La Thrombospondine-1 (TSP-1) représente l'un des principaux activateurs endogènes du TGF-?1 et possède des propriétés anti-angiogéniques et immunomodulatrices. L'un de ses récepteurs, le CD47, joue un rôle critique dans son effet anti-angiogénique et module l'inflammation. Après obstruction urétérale unilatérale (UUO), l'expression de la TSP-1 augmente, est corrélée à celle du TGF-?1 et du collagène III et décroît avec la réparation tissulaire qui accompagne la désobstruction urétérale. L'utilisation de souris knock-out pour la TSP-1 a permis de montrer qu'elle participe au développement des lésions tubulaires rénales en favorisant les altérations vasculaires et le recrutement des cellules inflammatoires. Cet effet pro-inflammatoire dépend, au moins en partie, du facteur chimiotactique MCP-1, de l'augmentation du rolling leucocytaire et de l'activation de la voie Th17. Les souris knock-out pour CD47 bénéficient également d'une protection tubulaire et vasculaire. Cependant, elles présentent une fibrose interstitielle accrue associée à une augmentation de l'expression de la TSP-1 et du TGF-?1 qui pourrait compromettre une éventuelle récupération rénale. L'étude préliminaire de modèles de néphroangiosclérose chez le rat et la souris révèle que la TSP-1 est surexprimée dans le parenchyme rénal au cours de l'hypertension artérielle. L'intensité de son expression est corrélée à la sévérité des lésions histologiques, suggérant son rôle physiopathologique. Ces résultats montrent que la TSP-1 et le récepteur CD47 participent au développement de la fibrose rénale et représentent donc des cibles thérapeutiques potentielles au cours des maladies rénales chroniques. / Thrombospondin-1 (TSP-1) is a major endogenous activator of TGF-β1 and has anti-angiogenic and immunomodulatory properties. One of its partners, receptor CD47, plays a critical role in its anti-angiogenic activity and also regulates inflammation. After unilateral ureteral obstruction (UUO), TSP-1 expression increases, correlates to TGF-β1 and collagen III expression and decreases with renal repair after desobstruction. Use of TSP-1 knock-out mice allowed us to demonstrate that TSP-1 favours renal injury by increasing vascular lesions and inflammatory cells recruitment. This pro-inflammatory effect depends, at least in part, on chemotactic factor MCP-1, increasing in leukocyte rolling and engagement of T cells in Th17 pathway. CD47 knock-out mice also benefit from tubular and vascular protection after UUO. However, they exhibit increased interstitial fibrosis associated with higher expression of TSP-1 and TGF-β1, which could compromise renal repair. Preliminary study of nephroangiosclerosis models in rats and mice revealed that TSP-1 expression is induced in renal tissue by arterial hypertension and is correlated to the severity of histological lesions, suggesting its physiopathological role. These results show that TSP-1 and CD47 are involved in renal fibrosis and that they represent potential therapeutic target in the management of chronic kidney disease.

In cardiovascular disease the repair response is insufficient to restore blood flow, leading to the death of muscle and loss of tissue function. Therefore, strategies to augment the endogenous cell response and its effects may help improve tissue recovery and function. In this study we explored the use of tissue-engineered collagen matrices for augmenting endogenous regenerative processes after myocardial infarction. Treatment with the sLeX-collagen matrix reduced inflammation and apoptosis and had a positive regenerative effect on the infarcted mouse heart, through improved vascular density and possibly enhanced cardiomyogenesis. Additionally, we investigated the effects of cryopreservation on generating circulating angiogenic cells (CACs) from peripheral blood mononuclear cells (PBMCs), as a potential source of stem cells that could be used in combination with our collagen scaffold. Our findings show that despite PBMCs experiencing phenotypic changes after cryopreservation, they may still be used to generate the same therapeutic CACs as freshly procured PBMCs.

The expanding field of tissue engineering provides a new approach to regenerative medicine for common ailments such as cardiovascular disease and type-I diabetes. Biomaterials can be administered as a delivery vehicle to introduce therapeutic cells to sites of damaged or diseased tissue. A specific class of biomaterials, termed hydrogels, is suitable for this application as they can provide a biocompatible, biodegradable scaffold that mimics the physical properties of the native soft tissue. Injectable hydrogels are increasingly being developed for biomedical applications due to their ability to be delivered in a minimally invasive manner. One potential use for such materials is in the delivery of therapeutics such as cells or growth factor-releasing particles. In this study, the first aim was to determine the interactive effects between collagen-based hydrogels and additives (cells and microspheres) for cardiac regeneration. The results demonstrated that the addition of either cells or microspheres to a collagen-based hydrogel decreased its gelation time and increased its viscosity. Increased cross-linker concentrations resulted in lower cell viability. However, this cell loss could be minimized by delivering cells with the cross-linker neutralizing agent, glycine. As a potential application of these materials, the second aim of this study was to develop a hydrogel for use as an ectopic islet transplant site. Specifically, collagen-chitosan hydrogels were synthesized and characterized, with and without laminin, and tested for their ability to support angiogenic and islet cell survival and function. Matrices synthesized with lower chitosan content (20:1 collagen:chitosan) displayed greater cell compatibility for both angiogenic cells and for islets and weaker mechanical properties, while matrices with higher chitosan content (10:1 collagen:chitosan) had the opposite effect. Laminin did not affect the physical properties of the matrices, but did improve angiogenic cell and islet survival and function. Overall the proposed collagen-based hydrogels can be tailored to meet the physical property requirements for cardiac and islet tissue engineering applications and demonstrated promising cell support capabilities.

京都大学 / 0048 / 新制・論文博士 / 博士(人間・環境学) / 乙第12951号 / 論人博第42号 / 新制||人||178(附属図書館) / 27||論人博||42(吉田南総合図書館) / 32250 / 三重大学大学院医学系研究科生命医科学専攻 / (主査)教授 石原 昭彦, 教授 神﨑 素樹, 准教授 久代 恵介, 准教授 月浦 崇, 教授 藤野 英己 / 学位規則第4条第2項該当 / Doctor of Human and Environmental Studies / Kyoto University / DGAM

OBJECTIF: Évaluer le rôle de l’endostatine, un nouveau marqueur anti-angiogénique, pour prédire le risque de prééclampsie (PE). METHODES: Il s’agit d’une étude cas témoins nichée dans deux cohortes prospectives. Les échantillons sanguins étaient collectés entre 11 et 17 semaines puis entre 18 et 26 semaines d’aménorrhée. L’hypertension gestationnelle était définie par une tension artérielle supérieure ou égale à 140/90mmHg à 2 reprises. Les cas de prééclampsie étaient définis par une hypertension gestationnelle associée à une protéinurie supérieure ou égale à 0.3 g /24h après 20 semaines de grossesse. La concentration d’endostatine était mesurée par une technique d’ELISA. Les résultats étaient exprimés en multiples de la médiane (MoM) et ajustés pour l’âge maternel, l’âge gestationnel, l’ethnie, et la cohorte d’origine. Une régression logistique était utilisée pour calculer des odds ratios (OR) ajustés et prédire le risque de PE. RESULTATS: Au total nous avons étudié 77 PE et 150 témoins chez des grossesses uniques. Parmi les PE 21 étaient de survenue précoce, avec un diagnostic avant 34 semaines et 41 étaient des PE sévères. Les cas avaient un IMC plus élevé que les témoins et étaient plus souvent Africaines. Les taux médians d’endostatine étaient significativement plus élevés chez les PE que chez les témoins au 1er trimestre (94.2 versus 90.7 ng/ml, p=0.004) et 2ème trimestre (105.8 versus 99.3 ng/ml p=0.002). Le taux d’endostatine entre 18 et 26 semaines était même plus élevé chez les patientes qui développaient une PE précoce. Lorsque l’endostatine était supérieure au 75èmepercentile (exprimée en MoM), le OR ajusté était de 1.33 95IC [0.68-2.58] à 11-17 semaines et 1.77 [0.94-3.34] à 18-26 semaines. L’OR ajusté pour les PE précoces était 3.51 [1.18-10.43] entre 11-17 semaines et 2.17 [0.67-7.06] entre 18-26 semaines. CONCLUSIONS: Un taux élevé d’endostatine dès le 1er trimestre est associé à une augmentation du risque de PE et surtout d’un risque de prééclampsie précoce. Toutefois l’endostatine seule a une trop faible valeur prédictive pour avoir une utilité clinique. / OBJECTIVE: To evaluate a new anti-angiogenic factor, endostatin, in relation to the risk of preeclampsia (PE). STUDY DESIGN: We performed a case control study nested in two separate prospective cohorts. Serum samples were collected at 11-17 weeks and 18-26 weeks of gestation. Maternal endostatin levels were measured by ELISA. Results were expressed as multiples of the median (MoM) adjusted for maternal age, gestational age, ethnicity, and cohort of origin. Logistic regression was used to calculate adjusted odds ratios (aORs) of PE. RESULTS: A total of 77 PE and 150 controls of singleton pregnancies were studied, including 21 early-onset PE (diagnosis before 34 weeks) and 41 severe PE. Cases had a higher pre-pregnancy BMI and were more likely of African ethnicity than controls. Endostatin levels were significantly higher in women with PE compared to controls at both the first and second trimester (median 94.2 vs. 90.7 ng/ml p=0.004 and 105.8 vs. 99.3 ng/ml p= 0.002 respectively). Endostatin levels were even higher in women with early-onset PE. At a cut-off level of 75th percentile of endostatin MoMs, the adjusted ORs for PE were 1.33, 95CI [0.68-2.58] at 11-17 weeks and 1.77 [0.94-3.34] at 18-26 weeks. The aORs for early-onset PE were 3.51 [1.18-10.43] at 11-17 weeks and 2.17 [0.67-7.06] at 18-26 weeks, respectively. CONCLUSION: Higher endostatin levels as early as in the first trimester may indicate an increased risk of subsequent PE, especially early onset PE. However endostatin alone has a poor predictive value for clinical usefulness.

De plus en plus d’études impliquent la signalisation Wnt/Frizzled (Wnt/Fzd) dans la formation des vaisseaux. La première partie de ce manuscrit démontre d’ailleurs que la signalisation Wnt, via son régulateur sFRP1 et un de ses ligands, Wnt4, potentialise les effets angiogéniques des cellules souches mésenchymateuses lors de l’angiogénèse. Le récepteur Frizzled4 (Fzd4), lui, est impliqué dans le développement vasculaire de la rétine puisque la délétion du gène fzd4 révèle une malformation du réseau vasculaire rétinien secondaire et tertiaire. Le but de ce travail a été d’étudier l’implication de Fzd4 dans la régulation de la morphogenèse vasculaire chez l’adulte. Il s’avère que Fzd4 présente un profil d’expression plutôt artériel, et que la délétion de ce gène empêche la formation d’un réseau artériel normal des organes périphériques. Des études in vitro réalisées sur des cellules vasculaires primaires ont mis en évidence plusieurs altérations de leurs propriétés angiogéniques. Cette étude a donc démontré un rôle central de Fzd4 dans la croissance vasculaire. Fzd4 régule les propriétés des cellules vasculaires mises en jeu dans l’angiogenèse, et régule la morphogenèse des ramifications vasculaires in vivo. Pour identifier et comprendre les mécanismes moléculaires induits par le récepteur Fzd4, une étude sur la protéine centrale du système Wnt/Fzd, l’isoforme Dishevelled (Dvl), et sur ses partenaires intracellulaires, a été initiée. Les premiers résultats suggèrent que les isoformes 1 et 3 de Dvl participent via Fzd4 à l’activation de la voie canonique nécessaire à la prolifération cellulaire. De plus, certains partenaires intracellulaires de Dvl3 ont pu être sélectionnés par une méthode de double hybride réalisée chez la levure. / Growing evidences link Wnt/Frizzled (Wnt/Fzd) pathway to proper vascular formation. The first part of this manuscript shows besides that Wnt pathway, via its regulator sFRP1 and one of its ligands, Wnt4, potentiates mesenchymal stem cells angiogenic properties during angiogenesis. An other Frizzled receptor (Fzd), Fzd4, has been shown to be implicated in retinal vascular formation because inactivation of the fzd4 gene revealed a malformation of the secondary and tertiary retinal vascular network. Here, we investigated the involvement of Fzd4 in adult vascular morphogenesis regulation. Fzd4 present an arterial vascular pattern, and the deletion of fzd4 impairs a normal arterial network formation in peripheral organs. In vitro studies on primary vascular cells show several alterations on their angiogenic properties. This study reveals a central role of Fzd4 in vascular growth. Fzd4 regulate angiogenic vascular cell properties and vascular branching morphogenesis in vivo. To further understand molecular mechanisms induced by Fzd4, we started to study the Wnt/Fzd central protein, Dishevelled (Dvl), and its intracellular partners. First results suggest that Dvl 1 and 3 isoforms would participate with Fzd4 to activate Wnt canonical pathway implicated in cell proliferation. Moreover, some Dvl3 partners could be selected by a yeast two-hybrid method.

Dans le glioblastome (GBM), la radiothérapie est un outil thérapeutique essentiel. Néanmoins, la récidive post-irradiation est quasiment inévitable, en raison de l’émergence d'une sous-population de cellules cancéreuses particulièrement radiorésistantes présentant une meilleure capacité proliférative, invasive et pro-angiogénique. Notre étude in vitro a cherché à déterminer comment les cellules cancéreuses survivantes à l’irradiation pouvaient affecter la fonction des cellules tumorales voisines et les cellules endothéliales non irradiées, en focalisant notre attention sur l’échange des signaux intercellulaires, à savoir, les facteurs solubles et les microvésicules tumorales (TMVs). La radiothérapie induit principalement un ralentissement de la prolifération des cellules de glioblastome (T98G et U87) et une mort cellulaire retardée (clonogénique) de 50-60%, sans engendrer d’apoptose. Grâce au suivi de la croissance cellulaire à long terme (via le système xCELLigence) et un test de blessure, nous avons confirmé que les cellules de glioblastome survivantes après irradiation libèrent des signaux qui peuvent modifier les fonctions de cellules endothéliales HUVEC et de cellules tumorales non-irradiées. Outre la sécrétion de certains facteurs solubles connus (VEGF, uPA), nous avons pu objectiver, en utilisant la microscopie électronique à balayage, le Nanoparticle Tracking Analysis (NTA), la libération de microvésicules tumorales (TMVs), dont la taille était globalement inférieure à 500 nm. Par NTA et cytométrie en flux, nous avons montré que cette libération de TMVs (exosome + "shedding vesicles"), peut être significativement stimulée par l’irradiation dans les 2 lignées, de façon temps-dépendant. D’après nos analyses protéomiques, les facteurs solubles tels que le VEGF ou l’IL-8, connus pour être des facteurs pro-angiogéniques, contribueraient plutôt à favoriser la survie, voire la prolifération, des cellules HUVEC, tandis que les TMVs libérées après irradiation, ont significativement modifié la capacité de migration des HUVEC et des cellules tumorales non-irradiées. Les propriétés pro-migratoires des TMVs pourraient en ce sens, contribuer aux processus de récidive dans les glioblastomes après irradiation / Radiation therapy is a major therapeutic tool for glioblastoma (GBM). However, the post-radiation recurrence is almost inevitable, due to the emergence of a subpopulation of radioresistant cancer cells with greater proliferative, invasive, and proangiogenic capacities. The objective of this study was to investigate in vitro how irradiated cancer cells affect the function of untreated neighboring tumor cells and endothelial cells, focusing on signals exchange initiated by irradiation, such as soluble factors and tumor microvesicles (TMVs). Radiotherapy has slowed down the proliferation of GBM cells (T98G, U87) and induced mitotic death of 50-60%, without significant apoptosis. Through long-term monitoring of cell growth (xCELLigence) and wound-healing assay, we have confirmed that surviving GBM cells after irradiation release signals that can change the functions of endothelial cells HUVEC and non-irradiated tumor cells. In addition to the secretion of known soluble factors (VEGF, uPA), we were able to show using scanning electron microscopy and the Nanoparticle Tracking Analysis (NTA), the release of tumor microvesicles (TMVS), whose size was generally less than 500 nm. By NTA and flow cytometry, we have shown that the release of TMVs (exosome + "shedding vesicles") can be significantly stimulated by irradiation in two lines, in a time-dependent manner. According to the proteomics analysis, soluble factors such as VEGF or IL-8, well known as pro-angiogenic factors, rather contribute to promote the survival or proliferation of HUVEC, while the released TMVs after irradiation, significantly altered the migration abilities of non-irradiated HUVEC and tumor cells. The pro-migratory properties of TMVs could thus contribute to glioblastoma recurrence after irradiation

Made available in DSpace on 2016-08-10T10:53:30Z (GMT). No. of bitstreams: 1 GUILHERME DE OLIVEIRA BESSA.pdf: 720852 bytes, checksum: 2a03c7ad65ffe25ca5a6debf0d09987e (MD5) Previous issue date: 2010-12-21 / Through this research we aim to demonstrate the effectiveness of the latex of E. tirucalli in wound care as an adjunct in the healing process. The latex was obtained from a specimen plant in the city of Goiania, and diluted to the concentration of 1mg/mL. Two experiments were conducted using experimental models: the chorioallantoic membrane of embryonated chicken eggs and wounds in rats. In the first experiment, we evaluated the angiogenic response of the use of latex membranes, compared with controls. In the second one, sequential measurements were made of the wounds of rats, with regular use of the substance and controls until complete healing of the lesions. The results showed effective healing and angiogenic stimuli, similar to the positive control used (the latex of rubber tree). / Por meio deste trabalho, objetiva-se demonstrar a eficácia do látex de E. tirucalli no tratamento de feridas como adjuvante no processo cicatricial. O látex foi obtido de um exemplar da planta, na cidade de Goiânia, e diluído para a concentração de 1mg/mL. Foram realizados dois experimentos, utilizando como modelos experimentais a membrana corio-alantóide de ovos embrionados de galinhas e feridas em dorsos de ratos. No primeiro experimento, avaliou-se a resposta angiogênica do uso do látex nas membranas, comparando-se com controles. No segundo, foram feitas mensurações seqüenciais dos ferimentos dos ratos, com o uso regular da substância e controles, até a cicatrização completa das lesões. Os resultados revelaram estímulos angiogênico e cicatricial efetivos, semelhantes ao controle positivo utilizado (a biomembrana de látex de seringueira).

O objetivo do atual trabalho, foi correlacionar os eventos celulares e teciduais com a expressão das proteínas VEGF, BMP-7, RANKL e OPG durante a osteogênese ectópica e ortotópica, induzida pela matriz óssea (MO) e dentinária (MD) alogênica. Matrizes alogênicas desmineralizada em HCl a 0,6N, obtidas de fêmur e incisivo de ratos, fori implantada entre as fáscias musculares da coxa e em defeito trans-ósseo de 8mm de diâmetro nos ossos parietais. As análises radiográfica e histomorfométrica da neoformação óssea e, a imunohistoquímica e o western blotting para as proteínas VEGF, BMP, RANKL e OPG, mostraram que: a) o volume da região do enxerto nos sítios ortotópicos reduziu 19% em 42 dias; b) em ambos tipos de enxerto e locais de implantação, ocorreu formação de tecido cartilaginoso e ósseo; c) nos sítios intramusculares, a reabsorção da matriz alogênica e a remodelação do tecido cartilaginoso, ósseo e medular foi mais acelerado, em relação a implantação ortotópico; d) o aumento na densidade de volume dos vasos sanguíneos e no número de osteoblastos/osteócitos e osteoclastos ocorreu simultaneamente e estava associado à maior reabsorção da matriz alogênica e à formação do tecido medular (hematopoiético); e) as proteínas VEGF, BMP-7, RANKL, OPG foram expressas em condrócitos, osteoblastos ativos, osteócitos recém aprisionados na matriz e em células estromais próximas aos osteoblastos ou às áreas da matriz alogênica reabsorvida; e f) a expressão das proteínas VEGF, BMP-7, RANKL e OPG foi maior no grupo MO. O pico de expressão dessas proteínas ocorreu nos períodos de 14 aos 21 dias no grupo da MO e 21 e 28 dias no grupo da MD. Concluímos que, a capacidade osteoindutora da matriz alogênica desmineralizada está relacionado a origem da matriz e ao sítio de implantação e que, as proteínas VEGF, BMP-7, RANKL e OPG estão associadas a maior reabsorção da matriz implantada, promovendo uma rápida e contínua liberação dos morfógenos contidos em seu interior que, induzem temporal e espacialmente a formação óssea/medular. / The aim of the present work was to correlate the cellular and tissue events with the expression of VEGF, BMP-7, RANKL and OPG during ectopic and orthotopic osteogenesis, induced by bone and dentin allogeneic matrix. Allogenic matrices obtained from femur and incisor of rats and demineralized in 0.6 N HCl were implanted into a intramuscular pocket and a 8mm-diameter bone defect in the skull. The radiographic and histomorphometric analysis of new bone formation, and immunohistochemistry and western blotting for VEGF, BMP, RANKL and OPG proteins, showed that: a) the total volume of the graft region in orthotopic site decreased 19% at 42 days b) in both graft types and implantation sites occurred formation of cartilaginous and bone tissues, c) in intramuscular sites, the resorption of allogenic matrix and remodeling of the new formed cartilage and bone were faster, in relation to orthotopic implantation sites; d) the increase in the volume density of blood vessels and in the number of osteoblasts/osteocytes and osteoclasts occurred simultaneously and was associated with greater reabsorption of the allogenic matrix and hematopoietic bone marrow formation; e) VEGF, BMP-7, RANKL, OPG proteins were expressed in chondrocytes, active osteoblasts, newly osteocytes confined and stromal cells located near the osteoblasts or in the surface of the reabsorbed matrix; and f) the VEGF, BMP-7, RANKL and OPG expression was higher in MO grafts than in the MD. The peak of expression of these proteins each occurred at 14 and 21 days in MO and 21 and 28 days in MD. We concluded that, the osteoinductive capacity of allogeneic demineralized matrix is related to matrix origin and implantation site and that the VEGF, BMP-7, RANKL and OPG proteins are associated with greater reabsorption of the implanted matrix, promoting rapid and continuous matrix-release morphogens that induces spatially and temporally the bone and bone marrow formation.

Les tumeurs endocrines digestives sont des tumeurs hypervasculaires, ce qui suggère l'existence d'un processus d'angiogenèse tumorale actif. Les mécanismes de l'angiogenèse tumorale associée aux tumeurs endocrines digestives sont complexes. Deux processus semblent coexister : les tumeurs les mieux différenciées sont capables de récapituler les propriétés pro-angiogéniques des cellules endocrines normales alors que les tumeurs moins différenciées et plus agressives sont associées à un processus d angiogenèse non spécifique, développé en réponse à l'hypoxie. Dans ces deux processus, le VEGF joue vraisemblablement un rôle important, dans la mesure où il fait partie intégrante du programme de différenciation endocrine. L'objectif de notre travail a été de mieux comprendre les mécanismes de sa régulation dans les cellules endocrines digestives tumorales et d'analyser son rôle dans la croissance tumorale, à travers une double approche expérimentale, in vitro et in vivo ; Nos résultats nous ont permis de : (a) montrer la complexité de la régulation de la synthèse et de la sécrétion du VEGF par les cellules endocrines néoplasiques, qui implique plusieurs voies de signalisation (PI3K/Akt/mTOR et p38/MAPK), dont les rôles respectifs varient selon le type de cellule étudiée ; (b) confirmer expérimentalement la dissociation entre expression du VEGF et capacités angiogéniques d'une part, propriétés invasives et métastatiques d'autre part ; (c) montrer expérimentalement que l'inhibition de l'angiogenèse peut contribuer à l'effet antitumoral de substances d'intérêt thérapeutique dans les tumeurs endocrines digestives / Digestive endocrine tumors are hypervascular tumors, likely to be associated with an active angiogenic process. The mechanisms of tumor angiogenesis in digestive endocrine tumors are complex. Two processes seem to coexist: well differentiated tumors are able to recapitulate the pro-angiogenic capacities of normal endocrine cells whereas less differentiated and more aggressive tumors are associated with a non specific angiogenic process, in response to hypoxia. In both processes, VEGF is likely to play an important role, since it is constitutively expressed by normal peptide-secreting endocrine cells, as part of their specific differentiation program. Our aim was to evaluate the mechanisms of regulation of VEGF synthesis and secretion by neoplastic digestive endocrine tumors and to analyze its role in tumor progression, through an in vitro and in vivo experimental approach. We were able to demonstrate that: (a) the regulation of VEGF synthesis and secretion is complex and involves several pathways (PI3K/Akt/mTOR and p38/MAPK), with different roles according to the cell studied; (b) there is a dissociation between VEGF expression and angiogenic capacities, on one hand, and invasive and metastatic properties, on the other hand; (c) the inhibition of angiogenesis may contribute to the anti-tumoral effect of several drugs of therapeutic interest in digestive endocrine tumors