La croissance tumorale et le processus de métastatisation dépendent de la néoformation de vaisseaux sanguins ou néoangiogenèse. Parmi les molécules intervenant dans ce processus, l'adrenomédul1ine (AM) est un peptide, dont l'expression est corrélée à l'agressivité de certaines tumeurs, et qui représente un maillon «clé» dans les interactions entre les cellules tumorales et les cellules du microenvironnement. Les résultats spectaculaires qu'offre le traitement des xénogreffes de cellules issues de glioblastomes (GBM) humains par les anticorps dirigés contre l'AM ou son récepteur sont très encourageants, puisque la tumeur traitée régresse en quelques semaines, la vascularisation tumorale s'en trouve touchée de manière spécifique. C'est dans ce contexte, que nous avons choisi de poursuivre notre travail sur les mécanismes d'action de l'AM dans la néoangiogenèse. Grâce à des études in vitro et in vivo, nous avons pu montrer que l'AM est impliquée dans plusieurs étapes de la néoangiogenèse tumorale : migration des cellules endothéliales, stabilisation des contacts endothéliaux et endothélio-péricytaires, recrutement des cellules mésenchymateuses. Nos résultats démontrent que nous sommes en présence d'une molécule d'AM qui agit sur diverses cibles moléculaires et cellulaires, régulant la stabilité du complexe d”adhésion intercellulaire VE-cadhérine/-caténine, nécessaire à la protection des interactions homotypiques et hétérotypiques de l°endothélium nouvellement formé. Ainsi, l'étude des mécanismes d'action de l'AM réalisée pennettra d'établir ue stratégie thérapeutique autour de l'AM. / Tumoral growth and process of metastatization depend on the formation of new blood vessels or angiogenesis. Among the molecules implicated in this process, adrenomedullin (AM) is a peptide, which expression is correlated with the aggressiveness of tumors, and which represents a "key" link in the interactions between tumoral cells and the microenvironment cells. The spectacular results offered by the treatment of human glioblastoma (GBM) xenograft by antibodies directed against the AM or its receptor are very encouraging, as the treated tumor declines in some weeks, and the tumoral vascularization is also touched in a specific way. In this context, we chose to pursue our work on the mechanisms of action of AM in angiogenesis. In vitro and in vivo studies showed that AM is involved in several stages of tumoral angiogenesis : migration of endothelial cells, stabilization of endothelial contacts, stabilization of the pericyte coverage, recruitment of multipotent cells. Our results demonstrate that we are in presence of a molecule of AM which acts on diverse molecular and cellular targets, regulating the stability of the VE-cadherin/β-catenin complex, required for the protection of the homotypics and heterotypics interactions of the newly formed endothelium. The study of the mechanisms of action of AM realized will allow us to establish a therapeutic strategy around AM.

L’irradiation cérébrale thérapeutique comporte un risque de neurotoxicité tardive irréversible en partie lié à l’effet de l’irradiation sur le compartiment vasculaire cérébrale. Les péricytes et les communications entre les péricytes et les cellules endothéliales jouent un rôle majeur dans la stabilisation des vaisseaux, dans la formation et la régulation de la barrière hématoencéphalique et dans le contrôle du flux sanguin cérébral. Nous montrons, dans un modèle murin d’irradiation cérébrale, que les péricytes sont une cible précoce de l’irradiation cérébrale. Après irradiation, la morphologie des péricytes est modifiée et des marqueurs d’activations du péricytes sont surexprimés. En conséquence, la communication entre péricyte et cellule endothéliale est rompue, ce qui se traduit par une diminution de la capacité du péricyte à induire une constriction vasculaire après stimulation électrique. De façon concomitante, la perméabilité de la barrière hémato-encéphalique est anormalement augmentée après irradiation. Un traitement par thalidomide, administré dans la semaine précédant et suivant l’irradiation, prévient les conséquences de l’irradiation sur les péricytes. Les communications entre péricytes et cellules endothéliales sont maintenues ainsi que les fonctions contractiles des péricytes. L’imperméabilité de la barrière hématoencéphalique est également préservée. La voie de signalisation PDGF-β/PDGFR-β essentielle au recrutement des péricytes par les cellules endothéliales, est, au moins partiellement, impliquée dans l’effet protecteur de la thalidomide. Des études supplémentaires sont nécessaires pour définir les mécanismes sous tendant l’effet de l’irradiation sur les péricytes ainsi que l’effet protecteur de la thalidomide. Nous avons en parallèle mis au point un modèle murin de lymphome cérébral luciférase positif pour vérifier, dans un premier temps, l’innocuité de l’association de la radiothérapie et de la thalidomide et de ses dérivés de la classe des immunomodulateurs (iMids), le lénalidomide et le pomalidomide. Ces trois molécules ne diminuent pas l’effet antitumoral de la radiothérapie, mais l’association de radiothérapie et de pomalidomide est synergique sur la décroissance tumorale mesurée par l’évolution des courbes de bioluminescence. Le concept de normalisation de la vascularisation tumorale fait référence aux molécules capables, non pas de faire régresser les vaisseaux tumoraux anormaux, mais de les « normaliser » pour améliorer, d’une part, la disponibilité des chimiothérapies au sein de la tumeur, et d’autre part, l’oxygénation tumorale pour accroitre l’efficacité de la radiothérapie. Nos résultats sont en faveur d’un tel effet exercé par le pomalidomide dans notre modèle murin de lymphome cérébral, caractérisé par une infiltration tumorale périvasculaire, une fuite capillaire mais sans néo angiogenèse. Nos travaux fournissent un rationnel biologique à de futurs essais cliniques avec les iMids dans le traitement des lymphomes cérébraux primitifs voire des tumeurs malignes cérébrales. / Therapeutic brain irradiation carries a risk of irreversible delayed neurotoxicity partly due to the effect of irradiation on the cerebral vascular compartment. Pericytes and communication between pericytes and endothelial cells play a major role in vessel stabilization in the formation and regulation of the blood-brain barrier and in the control of cerebral blood flow. We show in a murine model of brain irradiation, that pericytes are a target of early brain irradiation. After irradiation, the morphology of pericytes is altered and markers of activation of pericytes are overexpressed. Consequently, communication between the endothelial cell and pericyte is disrupted , which results in a decreased capacity of pericytes for inducing a vascular constriction after electrical stimulation. Concomitantly, the permeability of the blood - brain barrier is abnormally increased after irradiation. Treatment with thalidomide administered in the week before and after irradiation, prevents the effects of irradiation on pericytes . Communication between pericytes and endothelial cells are maintained as well as the contractile properties of pericytes. The impermeability of the blood brain barrier is also preserved. The PDGF-β/PDGFR-β signaling pathway, which is essential for the recruitment of pericytes by endothelial cells, is at least partially involved in the protective effect of thalidomide. Further studies are needed to define the mechanisms underlying the effect of irradiation on the pericytes and the protective effect of thalidomide. We have, in parallel, developed a model of murine luciferase positive CNS lymphoma to verify first, the safety of the combination of radiotherapy and thalidomide and its derivatives of the class of immunomodulators ( IMiDs ), lenalidomide and pomalidomide. These three molecules do not decrease the antitumor effect of radiotherapy, but the antitumoral effect of the association of radiotherapy and pomalidomide is synergistic. The concept of the normalization of the tumor vascularization refers to molecules capable not to induce regression of the abnormal tumor vessels but to "normalize" the tumoral vasculature in order to improve, on one hand , the availability of chemotherapy in the tumor , and on the other hand, the tumor oxygenation to increase the effectiveness of radiotherapy. Our results are in favor of such an effect exerted by pomalidomide in our murine model of cerebral lymphoma, characterized by perivascular tumor infiltration and capillary leak .Our work provide a biological rational for future clinical trials with IMiDs in the treatment of brain lymphomas or malignant brain tumors.

In the United Kingdom 1 in 6 - 8000 children are born missing one or both of their ears. The surgical technique most commonly used to reconstruct ears requires surgeons to remove ribs from the patient, and the cartilage from the ribs is used to carve a new ear. This procedure involves many risks including significant pain, punctured lung and chest deformity. Therefore the ability to ‘grow’ an ear would be a major advancement. Stem cells show huge promise in tissue engineering and regenerative medicine. Approved stem cell technology must be evaluated with regards to safety, purity, identity, potency and efficacy prior to biologic licensing and clinical use. Therefore, access to ethically sourced tissue for research is fundamental to the successful delivery of novel therapies. Adipose tissue provides an abundant and accessible source of stem cells for clinical translation. Within the first section of this thesis, the perceptions and attitudes of patients towards the donation and use of adipose tissue for research are sought. Based on this information, a tissue bank with all appropriate ethical approval to collect, process, store and distribute adipose tissue and adipose derived stem cells is established. The second part of this thesis demonstrates the specific identity, location and frequency of stem cells within adipose tissue; revealing them to reside in a perivascular niche. Using this data, protocols to rapidly purify stem cells from adipose tissue using Fluorescence Activated Cell Sorting are developed. The frequency of cells, and both the patient and procedure based variables that can affect this yield are also examined. The final section of this thesis uses a high-throughput microarray platform to screen thousands of polymers to identify potential substrates that can support the attachment, stable proliferation and subsequent differentiation of stem cells purified from adipose tissue. From the initial screen, 5 distinct polymers have been identified, characterised and their effects on the stem cells examined and quantified. Combined together, these elements provide significant advances in our understanding, and the basis for on going research to deliver a tissue engineered ear for use in human ear reconstruction.

As distrofias musculares progressivas (DMP) são um grupo de doenças genéticas hereditárias caracterizadas pela degeneração progressiva e irreversível da musculatura esquelética. A distrofia muscular de Duchenne (DMD) é a forma mais comum e mais grave de DMP, com prevalência de 1 a cada 3500 a 5000 meninos. Em geral, a perda da ambulação ocorre entre 9 a 12 anos e complicações respiratórias e cardíacas podem levar ao óbito a partir da segunda década. A pesquisa em terapia celular iniciou-se com o objetivo de reverter ou diminuir a progressão do processo distrófico através do repovoamento do músculo com células normais. Atualmente, acredita-se em um benefício terapêutico com base nas propriedades anti-inflamatórias, anti-fibróticas e imunomodulatórias das células tronco adultas (CTA). As CTAs mesenquimais são bastante heterogêneas quanto à sua composição celular o que ocasiona inconsistência de resultados. Por isso, a caracterização e separação de sub-populações através de marcadores específicos e o enriquecimento de culturas de CTA com um subtipo celular de interesse pode aumentar a robustez e o efeito das terapias. Uma dessas subpopulações é o pericito que, ao contrário das CTAs mesenquimais, foi bem descrito quanto à sua localização e função in vivo. Além disso, pericitos derivados de tecido adiposo humano aumentaram a sobrevida de camundongos duplo mutantes para distrofina e utrofina (dko). Dessa forma, este trabalho pretendeu comparar o potencial terapêutico de CTAs mesenquimais e pericitos de um mesmo tecido adiposo em camundongos dko. Conseguimos confirmar o resultado anterior, mostrando que os pericitos tendem a melhorar a sobrevida de animais tratados, sendo ainda melhores do que células mesenquimais, mas a melhora perdura somente durante o tratamento. A sobrevida é maior no começo do tratamento, sugerindo que o quanto antes o tratamento for iniciado, com animais mais jovens e sintomas mais leves, melhor poderá ser o resultado. Outras perguntas a serem pesquisadas na tentativa de melhorar o efeito terapêutico da terapia celular com pericitos são: número de injeções, quantidade de células a serem injetadas, tempo de tratamento e idade das células \"doadoras\" / Progressive muscular dystrophies (PMD) are inherited genetic diseases characterized by progressive muscle loss and weakness. Duchenne muscular dystrophy (DMD) is the most common and aggressive form of PMD, with incidence of 1 in every 3500-5000 boys. In general, patients with DMD are confined to wheelchairs around 9-12 years of age and death occurs due to respiratory and heart dysfunction after the second decade. Cell therapy research at first aimed to recover or slow down the dystrophic process by repopulating the patient\'s muscle with normal cells. However, nowadays it is believed also that therapeutic benefits occur by the anti-inflammatory, anti-fibrotic and immunomodulation properties of mesenchymal stem cells (MSC). MSC are constituted by an heterogeneous cell population and therefore, cell sorting of the subpopulation cell of interest is being done routinely. By doing this enrichment, the effect can be more robust and powerful. One of the cell populations of interest for research is pericyte, which are cells well defined regarding their in vivo function and location, as opposed to MSC. Besides that, pericytes derived from adipose tissue were successful in increasing survival of double knockout mice for dystrophin and utrophin (dko). The present work aimed to compare the therapeutic potential of MSC and pericytes derived from the same adipose tissue sample in the dko mouse model. We confirmed our previous results, showing that pericytes tend to improve the survival of treated mice, and are even better than MSC from the same source but the trend was statistically significant only during the treatment period. Additionally, we also observed that the survival was better in the beginning of treatment, suggesting that earlier treatment may lead to a better therapeutic effect. In an attempt to increase the therapeutic effect of these procedure other questions to be asked are: the number of injections and number of cells per injection, the duration of the treatment and the \"age\" of the donor cells

Angiogenesis, the growth of new blood vessels, is a complex process involving several cell types and molecular signals. Excessive vascular growth is a problem in tumors, and insufficient vascularization hampers the function of transplanted insulin-producing pancreatic islets. Understanding the mechanisms behind blood vessel growth generates increased means to control angiogenesis. In this thesis a model of pancreatic islet transplantation to muscle has been used to study the involvement of leukocytes in the development of new vasculature. Transplantation of isolated islets of Langerhans into mouse muscle promoted revascularization of the grafts to a level comparable to native islets in the pancreas. The complete and functional vascular restoration resulted in improved blood glucose control compared to the clinical standard implantation site, the liver. This proved muscle as a transplantation site to be a clinically relevant option for the treatment of type 1 diabetes. The rapid islet revascularization process was found to be dependent on a distinct subset of neutrophils characterized by high expression of the chemokine receptor CXCR4 and the enzyme matrix metalloproteinase 9 (MMP-9). These cells were recruited to recently transplanted and hypoxic grafts by islet-secreted vascular endothelial growth factor A (VEGF-A). Leukocyte migration and interactions in the engraftment area were monitored using a high-speed confocal microscope followed by software tracking. New software was developed to visualize migration statistics. This tool revealed areas around the islet graft where neutrophil gathering coincided with sites of angiogenesis. Macrophages in the engraftment area positioned themselves close to the newly formed vasculature and were shown to have a stabilizing effect on the vessels. When macrophages were removed, no pericytes were recruited to the forming vasculature. The perivascular macrophages also began to express a pericyte marker when in the graft, suggesting a close relationship between these cell types or macrophage plasticity. In conclusion, this thesis presents muscle as a proangiogenic transplantation site for pancreatic islets for the treatment of type 1 diabetes, where the revascularization of the grafts was dependent on the recruitment and actions of specialized immune cells.

Diabetes affects more than 382 million people worldwide and can lead to vision loss as a result of progressive degeneration of the neurovascular unit in the retina, a condition known as diabetic retinopathy (DR). Early stage DR is characterized by microangiopathies including microaneurysms, microhemorrhages, and hyperpermeability. Analyses of postmortem human retinal tissue and retinas from animal models indicate that degeneration of the pericytes, the cells that make up the outer layer of capillaries, is an early event in DR; however, the relative contribution of specific cellular components to DR pathobiology has been difficult to dissect due to the complexity of existing models.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [95]-111).

As distrofias musculares progressivas (DMP) são um grupo de doenças genéticas hereditárias caracterizadas pela degeneração progressiva e irreversível da musculatura esquelética. A distrofia muscular de Duchenne (DMD) é a forma mais comum e mais grave de DMP, com prevalência de 1 a cada 3500 a 5000 meninos. Em geral, a perda da ambulação ocorre entre 9 a 12 anos e complicações respiratórias e cardíacas podem levar ao óbito a partir da segunda década. A pesquisa em terapia celular iniciou-se com o objetivo de reverter ou diminuir a progressão do processo distrófico através do repovoamento do músculo com células normais. Atualmente, acredita-se em um benefício terapêutico com base nas propriedades anti-inflamatórias, anti-fibróticas e imunomodulatórias das células tronco adultas (CTA). As CTAs mesenquimais são bastante heterogêneas quanto à sua composição celular o que ocasiona inconsistência de resultados. Por isso, a caracterização e separação de sub-populações através de marcadores específicos e o enriquecimento de culturas de CTA com um subtipo celular de interesse pode aumentar a robustez e o efeito das terapias. Uma dessas subpopulações é o pericito que, ao contrário das CTAs mesenquimais, foi bem descrito quanto à sua localização e função in vivo. Além disso, pericitos derivados de tecido adiposo humano aumentaram a sobrevida de camundongos duplo mutantes para distrofina e utrofina (dko). Dessa forma, este trabalho pretendeu comparar o potencial terapêutico de CTAs mesenquimais e pericitos de um mesmo tecido adiposo em camundongos dko. Conseguimos confirmar o resultado anterior, mostrando que os pericitos tendem a melhorar a sobrevida de animais tratados, sendo ainda melhores do que células mesenquimais, mas a melhora perdura somente durante o tratamento. A sobrevida é maior no começo do tratamento, sugerindo que o quanto antes o tratamento for iniciado, com animais mais jovens e sintomas mais leves, melhor poderá ser o resultado. Outras perguntas a serem pesquisadas na tentativa de melhorar o efeito terapêutico da terapia celular com pericitos são: número de injeções, quantidade de células a serem injetadas, tempo de tratamento e idade das células \"doadoras\" / Progressive muscular dystrophies (PMD) are inherited genetic diseases characterized by progressive muscle loss and weakness. Duchenne muscular dystrophy (DMD) is the most common and aggressive form of PMD, with incidence of 1 in every 3500-5000 boys. In general, patients with DMD are confined to wheelchairs around 9-12 years of age and death occurs due to respiratory and heart dysfunction after the second decade. Cell therapy research at first aimed to recover or slow down the dystrophic process by repopulating the patient\'s muscle with normal cells. However, nowadays it is believed also that therapeutic benefits occur by the anti-inflammatory, anti-fibrotic and immunomodulation properties of mesenchymal stem cells (MSC). MSC are constituted by an heterogeneous cell population and therefore, cell sorting of the subpopulation cell of interest is being done routinely. By doing this enrichment, the effect can be more robust and powerful. One of the cell populations of interest for research is pericyte, which are cells well defined regarding their in vivo function and location, as opposed to MSC. Besides that, pericytes derived from adipose tissue were successful in increasing survival of double knockout mice for dystrophin and utrophin (dko). The present work aimed to compare the therapeutic potential of MSC and pericytes derived from the same adipose tissue sample in the dko mouse model. We confirmed our previous results, showing that pericytes tend to improve the survival of treated mice, and are even better than MSC from the same source but the trend was statistically significant only during the treatment period. Additionally, we also observed that the survival was better in the beginning of treatment, suggesting that earlier treatment may lead to a better therapeutic effect. In an attempt to increase the therapeutic effect of these procedure other questions to be asked are: the number of injections and number of cells per injection, the duration of the treatment and the \"age\" of the donor cells

Background: The urinary bladder requires a rich blood supply to maintain its functions, the storage and release of urine. Specialized properties of the bladder vasculature might be anticipated to ensure the integrity of this blood supply, because it is known that blood flow is reduced by distension during bladder filling. However, the bladder vasculature has been described in detail only at the gross level. A comprehensive, threedimensional view of the blood supply to the bladder wall is presented here. Methods: The microvasculature of the bladder of male New Zealand white rabbits was described using the combination of vascular corrosion casting, alkali digestion, light microscopy, and scanning and transmission electron microscopy. Following administration of an anticoagulant and an overdose of anesthetic, the abdominal aorta was cannulated just above the inferior mesenteric artery to permit flushing of the distal vasculature. The bladder vasculature was cleared of blood with buffered saline and then either perfuse‐fixed with buffered 2% glutaraldehyde and sectioned, or filled with “Mercox” resin to prepare vascular corrosion casts. Casts were cleaned with NaOH, formic acid, and water. In some cases fixed bladders were partially digested with NaOH to expose the mucosal capillary plexus. Results: The bladder is supplied with blood by single, left and right vesicular branches of the internal or external iliac arteries. The serpentine vesicular arteries extend along the lateral borders of the bladder from base to apex just deep to the serosal surface and send dorsal and ventral branches to supply the dorsal and ventral bladder walls. Veins accompany the arteries and exhibit numerous valves. A very dense complex of vessels at the apex of the bladder apparently serves to accommodate bladder distension. The muscularis and submucosa contains few vessels, but the mucosa is well vascularized. An especially dense capillary plexus is present in the lamina propria at its junction with the transitional epithelium. In the relaxed bladder these capillaries lie in grooves formed by the basal layers of the epithelium. The endothelial cells of these capillaries display few cytoplasmic vesicles and are continuous or fenestrated. These capillaries are often invested with pericytes. The mucosal capillary plexus may be associated with an epithelial transport function or may be necessary for urothelial metabolism or maintenance of the barrier function of the urothelium. Unusual capillary tufts, possibly associated with vascular lymphatic tissue, are found associated with the main vessels on the lateral walls in the basal half of the bladder. Conclusions: These methods present a clear, comprehensive, three‐dimensional view of the microvasculature of the bladder wall. They also identify several unique features of this vasculature and provide a basis for studies of the response of this vasculature to pathologic states and experimental manipulation.