[ES] La lesión medular traumática (LM) se refiere a una condición neurológica en la que un insulto mecánico interrumpe la adecuada comunicación de impulsos nerviosos a través del sistema nervioso central (SNC), resultando en la pérdida de función locomotora por debajo del área lesionada. Lamentablemente, en la actualidad aún no existe cura efectiva para restaurar la funcionalidad después de una LM. La búsqueda de un tratamiento eficiente sigue siendo un gran desafío debido a nuestra aún incompleta comprensión de la multitud de procesos biológicos desencadenados por la lesión. La terapia celular destaca como la aproximación más recurrente para el tratamiento de la LM. En las últimas décadas, se han explorado varias estrategias celulares, siendo una de las más prometedoras el trasplante de células progenitoras neurales (CPN). Muchos estudios preclínicos demostrado el potencial del trasplante de CPN para proporcionar una recuperación motora en modelos animales, sin embargo, las mejoras funcionales en ensayos clinicos humanos son limitadas. Por lo tanto, aún se deben realizar esfuerzos para descubrir la cascada precisa de procesos moleculares a lo largo de la fisiopatología de LM, así como el mecanismo subyacente de los CPN. En ese contexto, el Capítulo 1 del presente trabajo tuvo como objetivo proporcionar una caracterización de los cambios en el perfil transcripcional medular a lo largo de las diferentes etapas temporales de una lesión severa contusiva. Además, hemos descrito el impacto transcripcional del trasplante de CPN en ratas lesionadas. Hemos demostrado que mientras la LM conllevó una fuerte desregulación de varios componentes de señalización de AMPc (entre ellos EPAC2), el trasplante de CPN pudo restaurar estas alteraciones transcripcionales. Para explorar el papel de EPAC2 en el mecanismo terapéutico mediado por CPN, realizamos un experimento de inhibición sostenida de EPAC2 mediante la administración de ESI-05. En comparación con los animales solo trasplantados, los animales CPN +ESI-05 mostraron un aumento en el área de cicatriz, una exacerbación de la polarización de la microglía hacia un perfil inflamatorio y una ampliación de la brecha de neuronas preservadas a lo largo de la lesión, sugiriendo que el trnasplante de CPN en el contexto de LM implican un mecanismo dependiente de EPAC2, reduciendo la neuroinflamación y proporcionando un entorno neuro-permisivo. El Capítulo 2 explora el potencial del AMPc para la regeneración de la LM. Hemos diseñado una estrategia innovadora para inducir AMPc en las neuronas corticoespinales a través de la activación optogenética de un adenilato ciclasa foto-inducible (bPAC). La estimulación optogenética en ratas con una hemisección dorsal torácica promovió una recuperación locomotora en comparación con el grupo control. Además, la estimulación de bPAC aumentó el número de neuronas marcadas retrógradamente desde el segmento lumbar tanto en la corteza motora como en la formación rafe-reticular, pero no en el núcleo rojo.La inmunotinción del tracto rafespinal mostró que la estimulación de bPAC aumenta el ratio de axones serotonérgicos caudales a la lesión correlacionando con una mejora funcional. Por último, la depleción del sistema serotoninérgico mediante la administración de 5,7-Dihydroxytryptamina suprimió la abolió la mejora mediada por bPAC, confirmando la implicación de la vía serotoninérgica en la recuperación de los animales estimulados. En resumen, se han proporcionado nuevos conocimientos sobre los cambios transcripcionales que ocurren a lo largo de la progresión de la LM y tras el trasplante de CPN, con énfasis en la señalización de AMPc. La manipulación optogenética de AMPc en las neuronas corticoespinales después de la LM ha demostrado ser efectiva para la recuperación funcional y permitido descubrir una ruta cortical alternativa a través del tracto descendente serotoninérgico / [CA] Lesió medul·lar traumàtica (LM) es una condició neurològica en la qual un traumatisme interromp la comunicació adequada dels impulsos a través del sistema nerviós central (SNC), amb el resultat de la pèrdua de la funció locomotora per baix de la zona lesionada. Lamentablement, en l'actualitat encara no hi ha una cura efectiva per a restaurar completament la funcionalitat de la medul·la espinal després de la lesió. La recerca d'un tractament eficient per a la LM roman un repte complex a causa de la nostra comprensió encara incompleta de la gran quantitat de processos biològics desencadenats per la lesió primària. La teràpia cel·lular destaca com l'aproximació més recurrent per al tractament de la LM. En les dècades passades, s'ha explorat diverses estratègies basades en cèl·lules i una de les més prometedores és el trasplantament de cèl·lules progenitores neurals (CPN). Molts estudis preclínics han demostrat el potencial del trasplantament de CPN per proporcionar una recuperació motora en models animals, no obstant això, les millores funcionals en pacients humans tractats són limitades. Per tant, encara s'han de fer esforços per a descobrir la cascada precisa de processos moleculars al llarg de la fisiopatologia de la LM, així com el mecanisme subjacent dels CPN. El Capítol 1 del present treball va tindre com a objectiu proporcionar una caracterització dels canvis en el perfil transcripcional espinal al la llarga de les diferents etapes temporals de una lesió contusiva. A més, s'ha descrit l'impacte transcripcional del trasplantament d'CPN en animals lesionats. S'ha demostrat que mentre la LM va causar una forta desregulació de diversos components de senyalització de AMPc (sent EPAC2 el gen més regulat a la baixa), el transplantament de CPN van ser capaç de restaurar les alteracions derivades de la LM. Per a explorar el paper d'EPAC2 en el mecanisme terapèutic mediat per CPN, es va realitzar un experiment de inhibició sostinguda d'EPAC2 degut a l'administració d'ESI en animals lesionats. En comparació amb els animals només trasplantats, els animals CPN+ESI-05 van mostrar un augment de l'àrea de cicatriu, una exacerbació de la polarització de les micròglies cap a un perfil inflamatori i una ampliació de la bretxa de neurones preservades a través de la lesió.Aquests resultats suggereixen que el trasplantament de CPN en el context de la LM involucren un mecanisme depenent d'EPAC2, reduint la neuroinflamació i proporcionant un entorn més neuropermissiu. El Capítol 2 va tindre com objectiu explotar el potencial de regeneració de AMPc dissenyant una nova estratègia per a les induccions artificials de AMPc en les neurones corticoespinals mitjançant l'activació optogenètica d'una adenilat ciclasa fotoinduïble (bPAC). L'estimulació diària de AMPc en rates que pateixen una hemisècció dorsal toràcica va promoure una recuperació en comparació amb els control. L'estimulació de bPAC va augmentar el nombre de neurones marcades retrògradament des del segment lumbar, tant a l'escorça motora com a la formació rafe-reticular, però no al nucli roig. A més, la immunotinció del tracte rafespinal va mostrar que l'estimulació de bPAC va augmentar la ràtio d'axons serotoninèrgic cabals a la lesió, cosa que es va correlacionar significativament amb una millora dels paràmetres funcionals. Finalment, la depleció del sistema serotoninèrgic mitjançant l'administració de 5,7-Dihydroxytryptamina va abolir la millora mediada per bPAC, confirmant la implicació de la via serotoninèrgica en la recuperació. En resum, la investigació ha proporcionat coneixements sobre els canvis transcripcionals que tenen lloc a la llarga de la progressió de la LM i després del trasplantament de CPN, amb un èmfasi especial en la senyalització d'AMPc. La manipulació optogenètica d'AMPc a les neurones corticoespinals després de la LM ha demostrat ser efectiva per a la recuperació funcional i ha permès descobrir una ruta cortical alternativa a través del tracte descendent serotoninèrg / [EN] Traumatic spinal cord injury (SCI) refers to a neurological condition in which a mechanic insult disrupts the proper communication of the impulses through the central nervous system (CNS), resulting on the loss of locomotor function below the injured area. Unfortunately, nowadays there is still no effective cure to completely restore the functionality of the spinal cord after the injury. Cell therapy is the most recurring approach for SCI treatment. In the past decades several cell-based strategies have been explored, being one of the most promising the transplantation of neural progenitor cells (NPCs). Many pre-clinical studies evidenced the potential of the NPCs transplantation to provide a substantial motor recovery in animal models, yet functional improvements in clinical trials have been limited. Therefore, efforts still need to be made in disclosing the precise cascade of molecular processes along SCI pathophysiology as well as the NPCs underlying mechanism. In that context, Chapter 1 of the present work aimed to provide a comprehensive characterization of the spinal transcriptional changes along the different temporal stages of rats suffering a severe contusive injury. Additionally, we have described the transcriptional impact of acute and subacute NPCs transplantation in injured animals. Interestingly we have shown that while SCI caused a strong dysregulation of several cAMP-signaling components (being EPAC2 the most downregulated gene), NPCs was able to restore SCI-derived alterations over this pathway with EPAC2 significant upregulation. In order to further explore EPAC2 role in NPCs-mediated therapeutical mechanism we performed a loss-of-function experiment by sustained EPAC2 inhibition via ESI-05 administration along with NPCs transplantation after SCI. Compared with only transplanted animals, NPCs+ESI-05 animals showed increased scar area, exacerbated microglia polarization into an inflammatory profile and widened gaps of preserved neurons across the lesion. Overall, these results suggest that NPC therapeutic mechanisms in the context of SCI involve an EPAC2-dependent mechanism, reducing neuroinflammation and providing a neuro-permissive environment. Chapter 2 aimed to further explore cAMP potential for SCI regeneration. We designed a novel strategy for artificial cAMP inductions in corticospinal neurons via optogenetic activation of a photoinducible adenylyl cyclase (bPAC). Daily optogenetic cAMP stimulation in rats suffering a thoracic dorsal hemisection, which completely disrupt the dorsal aspect of the corticospinal tract (CST), promoted and early and sustained locomotor recovery compared to non-treated control animals. We have shown that bPAC stimulation increased the number of retrograde traced neurons from the lumbar segment both in the motor cortex and the raphe-reticular formation, but not in the red nuclei. Moreover, immunolabelling of the raphespinal tract by 5-HT showed that bPAC stimulation increased the ratio of descending serotoninergic axons caudal to the injury which significantly correlated with improved functional parameters. Our results from corticobulbar projection study, WGA trans-synaptic tracing, and P-CREB analysis suggest that bPAC modulation of cortico-serotonergic pathway might occurs at the brainstem level. Lastly, the serotonergic system depletion by 5,7-Dihydroxytryptamine administration suppressed bPAC-mediated recovery, confirming the implication of the serotonergic tract in the recovery of stimulated animals. In summary, our research has provided new insights into the transcriptional changes that occur along SCI progression and after NPCs transplantation with a special emphasis on cAMP signaling. Optogenetic cAMP manipulation in corticospinal neurons after SCI has proven to be effective for functional recovery and allowed to unveil a cortical rerouting pathway through the serotonergic descending tract. / This research was funded by FEDER/Ministerio de Ciencia e Innovación – Agencia Estatal de Investigación [RTI2018-095872-BC21/ERDF]. Part of the equipment employed in this work was funded by Generalitat Valenciana and cofinanced with ERDF funds (OP ERDF of Comunitat Valenciana 2014– 2020) and the UE; Fondo Europeo de Desarrollo Regional (FEDER) incluido en el Programa Operativo FEDER de la Comunidad Valenciana 2014-2020. B. MartinezRojas was supported by a grant from the Conselleria de Educación, Investigación, Cultura y Deporte de la Generalitat Valenciana and the European Social Fundation ACIF/2019/120. / Martínez Rojas, B. (2024). Transcriptional Insights for Spinal Cord Injury and Neural Precursor Cell Therapy: Toward a Novel Optogenetics-Based Treatment for cAMP Neuronal Induction [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/202972

Umbilical cord blood (UCB) is used as a source of haematopoietic stem cells (HSCs) for transplantation but shows defective homing to the bone marrow niche and delayed haematological reconstitution. Following transplantation, HSCs will home to the bone marrow in response to the CXCL12 chemokine, adhere to the bone marrow sinusoidal endothelial cells and then migrate into and lodge in bone marrow niches. In addition to CXCR4, a variety of molecules have been described as being important in these processes. In this laboratory, junctional adhesion molecule-A (JAM-A) was shown to be expressed on human UCB CD133⁺/CD34⁺ cells and regulated by hypoxia. In this thesis, further phenotypic studies show that this molecule is most highly expressed on human CD41a⁺ megakaryocytes and CD14⁺ monocytes/macrophages in UCB. JAM-A was also found to be expressed on all human UCB CD133⁺ cells, which have been shown by others to encompass the HSCs and early myeloid-lymphoid precursors and on the majority of CD34⁺ haematopoietic progenitor cells (HPCs). While it is also present on bone marrow sinusoidal endothelium (BMEC), JAM-A is not detected on cultured bone marrow mesenchymal stromal cells (MSCs). JAM-A blockade, silencing and overexpression experiments showed that JAM-A contributes to, but is not solely responsible for, the adhesion of CD34⁺ haematopoietic progenitor cells to IL-1β activated BMEC-60 cells and fibronectin. Lack of significance in cell migration suggested that JAM-A is more likely to act as an adhesion molecule or a regulator of adhesion rather than as a migratory molecule in such cells. Further functional studies using the proximity ligation assay highlight a potential association of JAM-A with CXCR4 and the adhesion molecules, tetraspanin CD82 and integrin β1. Mechanistic studies were commenced to establish if JAMA could modulate CXCR4 signalling following CXCL12 stimulation, but time constraints prevented these from being completed. These preliminary experiments which were carried out first in the Jurkat cell line lacking JAM-A or transduced to express JAM-A, however, suggest that JAM-A may modulate CXCL12-induced Rap1 phosphorylation and ERK1/2 phosphorylation. The former pathway is important for integrin function and the latter pathway is important in cell adhesion. The results described here, although requiring finalisation, support the hypothesis that JAM-A acts as an adhesion molecule and also may fine tune CXCR4 and integrin mediated functions on human CD34⁺ cells, thereby potentially regulating engraftment of these cells to the bone marrow niche.

Thesis (PhD (Physiology (Human and animal))-- University of Stellenbosch, 2007. / Definition: Stem cells are unspecialised cells with the capacity for long-term self-renewal and the ability to differentiate into multiple cell-lineages. The potential for the application of stem cells in clinical settings has had a profound effect on the future of regenerative medicine. However, to be of greater therapeutic use, selection of the most appropriate cell type, as well as optimisation of stem cell incorporation into the damaged tissue is required. In adult skeletal muscle, satellite cells are the primary stem cell population which mediate postnatal muscle growth. Following injury or in diseased conditions, these cells are activated and recruited for new muscle formation. In contrast, the potential of resident adult stem cell incorporation into the myocardium has been challenged and the response of cardiac tissue, especially to ischaemic injury, is scar formation. Following muscle damage, various growth factors and cytokines are released in the afflicted area which influences the recruitment and incorporation of stem cells into the injured tissue. Transforming Growth Factor-β (TGF-β) is a member of the TGF-β-superfamily of cytokines and has at least three isoforms, TGF-β1, -β2, and -β3, which play essential roles in the regulation of cell growth and regeneration following activation and stimulation of receptor-signalling pathways. By improving the understanding of how TGF-β affects these processes, it is possible to gain insight into how the intercellular environment can be manipulated to improve stem cell-mediated repair following muscle injury. Therefore, the main aims of this thesis were to determine the effect of the three TGF-β isoforms on proliferation, differentiation, migration and fusion of muscle progenitor cells (skeletal and cardiac) and relate this to possible improved mechanisms for muscle repair. The effect of short- and long-term treatment with all three TGF-β isoforms were investigated on muscle progenitor cell proliferation and differentiation using the C2C12 skeletal muscle satellite and P19 multipotent embryonal carcinoma cell-lineages as in vitro model systems. Cells were treated with 5 ng/mℓ TGF-β isoforms unless where stated otherwise. In C2C12 cells, proliferating cell nuclear antigen (PCNA) expression and localisation were analysed, and together with total nuclear counts, used to assess the effect of TGF-β on myoblast proliferation (Chapter 5). The myogenic regulatory factors MyoD and myogenin, and structural protein myosin heavy chain (MHC) were used as protein markers to assess early and terminal differentiation, respectively. To establish possible mechanisms by which TGF-β isoforms regulate differentiation, further analysis included determination of MyoD localisation and the rate of MyoD degradation in C2C12 cells. To assess the effect of TGF-β isoforms on P19 cell differentiation, protein expression levels of connexin-43 and MHC were analysed, together with the determination of embryoid body numbers in differentiating P19 cells (Chapter 6). Furthermore, assays were developed to analyse the effect of TGF-β isoforms on both C2C12 and P19 cell migration (Chapter 7), as well as fusion of C2C12 cells (Chapter 8). Whereas all three isoforms of TGF-β significantly increased proliferation of C2C12 cells, differentiation results, however, indicated that especially following long-term incubation, TGF-β isoforms delayed both early and terminal differentiation of C2C12 cells into myotubes. Similarly, myocyte migration and fusion were also negatively regulated following TGF-β treatment. In the P19 cell-lineage, results demonstrated that isoform-specific treatment with TGF-β1 could potentially enhance differentiation. Further research is however required in this area, especially since migration was greatly reduced in these cells. Taken together, results demonstrated variable effects following TGF-β treatment depending on the cell type and the duration of TGF-β application. Circulating and/or treatment concentrations of this growth factor could therefore be manipulated depending on the area of injury to improve regenerative processes. Alternatively, when selecting appropriate stem or progenitor cells for therapeutic application, the effect of the immediate environment and subsequent interaction between the two should be taken into consideration for optimal beneficial results.

L’athérosclérose est la principale cause d’infarctus du myocarde, de mort subite d’origine cardiaque, d’accidents vasculaires cérébraux et d’ischémie des membres inférieurs. Celle-ci cause près de la moitié des décès dans les pays industrialisés. Lorsque les obstructions artérielles athérosclérotiques sont tellement importantes que les techniques de revascularisation directe ne peuvent être effectuées avec succès, la sévérité de l’ischémie tissulaire résiduelle dépendra de l’habilité de l’organisme à développer spontanément de nouveaux vaisseaux sanguins (néovascularisation). La néovascularisation postnatale est le résultat de deux phénomènes : la formation de nouveaux vaisseaux à partir de la vasculature existante (angiogenèse) et la formation de vaisseaux à partir de cellules souches progénitrices (vasculogenèse). Notre laboratoire a démontré que plusieurs facteurs de risque associés aux maladies cardiovasculaires (tabagisme, vieillissement, hypercholestérolémie) diminuaient également la réponse angiogénique suite à une ischémie. Cependant, les mécanismes précis impliqués dans cette physiopathologie sont encore inconnus. Un point commun à tous ces facteurs de risque cardiovasculaire est l’augmentation du stress oxydant. Ainsi, le présent ouvrage visait à élucider l’influence de différents facteurs de risque cardiovasculaire et du stress oxydant sur la néovascularisation. Nos résultats démontrent que l’exposition à la fumée de cigarette et le vieillissement sont associés à une diminution de la néovascularisation en réponse à l’ischémie, et que ceci est au moins en partie causé par une augmentation du stress oxydant. De plus, nous démontrons que les acides gras dérivés de la diète peuvent affecter la réponse à l’ischémie tissulaire. La première étude du projet de recherche visait à évaluer l’impact de l’exposition à la fumée de cigarette sur la néovascularisation post-ischémique, et l’effet d’une thérapie antioxydante. L’exposition à la fumée de cigarette a été associée à une réduction significative de la récupération du flot sanguin et de la densité des vaisseaux dans les muscles ischémiques. Cependant, une récupération complète de la néovascularisation a été démontrée chez les souris exposées à la fumée de cigarette et traitées au probucol ou aux vitamines antioxydantes. Nous avons démontré qu’une thérapie antioxydante administrée aux souris exposées à la fumée de cigarette était associée à une réduction significative des niveaux de stress oxydant dans le plasma et dans les muscles ischémiques. De plus, les cellules endothéliales progénitrices (EPCs) exposées à de l’extrait de fumée de cigarette in vitro présentent une diminution significative de leur activité angiogénique (migration, adhésion et incorporation dans les tissus ischémiques) qui a été complètement récupérée par le probucol et les vitamines antioxydantes. La deuxième étude avait pour but d’investiguer le rôle potentiel de la NADPH oxydase (Nox2) pour la modulation de la néovascularisation post-ischémique dans le contexte du vieillissement. Nous avons trouvé que l’expression de la Nox2 est augmentée par le vieillissement dans les muscles ischémiques des souris contrôles. Ceci est associé à une réduction significative de la récupération du flot sanguin après l’ischémie chez les vieilles souris contrôles comparées aux jeunes. Nous avons aussi démontré que la densité des capillaires et des artérioles est significativement réduite dans les muscles ischémiques des animaux vieillissants alors que les niveaux de stress oxydant sont augmentés. La déficience en Nox2 réduit les niveaux de stress oxydant dans les tissus ischémiques et améliore la récupération du flot sanguin et la densité vasculaire chez les animaux vieillissants. Nous avons aussi démontré que l’activité fonctionnelle des EPCs (migration et adhésion à des cellules endothéliales matures) est significativement diminuée chez les souris vieillissantes comparée aux jeunes. Cependant, la déficience en Nox2 est associée à une récupération de l’activité fonctionnelle des EPCs chez les animaux vieillissants. Nous avons également démontré une augmentation pathologique du stress oxydant dans les EPCs isolées d’animaux vieillissants. Cette augmentation de stress oxydant dans les EPCs n’est pas présente chez les animaux déficients en Nox2. La troisième étude du projet de recherche a investigué l’effet des acides gras dérivés de la diète sur la néovascularisation postnatale. Pour ce faire, les souris ont reçu une diète comprenant 20% d’huile de maïs (riche en oméga-6) ou 20% d’huile de poisson (riche en oméga-3). Nos résultats démontrent qu’une diète riche en oméga-3 améliore la néovascularisation post-ischémique au niveau macro-vasculaire, micro-vasculaire et clinique comparée à une diète riche en oméga-6. Cette augmentation de la néovascularisation postnatale est associée à une réduction du ratio cholestérol total/cholestérol HDL dans le sérum et à une amélioration de la voie VEGF/NO dans les tissus ischémiques. De plus, une diète riche en acides gras oméga-3 est associée à une augmentation du nombre d’EPCs au niveau central (moelle osseuse) et périphérique (rate). Nous démontrons aussi que l’activité fonctionnelle des EPCs (migration et incorporation dans des tubules de cellules endothéliales matures) est améliorée et que le niveau de stress oxydant dans les EPCs est réduit par la diète riche en oméga-3. En conclusion, nos études ont permis de déterminer l’impact de différents facteurs de risque cardiovasculaire (tabagisme et vieillissement) et des acides gras dérivés de la diète (oméga-3) sur la néovascularisation post-ischémique. Nous avons aussi identifié plusieurs mécanismes qui sont impliqués dans cette physiopathologie. Globalement, nos études devraient contribuer à mieux comprendre l’effet du tabagisme, du vieillissement, des oméga-3, et du stress oxydant sur l’évolution des maladies vasculaires ischémiques. / Atherosclerosis is the main cause of myocardial infarction, sudden cardiac death, stroke and lower limb ischemia. It is responsible for nearly half of all deaths in industrialized countries. When atherosclerotic arterial obstructions are so important that direct revascularization techniques cannot be successfully performed, the severity of residual tissue ischemia depends on the ability of the organism to spontaneously develop new blood vessels (neovascularization). Postnatal neovascularization is the result of two phenomena: the formation of new bloods vessels from the existing vasculature (angiogenesis) and vessel formation from progenitor cells (vasculogenesis). Our laboratory has demonstrated that several cardiovascular risk factors (smoking, aging, and hypercholesterolemia) also impair the angiogenic response after ischemia. However, the precise mechanisms involved in that pathophysiology are still unknown. A common feature of all the cardiovascular risk factors is increased oxidative stress. Therefore, the purpose of the present work was to elucidate the influence of cardiovascular risk factors and oxidative stress on neovascularization. Our results demonstrate that exposure to cigarette smoke and aging are associated with impaired neovascularization in response to ischemia, and that this is at least in part due to increased oxidative stress. In addition, we demonstrate that fatty acids derived from the diet can modulate the response to tissue ischemia. The first study of the research project evaluated the effect of cigarette smoke exposure on neovascularization in response to ischemia, and the effect of an antioxidant therapy. Exposure to cigarette smoke was associated with a significant reduction in the recovery of blood flow perfusion and vessel density in ischemic muscles. However, a complete recovery of neovascularization was demonstrated in mice exposed to cigarette smoke that were treated with probucol or antioxidant vitamins. We found that antioxidant therapy in mice exposed to cigarette smoke was associated with a significant reduction of oxidative stress levels in the plasma and in ischemic muscles. In addition, endothelial progenitor cells (EPCs) exposed to cigarette smoke extracts in vitro showed a significant decrease in their angiogenic activities (migration, adhesion and homing into ischemic tissues) that was completely rescued by probucol and antioxidants vitamins. The goal of the second study was to investigate the potential role of NADPH oxidase (Nox2) in the modulation of ischemia-induced neovascularization in the context of aging. We found that the expression of Nox2 is increased by aging in ischemic muscles of control mice. This is associated with a significant reduction of blood flow recovery after ischemia in older compared to young control mice. We also demonstrated that the density of capillaries and arterioles is significantly reduced in ischemic muscles of older animals, whereas oxidative stress levels are increased. Nox-2 deficiency reduces oxidative stress levels in ischemic tissues and improves blood flow recovery and vascular densities in older animals. We also demonstrated that the functional activities of EPCs (migration and adhesion to mature endothelial cells) were significantly reduced in older compared to young mice. However, Nox2 deficiency is associated with preserved EPCs functional activities in older animals. We also demonstrated an age-dependent pathological increase of oxidative stress in EPCs that is not found in Nox2-deficient animals. The third study of the research project investigated the effect of fatty acids derived from the diet on postnatal neovascularization. To this end, mice received a diet containing either 20% corn oil (rich in omega-6) or 20% fish oil (rich in omega-3). Our results demonstrate that an omega-3 rich diet increases neovascularization in response to ischemia at the macrovascular, microvascular and clinical level compared to an omega-6 rich diet. This increased postnatal neovascularization is associated with decreased total cholesterol/HDL cholesterol ratio in the serum and improved VEGF/NO pathway in ischemic tissues. In addition, the omega-3 rich diet is associated with a significant increase of central (bone marrow) and peripheral (spleen) EPCs. We also show that the functional activities of EPCs (migration and incorporation into tubules) are improved and oxidative stress level in EPCs is reduced by the omega-3 rich diet. In conclusion, our studies have clarified the impact of cardiovascular risk factors (smoking and aging) and fatty acids derived from the diet (omega-3) on ischemia-induced neovascularization. We have also identified several mechanisms involved in that physiopathology. Globally, our studies should contribute to a better understanding of the effects of cigarette smoking, aging and omega-3 on the evolution of ischemic vascular diseases.

Le faible poids de naissance (FPN) est un facteur de risque indépendant reconnu de maladies cardiovasculaires et d'hypertension à l'âge adulte, mais les mécanismes physiopathologiques sous-tendant cette programmation précoce ne sont que partiellement connus. Chez l'adulte, la dysfonction endothéliale et l'altération tant quantitative que qualitative de la cellule progénitrice endothéliale (PEC) sont un marqueur précoce et sensible de risque cardiovasculaire. Des altérations vasculaires (raréfaction microvasculaire, anomalies de la structure vasculaire) et une dysfonction endothéliale (altération de la vasodilatation endothélium-dépendante) sont retrouvées chez le nouveau-né de FPN. Cependant, l'hypothèse d'une altération de la cellule progénitrice endothéliale chez le nouveau-né de FPN reste à être démontrée. Au cours de notre travail, nous avons montré une altération des capacités clonogéniques et angiogéniques des PECs des nouveau-nés de FPN, tant in vitro qu'in vivo. Cette dysfonction pourrait être liée à un déséquilibre antiangiogénique d'origine environnementale conduisant à un profil antiangiogénique d'expression génique de la PEC. Ainsi, nous avons pu montrer qu'une surexpression du gène de la thrombospondine-1 pouvait en partie expliquer la réduction du potentiel angiogénique des PECs du nouveau-né de FPN via une inhibition de la transduction du signal de la voie Akt/PI3K. D'autre part, une diminution des concentrations circulantes de VEGF, dont le rôle critique dans la néovascularisation est bien connu, peut-être liée à une augmentation de son inhibiteur circulant, sFlt1 (récepteur soluble au VEGF), a été retrouvée chez le nouveau-né de FPN. / Low birth weight (LBW) is a risk factor for cardiovascular disease in adulthood. However, the mechanisms explaining cardiovascular programming are incompletely understood. In adults, a reduced level of circulating endothelial progenitor cells (EPCs) is correlated with cardiovascular disease and independently predicts atherosclerosis disease progression. Recent studies demonstrated an impairment of vascular structure (microvascular rarefaction) and function (impaired vasodilation) in LBW neonates. Thus, we hypothesized that LBW infants display an EPCs impairment.We demonstrated an alteration of clonogenic and angiogenic capacities of EPCs fropm LBW infants, both in vitro and in vivo. This could be due to a fetal antiangiogenic imbalance and a subsequent antiangiogenic gene expression profile in EPCs of LBW infants. Through an inhibition of Akt/PI3K signaling, an upregulation of thrombospondin-1 expression could partially explain such observations. Moreover, VEGF pathway, the main angiogenesis regulator, could be involved as we found reduced circulating levels of VEGF, probably due to an increase of its main inhibitor, sFlt1 (soluble receptor of VEGF 1) in LBW infants. The addition of VEGF reversed the in vitro negative effect of LBW infants' sera on EPCs angiogenic function.This investigation opens the way for more studies of EPCs function in LBW subjects. Indeed, many questions emerged about the impact of such dysfunction on the future health of LBW infants.

INTRODUÇÃO: As células-tronco hematopoiéticas (CTH) do sangue do cordão umbilical (SCU) têm sido utilizadas com sucesso para o tratamento de doenças malignas e não malignas. No entanto, algumas unidades de SCU podem apresentar baixa quantidade de células nucleadas totais (CNT). Algumas abordagens têm sido sugeridas para evitar problemas em relação à baixa concentração de CTH no transplante, como a administração de duas unidades de SCU para o paciente e a expansão ex vivo de CTH. OBJETIVO: Avaliar as taxas de proliferação celular na expansão ex vivo do SCU em sistema de cocultura com células-tronco mesenquimais (CTM) obtidos a partir de diferentes fontes com alta e baixa confluência e adicionando-se ou não coquetel de citocinas no meio de cultura. MÉTODOS: Este estudo foi aprovado pelo Comitê de Ética de Pesquisa (CAPPesq) do Hospital das Clínicas da Faculdade de Medicina da USP. A coleta do SCU (n =10) foi realizada após o nascimento do bebê e expulsão da placenta. O processamento foi realizado utilizando o método de redução de volume, o qual consiste em depleção de eritrócitos. As amostras de CTM provenientes do endotélio vascular do cordão umbilical foram obtidas de doadores diferentes (n=3) e o tecido adiposo (n=3) do inventário do LIM-31. A expansão das CNT e das células com expressão de marcadores CD133+/CD34+ foram observados depois de sete dias de cultura. Além disso, o ensaio para análise de unidades de formadoras de colônias (UFC) foi realizado em todas as amostras antes e depois da expansão do SCU. Para a expansão em sistema de cocultura foi separado dois grupos para ambas as fontes de CTM (Grupo I - cocultura com adição de coquetel de citocinas vs. Grupo II - cocultura sem citocinas). RESULTADOS: Após sete dias, no grupo I com cocultura confluente, a taxa de proliferação de CNT foi duas vezes maior ao comparar com cocultura subconfluente (35 vs. 16 vezes). No mesmo grupo também foi possível evidenciar elevada taxa de proliferação de células CD133+/CD34+. O índice de proliferação das UFC no grupo I aumentou até oito vezes. A cocultura subconfluente tanto do endotélio vascular do cordão umbilical como do tecido adiposo apresentou menor rendimento em comparação as CTM confluentes. A expansão das células na presença de citocinas apresentou maior proliferação celular ao comparar às coculturas sem adição de citocinas. CONCLUSÃO: Este estudo mostrou que para alto rendimento de células do SCU, o sistema de cocultura requer adição de coquetel de citocinas e CTM confluente independentemente da fonte utilizada / INTRODUCTION: Umbilical cord blood (UCB) hematopoietic stem cells have been successfully used for the treatment of both malignant and non-malignant diseases. Nevertheless, some UCB units could have low total nucleated cells (TNC) dose. Several approaches have been suggested to avoid inadequacy problems of hematopoietic stem cells (HSC) number for transplantation, such as administration of two UCB units to the patient and HSC ex vivo expansion. OBJECTIVE: Evaluate UCB ex vivo expansion proliferative rates in a high and low mesenchymal stem cells (MSC) confluence feeder layer obtained from different MSC sources and by adding or not cytokines cocktail into the medium. METHODS: This study was approved by the Research Ethic Committee (CAPPESQ) of Hospital das Clínicas da Faculdade de Medicina da USP. The collection of UCB (n=10) was made after delivery of the infant and the expulsion of placenta. Processing was performed using volume reduction method which consists in red blood depletion. MSC samples from umbilical cord endothelium were obtained from three different donors and adipose tissue (n=3) obtained from LIM31\'s pattern inventory. The total nucleated cell (TNC), expression of hematopoietic surface markers such as CD133+/CD34+ were observed after seven days of culture. Beyond that, colony forming unit assay (CFU) was performed before and after UCB expansion. The expansion by coculture method was observed in two groups (Group I - coculture with cytokines cocktail added vs. Group II- coculture without cytokines cocktail) for both MSCs sources. RESULTS: After seven days, analysis of confluent coculture showed that TNC proliferation rate ware almost 2 times higher than in subconfluent coculture (35 vs. 16-fold) in Group I and also revealed higher proliferative rate in CD133+/CD34+ cells considering. CFU showed similar increase after seven days of culture in comparison of day 0 (up to 8-fold). Subconfluent coculture for both umbilical cord endothelium and adipose tissue showed lower yield compared with those with high MSC confluence. The expansion in the presence of cytokines showed higher cell proliferation compared to the cocultures without addition of cytokines. CONCLUSION: This study showed that coculture system may require the addition of cytokines cocktail in the media and confluent MSC regardless of source for high yield of UCB cells

Es wurde Zellneubildung im erwachsenen cerebralen Cortex der Maus in Abhängigkeit von Umweltbedingungen und Aktivitätsgrad untersucht. Es war bekannt, dass eine reizreiche Umgebung und körperliche Aktivität die Neubildung von Nervenzellen im erwachsenen Hippokampus steigern. Als Zellproliferationsmarker wurde BrdU appliziert und BrdU-inkorporierende Zellen 1 Tag und 4 Wochen nach BrdU-Gabe unter Verwendung immunhistochemischer Methoden zur Detektion BrdU-inkorporierender Zellen in verschiedenen kortikalen Regionen und Schichten quantifiziert. Die phänotypische Charakterisierung BrdU+ Zellen wurde durch kombinierte Verwendung immunhistochemischer Methoden und konfokaler Mikroskopie vorgenommen. Die im adulten murinen cerebralen Cortex proliferierenden Zellen differenzierten weit überwiegend glial. Keine der kortikalen BrdU+ Zellen zeigte zweifelsfreie Zeichen einer neuronalen Differenzierung. Damit scheint die adulte Nervenzellneubildung unter physiologischen Bedingungen eine regionale Spezialität des Hippokampus und anderer Strukturen zu sein. Weder körperliche Aktivität (RUN) noch eine reizreiche Umgebung (ENR) führten 1 Tag oder 4 Wochen nach BrdU zu einem signifikanten Unterschied zur Kontrollgruppe (CTR), was die Anzahl BrdU+ Zellen im gesamten Cortex zusamengefaßt betrifft. Dagegen konnten die vorbeschriebenen Effekte von RUN und ENR auf hippokampale BrdU-inkorporierende Zellen repliziert werden. Dies ist ein starker Hinweis darauf, dass die Verstärkung adulter Neurogenese durch RUN und ENR im Gyrus dentatus des Hippokampus eine hippokampus-spezifische Reaktion und nicht etwa Teil einer generalisierten zentralnervösen Reaktion ist. Jedoch konnte gezeigt werden, dass körperliche Aktivität und eine reizreiche Umgebung zur lokalen Beeinflussung kortikaler Zellneubildung in bestimmten Schichten und Regionen führten. So konnten bei RUN-Tieren signifikant mehr BrdU+ Zellen in Schicht I des cingulären, motorischen und visuellen Cortex als bei CTR-Tieren gefunden werden. ENR-Tiere hatten 4 Wochen nach BrdU signifikant mehr BrdU+ Zellen in Schicht II/III des visuellen Cortex als CTR-Tiere. Die Phänotypisierung BrdU+ Zellen in diesen kortikalen Bereichen ergab, dass RUN zu einer lokalen, deutlich ausgeprägten Verstärkung der Neubildung von Mikroglia führte, während ENR tendentiell lokal kortikale Astrozytogenese verstärkte (signifikant in Schicht I des motorischen Cortex 4 Wochen nach BrdU). Damit konnte erstmals berichtet werden, dass körperliche Aktivität zelltypspezifisch die Neubildung kortikaler Mikroglia stimuliert. Dieses Ergebnis ist zunächst überraschend, da mikrogliale Proliferation und Aktivierung klassischweise im Zusammenhang mit Schadenszuständen des ZNS gesehen werden. In der Tat ist dies einer der ersten Befunde, der eine mikrogliale Reaktion mit nicht-pathologischen, vollkommen physiologischen Bedingungen in Verbindung bringt. Dies könnte einen neuen Blickwinkel auf mikrogliale Funktionen eröffnen. / The effect of physical activity and enriched environment on cell genesis in the cerebral cortex of adult mice were investigated. It is well known that living under the conditions of an enriched environment and physical activity both enhance the generation of new neurons in the adult murine hippocampus. To label proliferating cells mice were injected with bromodesoxyuridine (BrdU). The number of BrdU incorporating cells in different regions and layers of the cerebral cortex was determined 1 day and 4 weeks after BrdU administration. To characterize cortical BrdU+ cells phenotypically immunohistochemistry and confocal microscopy were used. Adult-generated cortical cells were glial cells. None of all the examined cortical BrdU+ cells showed immunoreactivity for NeuN (expressed in mature neurons) unambiguously indicating that the generation of new neurons in the adult brain is a speciality of the hippocampus and other brain structures. Physical activity (RUN) and enriched environment (ENR) did not affect the number of BrdU+ cells in all cortical regions taken together compared to control animals (CTR), both 1 day and 4 weeks after BrdU. However, the known effects of RUN and ENR on hippocampal cell genesis were replicated suggesting that the enhancement of adult hippocampal neurogenesis by RUN and ENR is a hippocampus-specific reaction and not part of a generalized reaction of the adult cns. It was shown that physical activity and enriched environment had effects on cell genesis in distinct cortical layers and regions. RUN-animals had significantly more BrdU+ cells in layer I of the cingulate, motor and visual cortex than CTR. ENR-animals had significantly more BrdU+ cells in layer II/III of the visual cortex than CTR 4 weeks after BrdU. Phenotyping of BrdU+ cells in these cortical parts revealed that RUN led to a marked increase of the generation of microglia. ENR tended to enhance astrocytogenesis in several cortical parts (reaching significance in layer I of the motor cortex 4 weeks after BrdU). This is the first report that physical activity stimulates the generation of cortical microglia in a cell-type-specific and to some degree region-specific manner. This result is surprising because microglial proliferation and activation are generally thought to occur under conditions involving damage to the nervous system. In fact, this is one of the first reports linking a microglial reaction with an entirely physiological condition. This might shed a new light on microglial function.

Les taux élevés de mortalité et de morbidité associés aux maladies vasculaires en font des pathologies dont les conséquences physiopathologiques, chirurgicales et socio-économiques sont d’une importance majeure pour le système de santé. Malgré leurs avantages, la disponibilité limitée des vaisseaux autologues a conduit au développement de prothèses synthétiques. Cependant, leur surface hautement thrombogène limite leur utilisation dans la substitution des vaisseaux de petit calibre (< 6 mm). De ce fait, à cause de leur obstruction précoce, la reconstitution d’une surface luminale proche de l’endothélium natif est incontournable. Pourtant, les revêtements de surface actuellement disponibles possèdent de médiocres qualités de rétention des néo-endothélium lorsqu’ils sont soumis à des contraintes de cisaillement physiologiques. Dans ce travail, nous proposons un substitut vasculaire de petit calibre endothélialisé réalisé à partir de trois éléments : 1) une matrice préparée à partir d’une artère ombilicale désendothélialisée, 2) un recouvrement de surface innovant constitué du film multicouche de polyélectrolytes (MPE) (PAH-PSS)3-PAH, et 3) un néo-endothélium constitué de cellules endothéliales matures ou progénitrices. Les études in vitro menées sur ces substituts ont montré que la formation, la rétention sous contraintes de cisaillement et la fonctionnalité du néoendothélium élaboré sur la surface luminale étaient améliorées par le film MPE. L’implantation du substitut par pontage termino-latéral sur le lapin a montré que le cahier des charges imputé aux substituts de petit calibre était rempli, principalement en termes de perméabilité et de diamètre, mais aussi de résistance à la suture et aux infections. En conclusion, le film MPE favorise le développement d’un substitut vasculaire de petit diamètre perméable à « long » terme et qui pourrait répondre aux exigences des chirurgiens / Vascular diseases with their high rate of mortality and morbidity belong to the pathologies involving important socio-economic factors for health system. Despite the advantages of autografts, the limited availability of autologous vessels has led to the development of synthetic prostheses. However, their high thrombogenic surface limits their use as small calibre vascular substitutes (< 6 mm). To prevent narrowing of small diameter vascular grafts, the reconstruction of a luminal surface close to the native endothelium is essential. However, the retention of the neo-endothelium subjected to shear stress is poor on the coatings currently available. In this work, we developed a small calibre endothelialized vascular substitute thanks to three elements: 1) a natural matrix prepared from umbilical artery, 2) an innovative coating based on the polyelectrolytes multilayer film (PEM) (PAH-PSS)3-PAH, and 3) used for cell culture of mature or progenitor endothelial cells. In vitro studies have shown that the formation, the retention under shear stress and the endothelial function of the neoendothelium on the luminal surface were improved by PEM film. The anastomosis of this substitute on rabbits has shown that the specifications essential to small calibre vascular grafts were reached, mainly in terms of permeability and diameter but also of resistance to suture and infections. In conclusion, PEM films helped us to develop a small diameter vascular substitute with long term patency

Bei Patienten mit Keimzelltumoren werden Mobilisation und Separation peripherer Blut-stammzellen durch das Alter, die zytostatische Vorbehandlung und Art der Mobilisations-chemotherapie statistisch signifikant beeinfluát. Der beste pr diktive Parameter f r die gesammelten Stamm- und Vorl uferzellen ist die Anzahl der peripheren CD34+ Zellen, die am Tag der Leukapherese im Blutkreislauf zirkulieren. F r die Rekonstitution der Granulo- und Thrombozytopoese nach Hochdosischemotherapie ist die Dosis der trans-fundierten CD34+ Zellen von signifikantem Wert. Bei der Transplantation von mehr als 2,5 x 10 hoch 6 CD34+ Zellen/kg kann mit einer schnellen und sicheren Regeneration der H matopoese, einem niedrigeren Bedarf an Antibiotika, Erythrozyten- und Thrombozy-tenkonzentraten sowie einem k rzeren Krankenhausaufenthalt gerechnet werden. / In patients with germ cell cancer the mobilization and collection of peripheral blood progenitor cells are significantly influenced by patient's age, cytotoxic pretreatment and the mobilization chemotherapy used. The best predictive factor for harvested progenitor cells is the number of CD34+ cells circulating in the peripheral blood on the day of leukapheresis. The dose of transfused CD34+ cells has a significant impact on the reconstitution of granulocytes and platelets after high-dose chemotherapy. Transplantation of more than 2,5 x 10 to the power of 6 CD34+ cells/kg results in a rapid and safe regeneration of hematopoiesis, less antibiotics and transfusion requirements (red blood cell and platelet concentrates) and a shorter hospital stay.

EINLEITUNG: Eine Trachealrekonstruktion bedingt Komplikationen wie z.B. Infektionen und Stenosierungen durch Granulationsgewebe. Diese werden durch ein differenziertes respiratorisches Epithel, das eine mukoziliäre Clearance ermöglicht, deutlich reduziert. Die Basalzellen gelten als die Vorläuferzellen des humanen respiratorischen Epithels (HRE), d.h. sie können sich teilen und besitzen das Potenzial zur Differenzierung. Durch die magnetische Zellseparation (MACS) sollen Vorläuferzellen aus dem HRE angereichert und anschließend kultiviert werden. METHODEN: Die Conchae nasales inferiores von 80 Patienten (mittleres Alter 40 ± 14 Jahre) dienen als Zellquelle für HRE-Zellen, die mittels enzymatischen Verdaus mit Dispase II (2,4 U/ml) aus dem Gewebeverband isoliert werden. Die Kultivierung der HRE-Zellen erfolgt auf Kollagen-A-beschichteten Kulturgefäßen in serumfreiem AECG-Medium. Die Bindungsspezifität von verschiedenen extrazellulären Zellmarkern wie GSA I B4, CD44S und CD44v6 wird immunhistochemisch überprüft. Dabei erweist sich nur CD44v6 als spezifisch für Basalzellen. Die Vorläuferzellen aus dem HRE-Zellgemisch werden durch monoklonale Antikörper gegen CD44v6 und Goat-Anti-Mouse-Microbeads magnetisch konjugiert. Anschließend werden sie mittels MACS positiv selektiert. ERGEBNISSE: Die Präparation der Nasenmuscheln ergibt Einzelzellsuspensionen aus vitalen HRE-Zellen (Vitalität > 80%, n = 30). Eine Beschichtung der Kulturgefäße mit Kollagen A steigert die Adhärenz der HRE-Zellen signifikant (p < 0,0145, n = 5). Die Proliferationskinetik der HRE-Zellkulturen lässt sich durch die Populationsverdoppelungszeit charakterisieren (tPD = 23 ± 3h, n = 3). Während eines Monats wird die Proliferationskapazität der HRE-Zellen durch Zellvermehrung (383fach, n = 6) ermittelt. Die magnetische Separation ergibt eine Zellfraktion (20 ± 2%, n = 5), die sich positiv zu CD44v6 verhält. Anschließend werden die separierten Zellen eine Woche auf Kollagen A kultiviert, wobei sie alle ein adäquates Proliferationsverhalten aufweisen. SCHLUSSFOLGERUNG: Die Ergebnisse zeigen, dass CD44v6 ein spezifischer Marker für Basalzellen ist, der sich für die Anreicherung einer positiven Zellfraktion mittels MACS eignet. In weiteren Studien muss überprüft werden, inwiefern eine solche magnetisch separierte Population aus Basalzellen die Besiedelung eines Trachealersatzes mit einem differenzierten respiratorischen Epithel ermöglicht. / OBJECTIVE: Common problems affecting patients with tracheal replacement are infections and stenosis caused by granulation tissue. These complications can be minimized by establishing a differentiated respiratory epithelium, which facilitates mucocilliary clearance. The basal cells are regarded as the progenitor cells of the human respiratory epithelium (HRE). They are known to divide and possess the ability to differentiate. These cells can be enriched by means of magnetic cell sorting (MACS) for the purpose of cultivation. METHODS: The inferior nasal turbinates of 80 patients (mean age 40 ± 14 years) are used as cell source. The HRE-cells are isolated by a standard preparation using an enzymatic digestion with Dispase II (2,4 U/ml). The HRE-cells are plated on culture dishes coated with Collagen A in serum-free AECG-Medium. Several extracellular cell markers including GSA I B4, CD44S and CD44v6 are tested by immunohistochemistry. Only CD44v6 shows specific staining of basal cells. The progenitor cells of mixed single cell suspensions of HRE-cells are marked with monoclonal antibodies against CD44v6 and are conjugated with Goat-Anti-Mouse-Microbeads. Enrichment of progenitor cells is achieved by MACS using a positive selection protocol. RESULTS: The preparation of the nasal turbinates yields viable single cell suspensions of HRE-cells (viability > 80%, n = 30). Adhesion of HRE-cells is enhanced significantly (p < 0,0145, n = 5) by coating the culture dishes with Collagen A. The kinetics of proliferation of HRE-cell-cultures can be characterized by the population doubling time (tPD = 23 ± 3h, n = 3). In the course of one month the capacity of proliferation is approximated by cell expansion (383fold, n = 6). Magnetic cell sorting results in a cell fraction (20 ± 2%, n = 5) positive for CD44v6. The separated cells are cultured on Collagen A for one week, where they all show adequate proliferation. CONCLUSIONS: The results indicate that CD44v6 is a specific marker for basal cells and enables the enrichment of a positive cell fraction via application of MACS. Further studies will be required to investigate the potential of such a magnetically separated population of basal cells to generate a differentiated respiratory epithelium on a tracheal prosthesis.