The abdominal aorta (AA) in man is a vulnerable artery prone to atherosclerosis as well as aneurysmatic dilation. The underlying aortic composition, mechanical properties as well as the mechanisms responsible for age-related changes and vascular disease are however largely unknown. The aims of this study were 1) to characterize the age- and gender-related changes of the aortic wall components in vivo, using a mechanical model based on ultrasound measurements of pulsatile aortic diameter changes combined with intra-arterial pressure; 2) to validate ultrasound measurements of diameter and intima-media thickness (IMT) of the AA in order to calculate wall stress; 3) to study the stress driven remodeling response of the aortic wall in healthy individuals and the influence of age and gender; and 4) to study wall stress and remodeling of the AA in diabetic patients in order to elucidate the protective influence of diabetes on abdominal aortic aneurysm formation. The stiffness of the isotropic material (mainly elastin) increased in males despite the known decrease in elastin content with age. Further, an exponential increase in stiffness of the anisotropic material (mainly collagen) in males at high physiological pressure was found. This might be due to changed isoforms of collagen and increased glycation with age. Females were less affected than males. The reproducibility of the ultrasound measurements of diameter and IMT in the AA was acceptable (CV; 4% and 11% respectively), making it possible to calculate circumferential aortic wall stress in vivo. The age-related remodeling of the arterial wall led to increased diameter, and compensatory thickening of the wall preventing the circumferential wall stress from increasing in the common carotid artery of males and females, and the AA of females. However, the compensatory increase in wall thickness was defect in the male AA, where stress increased with age. Pulsatile stress influenced the material parameters of the AA, leading to increased stiffness of anisotropic material (mainly collagen), whereas stiffness of isotropic material (mainly elastin) was unaffected. Patients with diabetes mellitus had increased aortic wall thickness than controls, generating less circumferential stress. This coincides with the known reduction of abdominal aortic aneurysms in diabetic patients and may act as a protective factor.

Cardiovascular diseases form one of the most dangerous events that affect human life. They are usually the result of high blood pressure. Thus controlling blood pressure within patient specific healthy limits is a goal that we must target. There are two control loops for blood haemostasis inside the body either long term or short term. Baroreceptors control the short term blood pressure regulation. They are nerve endings that exist in certain locations within the blood vessel walls and they report blood pressure into the brain and the central nervous system. However the basics of their function are not yet known. We propose here that the baroreceptors work by converting circumferential and axial pressure into a stress into their respective direction and they start to send nerve signals based on a threshold of strain energy of the location they are embedded in. Thus baroreceptors A fibre is highly likely to exist in the stiffer adventitia, while the media will contain C fibres. This explains the reason behind having identical fibres with different threshold. We were able to arrive to this solution by getting a relationship between stress–strain relationship for the whole wall and for the arterial vessels. These findings are quiet significant as they allow a method to identify different stress in the arterial wall layers using whole wall experimental data and also as they were able to differentiate between different fibres based on their locations inside the arterial wall. A complete modelling of the baroreceptors function might lead to the formation of biosynthetic material that could interact with the body on the cellular level, so as to give humans the mean to the control of short term blood regulation thus preventing hypertension and its accompanying diseases such as atherosclerosis.

Elastin-like peptides (ELPs) are stimulus-responsive protein-based polymers which are attractive material for biomedical research due to their biocompatibility and unique properties. The physical properties of ELPs are dependent on the chain length and the chosen amino acid at the guest residue position. This imparts unlimited flexibility in designing ELP based biomaterials with the desired physical properties. We have shown that in addition to their physical properties, ELPs have biological activities that are conducive to tissue regeneration. Specifically, we found that ELPs induce fibroblast proliferation via cell surface heparan sulfate proteoglycans (HSPG). Furthermore, our data suggests that ELP based materials with differential proliferative potential can be designed by controlling the interaction of ELPs with HSPGs by incorporating either hydrophobic or positively charged residues within the ELP sequence. Fibroblast proliferation is important for granulation tissue formation which is important in chronic wounds as well as in healing of other tissues. The customizable biological activity of ELPs coupled with their unique physical properties will enable us to design novel, sustainable and cost effective therapies for different tissue regeneration applications. ELPs can be genetically fused to biologically active peptides or proteins. These fusions can be expressed and readily purified since they maintain the phase transitioning property of the fused ELP domain. Moreover, depending on the ELP sequence chosen the chimeric fusion sequences can self-assemble into unique structures such as nanoparticles. These structures can then be applied to the injury site where they not only provide unique topographical cues or structural support but also act as delivery vehicles for the fused bioactive protein. We developed a multifunctional nanoparticle that is comprised of PMP-D2-ELP fusion protein and different functional peptide ELP fusion proteins to preserve the bioactivity of the functional group with the existence of elastase. These heterogeneous particles will be beneficial for the delivery of combination therapies to solve multiple problems that often existed in chronic wound healing or other tissue regeneration process. In summary, this study adds to our understanding of the biological activity of ELP and the interaction mechanism that allow the regulation of cellular behavior. Furthermore this work also investigated the potential therapeutic application of ELP as a delivery platform for chronic wound healing.

Tendon disorders are painful, disabling, and a major healthcare problem, with millions of people affected by tendon injuries each year. Current treatment strategies are inadequate and knowledge of the underlying mechanobiological mechanisms is required to develop novel therapies. Although the tissue–level properties of tendon are well–documented there remains a lack of understanding of the deformation mechanisms of this complex tissue. Therefore, the aim of this thesis is to characterize the microstructural deformation of tendon through biological imaging, mechanical testing, and computational modeling. Emphasis is placed on the structure and function of elastic fibers in tendon, whose role is poorly understood. First, histology, immunohistochemistry, and multiphoton microscopy are used to characterize the organization of elastic fibers in healthy and damaged tendon providing detailed microstructural information on their morphology and location for the first time. Elastic fibers are found to have a sparse distribution in the extracellular matrix, but are highly concentrated in the endotenon sheath and pericellular matrix. Moreover, damaged specimens are found to have a severely disrupted elastic fiber network. Elastic fibers likely contribute to fascicular deformation mechanisms and the micromechanical environment of tenocytes, which are expected to be disrupted in damaged tendon. Second, mechanical testing and enzyme treatments are used to analyze the mechanical contribution of elastic fibers to tendon. Elastase is found to significantly affect the mechanical properties of the tissue and remove the elastin component of both tendon and a control collagen–elastin biomaterial. However, elastase is also found to degrade non–elastin structural molecules that may contribute to tendon mechanics. The mechanical changes associated with the elastase treatment suggest that elastic fibers do not contribute to the elastic recoil of tendon as previously hypothesized. Third, multiphoton microscopy in combination with a novel microtensile testing machine is used to observe the deformation of collagen fibrils and tenocytes in tissue exposed to load. Tissue displacement is consistent with a helical arrangement of fibrils and nuclei experience significant elongation under physiological conditions. These results suggest that a helical arrangement of fibrils is responsible for the nonlinear stress–strain response of tendon and that nuclei are prime candidates for sensing mechanical forces in tendon. Finally, computation modeling and structural imaging are used to generate a microstructural finite element model of tendon. A helical model with embedded pericellular matrix is able to reproduce the stress–strain response and cell–level deformation of the tissue. The pericellular matrix is found to amplify mechanical forces exposed to cells, which is required to initiate mechanobiological stimulation of tenocytes under physiological conditions. Therefore, the structure and composition of the PCM during health and disease is expected to significantly affect mechanobiological mechanisms of tendon. The work presented in this thesis has used new experimental methods to provide novel insight into the structure, function, and deformation mechanisms of tendon. The techniques and concepts developed are widely applicable to the study of collagenous tissues in health and disease. In particular, observations regarding the pericellular matrix may lead to the development of new tissue–engineered and pharmacological strategies for the treatment of tendon disorders.

L’élastine est la protéine de la matrice extracellulaire (MEC) responsable des propriétés de résilience et d'élasticité des tissus élastiques. Durant le vieillissement, les protéines de la MEC vasculaire sont exposées à des réactions délétères qui altèrent leurs propriétés structurales et fonctionnelles. Une des caractéristiques principales des protéines de la MEC est leur longue demi-vie, associée à un renouvellement très lent, comme pour l'élastine. Ainsi, tout dommage survenant sur l'élastine est essentiellement irréparable. La fragmentation des fibres élastiques génère des peptides d’élastine (EDP) bioactifs capables de modifier le comportement des cellules environnantes en se liant au complexe récepteur de l’élastine (CRE), composé de trois sous-unités dont la neuraminidase-1 (Neu-1), sous-unité catalytique du CRE. Cette thèse a consisté en l'étude, chez la souris, du rôle des EDP dans le développement du diabète de type 2 et dans la thrombose, deux pathologies vasculaires liées à l'âge, et s'est également focalisée sur la sous-unité Neu-1 du CRE. Dans un premier temps, nous avons montré que les EDP favorisent le développement d’une insulinorésistance et d’un diabète de type 2. Cet effet implique l'interaction de Neu-1 avec la sous-unité β du récepteur à l'insuline qui diminue son niveau de sialylation altérant ses voies de signalisation. Dans un second temps, nous avons identifié un mécanisme d'action des EDP à deux niveaux (matriciel et plaquettaire) et mis en évidence la présence d'un CRE fonctionnel dans les plaquettes régulant la thrombose. Enfin, nous avons étudié la topologie membranaire de Neu-1 par différentes approches technologiques et identifié un domaine transmembranaire potentiel jouant un rôle important pour sa dimérisation et son activité sialidase. En conclusion, les EDP sont des acteurs clefs du remodelage vasculaire physiopathologique et des pathologies vasculaires associées et de contribuer à faire avancer nos connaissances sur l'organisation de Neu-1 à la membrane plasmique. / Elastin is the extracellular matrix (ECM) protein responsible for resilience and elasticity of tissues such as arteries. During ageing, vascular ECM proteins are subjected to deleterious reactions that alter their structural and functional properties (addition reactions, proteolysis). One of the main features of ECM proteins is their long half-life, associated with a low, or even, inexistent turnover. This is the case for elastin with an estimated half-life at 70 years. Therefore, any damage occurring on elastin will be mostly irreparable. Fragmentation of elastic fibers produces bioactive elastin-derived peptides (EDP) able to modify the behavior of surrounding cells by binding to the elastin receptor complex (ERC). This receptor is composed of three subunits, among which neuraminidase-1 (Neu-1) is the catalytic subunit. The aim of this thesis was to study, in mice, the role of EDP in the development of type 2 diabetes and in thrombosis, two age-related vascular diseases, and to focus on the Neu-1 subunit of the ERC. In a first time, we have shown that EDP promote the development of insulin resistance and type 2 diabetes. This effect involves Neu-1 interaction with the β subunit of the insulin receptor and leads to its reduced sialylation level and signaling. In a second time, we have demonstrated that EDP are regulators of thrombosis. We identified a two-level mechanism (matrix and platelet) and the presence of a functional ERC in platelets. Finally, we have studied the membrane topology of Neu-1 by different biophysical, biochemical and molecular biology approaches, and identified a potential transmembrane domain involved in the dimerization and sialidase activity of Neu-1. In conclusion, this thesis consolidates the concept that EDP are crucial actors of pathophysiological vascular remodeling and related vascular diseases, and expands our knowledge on the plasma membrane organization of Neu-1.

La thèse présentée porte sur la préparation de dérivés de polypeptides recombinant à base de motifs élastine (ELPs) ainsi que sur l'étude de leurs propriétés physicochimiques et biologiques. Des ELPs contenant des résidus méthionine ont été modifiés de manière chimiosélective soit en utilisant des halogénures d'alkyle ou différents époxydes, soit par oxydation des résidus méthionine. La caractérisation par RMN et par spectrométrie de masse des composés obtenus a permis de confirmer leur fonctionnalisation quantitative. L'étude de la réponse en température de ces dérivés d'ELP par des mesures de turbidité ou par des mesures de diffusion de la lumière a montré le fort impact des modifications entreprises sur la température de transition (TI) des ELPs. Il a également été montré que la n peut être modifiée par échange des contre-ions des dérivés cationiques. Enfin, des monosaccharides ont été conjugué aux ELPs contenant des groupements alcyne par cycloaddition de Huisg en afin d'obtenir des glycopolypeptides. Les propriétés thermosensibles ainsi que les propriétés biologiques de ces conjugués ont été testées et ces dernières ont permis de montrer leur capacité à se lier sélectivement à des lectines. Leur utilisation pour trier des protéines d'intérêt et les redisperser est finalement évaluée de façon préliminaire. / This thesis describes the preparation of elastin-like polypeptides (ELPs) derivatives and the study of their physico-chemical and biological properties. Methionine-containing ELPs were chemoselectively modified using either alkyl halides or epoxides or by oxidation of their methionine residues. The successful functionalization was assessed by NMR and mass spectrometry analysis of the resulting compounds. The thermoresponsive properties of these ELP derivatives were evaluated either by light scattering or by turbidity measurements showing the strong effect of these modifications on the ELPs transition temperature (TI). The counterion affect on the thermosensitivity of the polycationic derivatives was also studied. The synthesis of ELP glycopolypeptides was finally achieved by conjugating monosaccharides to the ELP alkyne derivatives through Huisgens cycloaddition. Along with the thermoresponsive properties, the bioactivity of the ELP glycoconjugates was studied and proved their ability to specifically bind lectins. Their use for protein sorting and release was preliminary evidenced.

L'élastine est la protéine de la matrice extracellulaire responsable de l'élasticité des tissus. Elle est synthétisée en abondance dans les tissus soumis à de fortes contraintes mécaniques tels que la peau, les poumons, et les artères. Ce biopolymère présente une demi-vie longue de 70 ans et est par conséquent sujet aux altérations liées à l'âge. De plus, l'élastine est dégradée lors de la progression tumorale, notamment celle du mélanome. Sa dégradation entraîne la libération de peptides dérivés de l'élastine (EDP) dotés d'activités biologiques propres et pouvant être impliqués dans l'invasion tumorale (prolifération, angiogenèse, sécrétion de protéases, invasion, survie). Nous avons été les premiers à démontrer que les EDP sont capables de fortement promouvoir le développement de mélanome in vivo dans un modèle murin au travers de l'augmentation de la prolifération et de l'invasion des cellules tumorales impliquant la collagènase-1 ou MMP-1. Ces données suggèrent ainsi que le récepteur de ces peptides, le complexe récepteur de l'élastine (CRE), pourrait constituer une cible thérapeutique innovante afin de lutter contre ce cancer pour lequel un manque flagrant de thérapies efficientes prévaut à l'heure actuelle. Dans le cadre de cette étude, nous avons développé différentes stratégies thérapeutiques basées sur le ciblage du récepteur CRE. Nous démontrons pour la première fois que le ciblage thérapeutique du CRE permet de limiter de manière efficace la progression du mélanome in vitro et in vivo dans un modèle murin. De plus, le vieillissement étant un des facteurs de risque majeur du mélanome, nous avons évalué l'impact de l'âge du stroma sur les effets des EDP et le développement tumoral associé. / Elastin is the extracellular matrix protein responsible for tissue elasticity. It is abundant in tissues experiencing important mechanical constraints such as skin, lungs and arteries. This biopolymer possesses a half-life of 70 years and, consequently, it is subjected to age-related alterations. Moreover, elastin is degraded during tumor progression, notably that of melanoma. Its degradation results in the release of elastin-derived peptides (EDP) which possess their own biological activities that can be linked to tumor invasion (proliferation, angiogenesis, proteases secretion, invasion, survival). We have been the first to show that EDP can promote melanoma development in an in vivo mouse model by raising the proliferation and invasion of tumor cells through collagenase-1 (MMP-1) up-regulation. These data thus suggest that the receptor for these peptides, the elastin receptor complex, could constitute a novel therapeutic target to fight against this type of cancer for which no therapies are efficient now. In this study, we have developed therapeutic strategies targeting the ERC complex. We show for the first time that the therapeutic targeting of the ERC could efficiently limit melanoma progression in an in vitro and in vivo murine model. Additionally, as aging is one of melanoma major risk factors, we have evaluated the impact of stromal age on EDP effects and the associated tumor development.

La Broncho-Pneumopathie Chronique Obstructive (BPCO) est une pathologie inflammatoire chronique caractérisée par une détérioration irréversible des voies aériennes et des espaces alvéolaires. L'emphysème est la composante principale de la BPCO et l'exposition à la fumée de cigarette en est le principal facteur étiologique. La réponse inflammatoire au cours de la BPCO est caractérisée par une infiltration massive du tissu respiratoire par les cellules inflammatoires qui sécrètent de nombreuses protéases. Ces protéases participent à la dégradation des composants de la matrice extracellulaire et en particulier à la dégradation de l'élastine. Cette dégradation conduit à la genèse de peptides solubles d'élastine (PE) retrouvés dans les différents liquides biologiques de patients BPCO. Si les lymphocytes T CD4+ et les PE sont décrits dans la littérature comme impliqués dans la physiopathologie de la BPCO, en revanche, l'impact de leur interaction sur la polarisation Th reste à élucider. Le travail réalisé au cours de cette thèse a permis de mettre en évidence une polarisation de la réponse lymphocytaire T vers un profil de type Th1 et Th17 au cours de l'emphysème murin induit par le peptide d'élastine VGVAPG. Cette polarisation est dépendante de l'interaction du VGVAPG avec son récepteur spécifique à la surface des lymphocytes T, et est inhibée en présence d'analogues antagonistes du récepteur à l'élastine. Ces résultats permettent d'envisager l'utilisation de tels antagonistes comme des agents pharmacologiques régulant les effets délétères pro-inflammatoires de la réponse T helper au cours de la BPCO. / Chronic Obstructive Pulmonary Disease (COPD) is a chronic inflammatory disease characterized by irreversible damage to airways and alveolar spaces. Emphysema is the main component of COPD and exposure to tobacco smoke is the main etiological factor. The inflammatory response in COPD is characterized by a massive infiltration of respiratory tissue by inflammatory cells that secrete many proteases. These proteases participate in the degradation of extracellular matrix components and particularly in elastin degradation. This elastin breakdown leads to the genesis of soluble elastin peptides (EP) found in various biological fluids of COPD patients. If CD4+ T cells and EP are described in the literature as being involved in the pathophysiology of COPD, consequence of their interaction on the T helper polarization remains to elucidate. The work done during this thesis allowed highlighting the polarization of T cell response towards a Th1 and Th-17 profile during VGVAPG-induced murine emphysema. This polarization, which is dependent of the VGVAPG interaction with its specific receptor expressed on T cells, is inhibited using analogous antagonist elastin receptor peptides. These results allowed us to envisage such antagonist peptides as pharmacological agents that regulate the deleterious pro-inflammatory effects of the T helper cell response during COPD.

Devido as limitações relacionadas ao emprego do enxerto autólogo, o uso dos biomateriais poliméricos naturais tornou-se uma opção viável em terapias regenerativas do tecido ósseo. O objetivo desse trabalho é avaliar de forma qualitativa e quantitiva a contribuição da membrana de elastina utilizada isoladamente ou em associação a hidroxiapatita e proteína morfogenética óssea, no reparo de defeitos ósseos no crânio de ratos. Foram utilizados 49 ratos (Rattus norvegicus, Wistar), machos, com peso aproximado de 330 gramas e 4 meses de idade. Os animais foram submetidos ao procedimento cirúrgico para a criação do defeito ósseo no osso parietal esquerdo e divididos em 7 grupos com 7 animais cada. Os grupos foram implantados com os seguintes bioamateriais: grupo 1 controle (G1-C) sem implante, grupo 2 (G2-E24h) membrana de elastina 24 h, grupo 3 (G3-E24h/HA) membrana de elastina 24 h com hidroxiapatita, grupo 4 (G4-E24h/BMP) membrana de elastina 24 h com proteína morfogenética óssea, grupo 5 (G5-E96h) membrana de elastina 96 h, grupo 6 (G6-E96h/HA) membrana de elastina 96 h com hidroxiapatita, grupo 7 (G7-E96h/BMP) membrana de elastina 96 h com proteína morfogenética óssea. Após a morte indolor induzida em 6 semanas, as calotas cranianas foram retiradas para análise macroscópica, radiográfica, histológica e morfométrica. As análises macroscópicas, radiográficas e histológicas demonstraram a biocompatibilidade dos biomateriais utilizados. As médias e desvios-padrão do volume percentual relativo de osso neoformado nos defeitos cranianos dos grupos G1 a G7 foram 7,87±2,53; 24,01±0,55; 9,59±1,27; 31,31±6,37; 19,77±2,62; 7,31±2,43; 43,25±3,72, respectivamente. Os biomateriais mostraram-se biocompatíveis e o grupo 7 (G7-E96h/BMP) resultou na maior neoformação óssea. / Due to the limitations related to the use of autologous grafts, the use of natural polymeric biomaterials has become a viable option in regenerative therapies of bone tissue. The objective of this dissertation is to evaluate in qualitative and quantitative way the contribution of the elastin matrice used alone or in combination with hydroxyapatite and bone morphogenetic protein in the repair of bone defects in the skull of rats. Were use 49 Mices (Rattus norvegicus, Wistar), weighting approximately 330 grams and 4 months of age, were used. The animals were submitted to the surgical procedure to create the bone defect in the left parietal bone and divided into 7 groups with 7 animals each. The groups were implanted with the following biomaterials: group 1 control (G1-C) without biomaterial, group 2 (G2-E24h) 24 h elastin membrane, group 3 (G3-E24h/HA) 24 h elastin membrane with hydroxyapatite, Group 4 (G4-E24h/BMP) elastin membrane 24 h with bone morphogenetic protein, group 5 (G5-E96h) elastin membrane 96 h, group 6 (G6- E96h/HA) elastin membrane 96 h with hydroxyapatite, group 7 (G7-E96h/BMP) 96 h elastin membrane with bone morphogenetic protein. After painless death induced at 6 weeks, the skull caps were removed for macroscopic, radiographic, histological and morphometric analysis. Macroscopic, radiographic and histological analysis demonstrated the biocompatibility of the biomaterials used. The mean and standard deviations of the relative percentage volume of newly formed bone in the cranial defects of the G1 to G7 groups were 7,87±2,53; 24,01±0,55; 9,59±1,27; 31,31±6,37; 19,77±2,62; 7,31±2,43; 43,25±3,72, respectively. The implanted biomaterials were shown to be biocompatible and the group 7 (G7-E96h/BMP) resulted with greater bone neoformation.

No description available.