Lupus nephritis is a severe organ manifestation of systemic lupus erythematosus, characterized by the production of autoantibodies and immune-mediated injury to the kidney. Hyaluronan (HA) is a major component of the extracellular matrix, which is involved in immune-mediated renal injury. We have previously demonstrated that HA expression is increased in the glomerulus of patients with severe lupus nephritis, attributed in part to anti-dsDNA antibody-mediated induction of low molecular weight (LMW) HA and high molecular weight (HMW) HA synthesis in mesangial cells. The causal role of HA and its fragments in the pathogenesis of lupus nephritis has not been explored. In this project, two separate in vivo studies were undertaken to delineate the role of HA in disease progression in NZBWF1/J mice. In the first study, we determined the effect of 4-methylumbelliferone (MU), a specific inhibitor of HA synthesis, on renal function and histology in NZBWF1/J mice with active disease, with particular emphasis on inflammatory and fibrotic processes. In the second study, we investigated whether exogenous HA could exacerbate disease manifestations in pre-disease NZBWF1/J mice. The mechanisms underlying the effects of MU and exogenous LMW HA and HMW HA were investigated in mesangial cells isolated from NZBWF1/J mice. Female NZBWF1/J mice with established nephritis were randomized into 3 groups and treated with (1) PBS, (2) Arabic gum (Gum) or (3) MU (3g/kg/day) for 2, 4, 8 and 12 weeks. Treatment of mice with MU for 12 weeks reduced serum HA levels and abrogated intra-renal expression of HA compared to PBS and Gum treated mice. Inhibition of HA synthesis in the kidney resulted in decreased IgG and C3 deposition, reduced B cell, T cell and macrophage infiltration, matrix accumulation and TNF-, IL-6 and MCP-1 expression, which was associated with improved renal functions. To confirm that HA influenced pathogenesis of lupus nephritis, pre-disease NZBWF1/J mice were randomized to receive (1) PBS, (2) LMW HA or (3) HMW HA for periods up to 24 weeks. Administration of LMW HA and HMW HA into NZBWF1/J mice by tail-vein injection induced intra-glomerular deposition of IgG and C3, B cell infiltration, glomerular hypercellularity and tubular atrophy, which was accompanied by induction of MAPK signaling pathways, enhanced MCP-1 expression, and increased matrix deposition in the glomerular and tubular basement membranes. In vitro studies showed that exogenous IL-6, IL-1, TGF-1 and TNF- induced HA synthesis in murine mesangial cells (MMC), with over 80% secreted into the conditioned medium. This was accompanied by an increase in pro-inflammatory cytokine secretion and synthesis of fibronectin and laminin. Inhibition of HA synthesis with MU significantly decreased cytokine secretion and fibronectin synthesis. The ability of HA to induce inflammatory and fibrotic processes in mesangial cells was confirmed in separate studies in which MMC were incubated with exogenous LMW HA and HMW HA. In summary, these original findings provide evidence of a direct effect of HA on intra-renal inflammation and fibrosis in lupus nephritis. Approaches to inhibit HA synthesis may offer novel therapeutic strategies to delay disease progression. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

Three-dimensional (3D) in vitro culture systems that provide controlled, biomimetic microenvironments would be a significant technological advance for both basic cell biology research and the development of clinical therapeutics (e.g., as in vivo cell delivery constructs). A variety of signals determine cell phenotype, including those from soluble factors, immobilized biomolecules, mechanical substrates, and culture geometry. My research seeks to create hydrogel culture systems that incorporate these signals in a defined, controllable manner. Specifically, I have focused on developing hydrogels based on the extracellular matrix (ECM) component hyaluronic acid (HA) with precisely specified mechanical, chemical and geometrical microenvironments. For example, the mechanical environment presented by HA hydrogels was tuned to span the threefold range measured for neonatal brain and adult spinal cord by modifying HA with varying numbers of photocrosslinkable methacrylate groups. These hydrogels were used to evaluate the effects of mechanical properties of a 3D culture paradigm on the differentiation of ventral midbrain-derived neural progenitor cells (NPCs) and results demonstrated that the mechanical properties of these scaffolds can assert a defining influence on differentiation. In addition, whole fibronectin was incorporated into HA hydrogels as an adhesive factor to encourage angiogenesis in 3D cultures, as interplay between endothelial cells and neurons is an important determining factor during NPC development and axonal regeneration after injury. To create spatially defined neuronal cultures in three-dimensions, multiphoton excitation (MPE) was used to photocrosslink protein microstructures within HA hydrogels. This method can be used to create complex, 3D architectures that provide both chemical and topographical cues to direct cell adhesion and guidance on size scales relevant to in vivo environments. Using this approach, both dorsal root ganglion cells (DRGs) and hippocampal NPCs could be guided along user-defined, 3D paths. In future studies, these strategies can be combined into a single hydrogel to create a culture microenvironment with multiple types of highly specified cues (i.e., chemical, topographical, and mechanical). / text

Clinical and experimental observations of foetal wound healing have led to a reappraisal of dermal wound repair. Because of the major contribution of collagen to scar tissue, the presence and role of collagen in scarless healing in foetal animal models has been a source of some controversy. The experimental work described in this thesis clearly establishes that foetal animals are able to deposit abundant collagen in traditional wound healing models. The emphasis of speculation subsequently shifted to consider the role of other matrix elements, particularly hyaluronan, which has particularly high levels in foetal wounds. Some investigators have proposed that the more 'fluid' nature of the matrix facilitates the remodelling of the deposited collagen. Experimental evidence presented in this thesis suggests, however, that while the physico-chemical properties of hyaluronan are important in influencing cell and matrix interactions, more specific and controllable interactions may be due to the action of proteins associated with the hyaluronan.

Thesis (M.D.S.)--University of Hong Kong, 2004. / Also available in print.

Spinal Cord Injury (SCI) often causes cell death, demyelination, axonal degeneration and cavitation, resulting in functional motor and sensory loss below the site of injury. In an attempt to overcome SCI, the regenerating neurons require a permissive environment to promote their ability to reconnect. We report a novel thiolated hyaluronic acid (HA) hydrogel scaffold that can be used to repair the injured spinal cord. More specifically, thiolated hyaluronic acid hydrogels with varying thiol concentrations were successfully synthesized. The amount of thiol groups was measured spectrophotometrically using Ellman’s test. HA gels with different crosslinking densities were synthesized and the water content of the hydrogels was determined. The thermal behavior of the HA gels were studied by DSC. The strength of the hydrogels with varying thiol group content was evaluated by a rheometer. In addition, in vitro enzymatic degradation was performed through submerge the hydrogels in 200U/ml of hyaluronidase solution and incubate at 37°C. According to the result of the present study, this novel hydrogel shows great potential to serve as a 3D cell-patterning scaffold which can be inserted into a hollow fiber channel that could be used to promote regeneration after the SCI.

The chondroitin sulfate proteoglycan (CSPG) aggrecan forms link protein-stabilised complexes with hyaluronan (HA), via its N-terminal G1-domain, that provide cartilage with its load bearing properties. Similar aggregates (potentially containing new members of the link protein family), in which other CSPGs (i.e., versican, brevican and neurocan) substitute for aggrecan, may contribute to the structural integrity of many other tissues including skin and brain. In this thesis, cartilage link protein (cLP) and the G1-domains of aggrecan (AG1) and versican (VG1) were expressed in Drosophila S2 cells, purified to homogeneity and functionally characterised. The recombinant human proteins were found to have properties similar to those described for the native molecules. For example cLP formed dimers, and HA decasaccharides (HA 10-mers) were the minimum size that could compete effectively for their binding to polymeric HA. In addition, gel filtration and protein cross-linking/MALDI-TOF peptide fingerprinting showed that cLP and AG1 interact in the absence or presence of HA. Conversely, cLP and VG1 did not bind directly to each other hi solution yet formed ternary complexes with HA24. N-linked glycosylation of VG1 and AG1 was demonstrated to be unnecessary for either HA binding or the formation of ternary complexes. Additionally, the length of HA required to accommodate two G1-domains was found to be significantly larger for aggrecan than versican, which may reflect differences hi the conformation of HA stabilised on binding these proteins. To further investigate protein-HA interactions, fluorescent HA oligosaccharides were prepared and characterised. HA oligosaccharides labelled with the fluorophore 2-aminobenzoic acid (2AA) from four to 40 residues hi length were purified to homogeneity by ion exchange chromatography using a logarithmic gradient. Molecular weight and purity characterisation of HA oligosaccharides was facilitated by 2AA derivitisation since it enhanced signals in MALDI-TOF mass spectrometry and improves fluorophore-assisted carbohydrate electrophoresis (FACE) analysis by avoiding the inverted parabolic migration characteristic of 2-aminoacridone (AMAC) labelled sugars. The small size and shape of the fluorophore maintains the biological activity of the derivatised oligosaccharides, as demonstrated by their ability to compete for polymeric hyaluronan binding to VG1, AG1 and cLP. An electrophoretic mobility shift assay was used to study VG1 binding to 2AA-labelled HA 8-, 10-, 20-, 30- and 40-mers and although no stable VG1 binding was observed to labelled 8-mers, the equilibrium dissociation constant (100 nM) for VG1 with HA 10-mers was estimated from densitometry analysis of the free oligosaccharide. Interactions involving 2AA labelled HA 20-, 30-, and 40-mers with VG1 also displayed positive cooperativity. Therefore, oligosaccharides labelled with 2-aminobenzoic acid are biologically active and show excellent potential as probes in fluorescence-based assays that investigate protein-carbohydrate interactions.

No description available.

Three samples of the biopolymer hyaluronic acid (HA) were characterized in the following manner: the molecular weights were obtained via multi-angle laser light scattering; the intrinsic viscosities were calculated through dilute solution viscometry, and the rheology of HA solutions was determined with constant rate rotational viscometry and dynamical mechanical testing. In addition, the highly debated role of hyaluronic acid in wound healing was examined by studying the effect that HA has upon thrombin-catalyzed fibrin clotting. Fibrin, in phosphate-buffered saline, was clotted both alone and after being incubated with HA. It was determined that the presence of hyaluronic acid resulted in a slower clotting process; in effect, HA acts as an anti-coagulant. Based upon the experimental evidence, it is proposed that this anti-coagulant phenomenon arises through a combination of two mechanisms: 1) specific binding between HA and fibrin, which acts to retard fibrin clotting through steric hindrance, and 2) the formation of an HA network which slows fibrin clotting by hindering free diffusion of fibrin and thrombin. Finally, creation of a synthetic replacement for synovial fluid was attempted using xanthan gum and locust bean gum in phosphate-buffered saline. The phenomenon of gum synergism was utilized in an effort to exert some degree of fine-tuning over the final rheological properties of the solution. This also would provide the side benefit of reducing the weight of gum required per unit volume. By mixing the solutions at different temperatures, it was possible to exploit the tendency of xanthan gum to uncoil at higher temperatures and therefore bind more strongly to locust bean gum. However, it was determined that no combination of gum concentrations and processing conditions resulted in a gum solution that adequately mimicked the rheology of a hyaluronan solution. / Graduation date: 2003

Thesis (M.S.)--Oregon State University, 2007. / Printout. Includes bibliographical references (leaves 56-62). Also available on the World Wide Web.

Not available / text