Dissertations / Theses on the topic 'Extracellular matrix'

Dystroglycan and the integrins are two types of extracellular matrix receptors involved in astrocyte adhesion and migration. The molecular mechanisms that control their distribution to signaling platforms of the plasma membrane remain poorly understood. In the present study, we define four cell spreading stages for adherent astrocytes, termed stage 1 to 4, and defined by the state of spreading of the cell in culture. Immunocytochemistry analysis reveals that DG and integrins distribute differentially depending on the stage, and both receptors get recruited to leading edge of the cell, possibly to GM1-rich lipid rafts, in scratch-induced migration. Treatment of reactive astrocytes with cytochalasin-D or colchicine confirms that the formation of MT-rich protrusions is not actin-independent. However, cortical actin localization is independent of the polarized MT network. We confirm scratch-induced Cdc42/Rac1 activation early after wounding and further investigated the role of DG in Rho GTPases signaling. We conclude that ECM receptors localization is tightly regulated along with the cytoskeletal reorganization machinery in astrocyte proliferation.
Dystroglycan et les intégrines sont deux types de récepteurs de la matrice extracellulaire (RMEC) impliqués dans l'adhésion et la migration des astrocytes. Les mécanismes moléculaires qui contrôlent leur distribution aux plates-formes de signalisation de la membrane plasmique demeurent peu étudiés. Dans la présente étude, nous définissons quatre stages d'adhésion cellulaire pour les astrocytes dans lesquels DG et les intégrines sont distribués différemment. Dans la cellule en migration, les deux récepteurs sont recrutés à l'avant, possiblement dans des îlots riches en GM1. En combinant la microscopie à fluorescence et l'usage de perturbateurs du cytosquelette dans notre modèle de lésion in vitro, nous démontrons aussi que la formation des protrusions riches en microtubules n'est pas actine-indépendante. Cependant, la formation d'actine corticale est indépendante du réseau polarisé de microtubules. Nous confirmons l'activation rapide de Cdc42 et Rac1 suite à une lésion in vitro et avons développé une lignée d'astrocytes DG-nulles pour étudier le rôle de DG dans l'activation des Rho GTPases. Nous concluons que la distribution des RMEC est étroitement régulée avec l'appareil de réorganisation cytoskelettique dans la prolifération astrocytaire.

Thesis (Ph. D.)--Illinois State University, 1997.
Title from title page screen, viewed June 5, 2006. Dissertation Committee: H. Tak Cheung (chair), Sean Arkins, Herman E. Brockman, Paul A. Garris, Brian J. Wilkinson. Includes bibliographical references (leaves 113-121) and abstract. Also available in print.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Materials Science and Engineering, 2007.
Includes bibliographical references (p. 187-201).
In this thesis, the shear and self-adhesion nanomechanical properties between opposing cartilage aggrecan macromolecules were probed. In addition, nanoscale dynamic oscillatory mechanical properties of cartilage and its type II collagen network was measured. Aggrecan shear nanomechanics was assessed via microcontact printing and lateral force microscopy. Lateral force between aggrecan and the probe tip, and compression of aggrecan was simultaneously measured in 0.001 - 1.0 M NaCl aqueous solutions. Using the microsized tip (Rtip ~ 2.5 [mu]m) enabled a large assembly of ~ 103 aggrecan molecules to interact simultaneously, closely mimicking the in vivo conditions.Both electrostatic and nonelectrostatic components were identified to importantly contribute to aggrecan shear. At near physiological IS (0.1 M), significant rate dependence was observed, suggestive of visco/poroelastic interactions within the aggrecan layer. By using an aggrecan end-functionalized colloidal tip, shear of two opposing aggrecan layers was assessed in a similar fashion. Lower lateral force and a more marked rate dependence were measured compared to the shear of a single layer, due to the aggrecan inter-layer molecular interpenetration and the different local z-dependent charge density distribution. The addition of Ca2+, at physiological-like 2 mM concentration, significantly affects cartilage shear by its electrostatic screening and binding effects. Marked aggrecan self-adhesion upon separation was discovered after static compression in the presence of electrostatic repulsion in physiological-like conditions.
(cont.) Aggrecan self-adhesion increases as increasing equilibration time and bath IS. Molecular origins of the adhesion, also present in vivo, include van der Waals, hydrogen bonding, Ca2+-mediated bridging, and molecular entanglements between the glycosaminogly-can branches of aggrecan. This self-adhesion could be an important factor in protecting cartilage matrix structural integrity and function via these energy-dissipative mechanisms. The nanoscale oscillatory dynamic deformation properties of both nontreated and proteoglycan(PG)-depleted (left mostly type II collagen) calf knee cartilage disks (- 0.5 mm thick) was measured by connecting an external electronic wave generator to the AFM. A significant increase in effective stiffness E and phase lag A (deformation with respect to force) as increasing frequency for both disks suggests poro/viscoelasticity are more critical at higher frequency. The PG-depleted disk shows a more marked dependence of E and A on deformation amplitude - 2 - 100 nm, as the nanostructure and nanomechanical properties of porous collagen network are more heterogeneous without the entrapment of aggrecan motif. A unique - 23 nm banding pattern along the type II collagen fibrils was observed, which may be relative to the cartilage swelling properties and the molecular interaction between aggrecan and the collagen network. Taken together, this study provides insights into molecular-level deformation of cartilage extracellular matrix (ECM) macromolecules (e.g., aggrecan, type II collagen) that are important to the understanding of cartilage biomechanical function. Ongoing studies are probing the age, disease (osteoarthritis), source and species related variations of cartilage ECM properties at the molecular level.
by Lin Han.
Ph.D.

The perineuronal net (PN) is a specialised region of extracellular matrix around some neurons, particularly GABAergic neurons that contain the calcium binding protein, parvalbumin (PV). The negatively charged glycosaminoglycan side chains of chondroitin sulphate proteoglycans, a major constituent of the PN, create a polyanionic environment around neurons that is thought to be important in the buffering of ions. Maintaining ion homeostasis around the inhibitory PV-positive neurons is critical in order to sustain their fast-firing rates. Loss or disruption of inhibitory input, particularly to glutamatergic cells could result in excitotoxicity and cell death. The PN is also important in the development, stabilisation and remodelling of synapses, in maintaining the trophic microenvironment around neurons and in synaptic plasticity. Neurodegeneration, the considerable loss of synapses and the inflammatory reaction that occurs in Alzheimer's disease (AD) is likely to affect the PN. Glial cells, activated as part of the inflammatory response to the deposition of β-amyloid (Aβ) and formation of neurofibrillary tangles, release matrix metalloproteinases (MMPs) that are capable of degrading the PN. This thesis describes studies of PN N-acetylgalactosamine and PV-positive neurons in AD, and their relationship to parenchymal tau, Aβ, microglia, astrocytes, and MMPs-2, -3 and -9. The outcome of these studies showed that the PV-positive neurons tend to be spared in AD but there is degradation of surrounding PNs.

Exposure of the skin to ultraviolet radiation (UVR) results clinically in the formation of deep wrinkles and mottled pigmentation and histologically, in a vast remodelling of the dermal extracellular matrix (ECM), in particular the elastic fibre network. Fibrillin microfibrils and fibulin-5 are early biomarkers of photoageing, where a loss of these fibres from the dermal epidermal junction is apparent. A study by our group showed that isolated fibrillin microfibrils and fibronectin which are rich in amino acids which absorb energy from UVR (UV-chromophores) are susceptible to UVR-induced damage, whilst UV-chromophore poor collagen type I is not. This research, with other earlier studies, indicates that acellular mechanism may work in tandem with cell-mediated up-regulation of matrix metalloproteinases (MMPs) in the progression of photoageing. This thesis aims to: i) test whether acellular mechanisms of photoageing are a result of direct photon absorption and/or the photodynamic production of reactive oxygen species (ROS); ii) assess the functional consequences of UVB degradation on the susceptibility of fibrillin microfibrils to MMPs and; iii) assay whether ECM proteins are differentially susceptible to solar simulated radiation (SSR) or UVA (315-400nm) alone using physiologically relevant doses of irradiation. Isolated proteins were exposed to UVB (280-315nm) in depleted-O2 conditions and in the presence of deuterium oxide. Depleted-O2 conditions decreased and deuterium oxide conditions increased UVR-induced degradation. Isolated proteins also show a similar pattern of degradation when exposed to H2O2 as an exogenous source of ROS. These results indicate that ROS play an important role in the differential degradation of dermal proteins. MMPs-3 and -9 are both upregulated in the skin after exposure to UVR and have the ability to degrade elastic fibre components. After exposure to UVB, damaged fibrillin microfibrils become more susceptible to degradation by both MMPs-3 and -9. Chromophore-rich fibrillin microfibrils and fibronectin are susceptible to degradation by both SSR and UVA alone, whereas chromophore-poor collagens type I and VI and tropoelastin are not. These results support our previous findings that amino acid composition of proteins is a good indicator of their relative susceptibility to UV-induced damage with a physiologically relevant irradiation system. In conclusion this work shows that ROS are an important mediator of acellular mechanisms of photoageing and that amino acid composition is a good indication of relative susceptibility of proteins to both ROS and UVR. The ability to predict ROS-susceptible proteins also has wider implications for human ageing as a whole.

Chronic Obstructive Pulmonary Disease (COPD) is a leading cause of death worldwide. Alveolar wall destruction is a significant contributor to COPD. Inflammatory macrophages are a major source of the Extracellular Matrix (ECM) proteolysis. ECM breakdown causes air to get trapped in the alveoli, obstructing airflow. One step in curing COPD may be to convert inflammatory to pro-regenerative macrophages. Recently, decellularized ECM scaffolds have shown the ability to induce a pro-regenerative phenotype. Yet these scaffolds are incapable for reaching the alveolar region of the lungs. To reach the alveolar region particles need a diameter of 1-5 μm or smaller than 300 nm. We used protein from decellularized lung tissue to create nanoparticles. By first digesting the protein in acid, we electrosprayed the solution into nanoparticles. The average size of the nanoparticles is 225 (± 67) nm, within the requirements to reach alveoli. However, another barrier exists for treating this disease. That barrier is mucus; mucus hypersecretion is another sign of COPD. The formed particles are capable of penetrating the mucus layer in COPD. After characterizing the particles, we began in vitro investigations. First, we measured cytotoxicity of the nanoparticles. In alveolar epithelial cells, adding nanoparticles to the media increased cellular proliferation. We then added the nanoparticles to isolated murine macrophages. The nanoparticles induced a pro-regenerative phenotypic shift in murine macrophages. These experiments reveal that these nanoparticles may become an effective treatment for degenerative lungs diseases, such as COPD, after further investigation.

L’interaction épithélium/mésenchyme est cruciale pour de nombreux processus physiopathologiques. Lors de ma thèse, je me suis intéressée aux signaux mésenchymateux régulant les cellules épithéliales en utilisant comme modèle la peau, qui est composée de 2 compartiments : l’épiderme (épithélium) et le derme (mésenchyme). Les intégrines sont impliquées ces interactions. CD98hc est une protéine transmembranaire à double fonction qui chaperonne des transporteurs d’acides aminés et régule la signalisation des ß intégrines. Elle est exprimée dans les cellules prolifératives telle les cellules épithéliales. Dans la peau, CD98hc est exprimée l’épiderme mais également dans les fibroblastes, cellules post-mitotiques. Mon hypothèse a été que CD98hc participe aux régulations des interactions derme/épiderme. Grâce à un modèle de KO conditionnel de CD98hc dans les fibroblastes dermiques, j’ai mis en évidence que CD98hc permet le maintient des propriétés mécaniques et biologiques du derme, et, de ce fait, régule l’épiderme en conditions d’homéostasie, de perturbation de la barrière et lors de la formation de cancer. De plus, le rôle de CD98hc dans cette interaction apparait comme étant lié à l’âge. En conclusion, mes travaux de thèse montrent le rôle central de l’expression dermique de CD98hc dans le maintien de l’homéostasie cutanée au cours du vieillissement ainsi que lors de la tumorigenèse
The epithelial/mesenchymal interaction is crucial for many physiopathological processes. During my PhD, I focused on mesenchymal signals that regulate epithelial cells behavior using the skin as model. The skin is composed of 2 main compartments: the epidermis (epithelium) and the dermis (mesenchyme). While this crosstalk involves integrins, its regulations are poorly understood. The transmembrane protein CD98hc interacts with amino acid transporter and regulates integrin signaling. CD98hc which is expressed at the cell membrane of proliferative cells, specifically epithelial cells, is required for tissue homeostasis. We found that besides its expression in keratinocytes, CD98hc is also expressed in post-mitotic dermal fibroblast. Hence, I hypothesized that CD98hc is involved in epidermis/dermis crosstalk. Using a conditional KO mouse model that harbor a CD98hc deletion in dermal fibroblast, I have shown that dermal CD98hc is required to maintain mechanical and biochemical properties of the dermis. Moreover, I have shown that those CD98hc-dependent dermal properties are implicated in the regulation of the epidermal cell behavior during homeostasis, cutaneous barrier disruption and tumorigenesis. Moreover, the role of CD98hc in those processes seems to be age-related. To conclude, during my PhD, I have revealed a major role of CD98hc in the maintenance of skin homeostasis during aging and tumorigenesis

The extracellular matrix is (ECM) is a network of large, structural proteins and polysaccharides, important for cellular behavior, tissue development and maintenance. Present thesis describes work exploring ECM as scaffolds for tissue engineering by manipulating cells cultured in vitro or by influencing ECM expression in vivo. By culturing cells on polymer meshes under dynamic culture conditions, deposition of a complex ECM could be achieved, but with low yields. Since the major part of synthesized ECM diffused into the medium the rate limiting step of deposition was investigated. This quantitative analysis showed that the real rate limiting factor is the low proportion of new proteins which are deposited as functional ECM. It is suggested that cells are pre-embedded in for example collagen gels to increase the steric retention and hence functional deposition. The possibility to induce endogenous ECM formation and tissue regeneration by implantation of growth factors in a carrier material was investigated. Bone morphogenetic protein-2 (BMP-2) is a growth factor known to be involved in growth and differentiation of bone and cartilage tissue. The BMP-2 processing and secretion was examined in two cell systems representing endochondral (chondrocytes) and intramembranous (mesenchymal stem cells) bone formation. It was discovered that chondrocytes are more efficient in producing BMP-2 compared to MSC. The role of the antagonist noggin was also investigated and was found to affect the stability of BMP-2 and modulate its effect. Finally, an injectable gel of the ECM component hyaluronan has been evaluated as delivery vehicle in cartilage regeneration. The hyaluronan hydrogel system showed promising results as a versatile biomaterial for cartilage regeneration, could easily be placed intraarticulary and can be used for both cell based and cell free therapies.

Bone fractures and pathologic conditions like chronic wounds significantly reduce the quality of life for the patients, which is especially dramatic in an elderly population with considerable multi-morbidity and lead to substantial socio-economic costs. To improve the wound healing capacity of these patients, new strategies for the design of novel multi-functional biomaterials are required: they should be able to decrease extensive pathologic tissue degradation and specifically control angiogenesis in damaged vascularized tissues like bone and skin. Glycosaminoglycans (GAGs) like hyaluronan (HA) and chondroitin sulfate (CS) as important extracellular matrix (ECM) components are involved in several biological processes such as matrix remodeling and growth factor signaling, either by directly influencing the cellular response or by interacting with mediator proteins. This could be useful in functionalizing biomaterials, but native sulfated GAGs (sGAGs) show a high batch-to-batch variability and are limited in their availability. Chemically modified HA and CS derivatives with much more defined characteristics regarding their carbohydrate backbone, sulfate group distribution and sulfation degree are favorable to study the structure-function relationship of GAGs in their interaction with mediator proteins and/or cells and this might be used to precisely modulate activity profiles to stimulate wound healing. By combining collagen type I as the main structural protein of the bone and skin ECM with these GAG derivatives, 2.5-dimensional (2.5D) and 3D artificial ECM (aECM) coatings and hydrogels were developed. These biomaterials as well as the respective GAG derivatives alone were compared to native GAGs and used to analyze how the sulfation degree, pattern and carbohydrate backbone of GAGs influence: i) the activity of tissue inhibitor of metalloproteinase-3 (TIMP-3) and vascular endothelial growth factor-A (VEGF-A) as main regulators of ECM remodeling and angiogenesis, ii) the composition and characteristics of the developed 2.5D and 3D aECMs, iii) the enzymatic degradation of collagen-based aECMs and HA/collagen-based hydrogels, iv) the proliferation and functional morphology of endothelial cells. Surface plasmon resonance (SPR) and enzyme linked immunosorbent assay (ELISA) binding studies revealed that sulfated HA (sHA) derivatives interact with TIMP-3 and VEGF-A in a sulfation-dependent manner. sHA showed an enhanced interplay with these proteins compared to native GAGs like heparin (HEP) or CS, suggesting a further impact of the carbohydrate backbone and sulfation pattern. sGAGs alone were weak modulators of the matrix metalloproteinase-1 and -2 (MMP-1 and -2) activity and did not interfere with the inhibitory potential of TIMP-3 against these proteinases during enzyme kinetic analyses. However, the formation of TIMP 3/GAG complexes reduced the binding of TIMP-3 to cluster II and IV of its endocytic receptor low-density lipoprotein receptor-related protein-1 (LRP-1, mediates the up-take and degradation of TIMP-3 from the extracellular environment) in a sulfation- and GAG type-dependent manner. It is of note that the determined complex stabilities of TIMP-3 with cluster II and IV were almost identical indicating for the first time that both clusters contribute to the TIMP-3 binding. Competitive SPR experiments demonstrated that GAG polysaccharides interfere stronger with the TIMP 3/LRP-1 interplay than GAG oligosaccharides. The importance of the position of sulfation is highlighted by the finding that a sHA tetrasaccharide exclusively sulfated at the C6 position of the N-acetylglucosamine residues significantly blocked the receptor binding, while CS and HEP hexasaccharides had no detectable effects. Thus, sHA derivatives as part of biomaterials could be used to sequester and accumulate TIMP 3 in aECMs in a defined manner where sHA-bound TIMP-3 could decrease the matrix breakdown by potentially restoring the MMP/TIMP balance. GAG binding might extend the beneficial presence of TIMP-3 into wounds characterized by excessive pathologic tissue degradation (e.g. chronic wounds, osteoarthritis). Mediator protein interaction studies with sHA coated surfaces showed the simultaneous binding of TIMP-3 and VEGF-A, even though the sHA/VEGF-A interplay was preferred. Moreover, kinetic analysis revealed almost comparable affinities of both proteins for VEGF receptor-2 (VEGFR-2), explaining their competition that mainly regulates the activation of endothelial cells. Additional SPR measurements demonstrated that the binding of sGAGs to TIMP-3 or VEGF-A decreases the binding of the respective mediator protein to VEGFR-2. Likewise, a sulfation-dependent reduction of the binding signal was observed after pre-incubation of a mixture of TIMP-3 and VEGF-A with sGAG poly- and oligosaccharides. The biological consequences of GAGs interfering with VEGF-A/VEGFR-2 and TIMP-3/VEGFR 2 were assessed in vitro using porcine aortic endothelial cells stably transfected with VEGFR 2 (PAE/KDR cells). The presence of sHA both decreased VEGF-A activity and the activity of TIMP-3 to inhibit the VEGF-A-induced VEGFR-2 phosphorylation. The same decreased activities could be observed for the migration of endothelial cells. However, if sHA, TIMP-3 and VEGF-A were present simultaneously, sHA partially restored the TIMP-3-mediated blocking of VEGF-A activity. These findings provide novel insights into the regulatory potential of sHA during endothelial cell activation as an important aspect of angiogenesis, which could be translated into the design of biomaterials to treat abnormal angiogenesis. These sHA-containing materials might control the angiogenic response by modulating the activity of TIMP 3 and VEGF-A. The in vitro fibrillogenesis of collagen type I in the presence of sHA derivatives led to 2.5D collagen-based aECM coatings with stable collagen contents and GAG contents that resemble the organic part of the bone ECM. A burst release of GAGs was observed during the first hour of incubation in buffer with the GAG content remaining almost constant afterwards, implying that the number of GAG-binding sites of collagen restricts the amounts of associated GAGs. Moreover, two differently sulfated HA derivatives could for the first time be incorporated into one multi-GAG aECM as verified via agarose gel electrophoresis and fluorescence measurements. This illustrates the multiple options to modify the aECM composition and thereby potentially their functionality. Atomic force microscopy showed that the presence of sHA derivatives during fibrillogenesis significantly reduced the resulting fibril diameter in a concentration- and sulfation-dependent manner, indicating an interference of the GAGs with the self-assembly of collagen monomers. In line with enzyme kinetic results, none of the GAGs as part of aECMs altered the enzymatic collagen degradation via a bacterial collagenase. Thus aECMs were proven to be biodegradable independent from their composition, which is favorable concerning a potential biomedical usage of the aECMs e.g. as implant coatings. HA/collagen-based hydrogels containing fibrillar collagen embedded into a network of crosslinked HA and sGAGs were developed as 3D aECMs. Scanning electron microscopy demonstrated a porous structure of the gels after lyophilization, which could favor the cultivation of cells. The presence of collagen markedly enhanced the stability of the gels against the enzymatic degradation via hyaluronidase, something beneficial to clinical use as this is often limited by the generally fast breakdown of HA. Binding and release experiments with lysozyme, as positively charged model protein for e.g. pro-inflammatory cytokines, and VEGF A revealed that the sulfation of GAGs increased the protein binding capacity for pure GAG coatings and retarded the protein release from hydrogels compared to hydrogels without sGAGs. Moreover, the additional acrylation of sHA was shown to strongly reduce the interaction with both proteins when the primary hydroxyl groups were targets of acrylation. This stresses the influence of the substitution pattern on the protein binding properties of the GAG derivatives. However, hydrogel characteristics like the elastic modulus remained unaffected. The different interaction profiles of lysozyme and VEGF-A with GAGs demonstrated a protein-specific preference of different monosaccharide compositions, suggesting that the mediator protein binding could be simultaneously adjusted for several proteins by combining different GAG derivatives. This might allow the scavenging of pro-inflammatory cytokines and at the same time a binding and release of wound healing stimulating growth factors. Since there is a growing demand for biomaterials to regenerate injured vascularized tissues like bone and skin, endothelial cells were used to examine the direct effects of solute GAGs and hydrogels containing these GAGs in vitro. In both cases, sHA strongly enhanced the proliferation of PAE/KDR cells. A VEGFR-2-mediated effect of GAGs on endothelial cells as underlying mechanism is unlikely since GAGs alone did not bind to VEGFR-2 and had no influence on VEGFR-2 phosphorylation. Other factors like GAG-induced alterations of cell-matrix interactions and cell signaling could be responsible. In accordance with SPR results, a decreased endothelial cell proliferation stimulating activity of VEGF-A was observed in the presence of solute GAGs or after binding to hydrogels compared to the respective treatment without VEGF-A. However, tube formation could be observed in the presence of solute VEGF A and GAGs and within hydrogels with sGAGs that released sufficient VEGF-A amounts over time. Overall the presence of GAGs and VEGF-A strongly promoted the endothelial cell proliferation compared to the treatment with GAGs or VEGF-A alone. Thus, HA/collagen-based hydrogels functionalized with sHA derivatives offer a promising option for the design of “intelligent” biomaterials that direct and regulate the cellular behavior instead of simply acting as inert filling material. They could be used for the controlled delivery and/or scavenging of multiple mediator proteins, thus enhancing the local availability or reducing the activity of these GAG-interacting mediator proteins, or by directly influencing the cellular response. This might be applied to a range of pathological conditions by tuning the biomaterial compositions to patient-specific needs. However, extensive in vivo validation is required to show whether these in vitro findings could be used to control the biological activity of for instance TIMP-3 and VEGF-A, especially under the pathological conditions of extended matrix degradation and dysregulated angiogenesis.

Remodelling of extracellular matrix (ECM) is a prominent feature of atherosclerotic lesions and contributes to lipoprotein retention as well as smooth muscle cell (SMC) activation. To gain further knowledge about ECM, certain ECM components and their degrading enzymes were studied in injury-induced arterial neointima, which shares features with early atherosclerotic lesions.
It has been shown that synthesis of collagen and syndecan-1, a hybrid heparan/chondroitin sulfate proteoglycan, is enhanced. In situ hybridization indicates that syndecan positive cells are restricted to the arterial neointima. These data confirm the importance of arterial SMC in ECM metabolism and indicate that increased synthesis contributes to ECM accumulation in neointima.
Remodelling of ECM in atherogenesis refers not only to increased ECM deposition, but also involves enhanced ECM catabolism. A family of zinc-containing proteinases, termed matrix metalloproteinases (MMPs) has recently been implicated in atherosclerosis. Subsequently, we examined expression of two common MMPs, MMP-2 and MMP-9 in our model. The mRNAs for both MMPs are up-regulated, but their tissue distribution is different: MMP-2 positive cells are visible in neointima and in aortic media; whereas cells positive for MMP-9 are located only in neointima. MMPs are active at neutral pH and in tissue, their activity is regulated by tissue inhibitors of metalloproteinases (TIMPs) including TIMP-1. The enhanced MMP expression in neointima makes it relevant to examine the simultaneous expression of TIMP-1. To do this, we cloned rabbit TIMP-1 from neointima using a PCR-cloning technique. Transformation of the cloned gene resulted in synthesis of a TIMP-1 protein in E. Coli. The concentration of TIMP-1 in neointima was examined and a significant increase of both mRNA and protein levels was observed. It is suggested that the proteolytic activity of MMPs contributes to ECM breakdown. However, this digestion is limited, as continuous augmentation of TIMP-1 expression is observed after aortic de-endothelialization.

Dental pulp contains mesenchymal stem cells (MSCs) similar to those present within bone marrow. Several factors are postulated to contribute to the signalling involved in regulating these cells. This project aimed to investigate the role of pulp and dentine extracellular matrix (pECM/dECM) in the regulation of pulp cell behaviour during health and disease. pECM/dECM molecules were extracted using 10% EDTA, pH7.2 followed by 0.5M-NaCl, pH11.7 and 0.1M tartaric acid, pH2.0, respectively containing protease inhibitors. Proteomic analysis demonstrated the complexity of the ECM extractions. pECM-coated cultureware reduced pulp cell proliferation rates and increased stem cell marker expression compared with controls. Pulp cells exhibited multipotential capacity, with pECM-coated culture surfaces enhancing differentiation activity. pECM and dECM promoted pulp cell migration through an active rho dependent pathway and the chemotactic effects of these ECM molecules were enhanced following acidic/proteolytic degradation. Recruited cells exhibited increased stem cell marker expression. dECM and pECM possessed demonstrable bacteriostatic activity against three anaerobic bacteria associated with dental disease. Dental pulp cells were shown to be viable and capable of secreting mineral when encapsulated within a pECM/alginate scaffold and exposed to dECM molecules. Dental ECMs play important roles in regulating cellular and tissue responses during health and disease.

The introduction to the thesis discusses the control of phosphate balance and considers recent developments in the field. The concept of novel circulating substances which influence, and perhaps regulate, plasma phosphate (‘phosphatonins’), and which are important in tumour-induced hypophosphataemia, hereditary hypophosphataemias and, potentially, in normal phosphate physiology, is introduced. Finally, the isolation of the putative phosphatonin MEPE, and the current state of knowledge about this molecule, is described. The initial practical studies deal with the development and validation of a novel micromethod for measurement of phosphate and, simultaneously, other anions in biological fluids, using capillary electrophoresis. The accuracy and reproducibility of the method are demonstrated, and the method is compared with an established micromethod for measurement of individual ions. Finally, capillary electrophoresis is applied to the measurement of phosphate and other anions in urine, plasma and tubular fluid samples. In the next section, renal clearance experiments are performed in anaesthetized rats, in order to assess the effects of intravenously administered MEPE and to compare them with those of parathyroid hormone. MEPE is shown to be markedly phosphaturic. It does not alter glomerular filtration rate, blood pressure, plasma phosphate concentration or filtered phosphate load, but does cause large, dose-dependent increases in absolute and fractional phosphate excretion. The final series of experiments uses micropuncture methods in anaesthetized rats, in which fluid is collected from individual proximal convoluted tubules. The phosphaturic effect of MEPE is confirmed, and is shown to result, at least in part, from a reduction in fractional phosphate reabsorption in the proximal convoluted tubule, without a change in filtered phosphate load. The results obtained in these studies are discussed in relation to previous knowledge.

The extracellular matrix (ECM) is defined as the many different proteins and secreted substances between cells. The ECM plays a major role in the signaling pathways that stimulate cells to perform many varied functions, ranging from control of gene expression at the cellular level to differentiation and development of tissues, organs, and ultimately the entire organism. A portion of this work describes the identification of the division abnormally delayed gene, which encodes a proteoglycan, that is involved in growth factor reception with important developmental consequences in Drosophila melanogaster. The remainder of this work deals with three Drosophila homologs of vertebrate proteins that may interact with integrins, a family of cell surface receptors for extracellular matrix ligands. The three integrin-interacting proteins are referred to by their vertebrate names, and include CD81, a member of the tetraspanin family, ILK, integrin-linked kinase, and CD98hc, a type II transmembrane glycoprotein which is the heavy chain of a multi-protein complex. In this work, the mutant phenotype of CD98hc is larval lethal and not temperature sensitive. Clonal analyses of CD98hc mutants show no phenotype of mutant clones in the eye. Genetic interactions in adult tissues or interactions affecting larval lethality between CD98hc and Drosophila integrin mutants were not observed, and it remains unclear whether CD98hc physically interacts with Drosophila integrins in tissue culture cells. Since no correlation was seen between the interactions of CD98hc and integrins in vertebrate cells and similar putative interactions in flies, this raises the question as to what role, if any, does CD98hc play as an integrin modulator in this organism.

This project explored using dermal fibroblast-derived extracellular matrices (ECMs) to recapitulate the keratinocyte microenvironmental niche in vitro. The data indicated that fibroblast-derived matrices better supported keratinocytes expansion than substrates of type I collagen. ECM composition was found to profoundly alter the gene expression profiles of keratinocytes grown on these matrices, in that foetal matrices triggered expression of cell cycle genes, whereas adult matrices triggered the expression of a balance of differentiation and cell cycle genes.

Transfer RNAs (tRNAs) are key adaptor molecules that mediate decoding of messenger RNAs (mRNAs) into proteins by complementary pairing of their anticodons with mRNA codons. tRNAs that undergo adenosine to inosine editing at the wobble base, or position 34, display expanded codon decoding capacity as inosine enables pairing not only with uridine, but also with cytosine and adenosine. The essential heterodimeric enzyme Adenosine Deaminase Acting on Transfer RNA (ADAT) catalyzes this post-transcriptional modification in eukaryotes and is comprised of subunits ADAT2 and ADAT3. Emergence of heterodimeric ADAT has been proposed to have shaped both tRNA gene content and codon composition of eukaryotic genomes in such a way that these two features became mirrored. Although the exact contribution of wobble inosine (I34) to translation elongation has not been established, previous reports have suggested that it might play a role in improving translational efficiency and accuracy of genes enriched in codons recognized by I34-modified tRNAs. To further understand the role of the inosine modification in translation, we generated cell lines depleted in the catalytic subunit ADAT2. Silencing of ADAT2 lead to impaired cellular proliferation and had a variable impact on the expression of genes coding for extracellular matrix (ECM) proteins such as mucins. Notably, ADAT2 deficiency did not have major effects on the post-translational glycosylation of mucins, neither did it trigger the unfolded protein response. Supported by the absence of clear defects in decoding rates in ADAT2 depleted cells, as measured by ribosome profiling, our findings suggest that a reduced pool of I34-modified tRNAs might suffice to carry out cellular functions in steady-state conditions. However, we found that, under circumstances involving a high demand for these tRNAs such as airway remodeling, ADAT2 is required for the proper translation of an ECM gene enriched in stretches of codons read by I34-tRNAs. Taken together, our results suggest that the inosine modification is particularly relevant for the synthesis of ECM proteins during specialized processes including neural development and airway remodeling. The importance of the inosine modification has been recently underscored by the identification of pathogenic mutations in the gene encoding ADAT3, all of which share common neurodevelopmental phenotypes. The most prevalent mutation identified to date is a valine to methionine (V144M) substitution that is linked to intellectual disability and strabismus. In the present study we characterized human ADAT in terms of activity and quaternary structure, and investigated the effect of the ADAT3 V144M mutation on the enzyme. We showed that the V144M substitution leads to decreased enzymatic activity of ADAT, which might result from alterations in the tertiary structure and subcellular localization of ADAT3 that were found to be associated to the mutation.
Els ARNs de transferència (ARNt) són molècules que tenen un paper clau en el procés de traducció dels ARN missatgers (ARNm) en proteïnes mitjançant la interacció del seu anticodó amb codons d’ARNm. Els ARNt que passen per un procés d’edició d’adenosina a inosina a la base wobble, o posició 34, són capaços de llegir més d’un codó d’ARNm gràcies a la capacitat de la inosina de reconèixer els tres nucleòtids uridina, citidina i adenosina. L’enzim responsable d’aquesta modificació post-transcripcional en eucariotes s’anomena Adenosina Deaminasa específica per l’ARNt (ADAT), es tracta d’un complex heterodimèric format per les subunitats ADAT2 i ADAT3 que és essencial per a la viabilitat de l’organisme. Estudis previs han proposat que l’aparició d’ADAT va determinar el nombre de còpies gèniques de cada ARNt així com la composició de codons presents als genomes eucariòtics de tal manera que aquests dos factors estiguessin mútuament balancejats. Tot i que la contribució precisa de la inosina 34 (I34) a la traducció de proteïnes durant la fase d’elongació encara s’ha determinat experimentalment, algunes investigacions han suggerit que podria jugar un rol en l’eficiència i fidelitat de traducció de gens enriquits en codons reconeguts per ARNt modificats amb I34. Amb l’objectiu d’investigar el rol de la inosina en la traducció, hem generat línies cel·lulars on el gen codificant per ADAT2 ha estat silenciat. La depleció d’ADAT2 comporta un retard en el creixement cel·lular i té un efecte variable en l’expressió gènica de proteïnes de la matriu extracel·lular. El patró de modificacions post-traduccionals de glicosilació d’aquestes proteïnes no resulta alterat per la deficiència d’ADAT2, que tampoc activa la resposta a proteïnes desplegades. Juntament amb l’absència de defectes en la velocitat d’elongació analitzada per ribosome profiling, aquestes observacions suggereixen que la cèl·lula és capaç de dur a terme les seves funcions amb un nombre reduït d’ARNt modificats amb inosina. Hem vist, però, que en condicions que requereixen majors quantitats d’ARNt inosinats, la depleció d’ADAT2 dóna lloc a la traducció ineficient d’un gen de matriu extracel·lular altament enriquit en codons sensibles llegits per ARNt modificats. Així doncs, els nostres resultats indiquen que la inosina pot exercir un rol important en la síntesi de proteïnes de la matriu extracel·lular, particularment durant processos de desenvolupament neuronal i de remodelat de les vies respiratòries. La rellevància de la modificació I34 s’ha vist reforçada recentment per la identificació de mutacions de caire patogènic localitzades al gen que codifica ADAT3. Totes elles tenen en comú la presència de fenotips relacionats amb el desenvolupament neurològic. La mutació d’ADAT3 més comuna consisteix en la substitució d’un residu valina per un metionina (V144M) i està associada a la manifestació de discapacitat intel·lectual i estrabisme. En el present estudi hem caracteritzat l’activitat enzimàtica i l’estructura quaternària de l’ADAT humà, així com l’impacte de la mutació V144M d’ADAT3 en el complex heterdimèric. Els nostres revelen que la substitució V144M dóna lloc a una menor activitat enzimàtica d’ADAT. És possible que aquesta reducció es vegi influïda per les alteracions en l’estructura terciària i en la localització cel·lular d’ADAT3 que indueix la mutació.

Le muscle strié squelettique possède de grandes capacités de régénération grâce à ses cellules souches, les cellules satellites. Après une lésion, le processus de régénération musculaire qui se met en place est finement régulé dans le temps et l’espace par le microenvironnement, constitué de cellules avoisinantes mais également par des éléments de la matrice extracellulaire (MEC). Cette dernière se compose de molécules structurales comme les collagènes et de composants possédant un rôle trophique comme les glycosaminoglycanes (GAGs). La MEC musculaire est peu étudiée à cause d’une organisation tridimensionnelle complexe rendant son exploration difficile. Lors d’une lésion avec perte de substance musculaire, la régénération est altérée, associée à une fibrose et une inflammation chronique. Ce type de lésion est fréquemment rencontré en traumatologie mais survient également chez le blessé de guerre. Malgré un traitement optimal, une invalidité fonctionnelle persiste chez ces patients. L’utilisation d’un biomatériau décellularisé, constitué de MEC pourrait fournir ce support physique et trophique faisant défaut dans ce type de lésion. Dans ce travail, nous avons entrepris l'établissement d'une MEC d’origine musculaire et nous avons établi un protocole de décellularisation permettant d’obtenir un biomatériau conservant l’architecture spécifique de la MEC musculaire avec une élimination de la majorité des antigènes cellulaires afin d'éviter une réponse immunitaire délétère après implantation. Néanmoins, le protocole retenu ne permet de conserver certaines molécules trophiques d’intérêt comme les GAGs. Les « ReGeneRaTing Agent®» (RGTA®) sont des mimétiques fonctionnels de ces GAGs, utilisés en clinique pour améliorer la cicatrisation cutanée et cornéenne. Ces mimétiques conservent une capacité de liaison aux facteurs de croissance avec une résistance aux dégradations enzymatiques. Nous avons évalué l’utilisation de ces molécules au cours de la réparation musculaire, dans un modèle in vivo chez le rongeur. Nous avons réalisé une analyse histologique précoce (8e jour de régénération) mettant en évidence une augmentation du nombre de noyaux par myofibre en faveur d’une augmentation de la fusion, validée également in vitro sur des progéniteurs musculaires. Nous avons également observé une augmentation du nombre de vaisseaux, suggérant une amélioration de l’angiogenèse. Le nombre de gouttelettes lipidiques, marqueur d’une mauvaise régénération, était en diminution. L’exploration histologique plus tardive (28e jour de régénération) n’a retrouvé que l’augmentation du nombre de vaisseaux en faveur d’un effet durable sur l’angiogenèse. Ces RGTA® peuvent être couplés aux biomatériaux et sont particulièrement résistants dans un environnement inflammatoire pouvant être rencontré dans les lésions avec perte de substance musculaire. Des chimiokines et des facteurs de croissance pourront également être ajoutés au biomatériau matriciel afin de favoriser la migration des différents progéniteurs nécessaires à une néoformation musculaire. L’efficacité thérapeutique de ces biomatériaux optimisés nécessitera d’être évaluée dans un modèle in vivo de perte de substance
Skeletal muscle exhibits high capacity for regeneration after an injury that relies on resident stem cells. Muscle regeneration is tightly regulated by both the immune response and other resident cells, as well as by cues from the local extracellular matrix (ECM), contributing to a coordinated repair process. Muscle ECM is a network of structural macromolecules with a large majority of collagens and trophic molecules such as glycosaminoglycans (GAGs). In the skeletal muscle tissue, ECM was overlooked due to its complex organization making investigations difficult. Muscle regenerative ability can be overtaken in large muscle wasting, such as in volumetric muscle loss (VML), leading to fibrosis formation and chronic inflammation. This type of injury predominantly occurs in traumatology and in war-wounded patients, with functional disability despite an optimal treatment. The use of biomaterials could provide the biochemical and physical cues that are missing in this pathologic repair. In this work we have focused on obtaining a biomaterial composed of skeletal muscle ECM. We have tested several decellularization protocols both to preserve the three-dimensional architecture of the muscle ECM and to completely remove cell components in order to avoid a deleterious immune response after implantation. However, the protocol did not allow the preservation of trophic molecules such as GAGs, in the scaffold.“ReGenerating Agents” (RGTA®) are functionally analogous of GAGs with a crucial property to resist enzymatic degradation. They function to restore a proper microenvironment for tissue healing with already a clinical application in skin and corneal repair. We have explored the effects of RGTA® in muscle regeneration using an in vivo model in mouse. At early time of regeneration (day 8), we performed histologic analysis. We showed that regenerating myofibers contained more nuclei in the treated animals, in favor of an increase of progenitor fusion, which has been validated in vitro in myogenic cultures. The number of capillaries was higher in favor of a better angiogenesis. Lipid droplets, a marker of impaired regeneration, were reduced by RGTA® administration. At later time of regeneration (day 28), capillary number was still improved in favor of a durable effect of RGTA® on angiogenesis. RGTA® could be incorporated into biomaterials and are particularly resistant in an inflammatory environment, such as that occurring after a VML injury. Chemokines and growth factors could also be added in ECM-based scaffolds to promote the migration of progenitors that are essential for myofiber neoformation. Therapeutic efficacy of these optimized biomaterials will require to be evaluated in an in vivo model of VML

A number of purification strategies were performed in an attempt to produce highly purified, active TRAMP free from lysyl oxidase for fibrillogenesis and enzymatic studies. TRAMP was eventually purified by DEAE anion exchange followed by Superdex-75 size exclusion (in the presence of urea) and Mono Q FPLC anion exchange chromatography. Yields of 10μg of TRAMP per g wet weight starting material were obtained. TRAMP purified as above was active in fibrillogenesis assays using the 'warm start' technique, in buffers containing 30mM TES, 30mM Na₂HPO₄, 135mM NaCl pH 7.4. The acceleratory effect of TRAMP on collagen I fibril formation was also observed when the phosphate concentration was lowered to 10mM and when TES was removed. TRAMP has previously been shown to bind to in vitro reconstituted collagen fibrils if present during their formation. Reducing the phosphate concentration decreased the amount of TRAMP associated with collagen I fibrils in co-sedimentation assays, whilst subsequent removal of TES had no effect on TRAMP binding to collagen I fibrils. Preliminary observations also suggested that treatment of TRAMP with sulphatase had no effect on the ability of TRAMP to accelerate collagen I fibril formation. A solid phase assay showed TRAMP to bind collagen I monomers with a higher affinity than fibrils, suggesting a role for TRAMP in the early, nucleation phase of fibril formation. TRAMP was unable to reverse the inhibitory effect of decorin on fibril formation. The presence of decorin had no effect on TRAMP binding to collagen monomers in solid phase assays but led to an increase in the amount of TRAMP associated with collagen fibrils in co-sedimentation assays. Attempts to identify specific binding sites for TRAMP on in vitro reconstituted collagen I fibrils by immunogold labelling and electron microscopy were unsuccessful. Western blot analysis of murine tissues confirmed previous reports that TRAMP was present in lung and skeletal muscle and absent in brain and spleen.

The epithelial monolayer of cells surrounding the animal, the hypodermis, Synthesises five cuticles during the nematode life cycle. The first cuticle is formed within the egg, prior to hatching, and the remainder towards the end of each larval stage. Because of the structural role of the cuticle, mutations in genes involved in assembly of this ECM can cause a spectrum of effects from lethality late in embryogenesis to alterations in the nematode shape. The severity of phenotype correlates with the severity of cuticle synthesis defects. Accordingly, two distinct mutant alleles that cause death after embryonic elongation, possibly due to failure in synthesising an intact cuticle, were characterised . One mutant, ij15, was isolated from a forward genetic screen previously performed (I. Johnstone, Glasgow University, Glasgow, UK). ij15 defines mutationally the gene stc-1, which encodes a HSP70-like protein possibly localised in the secretory pathway. The other mutant, h402, defines mutationally the gene let-607. A second let-607 allele, h189, which results in larval lethaity at the L2 stage was also analysed in this study. let-607 corresponds to the predicted gene F57B10.1, which encodes a putative bZIP transcription factor. Both stc-1 and let-607 are expressed in the hypodermis at all developmental stages. Furthermore, disruption of the function of either stc-1 or let-607 by mutation or RNAi affects cuticle synthesis in different ways. Thus, stc-1 and let-607 encode for a HSP70-like protein and a putative bZIP transcription factor required for synthesis of the cuticular ECM in C. elegans. In addition, this study defines C. elegans mutant phenotypes that can be used as indicators for gene products with controlling roles in the synthesis of this ECM.

Glycation of the extracellular matrix has been linked to chronic vascular disease and endothelial cell dysfunction. Proper functioning of the ECM requircs both chemical and structural integrity, both of which arc compromised by glycation. The mechanism, extent and functional consequenc·cs of ECM glycation have not yet been clearly defined. The metabolic dysfunction underlying this vascular damage and disruption is unclear. Methylglyoxal, a dicarbonyl glycating agent increased in hyperglycaemia, is known to modify arginine residues in proteins. Increased modification of the vascular basement membrane type IV collagen by methylglyoxal formed arginine-derived hydroimidazolone residues at hotspot sites in RGD and GFOGER integrin-binding sites. Loss of functional contact between integrins and the cxtraccllular matrix activates anoikis and impairs angiogcnesis. Inc~bation of endothelial cclls in hyperglycacmia and cxperimental diabetes in vivo produced the same modifications in vascular collagcn and induced similar responses. Pharmacological scavenging ofmcthylglyoxal prevented anoikis and maintained angiogenesis. In normoglycaemia, thcse responses werc provoked by a cell permeable glyoxalase I inhibitor.

Il est de plus en plus évident que la matrice extracellulaire (MEC), au-delà de sa fonction d’échafaudage cellulaire, génère des signaux de nature biochimique et biophysique jouant un rôle primordial au cours du processus de différenciation des cellules souches. A l’heure actuelle, plus de 15 différents facteurs extrinsèques (environnementaux), incluant l’organisation spatiale de la MEC, sa topographie, rigidité, porosité, biodégradabilité et chimie ont été identifiés comme modulateurs potentiels de la différenciation des cellules souches en lignées cellulaires spécialisées. Ainsi, il est plausible que l’intégration d’un biomatériau au sein de l’organisme dépendra largement de sa capacité à mimer les propriétés de la MEC du tissu à remplacer. Récemment, les techniques de micro-ingénierie ont émergé comme outil innovant pour découpler les différentes propriétés de la MEC et étudier l’impact individuel ou combiné de ces facteurs sur le comportement des cellules souches. De plus, ces techniques de microfabrication ont un intérêt particulier dans une perspective de reconstruction de la MEC dans tous ses aspects, in vitro. Dans ce projet de thèse, le concept de déconstruction/reconstruction de la complexité de la MEC a été appliqué pour récapituler, in vitro, plusieurs aspects inhérents à la MEC osseuse et explorer leurs effets individuels ou combinés sur la différenciation ostéoblastique des cellules souches mésenchymateuses (CSMs) humaines. Trois principales composantes ont été utilisées tout au long du projet : un matériau modèle (verre borosilicate), des séquences peptidiques mimétiques dérivées de la MEC naturelle, favorisant à la fois l’adhérence cellulaire (peptide RGD) et la différenciation ostéoblastique (peptide BMP-2) des CSMs prélevées de la moelle osseuse des patients. La première étude du projet consiste à greffer, d’une manière aléatoire, les peptides RGD et/ou BMP-2 sur la surface du matériau. Brièvement, nous avons développé trois types de matériaux bioactifs : matériaux fonctionnalisés avec le peptide RGD, matériaux fonctionnalisés avec le peptide BMP-2 et matériaux bi-fonctionnalisés avec les peptides RGD/BMP-2. La caractérisation physicochimique de ces matériaux a été réalisée en utilisant la spectrométrie photoélectrique à rayons X (XPS) pour évaluer la composition chimique de la surface, la microscopie à force atomique (AFM) pour évaluer la topographie de la surface et la microscopie à fluorescence pour confirmer la présence des peptides sur la surface et évaluer leur densité. L’objectif de cette étude est d’évaluer le potentiel individuel et synergétique de ces peptides à induire et contrôler la différentiation ostéoblastique des CSMs. Dans un premier temps, la caractérisation physicochimique nous a permis de confirmer l’immobilisation covalente des peptides sur la surface et de mesurer leur densité. En effet, la densité des peptides, mesurée sur les surfaces greffées uniquement avec le peptide RGD ou BMP-2, était de 1.8 ± 0.2 pmol/mm² et 2.2 ± 0.3 pmol/mm², respectivement. Cependant, sur les surfaces bifonctionnalisées, la densité de chaque peptide a diminué de presque la moitié, atteignant 0.7 ± 0.1 pmol/mm² pour le peptide RGD et 1 ± 0.1 pmol/mm² pour le peptide BMP-2. Ensuite, l’évaluation biologique des différents matériaux fonctionnalisés a clairement révélé que contrairement au peptide RGD, le peptide BMP-2 induit la différenciation ostéoblastique des CSMs. Cependant, le greffage simultané des peptides RGD/BMP-2 améliore significativement la différenciation des CSMs en ostéoblastes et cela malgré la diminution significative de la densité de chaque peptide sur les surfaces bi-fonctionnalisées, comparativement aux surfaces contenant qu’un seul peptide. Ces résultats montrent que les peptides RGD et BMP-2 peuvent engendrer un effet synergétique pour améliorer la différenciation ostéoblastique des CSMs. Le second chapitre de thèse vise à déterminer si la microstructuration de la surface des matériaux avec des ligands bioactifs améliore la différenciation ostéoblastique des CSMs. En effet, les peptides RGD et BMP-2 ont été greffés séparément sur la surface du matériau sous forme de micro-motifs de différentes formes mais de taille similaire. En se basant sur des précédents travaux de littérature – discutés dans le chapitre II – nous avons sélectionné trois différentes formes de motifs peptidiques (triangle, carré et rectangle) dont la surface est de 50 μm². Ces micromotifs ont été créées grâce à une technique assez répondue et facile à utiliser qui est la photolithographie. Les surfaces microstructurées ont été caractérisées avec l’interférométrie optique et la microscopie à fluorescence. Les résultats montrent que les micromotifs peptidiques ont à la fois la forme et les dimensions prédéfinies. In vitro, les résultats de différenciation cellulaire ont révélé que la distribution spatiale des ligands à l’échelle micrométrique joue un rôle très important dans l’engagement et la différenciation des CSMs en ostéoblastes. En effet, contrairement aux micromotifs peptidiques en forme de rectangles, les micromotifs triangulaires et carrés améliorent significativement l’expression des marqueurs ostéogéniques (Runx-2 et Ostéopontine) comparativement à la distribution aléatoire des peptides. Il est important de noter que ce profile d’expression des marqueurs biologiques a été observé que sur les matériaux fonctionnalisés avec le peptide BMP-2, tant dis que les matériaux fonctionnalisés avec le peptide RGD n’ont induit aucun effet spécifique sur la différenciation des CSMs et cela peu importe la forme des micromotifs peptidiques. En conclusion, cette étude a permis d’identifier un nouveau facteur extracellulaire capable de contrôler la différenciation des CSMs. De plus, nous avons démontré que la distribution spatiale des ligands à l’échelle micrométrique affecte le devenir des CSMs, dépendamment de la nature du principe actif. Finalement, la troisième étude de ce projet de thèse est une suite logique de l’étude 1 et 2, puisqu’elle consiste à greffer simultanément les peptides RGD et BMP-2 sous forme de micromotifs. En effet, ces surfaces ont été développées afin de bénéficier à la fois de l’effet synergétique des peptides RGD/BMP-2, observé dans l’étude 1 (facteur 1), et de l’effet de la distribution spatiale contrôlée des ligands, observé dans l’étude 2 (facteur 2). Les différents types de matériaux ont été caractérisés avec les mêmes techniques de caractérisation de surface mentionnées dans l’étude 2. Les résultats montrent clairement que les surfaces microstructurées sont très bien définies et correspondent à un damier de micromotifs RGD, intercalé par un damier de micromotifs BMP-2. L’évaluation de la différenciation des CSMs sur ces matériaux a révélé que la combinaison des facteurs 1 et 2 améliore significativement la différenciation des CSMs vers le lignage ostéoblastique, comparativement à l’exposition des CSMs à un seul facteur extracellulaire (1 ou 2). De plus, cette étude confirme les résultats obtenus dans l’étude 2, puisque les micromotifs triangulaires et carrés ont permis une meilleure différenciation cellulaire, comparativement aux micromotifs rectangulaires. Il est important de noter également que l’évaluation biologique des différentes surfaces biomimétiques a été réalisée dans un milieu de culture basal qui ne contient pas de facteurs ostéogéniques solubles, afin d’étudier d’une manière assez précise et fiable les interactions des CSMs avec les différents microenvironnements in vitro développés dans ce projet. En conclusion générale, les travaux effectués jusqu’à présent ont permis d’identifier deux aspects de la MEC qui influencent considérablement la différenciation ostéoblastique des CSMs. De plus, nous avons démontré que ces deux facteurs peuvent coopérer pour induire une meilleure différenciation cellulaire. Cela révèle clairement l’intérêt des techniques de micro-ingénierie pour une meilleure et plus profonde compréhension des mécanismes d’interactions des cellules souches avec leurs niches, ce qui permettra éventuellement de concevoir des produits d’ingénierie tissulaire sur-mesure. Mots clés : Microstructuration de la surface des matériaux, matrice extracellulaire biomimétique, peptides mimétiques, BMP-2, cellules souches, ostéogenèse.
It is becoming increasingly appreciated that the role of extracellular matrix (ECM) extends beyond acting as scaffolds to providing biochemical and biophysical cues, which are critically important in regulating stem cell self-renewal and differentiation. To date, more than 15 different extrinsic (environmental) factors, including the matrix spatial organization, topography, stiffness, porosity, biodegradability and chemistry have been identified as potent regulators of stem cells specification into lineage-specific progenies. Thus, it is plausible that the behavior of biomaterials inside the human body will depend to a large extent on their ability to mimic ECM properties of the tissue to be replaced. Recently, nano- and microengineering methods have emerged as an innovative tool to dissect the individual role of ECM features and understand how each element regulates stem cell fate. In addition, such tools are believed to be useful in reconstructing complex tissue-like structures resembling the native ECM to better predict and control cellular functions. In the thesis project presented here, the concept of deconstructing and reconstructing the ECM complexity was applied to reproduce several aspects inherent to the bone ECM and harness their individual or combinatorial effect on directing human mesenchymal stem cells (hMSCs) differentiation towards the osteoblastic lineage. Three main components were used throughout this project: a model material (borosilicate glass), ECM derived peptides (adhesive RGD and osteoinductive BMP-2 mimetic peptides) and bone marrow derived hMSCs. All cell differentiation experiments were performed in the absence of soluble osteogenic factors in the medium in order to precisely assess the interplay between hMSCs and the different artificial matrices developed in the current study. First, RGD and/or BMP-2 peptides were covalently immobilized and randomly distributed on glass surfaces. The objective here was to investigate the effect of each peptide as well as their combination on regulating hMSCs osteogenic differentiation. The most important funding was that RGD and BMP-2 peptides can act synergistically to enhance hMSCs osteogenesis. Then, micropatterning technique (photolithography) was introduced to control the spatial distribution of RGD and BMP-2 at the micrometer scale. The peptides were grafted individually onto glass substrates, as specific micropatterns of varied shapes (triangular, square and rectangle geometries) but constant size (50 μm² per pattern). In this second part of the project, the focus was made on investigating the role of ligands presentation in a spatially controlled manner in directing hMSCs differentiation into osteoblasts. Herein, we demonstrated that the effect of microscale geometric cues on stem cell differentiation is peptide dependent. Finally, glass surfaces modified with combined and spatially distributed peptides were used as in vitro cell culture models to evaluate the interplay between RGD/BMP-2 crosstalk and microscale geometric cues in regulating stem cell fate. In this study, we revealed that the combination of several ECM cues (ligand crosstalk and geometric cues), instead of the action of individual cues further enhances hMSCs osteogenesis. Overall, our findings provide new insights into the role of single ECM features as well their cooperation in regulating hMSCs fate. Such studies would allow the reconstruction of stem cell microenvironment in all the aspects ex vivo, which may pave the way towards the development of clinically relevant tissue-engineered constructs. Keywords: Chemical micropatterning, bioactive surfaces, mimetic peptides, BMP-2, mesenchymal stem cells, stem-cell differentiation, stem-cell niche, osteogenesis.

L’angiogenèses par bourgeonnement est associée à une réorganisation majeure de la matrice extracellulaire (MEC). Nous avons déjà démontré que la lysyl oxydase-like 2 (LOXL2), une enzyme responsable du crosslinking de la MEC, régule la formation de vaisseaux intersomitiques dans les embryons de poisson zèbre et de capillaires en hydrogels 3D. Dans ce manuscrit, nous avons examiné les mécanismes impliqués dans cette régulation. Nous avons constaté l’association intracellulaire de LOXL2 avec la fibronectine et le collagène IV, avant d’être incorporée dans des structures fibrillaires dès l’exocytose. De plus, l’inhibition de l’expression de LOXL2 entraine des défauts de déposition de la MEC et diminue sa rigidité, inhibant secondairement la maturation des structures d'adhésion cellulaire. Alors que LOXL2 n‘est pas nécessaire pour la formation de capillaires dans un modèle 2D sur MEC de fibroblastes, les défauts de déposition de MEC sont corrélés à l'inhibition de formation des capillaires en hydrogel 3D. Ni l’addition de LOXL2 exogène, ni l’augmentation de la rigidité des hydrogels ne compense la perte d’expression de LOXL2. Enfin, nous avons pu montrer que ni l'activité catalytique ni le domaine catalytique de LOXL2 ne sont essentiels pour la formation de capillaire dans le poisson zèbre et dans les hydrogels et pour l’assemblage du collagène IV par des cellules endothéliales. L’ensemble de ces données suggère donc que les domaines SRCR de LOXL2 exprimés par des cellules endothéliales régulent l’échafaudage de fibronectine et de collagène IV dans la MEC qui est nécessaire à la formation de capillaires
Sprouting angiogenesis is associated with major extracellular matrix (ECM) remodelling, consisting in both degradation of the microenvironment and generation of a new basement membrane. We have previously reported that lysyl oxidase-like 2 (LOXL2), an enzyme responsible for ECM crosslinking, regulates formation of intersomitic vessels (ISV) of zebrafish embryos and of capillaries in 3D hydrogels. In this manuscript we investigated the mechanisms involved. We found that LOXL2 associates with fibronectin and collagen IV intracellulary before direct incorporation in fibrillar structures of the ECM upon exocytosis. In addition, silencing LOXL2 demonstrated its involvement in ECM deposition as both composition and stiffness of the ECM were affected, which subsequently altered maturation of cell adhesion structures. Whereas LOXL2 is not required for formation of capillaries on top of a fibroblast monolayer, in a 2D assay, ECM defaults were associated with altered formation of capillaries in 3D hydrogels.Neither addition of exogenous LOXL2, nor increasing the stiffness of hydrogels could restore capillary formation. Moreover, we could show that neither the catalytic activity nor the catalytic domain were required for capillary formation in vivo and in 3D hydrogels, and for collagen IV deposition by endothelial cells. Altogether, these data suggest that the SRCR domains of LOXL2 expressed by endothelial cells regulate 3D capillary morphogenesis through scaffolding of fibronectin and collagen IV in the ECM

La matrice extracellulaire (MEC) contrôle et maintient les principales activités biologiques telles que la survie, la prolifération et la différenciation cellulaire. Récemment, les hydrogels ont marqué une progression remarquable en tant que candidats dans le domaine de l'ingénierie tissulaire et de la médecine régénérative. Les hydrogels sont des réseaux polymériques hydrophiles ayant la capacité d’absorber une grande quantité d’eau et de fluide biologique. Les hydrogels présentent un support mécanique approprié pour les cellules tissulaires et fournissent des signaux chimiques et biologiques imitant la MEC native. Par conséquent, de nombreux hydrogels de nature biologique et chimique ont été développés dans le domaine de régénération tissulaire. Les propriétés mécaniques des hydrogels sont nécessaires pour induire les fonctions biologiques telles que l'adhésion, la prolifération et la différenciation cellulaire. L'objectif de la thèse était de développer des hydrogels à base de polymères et d'étudier l'effet de leurs propriétés physiques sur les activités biologiques des cellules podocytaires. Cette étude consiste de synthétiser et de développer des hydrogels à base de polyacrylamide hydrolysé (PAAm) où les propriétés physiques peuvent être adaptées et réglées sur une large gamme d’élasticité. Ces matériaux ont fourni une élasticité similaire à celle de la membrane basale glomérulaire (GBM) in vivo et ont représenté un candidat approprié pour la régulation des fonctions des cellules podocytaires. De même, la synthèse des hydrogels à la fois synthétiques et biologiques a pu imiter les propriétés biologiques et mécaniques de la MEC native. La combinaison des polymères à base de méthacrylate de gélatine et d'acrylamide (GelMA-AAm) a été synthétisée et analysée. Ces hydrogels ont montré des propriétés mécaniques ajustables imitant l'élasticité native du GBM du rein et une fixation significative des podocytes sans modification de surface par des protéines d'adhésion. Ce travail consiste à étudier la physiologie cellulaire et à développer un système microfluidique afin de suivre les fonctions rénales dans les états normales et défectés
Extracellular matrix (ECM), non-cellular component, regulates and maintains the main biological activities of cells such as cellular survival, proliferation and differentiation. Recently, hydrogels scaffolds have shown a remarkable advancement as candidates for tissue engineering and regenerative medicine. Hydrogels are defined as hydrophilic polymer network having the ability to hold a large amount of water and biological fluid. Various natural and synthetic hydrogels have been studied and developed in many tissue regeneration purposes. They provide an appropriate mechanical support, chemical and biological cues mimicking the native extracellular matrix (ECM). These artificial matrices characteristics contribute to induce the cellular functions as adhesion, proliferation and differentiation. The thesis aim was to develop polymers based hydrogels and to study the effect of their physical properties on podocyte kidney cells. Synthetic hydrolyzed polyacrylamide based hydrogel (PAAm) was the choice of study where the physical properties can be tailored and tuned over a wide range. These scaffolds have provided elasticity similar to the in vivo glomerular basement membrane (GBM) and have shown a suitable candidate for the regulation of podocyte functions. Moreover, the development of synthetic and biologic hybrid hydrogels was able to mimic the biological and mechanical properties of native ECM. The combination of gelatin methacrylate and acrylamide (GelMA-AAm) based hydrogels have been investigated and has shown tunable mechanical properties mimicking the native kidney GBM elasticity and a significant attachment of podocytes without any surface functionalization with adhesion proteins. This work permits to investigate the cellular physiology and to develop kidney-on-chip in order to study the functions of kidney on both healthy and diseased states

L’interaction épithélium/mésenchyme est cruciale pour de nombreux processus physiopathologiques. Lors de ma thèse, je me suis intéressée aux signaux mésenchymateux régulant les cellules épithéliales en utilisant comme modèle la peau, qui est composée de 2 compartiments : l’épiderme (épithélium) et le derme (mésenchyme). Les intégrines sont impliquées ces interactions. CD98hc est une protéine transmembranaire à double fonction qui chaperonne des transporteurs d’acides aminés et régule la signalisation des ß intégrines. Elle est exprimée dans les cellules prolifératives telle les cellules épithéliales. Dans la peau, CD98hc est exprimée l’épiderme mais également dans les fibroblastes, cellules post-mitotiques. Mon hypothèse a été que CD98hc participe aux régulations des interactions derme/épiderme. Grâce à un modèle de KO conditionnel de CD98hc dans les fibroblastes dermiques, j’ai mis en évidence que CD98hc permet le maintient des propriétés mécaniques et biologiques du derme, et, de ce fait, régule l’épiderme en conditions d’homéostasie, de perturbation de la barrière et lors de la formation de cancer. De plus, le rôle de CD98hc dans cette interaction apparait comme étant lié à l’âge. En conclusion, mes travaux de thèse montrent le rôle central de l’expression dermique de CD98hc dans le maintien de l’homéostasie cutanée au cours du vieillissement ainsi que lors de la tumorigenèse
The epithelial/mesenchymal interaction is crucial for many physiopathological processes. During my PhD, I focused on mesenchymal signals that regulate epithelial cells behavior using the skin as model. The skin is composed of 2 main compartments: the epidermis (epithelium) and the dermis (mesenchyme). While this crosstalk involves integrins, its regulations are poorly understood. The transmembrane protein CD98hc interacts with amino acid transporter and regulates integrin signaling. CD98hc which is expressed at the cell membrane of proliferative cells, specifically epithelial cells, is required for tissue homeostasis. We found that besides its expression in keratinocytes, CD98hc is also expressed in post-mitotic dermal fibroblast. Hence, I hypothesized that CD98hc is involved in epidermis/dermis crosstalk. Using a conditional KO mouse model that harbor a CD98hc deletion in dermal fibroblast, I have shown that dermal CD98hc is required to maintain mechanical and biochemical properties of the dermis. Moreover, I have shown that those CD98hc-dependent dermal properties are implicated in the regulation of the epidermal cell behavior during homeostasis, cutaneous barrier disruption and tumorigenesis. Moreover, the role of CD98hc in those processes seems to be age-related. To conclude, during my PhD, I have revealed a major role of CD98hc in the maintenance of skin homeostasis during aging and tumorigenesis

The bone marrow (BM) niche provides hematopoietic stem (HSC) and progenitor cells with many exogenous cues that tightly regulate homeostasis. These cues orchestrate cellular decisions, which are difficult to dissect and analyze in vivo. This thesis introduces a novel in vitro platform that permits systematic studies of BM-relevant factors that regulate homeostasis. Specifically, the role of 3D patterned adhesion ligands and soluble cytokines were studied in a combinatorial fashion. Analysis of human HSC differentiation and proliferation at both population and single cell level showed synergistic and antagonistic effects of adhesion- and cytokine-related signals. Those effects were dependent on the cytokine concentration and the distribution and number of adhesion ligands. The aim of this thesis was to model the in vivo bone marrow with its porous 3D structure and different sized niche compartments using a microcavity culture carrier. The developed culture system presented extracellular matrix (ECM) adhesion ligands to the HSCs in various defined dimensions ranging from single- to multi-cell capacity. The 3D open well geometry of the microcavity carriers also allowed HSCs to freely explore different scenarios including homing, migration, adhesion, or suspension. Furthermore, the developed setup offered straightforward accessibility to analytical methods like cytometry and quantitative microscopy. Single cell analysis of adherent HSCs showed decreased DNA synthesis and higher levels of stem cell marker expression within single cell microcavities under low cytokine conditions . This effect was reflected in a decline of proliferation and differentiation with decreasing microcavity size. When the cytokine concentration was increased2 beyond physiological levels the inhibitory effect on proliferation and differentiation due to single-cell-microcavity adherence was diminished. This result highlighted the fine balance between adhesion related and soluble cues regulating HSC fate. Within small microcavities more adhesion related receptors were engaged due to the 3D character of the culture carrier compared to multi-cell wells or conventional 2D cell culture plates. This study demonstrated that adhesion-related signal activation leads to reduced proliferation and differentiation. This geometry-based effect could be reversed by increased cytokine supplementation in the culture media. For plane substrates, HSCs attachment to fibronectin or heparin initiated early cell cycle entry compared to non-adherent cells during the initial 24h. Cytokine supplemented media favored integrin activation that induced fast adhesion, ultimately leading to early cell cycle activation. However, after prolonged cell culture the system balanced itself with a lower cycling rate of adherent versus non-adherent HSCs. Furthermore, HSCs within the 3-dimensionality of the microcavities cycled less than 2D adherent cells. These findings additionally supported the above stated idea of limited HSC proliferation as a consequence of more adhesion-related signals overwriting cytokine driven expansion. To complement the various in vitro studies, an in vivo repopulation study was performed. Cultured HSCs derived from single cell microcavities outperformed freshly isolated HSCs in a competitive repopulation assay, indicating that carefully engineered substrates are capable of preserving stem cell potential. Overall the reported findings provide a promising in vitro culture strategy that allows the stem cell field to gain a better understanding of the impact of distinct exogenous signals on human HSCs, which discloses new concepts for the wide scientific community working towards tissue engineering and regenerative medicine
Die Homöostase der Hämatopoietischen Stamm- und Vorläuferzellen (HSC) in der Knochenmark Nische wird von einer Vielzahl exogener Faktoren gezielt reguliert. Diese Faktoren orchestrieren intrazelluläre Vorgänge, deren in vivo Analyse kompliziert ist. Die vorliegende These widmet sich einem neuen biotechnologischen Ansatz, der systematische Studien von Knochenmark-relevanten Faktoren ermöglicht. Im Speziellen wurde die Rolle 3D-präsentierter Zell Adhäsionsliganden in Kombination mit verschiedenen Konzentrationen löslicher Zytokine untersucht. Die Auswertung der Proliferation und Differenzierung von humanen HSC auf Einzelzell- und Populationsebene offenbarte die synergistischen und antagonistischen Effekte von Adhäsions- und Zytokinsignalen in ihrer Abhängigkeit von der Verteilung und der Anzahl von Adhäsionsliganden sowie der Zytokinkonzentration. Um die poröse Struktur des Knochenmarks in vivo-ähnlich darzustellen, wurde eine Zellkultur Plattform mit Mikrokavitäten verschiedenster Dimensionen von Multi- bis Einzelzellgröße entwickelt und mit Molekülen der extrazellulären Matrix beschichtet. Die Vorteile dieser Plattform liegen in der offenen 3D-Geometrie dieses mikrokavitäten Kultursystems, die den Zellen ermöglichte verschiedene Wachstumsbedingungen bezüglich Homing, Migration, Adhäsion oder Suspension frei zu erkunden. Das leicht zugängliche Setup eignete sich zudem hervorragend für die zytometrische Analyse der Zellen oder die quantitative Mikroskopie. Die Einzelzellanalyse adhärenter HSC ergab eine Reduktion von DNA Synthese und eine höhere Expression von Stammzelloberflächenfaktoren innerhalb der Einzelzell-Mikrokavitäten bei niedrigen Zytokinkonzentrationen . Dieser Effekt spiegelte sich auch auf Populationsebene in verminderter Proliferation und Differenzierung mit abnehmender Größe der Mikrokavitäten wider. Wurde die Zytokinkonzentration jedoch weit über physiologische Bedingungen erhöht, verminderte sich der Effekt (reduzierte DNA Synthese und höhere Stammzellfaktorexpression) beschrieben für die Einzelzellmikrokavitäten. Dieses Ergebnis verdeutlicht die empfindliche intrazelluläre Balance, vermittelt durch Adhäsionsignale und löslichen Faktoren, die das Verhalten von HSCs regulieren. Aufgrund des 3D-Charakters des Zellkulturträgers wurden innerhalb kleiner Mikrokavitäten mehr Adhäsionsrezeptoren ringsum die Zelle aktiviert. Dieser Vorteil gegenüber den Multizellkavitäten oder der herkömmlichen 2D–Zellkultur ermöglichte eine hohe Anzahl adhäsionsvermittelter Signale mit entsprechend höherer Proliferations-inhibitorischer Wirkung. Je höher die Konzentration der Zytokine war, desto stärker erfolgte die Stimulation der Proliferation und Differenzierung. Auf 2D Substraten, initiierte Adhäsion zu Fibronektin und Heparin innerhalb der ersten 24h einen frühen Zell-Zyklus-Start im Gegensatz zu nicht adhärenten Zellen. Die Zytokine im Zellmedium förderten die Integrin Aktivierung, was zu einer schnellen Zelladhäsion führte. Die Adhäsionsrezeptoren wiederum kooperieren mit Zytokinrezeptoren im Zellinneren und begünstigten damit einen zeitigeren Zell-Zyklus- Start. Allerdings stellte sich danach ein Gleichgewicht im Kultursystem ein, wobei weniger adhärente Zellen als nicht-adhärente Zellen den Zellzyklus durchliefen. Des Weiteren war die Zellzyklusrate innerhalb von 3D Mikrokavitäten niedriger verglichen mit herkömmlichen 2D Substraten. Diese Ergebnisse bestätigen ferner obenstehende These, dass Zytokin-induzierte Zellexpansion durch erhöhte Zelladhäsions-vermittelte Signale überschrieben wird. Um die in vitro Studien zu komplettieren wurde ein in vivo Repopulationsversuch durchgeführt. HSC kultiviert auf Einzel-Zell-Mikrokavitäten übertrafen frisch isolierte Konkurrenz-Zellen in einem kompetitiven Repopulationsversuch. Dieses erste Ergebnis zeigt, dass sich der Zellgröße entsprechende Biomaterialien für die erfolgreiche Stammzell-Kultur eignen. Die Ergebnisse dieser Arbeit bieten eine vielversprechende in vitro Zellkulturstrategie, die ein besseres Verständnis der Einflüsse von exogenen Signalen auf HSC erlaubt und damit eine Grundlage für neue Erkenntnisse in Richtung erfolgreicheres Tissue Engineering und klinische Anwendungen im Bereich der regenerativen Medizin bildet

Glomerular epithelial cells (GECs) are intrinsic components of the kidney glomerulus and are in contact with extracellular matrix (ECM). Under normal conditions, there is little turnover of GECs, but in glomerular injury, apoptosis and proliferation of GECs, and expansion of ECM may lead to sclerosis and impaired glomerular function and/or permselectivity. In order to better understand how adhesion to ECM modulates GEC survival and proliferation, we monitored the activation of the intracellular signaling pathways activated by adhesion to ECM (collagen), along with their coordinate modulation of the growth factor signaling pathways. Adhesion to collagen resulted in the activation of a focal adhesion kinase (FAK)/Src/Grb2 complex, thus activating extracellular signal-regulated kinase (ERK), possibly within focal adhesions. The ERK pathway was necessary to promote survival and growth factor-dependent proliferation. ECM-induced ERK activation was also necessary to achieve and maintain a cortical F-actin structure. There appeared to be a reciprocal relationship between collagen-induced cortical F-actin assembly and collagen-dependent survival signaling, including ERK activation. Moreover, maintenance of the cortical F-actin architecture was associated with increased turnover of inositol phospholipids. Analogous signals for GEC survival are activated in experimental focal segmental glomerulosclerosis (FSGS) in vivo. These studies will provide insights into novel therapeutic approaches to preserving intact glomerular permselectivity.

Stem cell research has raised great interest in the scientific community as it has the potential to form multiple cell types and it is believed they hold the key to curing many diseases. However, there is a need for better understanding of how to control these cells and further research investigating methods for controlling and directing these cells is required. Pluripotent stem cells transplanted into immune-deficient mice 'spontaneously’ differentiate and proliferate to form a complex mass of differentiated and undifferentiated cells, teratomas - teratoma assay. Such tumours are generally haphazard in their organisation however; they do contain some structures similar to those observed in the embryo. Teratoma formation is a useful model to explore the developmental potential of stem cells and study aspects of tissue development. Examination of how the anatomical location into which human pluripotent stem cells are grafted influences their growth in situ allows investigation of how these cells are affected by different areas within the body: cells grafted into the liver rapidly produce large tumours containing predominantly immature cells whereas, subcutaneous implants were significantly slower growing and formed tumours composed of differentiated tissues. These different growth patterns indicate how environmental cues within the niche affect stem cell behaviour. One factor which contributes to the maintenance of a niche is the extracellular matrix (ECM). To investigate how endogenous ECM affects teratoma behaviour, co-transplantation is carried out with stem cells and ECM components. The ECM extract Matrigel™ dramatically increased the success rate of teratoma formation and size with no detectable affect on teratoma composition when compared to controls and removal of the growth factors from the co-transplanted ECM extract had no effect on teratoma success rate, growth rate, or composition. To study the effects of the ECM in vitro, components of the ECM are often used topcoat glass or plastic surfaces to enhance cell attachment in vitro. Fragments of ECM molecules can be immobilised on surfaces in order to mimic the effects seen by whole molecules. In this study a novel technology developed by Oria Protein Technologies for the immobilisation of peptide sequences from ECM proteins is evaluated. By examining: the adherence of cultured PC 12; neurite outgrowth from PC 12 cells; and neuronal differentiation of neural stem/progenitor cells (NSPCs) it is shown that peptides from collagen I, collagen IV, fibronectin and laminin can mimic surfaces coated with ECM proteins. Collectively, this data demonstrates that peptides from ECM proteins can be immobilised in functional fashion to control cell behaviour. Surfaces with adsorbed proteins and biomimetic surfaces presenting peptide motifs from ECM proteins are used to investigate and explain observations from in vivo teratoma experiments. In vivo, Matrigel™ increases the gene expression of the pluripotent stem cell marker Oct4, increasing the pluripotent cell percentage and thus increases the likelihood of teratoma formation. In vitro, Matrigel™ also increases the gene expression of the proliferative marker Ki67, indicating that large teratomas from by the co-transplantation of stem cells with Matrigel™ could be due to increased cell proliferation.

Intervertebral disc degeneration (IVDD) remains one of the commonest causes of chronic disability in the working years. It involves both structural disruption and cell mediated alteration in the extracellular matrix (ECM) composition of the disc. ECM changes differentiation between disc degeneration and the normal physiological ageing and between disrupted and non-disrupted discs is also not fully understood. The hypothesis of this dissertation, is that IVDD is initiated by micro-damage to disc tissues, followed by cellular attempts to repair which are impeded by some degree of tissue hypoxia, leading to synthesis of an "inferior collagen" and to progressive disc degeneration. The collagen turnover of the extracellular matrix in aged and degenerated as well as in disrupted and non-disrupted discs were determined by measuring the levels of proteolytic enzymes such as the matrix metalloproteinases (MMPs) and collagen cross links. Other ECM components such as sulphated glycosaminoglycans (sGAG) and water contents were also measured in both disc groups. The biochemical and thermal changes were compared in hypoxic and normoxic monolayer cell culture medium of cultured human fibroblasts. Aged discs were found to have greater level of matrix turnover associated with synthesis of poorly hydroxylated neo-collagen as well as different ECM composition, than degenerated discs which found to have increased level of MMPs that might facilitate cellular invasion into their disrupted matrix. Disrupted discs were found to have imbalanced collagen degradation (over synthesis) which might lead to a weak non- functional disc. Focal tissue disruption acquiesce swelling of tissue as well as GAG depletion, thus, altering the GAG/water ratio locally. Painful discs with structural disruption were found to have up-regulated MMPs which is thought to contribute to further annular weakening (radial annular tear) leading to the nuclear material to be forced outward (disc herniation) induced by trauma and/or axial load shift.

Previous work demonstrated that fibronectin is expressed in embryonic mouse detrusor smooth muscle cells (DSMC). Integrins are cell surface receptors for extracellular matrix (ECM) molecules, including fibronectin. The general hypothesis explored here is that ECM/integrin interactions are important in normal and pathological DSMC differentiation. The first specific hypotheses tested were that the candidate fibronectin receptor integrin a5pi and the laminin 1/2 candidate receptor integrin a7pi are expressed by developing DSMC, Using immunohistochemistry, Western blots, and flow cytometry, these proteins were found to be expressed during murine bladder differentiation. This led to the second specific hypothesis, namely that embryonic nascent DSMC adhere to fibronectin, a process mediated by integrin a5 31. Fibronectin substrate was shown to enhance the adhesion of disaggregated embryonic mouse bladder cells. Blocking integrin receptors using RGD oligopeptides modestly but significantly decreased adherent cells expressing desmin, and reduced cell spreading. To explore roles for fibronectin in whole bladders, rather than isolated cells, embryonic mouse organ cultures were established which recapitulated some in vivo differentiation features. However, no specific effects on growth or differentiation could be demonstrated using presumed Tibronectin-blocking' antibodies. Time did not allow testing of RGD oligopeptides. Last, the following hypotheses were explored: that fibronectin is expressed during normal human fetal DSMC differentiation, and that this pattern is altered in bladders from fetuses with presumed bladder outflow obstruction. Fibronectin was indeed found to be expressed during normal human detrusor differentiation and its expression was sometimes reduced in malformed human fetal bladders. These studies provide further descriptive, and hence circumstantial, evidence that ECM/integrin interactions may be important in normal DSMC development. Further studies are warranted to resolve the apparently conflicting/ambiguous organ and cell culture data regarding possible roles for fibronectin and its receptors. The studies also provide preliminary evidence of abnormal fibronectin expression in human congenital bladder anomalies.

The first main objective of this project was to isolate cellular fibronectin from cultures of skin fibroblasts derived from tuberous sclerosis (TS) patients and normals and to compare their structures, paying particular attention to the content and pattern of carbohydrate (glycosylation). The second main objective of this project was to establish the expression and localisation of glycoproteins fibronectin, laminin and tenascin of the extracellular matrix (ECM) in cultures of skin fibroblasts derived from patients with TS and normal individuals. In order to achieve these objectives, fibroblasts were established from primary cultures of skin explants of patients with TS. Control cells were cultured from skin explants donated by people not known to be suffering from any disorder. The purification of cellular fibronectin was achieved from conditioned medium of skin fibroblasts of TS patients and control fibroblasts using Prosep-gelatin affinity chromatography and gel filtration chromatography techniques. Analysis of purified cellular fibronectin by high pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) revealed that the carbohydrate portion of the fibronectin molecule was made up of galactose, mannose, glucosamine, galactosamine, sialic acid and fucose. An increased concentration of sialic acid, galactosamine, glucosamine, galactose and mannose was observed in purified fibronectin derived from neck and ungual fibromas of patients with TS. To provide a total increase of carbohydrates more than two fold in comparison to normal fibroblastsderived fibronectin. Purified cellular fibronectin from conditioned medium of fibroblasts grown from skin lesions of different TS patients and from normal skin fibroblasts did not express HNK-1 (anti-leu 7) carbohydrate epitope. Normal skin fibroblasts showed an altered morphology and less confluence when grown on cell culture plates coated with cellular fibronectin derived from TS fibroblasts compared with control fibronectin. This may be a consequence of an altered glycosylation of this protein. The amino acid composition of the purified fibronectin from TS fibroblasts was very similar to that purified fibronectins from normal fibroblasts and to standard commercial plasma and cellular fibronectins. Laminin and tenascin were partially purified from conditioned cell culture medium demonstrating their synthesis and secretion into the cell culture medium by dermal skin fibroblasts. Expression and distribution of fibronectin, tenascin and laminin by established TS and normal skin fibroblasts using immunofluorescence, ELISA, and flow cytometry techniques were analysed and presented qualitative and quantitatively in this thesis. Increased expression and altered distribution of fibronectin and tenascin were observed in the fibroblasts derived from ungual fibroma lesion of a TS patient, but not in fibroblasts of neck fibroma, forehead plaque lesion or unaffacted skin of TS patients in comparison to control fibroblasts. However, increased expression and altered distribution of laminin were observed in neck fibroma-derived fibroblasts in contrast to fibronectin and tenascin. Laminin expression was not changed in ungual fibroma and forehead plaque lesion-derived fibroblasts in comparison to control fibroblasts. Altered distribution of fibronectin was well observed by immunofluorescence particularly in large cells of ungual fibroma. Similar differences were observed with laminin of cells from neck fibroma of TS patients. These results suggest the abnormal assembly of ECM in different TS skin lesions. Abnormal migration of cells during early embryonic development and the hardening of tissues associated with TS may result from abnormal assembly of the ECM. Alterations in distribution and structure of these adhesive glycoproteins may cause functional disruption in their binding and interactions with cells and ECM macromolecules. Studies of these changes in the ECM components may contribute to the understanding of the mechanisms involved in the aetiology of hardened tissues of TS.

Tendon injury is common in humans and horses, and incidence increases with age. The high-strain energy storing equine superficial digital flexor (SDFT) is injured more frequently than the low-strain positional common digital extensor (CDET). However, previous work indicated that matrix turnover is greater in the CDET than in the SDFT. It was hypothesised that matrix turnover is programmed by the cells’ strain environment; therefore high-strain energy storing tendons would have a lower rate of matrix turnover than low-strain positional tendons and the rate of matrix turnover would decrease with increasing age. The rate of matrix turnover was investigated by measuring the potential of the cells to synthesise and degrade matrix proteins, measuring the half-life of the collagenous and non-collagenous matrix proteins and assessing collagen turnover at the protein level. In vitro cell phenotype was also assessed in 2D and 3D culture and the effect of load on cells within native tissue was determined. The results show that turnover of collagenous and non-collagenous matrix proteins is differentially regulated in the functionally distinct SDFT and CDET. CDET tenocytes show greater potential for collagen turnover, whereas SDFT tenocytes have a greater potential for proteoglycan turnover; differences that are also present at the protein level. The differences in cell phenotype identified in vivo were lost in 2D and 3D culture, but tendon organ culture resulted in the maintenance of tenocyte phenotype. The cells’ ability to turnover the matrix does not decrease with increasing age, but collagen within the SDFT appears to become more resistant to degradation with ageing. This results in the accumulation of partially degraded collagen within the SDFT which may have a detrimental effect on tendon mechanical properties. These findings will help to elucidate the mechanisms behind the development of age-related tendinopathy and will be of use when developing treatment regimes.

It has been well known that the extracellular matrix (ECM) is important to the aortic valve function and that mechanical forces and cell-cell communication can regulate ECM remodeling. However, it has not been determined which cells regulate ECM production by valve interstitial cells (VICs). Thus, this study aimed to investigate if the valve endothelial cells (VECs) can influence ECM production and whether there is a differential role of VECs from either surface of the valve. In addition, the study aimed to assess the suitability of adipose derived stem cells (ADSCs) to differentiate into VECs with side-specific characteristics, so that could be used to populate tissue engineered heart valves. Firstly, a reliable method of side-specific isolation and culture of VECs was developed. Consequently, the phenotypic characteristics of aortic VECs (aVECs) and ventricular VECs (vVECs) were investigated in vitro by immunostaining, western blots and protein array. Endothelial markers vWF, CD-31, VE-cadherin and eNOS and the release of cytokines IL-1β, IL-8, and TGF-β were assessed in cultured VECs from either side of the valve. However the distinct phenotypes between aVECs and vVECs in culture were not established. The differential ability of side-specific VECs to regulate the production of ECM components by VICs was observed when VICs were cultured with media containing molecules released from side-specific VECs, in static culture, and when aVECs or vVECs were co-cultured with VICs. The media collected from vVECs increased amount of collagen by VICs while the media from aVECs showed no significant changes in the content of ECM. Interestingly, in the co-culture system, vVECs-VIC co-culturing enhanced the amount of both collagen and glycosaminoglycans (GAGs) whereas aVECs did not affect the ECM proteins. Moreover side-specific VECs were exposed to the oscillatory and laminar shear stresses (flow patterns experienced by aortic and ventricular surface of the valve, respectively). Their distinct responses on the production of ECM by VICs were investigated. Aortic VECs exposed to oscillatory flow had higher content of collagen and GAGs while vVECs did not share this effect. The laminar shear stress on both sides of the valve maintained elastin content (as compared to fresh tissue). ADSCs were also exposed to side-specific patterns of flow to assess their ability to differentiate into side-specific VECs. With respect to these factors, the exposure of ADSCs to the oscillatory shear stress induced the differential expression of NOS III and SMαA, similar to reported differences found between porcine aVECs and vVECs. Both flow patterns also increased the endothelial function of ADSCs by up-taking low-density lipoprotein. In conclusion, this study reveals the unique functional phenotypes between the aortic and ventricular VECs, and the unique communication between VECs and VICs that is mediated by shear stress to regulate the specific production of ECM components. ADSCs appear to have the capacity to express endothelial markers, but a full functional assessment of their communication with VICs remains to be investigated. This information is important to the development of a living tissue engineered heart valve.