京都大学 / 新制・課程博士 / 博士(理学) / 甲第23726号 / 理博第4816号 / 新制||理||1689(附属図書館) / 京都大学大学院理学研究科化学専攻 / (主査)教授 杉山 弘, 教授 深井 周也, 教授 秋山 芳展 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

Studying molecular mechanisms underlying adipocyte differentiation is imperative to understanding adipocyte function and its role in obesity. However, the majority of research exploring adipogenesis is conducted with cell lines cultured directly on tissue culture plastic. Culturing cells on plastic may result in altered proliferation and differentiation, and subsequent change in pharmacological response. The extracellular matrix (ECM) plays a critical role in adipocyte development and survival. It is suggested that cells in vitro express high levels of ECM proteins to compensate for lack of an ECM. Differentiating preadipocytes on a substrate representative of the mature adipocyte extracellular environment may provide a more physiological response to drugs and environmental chemicals. The purpose of this study was to investigate the impact of Matrigel on 3T3-L1 cell growth, differentiation, lipid accumulation and responsiveness to Rosiglitazone. Matrigel decreased 3T3-L1 cell proliferation, enhanced lipid accumulation, and increased expression of adipogenic and lipogenic markers, including PPARγ, C/EBPα, SREBP1c, FAS, LPL, FABP4 and PLIN1. This was accompanied by a decrease in gene expression of ECM proteins, including fibronectin, collagen 1, collagen 3, collagen 4, laminin and collagen 6 in 3T3-L1 cells on Matrigel. Finally, Matrigel enhanced the response of 3T3-L1 cells to Rosiglitazone, which is a known PPARγ agonist and significantly increases lipid accumulation in 3T3-L1 cells. Our results suggest that enhanced lipid accumulation in 3T3-L1 cells on Matrigel is associated with decreased expression of ECM genes. Future studies require investigation of the cell-to-ECM interaction to confirm these findings. This study proposes that the nature of the ECM for cultured adipocytes alters temporal lipid accumulation patterns and response to various drugs as compared to 3T3-L1 cells grown on tissue culture plastic. / Thesis / Master of Science (MSc)

Resistin-like molecule alpha (RELMα) is a secreted protein implicated in murine models of allergen-induced asthma, bleomycin-induced pulmonary fibrosis, and helminth infection. Transient pulmonary overexpression of Oncostatin M by Adenovirus vector (AdOSM) induces lung inflammation biased toward Th2 cytokines, eosinophil and alternatively activated (AA/M2) macrophage accumulation. In AdOSM-treated C57Bl/6 and BALB/c mice, we observed RELMα mRNA and protein markedly induced. RELMα is recognized as a marker of AA/M2 macrophages, and we observed by chromogenic in situ hybridization that RELMα mRNA co-expresses with the macrophage marker CD68, and RELMα mRNA was also highly induced in columnar airway epithelial cells upon AdOSM treatment. Assessing IL-6 as a comparator gp130 cytokine, AdIL-6 induced RELMα at significantly lower levels, however maximal induction of RELMα by AdOSM in C57Bl/6 mice required IL-6, assessed in IL-6–/– mice. Maximal induction of RELMα by AdOSM also required IL-33 in C57Bl/6 mice but not in BALB/c mice, assessed in IL-33–/– mice. We investigated functions of RELMα in response to OSM, in RELMα–/– mice. Inflammatory cell infiltration and Th2-associated cytokine responses were not altered in RELMα–/– in comparison to wildtype mice. However, RELMα-deficiency resulted in less accumulation of CD206+ AA/M2 macrophages, IFNγ+ Th1 cells in the lung, reduced induction of extracellular matrix gene mRNAs for COL1A1, COL3A1, MMP13, TIMP1, and reduced parenchymal alpha smooth muscle actin. RELMα–/– mice also showed less airway epithelial hyperplasia, increased epithelial cell damage/death (assessed morphologically) and increased LDH and soluble CK18 in response to AdOSM. Our findings suggest that RELMα does not modulate Th2 cytokines, but does participate in matrix deposition, airway remodelling mechanisms, and protection from inflammation-induced damage due to OSM-overexpression in lungs of C57Bl/6 mice. / Dissertation / Master of Science (MSc)

The heart, as a vital organ, must pump continuously to deliver oxygenated blood to the tissues of the body. The physical stress of pumping is supported by the extracellular matrix (ECM), a dynamic protein scaffold inside and around the heart. While a regulated ECM is required to maintain heart function, aberrant or excessive ECM remodelling, called fibrosis, is associated with disease states and is a hallmark of cardiovascular disease. One major trigger of cardiovascular disease is obesity, and fibrotic remodelling is known to occur in this context. In order to study the impact of increased body size on heart function and the molecular and biophysical characteristics of the ECM, a larval overgrowth model for obesity in the genetic model Drosophila melanogaster has been developed and characterised. This model produces giant larvae twice as heavy as their wildtype counterparts, and allows a unique opportunity to study changes in the cardiac ECM in a simple genetic model. Results demonstrate a remarkable ability of the ECM to accommodate this increase in size. The muscles of the heart are particularly robust, and there are no obvious observable defects to the matrix. Preliminary results suggest Collagen fibres are thicker and more disperse. When observing heart functionality, the cross-sectional area of the heart lumen is increased significantly in giant larvae, both at diastole and systole. However, giant larvae display defects in contraction of the heart tube, characterised by an inability to contract fully at systole. This results in a less than proportional increase in stroke volume, and an increase in heart rate. Heart function of giant larvae is clearly affected by the increase in body size. To quantify the impact to the biophysical structure of the ECM, an atomic force microscopy protocol is being developed. / Thesis / Master of Science (MSc) / A known side effect of cardiovascular disease is fibrosis of the heart, a form of pathological extracellular matrix (ECM) remodelling. Fibrosis causes the stiffening of heart muscle, leading to impaired cardiac function. One of the main risk factors for the development of cardiovascular disease is obesity, and fibrosis is known to occur in this context. I have characterised changes in the morphology and physiology of the heart in a Drosophila model for obesity. The resulting cardiac hypertrophy reveals significant plasticity in the heart ECM, while heart contraction and output is compromised.

Extracellular matrix (ECM) modifications occur during plant growth, development, and in response to environmental stimuli. Key modulators of ECM modification in vertebrates, the extracellular matrix metalloproteinases (MMPs), have also been described in a few plants. Here, we report the identification of Loblolly pine (Pinus taeda) Pta1-MMP and its characterization during seed development and germination. The Pta1-MMP protein has the structural characteristics of other plant MMPs, and a recombinant protein (rPta-MMP) generated by using EST sequences for a seed-expressed MMP exhibits Zn2+-dependent protease activity, and is inhibited by the active site-binding hydroxamate inhibitor GM6001 and EDTA. The Pta1-MMP gene is expressed during embryo development, with transcript levels increasing from proembryo to early cotyledonary stage, then declining during late cotyledonary expansion and maturation drying. Protein extracts exhibited similar developmental-stage MMP-like activity. Seed imbibition in water facilited germination, which was stimulated by GA3 and inhibited by ABA. The timing of germination was mirrored by the presence of MMP-like protease activity in both water- and GA3-imbibed embryos. Pta1-MMP transcript levels increased in association with germination for both GA3- and water-treated embryos, in agreement with MMP-like activity. In contrast, by 10 days after imbibition, Pta1-MMP transcripts in ABA-treated embryos were at levels similar to the other treatments, although MMP-like activity was not observed. The application of GM6001 during Loblolly pine seed imbibition inhibited germination in a dose-dependent manner. Our results suggest that Pta1-MMP is required for ECM modification, facilitating the cell division and expansion required for both embryo development and germination. To our knowledge, this is the first report of an MMP in any gymnosperm and also its involvement in embryo development and subsequent germination. / Ph. D.

One of the principal impediments to the broad success of conventional chemotherapy is poor delivery to and transport within the tumor microenvironment (TME), caused by irregular and leaky vasculature, the lack of functional lymphatics, and underscored by the overproduction of extracellular matrix (ECM) proteins such as collagen. Coupled with limited specificity, the high chemotherapeutic doses needed to effectively treat tumors often lead to unacceptable levels of damage to healthy tissues. Bacteria-based cancer therapy (BBCT) is an innovative alternative. Attenuated strains of species such as Salmonella Typhimurium have been shown to preferentially replicate in the TME, competing for cellular resources and imparting intrinsic and immune-mediated cytotoxic effects on cancer cells. Nevertheless, the immense successes observed in in vitro and immunocompromised murine models have not translated to the clinic, attributable to the lack of sufficient tumor colonization. Synthetic biology today enables the precision engineering of microbes with traits for improved survival, penetration, and replication in the TME, rationally optimizable through computational modeling. In this dissertation, we explore several facets of rationally engineering of bacteria toward augmenting bacterial penetration and retention in the TME. Namely, we (1) develop a novel assay to interrogate the neutrophil migratory response to pathogens and characterize the effects of modifying the molecular structure of the outer membrane (OM) of S. Typhimurium, (2) develop a mathematical model of bacterial intratumoral transport and growth and explore the effects of nutrient availability and the tumor ECM on colonization, (3) engineer bacteria that constitutively secrete collagenase and show significantly augmented transport in collagen hydrogels and collagen-rich tumor spheroids, and (4) develop computational models to explore control schemes for the programmed behavior of bacteria-based biohybrid systems, which will leverage the engineered bacteria to deliver therapeutics to the TME. Our work serves as the foundation for the logical and efficient design of the next generation of BBCTs. / Doctor of Philosophy / Cancer is one of the deadliest diseases facing our world today not because of a lack of effective medications in most cases, but because of our inability to target the diseased sites with those treatments. Many tumors lie in deep and sensitive regions that render them untouchable by direct physical means. Poor vascularization leads to only small fractions of toxic, systemically injected drugs being deposited in tumors. State-of-the-art treatments such as so-called "nano-medicines" that can target features of the diseased tissues and immunotherapies that train the immune system to attack tumor cells have made tremendous strides, but for many types of cancer, the underlying challenge of reaching cells far from blood vessels and targeting immunologically cold tumors remains. Bacteria-based cancer therapy (BBCT) presents an exciting opportunity to address these challenges. Based on microorganisms that can self-propel, proliferate, and display a preference for diseased tissues, their potential not only to carry chemotherapeutic payloads but also to elicit directly toxic or immunotherapeutic effects on cancer cells is clear from experimental work. Nevertheless, the same delivery and transport barriers facing other treatments, as well as immune-mediated clearance, have limited BBCTs' clinical success. Advances in synthetic biology and computational modeling today make the precision engineering of BBCT for traits that favor targeted cancer therapy a reality. The central hypothesis of this dissertation is that endowing tumor-targeting bacteria with a tissue-degrading enzyme has the potential to enhance tumor penetration and colonization. This dissertation work has led to development of computational and experimental frameworks for the design, testing, and optimization of BBCTs through direct quantitative assessment of the immune response, simulations to both optimize nutrient consumption for optimal growth and for programming genetic control strategies, and characterization of transport in tissue. Our work serves as a foundation for engineering "intelligent" BBCT.

Peptide-based biomaterials have gained much interest in various applications in drug delivery and tissue engineering in recent years, in large part due to their typically excellent biocompatibility and biodegradability. Composed of different amino acids, peptides can be designed with numerous sequences, providing flexibility and tunability in biomaterials. Peptides are easy to modify with small molecule drugs, inorganic components, and polymer chains to access multiple functions and tune properties relevant to biology and medicine. Stimuli-responsive peptide-based biomaterials can respond to environmental stimuli, such as light and ultrasound, in addition to local environmental factors, such as temperature, enzyme activity, and pH. Under environmental changes, these materials can be triggered to release therapeutic payloads, change conformations, or induce self-assembly in the target sites. In this work, I introduce the design, synthesis, and potential applications of several stimuli-responsive peptide-based biomaterials. The first half of this dissertation is based on enzyme-responsive, peptide-based biomaterials as extracellular matrix (ECM) mimics in tissue engineering. We synthesized linear and dendritic elastin-like peptides (ELPs) as crosslinkers and conjugated them with hyaluronic acid (HA) to form hydrogels. Trypsin was used as the enzyme trigger for cleaving the C-terminal lysine and to study how crosslinker topology affects enzymatic degradation. Hydrogels with dendritic ELPs degraded more slowly than linear ELPs, providing a novel strategy to tune the degradation rate of hydrogels as ECM mimics by the molecular design of crosslinker topology. Building on this peptide-polysaccharide platform for synthetic ECM design, we subsequently prepared hydrogels embedded with bioactive cryptic sites. These novel polymeric hydrogels mimicked native ECM cryptic sites by using depsipeptides that undergo an enzyme-triggered molecular rearrangement, "switching" from a non-functional epitope to a bioactive sequence. Mass spectrometry, 1H and 13C NMR spectroscopy, and fluorescence studies were applied to track structural changes in the peptide. SEM was used to image these polymer-peptide hybrid hydrogels. Finally, in vitro studies were conducted to evaluate cell interactions with the hydrogels. Switch peptide-modified alginate hydrogels showed increased cell adhesion upon induction of enzymatic activity, which provided a "gain of function" of the synthetic ECM. Critically, enzymes associated with the cells themselves could trigger the peptide switch and change in synthetic ECM behavior. With knowledge of stimuli-responsive peptide-based biomaterials applied in tissue engineering, I then studied how this system could be used in drug delivery by designing peptide-hydrogen sulfide (H2S) donor conjugates (PHDCs). H2S is a gasotransmitter that is produced endogenously, which has been explored in recent years with many potential therapeutical applications. We studied H2S release profiles in dual-enzyme-responsive PHDCs, with a further investigation into PHDC–Fe2+ complexes for potential tumor treatments via chemodynamic therapy. The PHDC–Fe2+ complexes were examined in a C6 glioma cell line, exhibiting an improved cell-killing effect compared with controls, by inducing toxic hydroxyl radical generation (•OH) via a Fenton reaction. To this end, we further discovered how side chains influence self-assembling nanostructures, H2S release profiles, and biological activities via three constitutionally isomeric PHDCs. Different morphologies and varied H2S release rates were observed, paving the way for tuning the properties of PHDCs by simple changes in molecular design. Finally, this dissertation discloses conclusions and future directions on stimuli-responsive peptide-based biomaterials using similar platforms with different designs in the drug delivery and tissue engineering fields. / Doctor of Philosophy / Peptides, short sequences of two or more amino acids linked by chemical bonds, are smaller versions of proteins. Forming naturally in nature, peptides are promising candidates in the design of biocompatible and biodegradable materials. To make these peptide-based materials "smart", certain sequences or functional groups are installed in the peptides, making them responsive to environmental changes, or stimuli. These external stimuli include light, ultrasound, temperature, enzyme activity, and pH changes. In this work, we have explored the design and synthesis of stimuli-responsive peptide-based biomaterials and their potential applications in tissue engineering and drug delivery. The first half of this dissertation focuses on the design and synthesis of two enzyme-responsive, peptide-based materials that function as extracellular matrix (ECM) mimics. The ECM is a three-dimensional microenvironment where cells reside, providing structural support and adhesive anchor points for cells. In the first system, we synthesized peptide-polysaccharide hydrogels with different peptide crosslinkers, comparing their enzymatic degradation performance to evaluate how peptide topology (architecture) influences degradation. A more branched topology led to a slower hydrogel degradation rate. To introduce biofunctionality into the ECM mimics, we embedded the second system with a "switchable" peptide sequence, which transformed from a non-functional peptide into a functional, bioactive epitope after being triggered by an enzyme. The functional peptide after the switch provided cell adhesion and increased cell spreading. The latter half of this dissertation explores the possibility of stimuli-responsive peptide-based biomaterials in drug delivery. We designed peptides that release hydrogen sulfide (H2S), a signaling gas is commonly known for its foul smell and toxicity, and studied the biological behaviors in cells. The peptide-H2S donor conjugates (PHDCs) were activated by the enzyme legumain, which cancer cells overproduce, leading to H2S release. With the combined treatment with Fe2+, the PHDC-Fe2+ system reduced cancer cell viability due to the high amount of hydroxyl radicals (•OH) generated by the Fenton reaction. This system may be a potential design platform for precise tumor treatments.

Extracellular matrix (ECM) is responsible for tendon strength and elasticity. Healed tendon ECM lacks structural integrity, leading to reinjury. Porcine urinary bladder matrix (UBM) provides a scaffold and source of bioactive proteins to improve tissue healing, but has received limited attention for treating tendon injuries. The objective of this study was to evaluate the ability of UBM to induce matrix organization and tenogenesis using a novel in vitro model. We hypothesized that addition of UBM to tendon ECM hydrogels would improve matrix organization and cell differentiation. Hydrogels seeded with bone marrow cells (n = 6 adult horses) were cast using rat tail tendon ECM ± UBM, fixed under static tension and harvested at 7 and 21 days for construct contraction, cell viability, histology, biochemistry, and gene expression. By day 7, UBM constructs contracted significantly from baseline, whereas control constructs did not. Both control and UBM constructs contracted significantly by day 21. In both groups, cells remained viable over time and changed from round and randomly oriented to elongated along lines of tension with visible compaction of the ECM. There were no differences over time or between treatments for nuclear aspect ratio, DNA, or glycosaminoglycan content. Decorin, matrix metalloproteinase 13, and scleraxis expression increased significantly over time, but not in response to UBM treatment. Mohawk expression was constant over time. Cartilage oligomeric matrix protein expression decreased over time in both groups. Using a novel ECM hydrogel model, substantial matrix organization and cell differentiation occurred; however, the addition of UBM failed to induce greater matrix organization than tendon ECM alone. / Master of Science / Tendon injuries are common in horses and are painful and can be career- and life-ending. Tendons have a special structure and organization that enables them to withstand high tensile forces without permanent deformation. Injured tendons heal by forming stiff, disorganized scar tissue that makes the tendon more prone to re-injury. The lining of urinary bladders from pigs (UBM) provides a physical mesh and signaling factors that help heal injuries in a variety of tissues to a more normal state. However, UBM has not been evaluated in a laboratory model of tendon tissue formation to determine how it can help heal tendon injuries. Three-dimensional models of new tendon tissue formation (neotendons) were made with rat tail tendon matrix and stem cells collected from horse bone marrow. The neotendons were placed under steady tension for 3 weeks. The models were collected after 1 and 3 weeks to measure their width, numbers of live cells, cell and matrix organization, levels of tendon matrix components and expression of genes found in tendons. Most cells in the neotendons remained alive during the study period. Over time, UBM-treated and untreated neotendons became narrower compared to their starting width. The width of UBM-treated neotendons decreased faster than non-treated neontendons in the first week of the study. Cells became longer, narrower, and oriented along lines of tension. Expression of genes important in tendon development and structure either increased or was constant over time. UBM treatment did not change cell shape or increase levels of tendon-associated genes, DNA, or tendon matrix components. Our novel tendon model successfully created organized tendon-like tissue when placed under tension. However, UBM treatment did not improve formation of tendon-like tissue to a greater extent than controls.

It is estimated that approximately 2 million fires which occur in United States each year result in 1.2 million burn victims. Fibroblasts are responsible for responding to this tissue damage by breaking down the damaged extracellular matrix (ECM) and secreting a new ECM which aids in wound repair and supports the migration of immune cells. Pseudomonas aeruginosa is an opportunistic pathogen commonly associated with health-care infections (HCAIs) due to its ability to take advantage of immunocompromised hosts. However, little research has investigated how wound invading P. aeruginosa interacts with wound repairing fibroblasts. To address this lack of understanding, this thesis focuses on quantifying changes in fibroblast morphology, migratory behavior, and force exertion to investigate this host cell's response to representative cytotoxic (PAO1) and invasive (PA14) strains of P. aeruginosa. These assays study host cell-pathogen interactions on highly aligned nanofibers of varied spacing and diameter, which mimic the fibroblast deposited ECM and dictate fibroblast morphology. We discovered that the cytotoxic strain of P. aeruginosa induced significantly shorter fibroblast death times. Furthermore, two modes of death, sharp and gradual, were identified and found to be dependent on both fiber configuration and strain of P. aeruginosa. In addition, fibroblasts exposed to PAO1 migrating on the parallel formation were found to be significantly slower and less persistent than those exposed to PA14, however, fibroblasts exposed to both strains of bacteria were shown to exert similar forces. Lastly, exposure to PA14 led to the greatest change in actin, evident by increased actin punctae and less prominent actin stress fiber formation. / Master of Science / It is estimated that approximately 2 million fires which occur in United States each year result in 1.2 million burn victims. Fibroblasts respond to burn wounds by breaking down the damaged tissue fibers, termed extracellular matrix (ECM), and secreting a new ECM. Unfortunately, severe thermal injuries place hospitalized burn victims at high risk of infection. Pseudomonas aeruginosa is an opportunistic pathogen commonly associated with health-care infections (HCAIs) due to its ability to take advantage of immunocompromised hosts. However, little research has investigated how wound invading P. aeruginosa interacts with wound healing fibroblasts. To address this knowledge gap, this thesis focuses on quantifying changes in fibroblast shape, migratory behavior, and force exertion to investigate this host cell's response to two strains of P. aeruginosa, which employ different mechanisms of invasion. These interactions are studied on a platform of suspended nanofibers with controlled spacing and diameter, to dictate fibroblast shape and mimic the fibroblast deposited ECM. We discovered that the two strain of P. aeruginosa induced significantly different fibroblast death times. During death, it was observed that fibroblasts either balled up quickly, termed sharp death, or remained spread out, termed gradual death, dependent upon fibroblast shape and strain of P. aeruginosa introduced. In addition, significant differences in migration speed and persistence were found between fibroblasts exposed to the two strains of bacteria, however, both groups were shown to exert similar forces. Lastly, the fibrous proteins which make up the cytoskeleton of the cell, actin stress fibers, were found to vary among the control and bacteria treated cells.

The tissue microenvironment is a complex living system containing heterogeneous mechanical and biophysical cues. Cellular components are surrounded by extracellular matrix and interstitial fluid, while transport of nutrients and biochemical factors is achieved via the vasculature. Each constituent of the tissue microenvironment can play a significant role in its ability to function normally. Many diseases including cancer have been linked with dysfunction in the tissue microenvironment; therefore an improved understanding of interaction between components of this complex system is needed. In vitro platforms mimicking the tissue microenvironment appear to provide the most promising avenue for studies of cell-cell and cell-matrix interactions as well as elucidation of the mechanisms leading to disease phenomena such as tumor metastasis. However, successful recapitulation of all three primary components of the tissue microenvironment in three dimensions has remained challenging. In particular, matching mechanical cues and biochemical transport to in vivo conditions is difficult because of lack of quantitative characterization of the physical properties and parameters of such platforms. In this work, extensive characterization of collagen I hydrogels, popular for use as extracellular matrix mimics, was performed in order to enable tuning to specific in vivo conditions. Additionally, perfusion of blood in a 3D tissue microenvironment platform fabricated using collagen hydrogels was characterized to enable future advances in in vitro modeling of the in vivo microenvironment. Finally, the tissue microenvironment platform is modified to enable biochemical gradients within the hydrogel and used to examine directed migration (chemotaxis) of human breast cancer cells in response to gradients in growth factor combined with varied stiffness and pore diameter of the extracellular matrix. / Ph. D.