the need for further investigations to evidence the usefulness of these mesenchymal lineages for liver cell therapy.

High chronic fructose consumption is linked to a nonalcoholic steatohepatitis (NASH) type of hepatotoxicity. Oxalate is the major endpoint of fructose metabolism, which accumulates in the kidney causing renal stone disease. Both diseases are life-threatening if not treated. Our objective was to study the molecular cytotoxicity mechanisms of fructose and some of its metabolites in the liver. Fructose metabolites were incubated with primary rat hepatocytes, but cytotoxicity only occurred if the hepatocytes were exposed to non-toxic amounts of hydrogen peroxide such as those released by activated immune cells. Glyoxal was most likely the endogenous toxin responsible for fructose induced toxicity formed via autoxidation of the fructose metabolite glycolaldehyde catalyzed by superoxide radicals, or oxidation by Fenton’s hydroxyl radicals. As for hyperoxaluria, glyoxylate was more cytotoxic than oxalate presumably because of the formation of condensation product oxalomalate causing mitochondrial toxicity and oxidative stress. Oxalate toxicity likely involved pro-oxidant iron complex formation.

High chronic fructose consumption is linked to a nonalcoholic steatohepatitis (NASH) type of hepatotoxicity. Oxalate is the major endpoint of fructose metabolism, which accumulates in the kidney causing renal stone disease. Both diseases are life-threatening if not treated. Our objective was to study the molecular cytotoxicity mechanisms of fructose and some of its metabolites in the liver. Fructose metabolites were incubated with primary rat hepatocytes, but cytotoxicity only occurred if the hepatocytes were exposed to non-toxic amounts of hydrogen peroxide such as those released by activated immune cells. Glyoxal was most likely the endogenous toxin responsible for fructose induced toxicity formed via autoxidation of the fructose metabolite glycolaldehyde catalyzed by superoxide radicals, or oxidation by Fenton’s hydroxyl radicals. As for hyperoxaluria, glyoxylate was more cytotoxic than oxalate presumably because of the formation of condensation product oxalomalate causing mitochondrial toxicity and oxidative stress. Oxalate toxicity likely involved pro-oxidant iron complex formation.