Perivascular cells of microvascular niches are the prime candidates for being a reservoire of mesenchymal stem cell (MSC)-like cells in many tissues and organs that could serve as a potential source of cells and a target of novel cell-based therapeutic approaches. In the present study, by utilising typical markers of pericytes (neuronal-glial antigen 2, NG2, a chondroitin sulphate proteoglycan) and those of MSCs (CD146 and CD105) and primitive pluripotent cells (sex-determining region Y-box 2, Sox2), the phenotypic traits and the distribution of murine and rat retinal perivascular cells were investigated in situ. Our findings indicate that retinal microvessels of juvenile rodents are highly covered by NG2-positive branching processes of pericytic (perivascular) cells that are less prominent in mature capillary networks of the adult retina. In the adult rodent retinal vascular bed, NG2 labeling is mainly confined to membranes of the cell body resulting in a pearl-chain-like distribution along the vessels. Retinal pericytes, which were identified by their morphology and NG2 expression, simultaneously express CD146. Furthermore, CD146-positive cells located at small arteriole-tocapillary branching points appear more intensely stained than elsewhere. Evidence for a differential expression of the two markers around capillaries that would hint at a clonal heterogeneity among pericytic cells, however, is lacking. In contrast, the expression of CD105 is exclusively restricted to vascular endothelial cells and Sox2 is detected neither in perivascular nor in endothelial cells. In dissociated retinal cultures, however, simultaneous expression of NG2 and CD105 was observed. Collectively, our data indicate that vascular wall resident retinal pericytes share some phenotypic features (i.e. CD146 expression) with archetypal MSCs, which is even more striking in dissociated retinal cultures (i.e. CD105 expression). These findings might have implications for the treatment of retinal pathologies.

Les cellules murales du cerveau sont un groupe de cellules neurovasculaires qui présentent une hétérogénéité moléculaire, morphologique et fonctionnelle exceptionnelle. Celles en contact avec les plus petis vaisseaux du cerveau, les péricytes du lit capillaire moyen sont connues pour être essentielles à l'homéostasie cérébrale, bien que leur capacité contractile ait longtemps été débattue. Cependant, nombre de leurs propriétés physiologiques, telles que leurs mécanismes de signalisation calcique, n'ont pas encore été élucidées. Cette thèse vise donc à identifier les mécanismes cellulaires de la signalisation calcique des péricytes des capillaires cérébraux. Dans le chapitre 2, nous utilisons la pharmacologie et l'imagerie des péricytes cérébraux exprimant l'indicateur de calcium GCaMP6f (provenant de souris transgéniques PDGFRβ-Cre::GCaMP6f) pour découvrir ces mécanismes. Contrairement aux péricytes engainants dont la signalisation du calcique dépend des canaux calcique voltage-dépendants, nous constatons que les signaux calcique des péricytes capillaire moyen sont indépendants des canaux calcique voltage-dépendants. Au contraire, nous constatons que les signaux calciques transitoires des pericytes du lit capillaire moyen sont inhibés par l'élimination du Ca2+ extracellulaire, l'inhibition des canaux Orai opérés par les réserves, le blocage du remplissage des réserves du réticulum endoplasmique, ainsi que l'inhibition des récepteurs de la ryanodine (RyRs) et des récepteurs de l'inositol trisphosphate (IP3Rs). Nous constatons également que l'entrée de Ca2+ opérée par les réserves peut être induite par la déplétion des réserves du réticulum endoplasmique et inhibée par les bloqueurs d'Orai dans les pericytes du lit capillaire moyen, et que l'influx basal de Ca2+ est largement dépendant de la déplétion des réserves. Enfin, nous montrons que l'entrée de Ca2+ opérée par les réserves d'Orai amplifie les élévations de Ca2+ cytosolique en réponse au vasoconstricteur endothéline-1. Nous concluons que la signalisation calcique dans les pericytes du lit capillaire moyen, qu'elle soit spontanée ou induite de façon agoniste, est régulée par le couplage entre la libération des réserves du réticulum endoplasmique et les voies d'influx opérées par les réserves. / Brain mural cells are a grouping of neurovascular cells that display exceptional molecular, morphological, and functional heterogeneity. Mid-capillary pericytes, the mural cells which contact the smallest vessels of the brain, are known to be critical to brain homeostasis, and their contractile ability has long been debated. However, many of their physiological properties, such as their Ca2+ signaling mechanisms, have not been elucidated. This thesis aims to uncover the cellular mechanisms of brain mid-capillary pericyte Ca2+ signaling. In chapter 2, we harness pharmacology and imaging of brain pericytes expressing the calcium indicator GCaMP6f (from transgenic PDGFRβ-Cre::GCaMP6f mice) to uncover these mechanisms. In contrast to ensheathing pericytes whose Ca2+ signaling is dependent on voltage-gated Ca2+ channels (VGCCs), we find that mid-capillary pericyte Ca2+ signals are independent of VGCCs. Instead, we find that mid-capillary pericyte Ca2+ transients are inhibited by removal of extracellular Ca2+, inhibition of store-operated Orai channels, blockade of endoplasmic reticulum store filling, as well as inhibition of ryanodine receptors (RyRs) and inositol triphosphate receptors (IP3Rs). We further find that store-operated Ca2+ entry can be induced by endoplasmic reticulum store depletion and inhibited by Orai blockers in mid-capillary pericytes, and that basal Ca2+ influx is largely dependent on store depletion. Finally, we show that Orai store-operated Ca2+ entry amplifies cytosolic Ca2+ elevations in response to the vasoconstrictor endothelin-1. We conclude that both spontaneous and Gq-coupled protein receptor agonist-induced Ca2+ signaling in mid-capillary pericytes is regulated by coupling between endoplasmic reticulum store release and store-operated influx pathways.

Hemangiopericitomas (CHP) assim como schwanomas são neoplasias cutâneas de origem mesenquimal, sendo os hemangiopericitomas freqüentemente relatados em cães, ao contrário dos neurofibromas que são mais raros nos mesmos. Relata-se que o CHP origina-se de pericitos, ou células que se localizam ao redor de vasos sanguíneos. São observadas mais freqüentemente nos membros, como massas, bem circunscritas, firmes e grandes. Os hemangiopericitomas têm características histológicas comuns aos schwanomas (neurofibromas), sugerindo uma possível semelhante histogênese. Na realidade, na experiência deste Serviço de Anatomia Patológica, o diagnóstico diferencial entre hemangiopericitomas e neurofibromas apenas com base na morfologia é bastante difícil. O aspecto histopatológico do hemangiopericitoma e schwanoma correspondem à presença de agrupamentos celulares na forma de espiral pericapilar ou não respectivamente. Na tentativa de melhor caracterizar os hemangiopericitomas e distinguí-los de outros tumores mesenquimais, principalmente dos neurofibromas, propõe-se a caracterização histológica, epidemiológica e imunoistoquímica, dos hemangiopericitomas em animais da espécie canina. Com este, espera-se refinar o diagnóstico diferencial destes tumores, por tratar-se de neoplasias pouco e mal diagnosticadas devido à dificuldade de caracterizá-las morfologicamente. Procura-se estudar também, a proliferação celular nos três subtipos histológicos dos CHP e contagem dos critérios morfológicos de malignidade, correlacionando com o prognóstico clínico. Para isto, o presente estudo propôs realizar um levantamento criterioso dos casos de hemangiopericitomas e neurofibromas e/ou neurofibrossarcomas em cães nos Arquivos do Serviço de Necroscopia do Departamento de Patologia da Faculdade de Medicina veterinária e Zootecnia da Universidade de São Paulo (FMVZ/USP) durante o período de 1990 a 2003, reclassificando histologicamente estas neoplasias e graduando os subtipos dos CHP. Os resultados apontaram um total de 77 casos de CHP dos quais o subtipo perivascular correspondeu (35/77casos); o estoriforme (20/77) e o epitelióide (22/77).As conclusões da imunoistoquímica para tipo perivascular (HPV) revelam positividade de 100% para F8; 60% para S100; 100% para vimentina; 7% para GFAP e 0% para CD34 e citoqueratina. O tipo estoriforme (HES) revelou 70% de positividade para F8; 50% para S100; 100% para vimentina; 0% para citoqueratina e GFAP; 10% para CD34. Destaca-se para o epitelióide (HEP) 70% de positividade para o F8; 40 % para o S100; 90% para vimentina; 0% para citoqueratina; GFAP e CD34. A contagem estatística dos critérios de malignidade como PCNA (CL3); índice mitótico (CL1); índice apoptótico (CL4); células multinucleadas (CL0) revelaram nos subseqüentes (HPV, HE, HEP) subtipos de CHP, respectivamente: CL0 (6.135±4.038; 5.067±3.019; 11.217±5.729); CL1 (6.155±2.380; 7.545±1.941; 12.540±8.629); CL3 (30.042±10.824; 39.1.22±11.158; 41.945±9.705); CL4 (1.153±1.1443; 1.888±1.988; 2.400±1.648). O prognóstico clínico revelou 59% de taxa de recidiva para o tipo epitelióide. Assim, o presente estudo mostrou que os hemangiopericitomas devem ser graduados histologicamente por três subtipos dos quais o epitelióide parece ser o mais agressivo e o perivascular o mais incidente (45,5%) (35/77 casos) e que a imunoistoquímica pode ter papel importante na diferenciação entre os hemangiopericitomas e neurofibromas, devido à positividade e negatividade do Fator 8 respectivamente; no entanto não auxilia para distinção entre os subtipos dos CHP. Contudo o estabelecimento de critérios para diferenciar, graduar e caracterizar os CHP poderão ter implicações epidemiológicas, terapêuticas e prognósticas importantes como mostra o presente estudo / Haemangiopericytomas CHP like Schwanomas are cutaneous neoplasms of mesenchymal origin, frequently appearing in dogs, unlike neurofibromas, which are rare on the species. There are cases reported that CHP originates from pericytes, or cells located around blood vessels. They are observed more in limbs such as tissues, they are well defined, big and firm. Haemangiopericytomas have histologic characteristics common to schwanomas (neurofibromas), suggesting a possible similarity in histogenesis. In fact, at this service of Animal Pathology experience, the distinguishing final diagnosis between Haemangiopericytomas and neurofibromas, based only on morphology offers great difficulty. The hispathological aspects of the haemangiopericytons and of the schwanomas correspond to the presence or not (respectively) of spiral pericilar shape cell forms. Attempting to better identify and distinguish the haemangiopericytons from other mesenquimal tumors, mainly neurofiberns, the study indicates the histological, epidemiological and immunohistochemical characterization of Haemangiopericytomas in canine species. With this, it is expected to refine the distinguishing diagnosis of such tumors, for they are known to be neoplasms rarely and wrongly diagnosed due to difficulties to identify them morphologically. The work also studies cell proliferation at the three histological subtypes of CHP and the count for morphological malignity, correlating with the clinic prospect. In order to accomplish it, the work proposes a thoroughly case study of Haemangiopericytomas and neurofibromas and/or neurobrosarcomas in dogs; investigating on the archives of the Necrology Department of Pathology at the Veterinary Medical and Zoology at São Paulo University; from 1990 to 2003, to reclassify histologically these neoplasms and scale the subtypes of CHP. The results indicated a sum of 77 cases of CHP from which the subtype perivascular accounted for (35/77) cases; the storiform (20/77) and the epithelioid (22/77). The conclusions of immunohistochemical to perivascular type (HPV) reveal positiveness of 100% to F8; 60% to S100; 100% to vimentin; 7% to GFAP and 0% to CD34 and cytokeratin. The type estoriform (HES) revealed 70% positiveness to F8; 50% to S100; 100% to vimentin; 0% to cytokeratin and GFAP; 10% to CD34. Note that to epithelioid (HEP) 70% positiveness to F8; 40% to S100; 90% to vimentin; 0% to cytokeratin; GFAP and CD34. Statistical count of malignity criteria such as PCNA (CL3); miotic level (CL1); apoptotic level (CL4); multinucleous cells (CLO) reveal on subsequent (HPV, HE, HEP) subtypes of CHP, respectively: CLO (6.135 (6.135±4.038; 5.067±3.019; 11.217±5.729); CL1 (6.155±2.380; 7.545±1.941; 12.540±8.629); CL3 (30.042±10.824; 39.1.22±11.158; 41.945±9.705); CL4 (1.153±1.1443; 1.888±1.988; 2.400±1.648). The clinical prognostic revealed 59% receding rate to epithelioid. Therefore the study showed that Haemangiopericytomas must be histologically scaled by three subtypes of which the epithelioid appears to be the most AGGRESSIVE =INVASIVE) and perivascular the most incidental (45,5%) (35/77cases); immunohistochemical may have an important role on distinguishing between Haemangiopericytomas and neurofibrossarcomas due to positiveness and negativeness of Factor 8 respectively; notwithstanding it does not aid to distinguish between subtypes from CHP. Nevertheless, a criteria definition that enables to distinguish, scale and define CHP may have relevant epidemiologic, therapeutical and prognostical connotations as shown by the present study

In vitro, la formation de structures de type tubulaire avec des cellules endothéliales de veine ombilicale humaine (HUVEC) a été étudiée en combinant la fonctionnalisation de la chimie des matériaux et le développement de la géométrie tridimensionnelle. Le polycarbonate (PC) a été utilisé comme modèle pour le développement de l'échafaud. Le film de polysaccharide naturel, basé sur un dépôt alternatif couche par couche (LbL) d’acide hyaluronique (HA) et de chitosane (CHI), a d’abord été appliqué sur une surface PC et caractérisé en termes de croissance d’épaisseur microscopie à balayage lascar (CLSM). Cette première fonctionnalisation se traduit par un revêtement complet de la couche PC. Une biofonctionnalisation supplémentaire avec un peptide adhésif (RGD) et deux peptides angiogénétiques (SVV et QK) a été étudiée, immobilisant ces peptides sur le groupe carboxylique de HA précédemment déposé, en utilisant la chimie bien connue du carbodiimide. La version marquée de chaque peptide a été utilisée pour caractériser l’immobilisation et la pénétration des peptides dans les couches de polyélectrolytes, aboutissant à une greffe réussie avec une pénétration complète dans toute l’épaisseur du LbL. Des tests in vitro ont été effectués à l'aide de cellules HUVEC pour évaluer leur efficacité d'adhésion et leur activité métabolique sur la LbL avec et sans immobilisation de peptides, ce qui a permis d'améliorer l'activité préliminaire lorsque des combinaisons de peptides sont utilisées. Enfin, les micro-canaux PC (μCh) ont été développés et caractérisés pour la première fois, et les autres expériences ont été réalisées sur un micromètre de 25 μm de largeur, fonctionnalisé avec une architecture (HA / CHI) 12,5 (PC-LbL) avec des peptides RGD et QK -RGD + QK) ou avec des peptides RGD et SVV (PC-RGD + SVV). Notre première expérience de tubulogénèse a montré de manière surprenante la formation de structures de type tubulaire déjà après 2h d'incubation en utilisant la combinaison double-peptides, mais uniquement avec PC-RGD + QK. Les tubes étaient également présents après 3 et 4 heures de culture. L'expérience de co-culture avec des péricytes humains dérivés du placenta (hPC-PL) montre comment la stabilisation des tubes a été améliorée après 3 et 4 heures également pour l'échantillon de PC-RGD + SVV. Globalement, notre matériel bio-fonctionnel avec les peptides PC-RGD + QK et PC-RGD + SVV permet la formation d'une structure de type tubulaire à la fois dans une expérience de monoculture et de co-culture. / In vitro, tubular-like structures formation with human umbilical vein endothelial cells (HUVECs) was investigated by combining material chemistry functionalization and three-dimensional geometry development. Polycarbonate (PC) was used as a template for the development of the scaffold. Natural polysaccharide’s film based on alternate layer-by-layer (LbL) deposition of hyaluronic acid (HA) and chitosan (CHI), was first applied to PC surface and characterized in terms of thickness growth both, in dry conditions using ellipsometry, and confocal lascar scanning microscopy (CLSM). This first functionalization results in a complete coating of the PC layer. Further biofunctionalization with one adhesive peptide (RGD) and two angiogenetic peptides (SVV and QK) was investigated, immobilizing those peptides on the carboxylic group of HA previously deposited, using the well-known carbodiimide chemistry. The labeled version of each peptide was used to characterize the peptides’ immobilization and penetration into the polyelectrolytes layers, resulting in a successful grafting with complete penetration through the entire thickness of the LbL. In vitro tests were performed using HUVECs to assess their adhesion efficiency and their metabolic activity on the LbL with and without peptide immobilization, resulting in a preliminary improved activity when peptide-combinations is used. Finally, PC micro-channels (μCh) were first developed and characterized, and the rest of the experiments were performed on μCh of 25μm width, functionalized with (HA/CHI)12.5 architecture (PC-LbL) with RGD and QK peptides (PC-RGD+QK) or with RGD and SVV peptides (PC-RGD+SVV). Our first tubulogenesis experiment surprisingly showed the formation of tubular-like structures already after 2h of incubation using the double-peptides combination but only using PC-RGD+QK the tubes were present also after 3 and 4 hours of culture. The co-culture experiment with human pericytes derived from placenta (hPC-PL) demonstrates how the stabilization of the tubes was improved after 3 and 4 hours also for the PC-RGD+SVV sample. Globally our bio-functional material with PC-RGD+QK and PC-RGD+SVV peptides allow the formation of tubular-like structure in both mono and co-culture experiment.

Hemangiopericitomas (CHP) assim como schwanomas são neoplasias cutâneas de origem mesenquimal, sendo os hemangiopericitomas freqüentemente relatados em cães, ao contrário dos neurofibromas que são mais raros nos mesmos. Relata-se que o CHP origina-se de pericitos, ou células que se localizam ao redor de vasos sanguíneos. São observadas mais freqüentemente nos membros, como massas, bem circunscritas, firmes e grandes. Os hemangiopericitomas têm características histológicas comuns aos schwanomas (neurofibromas), sugerindo uma possível semelhante histogênese. Na realidade, na experiência deste Serviço de Anatomia Patológica, o diagnóstico diferencial entre hemangiopericitomas e neurofibromas apenas com base na morfologia é bastante difícil. O aspecto histopatológico do hemangiopericitoma e schwanoma correspondem à presença de agrupamentos celulares na forma de espiral pericapilar ou não respectivamente. Na tentativa de melhor caracterizar os hemangiopericitomas e distinguí-los de outros tumores mesenquimais, principalmente dos neurofibromas, propõe-se a caracterização histológica, epidemiológica e imunoistoquímica, dos hemangiopericitomas em animais da espécie canina. Com este, espera-se refinar o diagnóstico diferencial destes tumores, por tratar-se de neoplasias pouco e mal diagnosticadas devido à dificuldade de caracterizá-las morfologicamente. Procura-se estudar também, a proliferação celular nos três subtipos histológicos dos CHP e contagem dos critérios morfológicos de malignidade, correlacionando com o prognóstico clínico. Para isto, o presente estudo propôs realizar um levantamento criterioso dos casos de hemangiopericitomas e neurofibromas e/ou neurofibrossarcomas em cães nos Arquivos do Serviço de Necroscopia do Departamento de Patologia da Faculdade de Medicina veterinária e Zootecnia da Universidade de São Paulo (FMVZ/USP) durante o período de 1990 a 2003, reclassificando histologicamente estas neoplasias e graduando os subtipos dos CHP. Os resultados apontaram um total de 77 casos de CHP dos quais o subtipo perivascular correspondeu (35/77casos); o estoriforme (20/77) e o epitelióide (22/77).As conclusões da imunoistoquímica para tipo perivascular (HPV) revelam positividade de 100% para F8; 60% para S100; 100% para vimentina; 7% para GFAP e 0% para CD34 e citoqueratina. O tipo estoriforme (HES) revelou 70% de positividade para F8; 50% para S100; 100% para vimentina; 0% para citoqueratina e GFAP; 10% para CD34. Destaca-se para o epitelióide (HEP) 70% de positividade para o F8; 40 % para o S100; 90% para vimentina; 0% para citoqueratina; GFAP e CD34. A contagem estatística dos critérios de malignidade como PCNA (CL3); índice mitótico (CL1); índice apoptótico (CL4); células multinucleadas (CL0) revelaram nos subseqüentes (HPV, HE, HEP) subtipos de CHP, respectivamente: CL0 (6.135±4.038; 5.067±3.019; 11.217±5.729); CL1 (6.155±2.380; 7.545±1.941; 12.540±8.629); CL3 (30.042±10.824; 39.1.22±11.158; 41.945±9.705); CL4 (1.153±1.1443; 1.888±1.988; 2.400±1.648). O prognóstico clínico revelou 59% de taxa de recidiva para o tipo epitelióide. Assim, o presente estudo mostrou que os hemangiopericitomas devem ser graduados histologicamente por três subtipos dos quais o epitelióide parece ser o mais agressivo e o perivascular o mais incidente (45,5%) (35/77 casos) e que a imunoistoquímica pode ter papel importante na diferenciação entre os hemangiopericitomas e neurofibromas, devido à positividade e negatividade do Fator 8 respectivamente; no entanto não auxilia para distinção entre os subtipos dos CHP. Contudo o estabelecimento de critérios para diferenciar, graduar e caracterizar os CHP poderão ter implicações epidemiológicas, terapêuticas e prognósticas importantes como mostra o presente estudo / Haemangiopericytomas CHP like Schwanomas are cutaneous neoplasms of mesenchymal origin, frequently appearing in dogs, unlike neurofibromas, which are rare on the species. There are cases reported that CHP originates from pericytes, or cells located around blood vessels. They are observed more in limbs such as tissues, they are well defined, big and firm. Haemangiopericytomas have histologic characteristics common to schwanomas (neurofibromas), suggesting a possible similarity in histogenesis. In fact, at this service of Animal Pathology experience, the distinguishing final diagnosis between Haemangiopericytomas and neurofibromas, based only on morphology offers great difficulty. The hispathological aspects of the haemangiopericytons and of the schwanomas correspond to the presence or not (respectively) of spiral pericilar shape cell forms. Attempting to better identify and distinguish the haemangiopericytons from other mesenquimal tumors, mainly neurofiberns, the study indicates the histological, epidemiological and immunohistochemical characterization of Haemangiopericytomas in canine species. With this, it is expected to refine the distinguishing diagnosis of such tumors, for they are known to be neoplasms rarely and wrongly diagnosed due to difficulties to identify them morphologically. The work also studies cell proliferation at the three histological subtypes of CHP and the count for morphological malignity, correlating with the clinic prospect. In order to accomplish it, the work proposes a thoroughly case study of Haemangiopericytomas and neurofibromas and/or neurobrosarcomas in dogs; investigating on the archives of the Necrology Department of Pathology at the Veterinary Medical and Zoology at São Paulo University; from 1990 to 2003, to reclassify histologically these neoplasms and scale the subtypes of CHP. The results indicated a sum of 77 cases of CHP from which the subtype perivascular accounted for (35/77) cases; the storiform (20/77) and the epithelioid (22/77). The conclusions of immunohistochemical to perivascular type (HPV) reveal positiveness of 100% to F8; 60% to S100; 100% to vimentin; 7% to GFAP and 0% to CD34 and cytokeratin. The type estoriform (HES) revealed 70% positiveness to F8; 50% to S100; 100% to vimentin; 0% to cytokeratin and GFAP; 10% to CD34. Note that to epithelioid (HEP) 70% positiveness to F8; 40% to S100; 90% to vimentin; 0% to cytokeratin; GFAP and CD34. Statistical count of malignity criteria such as PCNA (CL3); miotic level (CL1); apoptotic level (CL4); multinucleous cells (CLO) reveal on subsequent (HPV, HE, HEP) subtypes of CHP, respectively: CLO (6.135 (6.135±4.038; 5.067±3.019; 11.217±5.729); CL1 (6.155±2.380; 7.545±1.941; 12.540±8.629); CL3 (30.042±10.824; 39.1.22±11.158; 41.945±9.705); CL4 (1.153±1.1443; 1.888±1.988; 2.400±1.648). The clinical prognostic revealed 59% receding rate to epithelioid. Therefore the study showed that Haemangiopericytomas must be histologically scaled by three subtypes of which the epithelioid appears to be the most AGGRESSIVE =INVASIVE) and perivascular the most incidental (45,5%) (35/77cases); immunohistochemical may have an important role on distinguishing between Haemangiopericytomas and neurofibrossarcomas due to positiveness and negativeness of Factor 8 respectively; notwithstanding it does not aid to distinguish between subtypes from CHP. Nevertheless, a criteria definition that enables to distinguish, scale and define CHP may have relevant epidemiologic, therapeutical and prognostical connotations as shown by the present study

La barrière hémato-encéphalique (BHE) est la structure formant les capillaires du système nerveux central (SNC). La BHE est responsable de maintenir l’homéostasie du cerveau en régulant précisé- ment les échanges entre le sang et le tissu cérébral. Elle est composée de trois types cellulaires : les cellules endothéliales (ECs), les péricytes (PCs) et les astrocytes (ACs). Du fait de ses propriétés très sélectives, la BHE est une des causes majeures des échecs observés dans le développement des médicaments destinés au SNC. En effet, ces médicaments en développement sont souvent in- capables de franchir la BHE pour atteindre leurs cibles thérapeutiques. C’est pour cette raison que la mise au point et l’utilisation de modèles de BHE sont cruciaux pour étudier la capacité de nou- veaux agents thérapeutiques à traverser la BHE mais également les mécanismes sous-jacents à leurs passages. Utilisé très tôt dans le développement pharmaceutique, un modèle de BHE infor- matif permettrait de réduire les échecs dans des phases de R&D plus avancées. Nous reportons dans cette thèse le développement et la validation d’un modèle de BHE réalisé sur un Transwell®, composé d’ECs extraites chez la souris. Les travaux ont été réalisés avec la perspective de fournir le plus d’informations possibles quant à la réalisation, l’utilisation et l’interprétation du modèle de BHE dans l’étude de la perméabilité de petites molécules en conditions saines et pathologiques. Dans un premier temps, nous avons établi un protocole permettant l’isolation chez la souris des trois types cellulaires composant la BHE, soit les ECs chez la souris adulte, et les PCs et les ACs chez les nouveau-nés. Les méthodes décrites sont efficaces pour obtenir rapidement un grand nombre de cellules pures à moindre coût. De plus, les essais préliminaires démontrent que les cellules endo- théliales isolées sont pertinentes pour la création d’un modèle de la BHE. Dans un deuxième temps, nous avons entrepris de valider un modèle sain de BHE. Pour ce faire, nous avons comparé les résultats de la perméabilité in vivo de 7 molécules à trois modèles de BHE composés respectivement d’ECs primaires, d’ECs immortalisées ou de lipides extraits du cerveau porcin. Le modèle de BHE composé des ECs primaires corrèle le mieux avec les résultats obtenus in vivo chez la souris. Dans un troisième temps, nous avons exploré la possibilité d’utiliser ce modèle de BHE validé comme modèle dans l’étude de la perméabilité lors d’une inflammation aiguë. Les résultats obtenus nous permettent de décrire les possibles voies empruntées par les molécules dont la perméabilité est augmentée lors de l’inflammation. Néanmoins, cette étude met aussi en lumière l’absence de généralisation possible quant à l’impact de l’inflammation aiguë sur le passage des petites molé- cules à travers la BHE. À l’issue de cette thèse, un modèle de BHE composé d’ECs primaire a été développé et validé. Ce modèle permettra l’étude systématique des nombreux paramètres impliqués dans le passage des molécules à travers la BHE très tôt dans le développement du médicament. / The blood-brain barrier (BBB) is the structure that forms the capillaries of the central nervous system (CNS). BBB major role is to maintain brain homoeostasis by precisely regulating exchange between blood and brain tissues. Three cellular types constitute the BBB, namely: endothelial cells (ECs), perycites (PCs) and astrocytes (ACs). Due to the BBB selectivity, this barrier is the major cause of failing in the drug development of molecules targeting the CNS. Indeed, drugs developed for CNS pathologies are often unable to cross the BBB to reach their therapeutic targets. The use of a relevant BBB model is therefore critical to study drug permeability when developing new drugs. Used earlier in drug development, this kind of BBB model could allow reducing failure rates in more advanced R&D phases. In this thesis, we report the development and validation of a BBB model made from ECs extracted from mouse and seeded in a Transwell®. This work aimed at providing as much information as possible regarding the production, use and interpretation of the BBB model in the study of small molecule permeability under healthy and pathological conditions. First, we established a protocol allowing the isolation of the three cell types from mice : endothelial cells from adult mice, and pericytes and astrocytes from newborns. The methods described herein are effective to quickly obtain a large number of pure cells at a low cost. Furthermore, preliminary tests show that isolated endothelial cells are relevant for the creation of a BBB model. Next, we focused on validating a healthy BBB model. We have compared the results of the in vivo permeability of 7 molecules with three models of BBB composed respectively of primary ECs, immortalized ECs or extracted porcine brain lipids. The BBB model composed of primary ECs best correlates with the results obtained in vivo in mice. Finally, we studied the possibility of using our BBB model as a model of permeability under acute inflammation. Our results allowed describing the possible routes used by the molecules whose permeability was increased during inflammation. The inflamed BBB model is relevant for studying the permeability of small molecules; however, this work also shows that one cannot generalize the impact of inflammation on the passage of small molecules through the BBB. Overall, we have developed and validated a BBB model composed of primary endothelial cells. This model allows studying drug permeability and provides a better understanding of the mechanisms involved in the permeability. This model could be used to study many parameters involved in the passage of molecules through the BBB at very early stages of drug development.

Cette thèse de doctorat englobe deux projets majeurs visant à étudier l'interaction entre les nanotubes à effet tunnel inter-péricytes (IP-TNT), le couplage neurovasculaire et la modulation des cellules gliales dans le contexte du glaucome. Le premier projet se concentre sur la caractérisation et l'importance fonctionnelle des IP-TNT dans la régulation du couplage neurovasculaire, tandis que le second projet explore le rôle des cellules gliales, en particulier S100Β, dans la modulation des réponses des péricytes pendant l'hypertension oculaire (HTO), un facteur de risque important pour le développement du glaucome. Dans le premier projet, nous avons étudié la présence et les implications fonctionnelles des IP-TNT dans l'unité neurovasculaire. Grâce à des techniques d'imagerie avancées et à des expériences d'imagerie en direct chez la souris, nous avons visualisé et caractérisé ces nanotubes à effet tunnel qui relient les péricytes voisins dans la rétine. Nous avons découvert que les IP-TNT jouent un rôle crucial en facilitant la communication intercellulaire et la signalisation calcique entre les péricytes. Ces nanotubes contribuent à la régulation du flux sanguin capillaire et au couplage neurovasculaire, assurant l'apport efficace d'oxygène et de nutriments aux neurones actifs. Nos résultats mettent en lumière les interactions cellulaires complexes au sein de l'unité neurovasculaire et élargissent notre compréhension des mécanismes qui sous-tendent le couplage neurovasculaire. Dans le second projet, nous nous sommes concentrés sur le rôle des cellules gliales, en particulier la protéine S100Β qui se lie au calcium, dans la modulation des réponses des péricytes au cours de l'HTO, une caractéristique pathologique clé du glaucome. Grâce à une combinaison d'expériences in vivo, d'analyses moléculaires et de techniques d'imagerie, nous avons étudié l'impact de la S100Β sur les niveaux de calcium des péricytes et sur le flux sanguin capillaire. Nous avons observé que la S100Β est régulée à la hausse dans les cellules gliales, y compris les cellules de Müller et les astrocytes, au cours de l'HTO. L'administration de la protéine recombinante exogène S100Β a exacerbé l'influx de calcium intra-péricyte et altéré le flux sanguin capillaire, tandis que le blocage de la fonction S100Β a amélioré les niveaux de calcium des péricytes et rétabli un flux sanguin basal. La neutralisation de la S100Β a également protégé les cellules ganglionnaires de la rétine de la mort induite par l'HTO. Ces résultats mettent en évidence le rôle critique des cellules gliales et de la S100Β dans les déficits du couplage neurovasculaire au cours du glaucome, et donnent un aperçu des cibles thérapeutiques potentielles pour préserver la santé et la fonction de la rétine. Collectivement, les résultats des deux projets contribuent à notre compréhension de l'interaction complexe entre les IP-TNT, le couplage neurovasculaire et la modulation des cellules gliales dans le contexte du glaucome. En élucidant le rôle des IP-TNT dans la régulation neurovasculaire et l'impact des cellules gliales, en particulier la S100Β, sur les réponses des péricytes, cette thèse fournit des informations précieuses sur les mécanismes sous-jacents de la pathogenèse du glaucome. Ces résultats peuvent ouvrir la voie au développement de stratégies thérapeutiques innovantes ciblant les IP-TNT et la modulation médiée par les cellules gliales afin de préserver la fonction rétinienne et de prévenir la perte de vision dans le glaucome et les maladies neurodégénératives associées / This PhD thesis encompasses two major projects aimed at investigating the interplay between interpericyte tunneling nanotubes (IP-TNTs), neurovascular coupling, and glial cell modulation in the context of glaucoma. The first project focuses on the characterization and functional significance of IP-TNTs in neurovascular coupling regulation, while the second project explores the role of glial cells, particularly S100Β, in modulating pericyte responses during ocular hypertension (OHT), an important risk factor for developing glaucoma. In the first project, we investigated the presence and functional implications of IP-TNTs in the neurovascular unit. Through advanced imaging techniques and live imaging experiments in mice, we visualized and characterized these tunneling nanotubes connecting neighboring pericytes in the retina. We found that IP-TNTs play a crucial role in facilitating intercellular communication and calcium signaling between pericytes. These nanotubes contribute to the regulation of capillary blood flow and neurovascular coupling, ensuring the efficient delivery of oxygen and nutrients to active neurons. Our findings shed light on the intricate cellular interactions within the neurovascular unit and expand our understanding of the mechanisms underlying neurovascular coupling. In the second project, we focused on the role of glial cells, specifically the calcium-binding protein S100Β, in modulating pericyte responses during OHT, a key pathological feature of glaucoma. Through a combination of in vivo experiments, molecular analyses, and imaging techniques, we investigated the impact of S100Β on pericyte calcium levels and capillary blood flow. We observed that S100Β is upregulated in glial cells, including Müller cells and astrocytes, during OHT. Administration of recombinant S100Β protein exacerbated intrapericyte calcium influx and impaired capillary blood flow, while blocking S100Β function improved pericyte calcium levels and restored normal blood flow. Notably, S100Β neutralization also protected retinal ganglion cells from OHT-induced death. These findings highlight the critical role of glial cells and S100Β in neurovascular coupling deficits during glaucoma, providing insights into potential therapeutic targets for preserving retinal health and function. Collectively, the results from both projects contribute to our understanding of the complex interplay between IP-TNTs, neurovascular coupling, and glial cell modulation in the context of glaucoma. By elucidating the role of IP-TNTs in neurovascular regulation and the impact of glial cells, particularly S100Β, on pericyte responses, this thesis provides valuable insights into the underlying mechanisms of glaucoma pathogenesis. These findings may pave the way for the development of innovative therapeutic strategies targeting IP-TNTs and glial cell-mediated modulation to preserve retinal function and prevent vision loss in glaucoma and related neurodegenerative diseases