Polysaccharides are found in all forms of life and serve diverse purposes. They are enzymatically synthesised from activated monosaccharide precursors, nucleotide sugars. One such nucleotide sugar is UDP-glucuronic acid, which is formed from UDP-glucose by the UDP-glucose dehydrogenase (UGDH) enzyme. UGDH has been proposed to have a regulatory role in the biosynthesis of polysaccharides. The aim of the studies presented in this thesis was to investigate the role of UGDH in the polysaccharide biosynthesis in three different systems: human cell culture, bacterial cultures and growing plants. The effects of UGDH-overexpression on polysaccharide biosyntheses and, when achievable, on UDP-sugar levels, were investigated. A mammalian UGDH was cloned from a kidney cDNA library. Transient expression of the cloned enzyme in mammalian cells led to an increased UGDH-activity. Northern blotting analyses revealed a single transcript of 2.6 kb in adult mouse tissues whereas human tissues expressed a predominant transcript of 3.2 kb and a minor transcript of 2.6 kb. Overexpression of the bovine UGDH in mammalian cells induced increased synthesis of the glycosaminoglycans; heparan sulphate, chondroitin sulphate and hyaluronan, without changing their relative proportions. The effects on glycosaminoglycan synthesis caused by an increased demand of UDP-glucuronic acid were studied by overexpression of hyaluronan synthase (Has3), which requires UDP-glucuronic acid as substrate. Overexpression of Has3 and coexpression of Has3 and UGDH resulted in highly augmented production of hyaluronan without noticeably affecting heparan sulfate and chondroitin sulfate synthesis. Expression of the bacterial UGDH in E. coli resulted in increased formation of UDP-glucuronic acid, but, unexpectedly, also to synthesis of fewer K5 polysaccharide chains. Overexpression of UGD1, one of four A. thaliana UGDH genes, in A. thaliana, resulted in dwarfism. Analysis of the cell wall polysaccharides showed alteration in saccharide composition. Paradoxically, the UDP-sugars derived from UDP-glucuronic acid decreased in amount.

Glycosaminoglycans (GAGs) such as hyaluronan (HA) and chondroitin sulfate (CS) are important, natural polysaccharides which occur in biological (connective) tissues and have various biotechnological and medical applications. Additionally, there is increasing evidence that chemically (over)sulfated GAGs possess promising properties and are useful as implant coatings. Unfortunately, a detailed characterization of these GAGs is challenging: although mass spectrometry (MS) is one of the most powerful tools to elucidate the structures of (poly)saccharides, MS is not applicable to high mass polysaccharides, but characteristic oligosaccharides are needed. These oligosaccharides are normally generated by enzymatic digestion. However, chemically modified (particularly sulfated) GAGs are extremely refractive to enzymatic digestion. This study focuses on the investigation of the digestibility of GAGs with different degrees of sulfation by bovine testicular hyaluronidase (BTH). It will be shown by using an adapted spectrophotometric assay that all investigated GAGs can be basically digested if the reaction conditions are carefully adjusted. However, the oligosaccharide yield correlates reciprocally with the number of sulfate residues per polymer repeating unit. Finally, matrix-laser desorption and ionization (MALDI) MS will be used to study the released oligosaccharides and their sulfation patterns.

Injury to the central nervous system (CNS) results in the formation of a highly inhibitory glial scar consisting mainly of chondroitin sulfate proteoglycans (CSPGs). CSPGs are comprised of a protein core with covalently attached chondroitin sulfate glycosaminoglycan (CS-GAG) side chains. CSPGs and CS-GAGs have been implicated in the regenerative failure of the CNS, though the mechanism underlying inhibition is unclear. Sulfation affects both the physical and chemical characteristics of CS-GAGs and, therefore, it has been hypothesized that certain sulfation patterns are more inhibitory than others. To investigate this hypothesis, specific chondroitin sulfate sulfotransferases (CSSTs), the enzymes responsible for CS-GAG sulfation, were knocked down in vitro using siRNA. C4ST-1, C4ST-2, and C46ST were chosen as targets for gene knockdown in this study based on their expression in neural tissue and the extent of inhibition caused by their respective CS-GAG. It was hypothesized that transfection of primary rat astrocytes with siRNAs designed to prevent the expression of C4ST-1, C4ST-2, and C46ST would decrease specific sulfation patterns of CSPGs, resulting in improved neurite extension in a neurite guidance assay. Through optimization of siRNA dose, astrocyte viability was maintained while successfully knocking down mRNA levels of C4ST-1, C4ST-2, and C46ST and significantly reducing total levels of secreted CS-GAGs. However, no increase in the incidence of neurite extension was observed using conditioned media collected from siRNA transfected astrocytes compared to non-transfected controls. These data suggest that sulfation does not contribute to CSPG-mediated neurite inhibition, though further investigation is necessary to confirm these findings. Significantly, this work has established a paradigm for investigating the role of CSPG sulfation patterns in CNS regeneration.

Abstract Chronic Chlamydia pneumoniae infections have been associated with cardiovascular diseases (CVD), but the treatment is difficult. Some drugs used for CVD have been found to have an inhibitory effect on the C. trachomatis infection, which is not considered to be associated with CVD. The purpose of this study was to investigate the effects of heparan sulfate-like glycosaminoglycans, COX inhibitors and rapamycin on the C. pneumoniae infection with cell culture methods. Almost any conceivable factors may affect the results of cell cultures. This study showed the complex interaction between temperature, time and medium during the pre-treatment before inoculation. The influences of these factors on the results overlapped and interlaced. The simple washing procedure could enhance the infectivity of C. pneumoniae although it is generally considered to cause the loss of chlamydial EBs and sequentially decrease the chlamydial infectivity. Although the detailed mechanisms were not studied, the results of this study showed that selective COX inhibitors and rapamycin can inhibit the infectivity of C. pneumoniae by inhibiting the growth and maturation, whereas heparan sulfate-like glycosaminoglycans perhaps inhibit the attachment of C. pneumoniae EBs onto the host cells. Recovery and repassage results showed that the growth can be only delayed by selective COX inhibitors, and it can recover to normal level once the drugs were removed. However, rapamycin inhibited the maturation of chlamydial EBs and therefore the infectivity fell down further even when the rapamycin was removed. This study also presented the variations of pathogenicity between different C. pneumoniae strains in vitro. This study is based on in vitro experiments with an acute infection model. Thus, any definite conclusions on the possible antichlamydial effects of the drugs tested in the treatment of cardiovascular diseases which are associated with chronic C. pneumoniae infections cannot be drawn on the basis of this study.

Signaling molecules are critically important to regulate cellular processes. Therefore, their incorporation into engineered biomaterials is indispensable for the applications in tissue engineering and regenerative medicine. In particular, the functionalization of highly hydrated polymer networks, so-called hydrogels, with the signaling molecules, has been quite beneficial to provide multiple cell-instructive signals. Following this strategy, the incorporation of sulfated glycosaminoglycans (GAGs) into such polymer networks offers unprecedented options to control the administration of signaling molecules via electrostatic interactions. Moreover, mathematical models can be instrumental in designing materials to tune the transport and adjust the local concentration of the signaling molecules to precisely modulate cell fate decisions. Accordingly, this study aims to systematically investigate the impact of different binary poly(ethylene glycol)-glycosaminoglycan hydrogel networks on the transport of signaling molecules by developing and applying mathematical modeling in combination with experimental approaches. The gained knowledge was then applied to modulate the bioactivities of pro-angiogenic growths factor within the binary hydrogel and rationally design a new class of cytocompatible GAG-based materials for the controlled administration of pro-angiogenic growth factors. Firstly, systematic studies on the mobility of signaling molecules within GAG-based polymer networks revealed differential effects of hydrogel network parameters such as mesh size, GAG content, and the sulfation pattern of the GAG building block on the transport of these signaling molecules. Secondly, the effect of the GAG content of the hydrogel and the sulfation pattern of the GAG building block on the bioactivity of hydrogel administrated vascular endothelial growth factor (VEGF) have been analyzed. Since VEGF is a GAG-affine protein that plays a major role in angiogenesis, its ability to promote vascular morphogenesis has been investigated. The simulation and experimental results demonstrated the determining impact of the availability of free (unbound) VEGF as well as the presence of GAGs with a specific sulfation pattern within the polymer network on the formation of the endothelial capillary network within the hydrogel. Finally, a rational design strategy has been applied to extend a GAG-hydrogel platform to allow for a far-reaching control of its cell instructive properties. The resulting materials are independently tunable over a broad range for their mechanical properties and GAG content. The GAG content of the hydrogel matrices, in particular, was shown to modulate the transport of pro-angiogenic growth factors most. Moreover, the hydrogel also supports endothelial vascular morphogenesis. In conclusion, the in here followed approach of combining experimental results and mathematical modeling for predicting the transport of signaling molecules and the rational design concept for customizing GAG-based hydrogel networks provide the fundamentals to precisely modulate cell fate decisions within GAG-based biohybrid polymer networks rationalizing their application for tissue engineering and regenerative medicine

Cell fate decisions in many physiological processes, including embryogenesis, stem cell niche homeostasis and wound healing, are regulated by secretion of small signaling proteins, called cytokines, from source cells to their neighbors or into the environment. Concentration level and steepness of the resulting paracrine gradients elicit different cell responses, including proliferation, differentiation or chemotaxis. For an in-depth analysis of underlying mechanisms, in vitro models are required to mimic in vivo cytokine gradients. We set up a microparticle-based system to establish short-range cytokine gradients in a threedimensional extracellular matrix context. To provide native binding sites for cytokines, agarose microparticles were functionalized with different glycosaminoglycans (GAG). After protein was loaded onto microparticles, its slow release was quantified by confocal microscopy and fluorescence correlation spectroscopy. Besides the model protein lysozyme, SDF-1 was used as a relevant chemokine for hematopoietic stem and progenitor cell (HSPC) chemotaxis. For both proteins we found gradients ranging up to 50 µm from the microparticle surface and concentrations in the order of nM to pM in dependence on loading concentration and affinity modulation by the GAG functionalization. Directed chemotactic migration of cells from a hematopoietic cell line (FDCPmix) and primary murine HSPC (Sca-1+ CD150+ CD48-) toward the SDF-1-laden microparticles proved functional short-range gradients in a twodimensional and three-dimensional setting over time periods of many hours. The approach has the potential to be applied to other cytokines mimicking paracrine cell-cell interactions in vitro

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21378号 / 農博第2302号 / 新制||農||1070(附属図書館) / 学位論文||H30||N5151(農学部図書室) / 京都大学大学院農学研究科食品生物科学専攻 / (主査)教授 橋本 渉, 教授 入江 一浩, 教授 保川 清 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

The extracellular matrix (ECM) of the aorta is a complex meshwork of elastin, collagen, and glycosaminoglycans (GAG). It also modulates the mechanical properties of the aorta, which in turn dictate lethal ruptures such as those caused by aneurysm and dissection. Amongst other roles, aortic stiffness controls the aorta’s ability to expand and recoil, and residual stresses, which are those existing in the absence of load, affect the magnitude and distribution of the mechanical stresses throughout the aortic wall. Mechanical stresses can be predicted via complex computer models, powerful tools that can also provide insight regarding the risk of rupture, given that ruptures occur when the mechanical stresses exceed the strength of the aorta. While this dissertation is primarily focused on the effect of GAG on residual stresses, other ECM (collagen, elastin) and mechanical (stiffness) factors are considered to expand our understanding of the structure-mechanics relationship in the aorta. This is important because the ECM undergoes extensive remodelling during aging and disease, but it is also critically important, as mentioned, in the context of aortic rupture. We first explored the mechanical roles of GAG in a finite element model by studying both the transmural residual stresses and the opening angle (an indicator of circumferential residual stresses) in ascending (AS) aortic ring models. Both were shown to be modulated by the GAG content, gradient, and the nature of the transmural distribution. While a heterogeneous GAG distribution led to the development of residual stresses which could be released by a radial cut, this was not the case when a homogeneous distribution was prescribed. Because the GAG distributions used in the first study were based on assumptions, and to get an in vitro understanding of the ECM role in modulating residual stresses, biomechanical mechanisms were explored in thoracic aortas from 5- to 6-month-old pigs. In a second study, we generated new detailed data on the distributions of collagen, elastin and GAG, throughout the aortic wall in the AS, arch (AR), and descending thoracic (DT) regions, and established correlations between the ECM constituents and the opening angle. The strongest correlations were observed between the opening angle and the total collagen:GAG ratio as well as the total GAG content. In line with our first in silico work, this in vitro investigation revealed that the GAG content and gradient modulate circumferential residual stresses and suggested that the interaction between GAG and the ECM fibers also plays a role in regulating residual stresses. In a third study, we examined the extent of contribution of GAG to circumferential residual stresses and to the radial compressive stiffness of the aortic wall, as well as the underlying mechanism through which GAG contribute to the mechanical properties using enzymatic GAG depletion. GAG depletion was associated with a decrease in the opening angle, by approximately 25%, 32%, 42% in the AS, AR, and lower DT regions respectively, and an increase in the radial compressive stiffness of the AS aorta. Glycation was also associated with a decrease in the opening angle, in which GAG depletion also had a similar effect. A small loss of water content was detected after GAG depletion, and the AS region was also associated with a significant loss of compressive deformation in the inner layer of the aorta following GAG depletion, suggesting that GAG interact with ECM fibers in their effect on aortic mechanics. The garnered experimental geometrical data and intramural GAG distributions were finally used to simulate animal-specific aortic rings from the AS, AR, and DT regions. The opening angle response was evaluated in solid matrices assuming one layer, and two layers to capture the different mechanical behaviors of the intima-media and the adventitia. A Holmes-Mow constitutive relationship was used and material parameters were obtained by curve fitting experimental stress-strain curves obtained from biaxial tests. Numerical results were evaluated by comparing simulated and experimental opening angles, revealing a notable overall agreement between the two.

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