Dissertations / Theses on the topic 'Blood retinal barrier'

Background: Diabetic vascular complications affect both the macro- and microvasculature. Microvascular pathology in diabetes may be mediated by biochemical factors that precipitate cellular changes at both the gene and protein levels. In the diabetic retina, vascular pathology is found mainly in microvessels, including the retinal precapillary arterioles, capillaries and venules. Macular oedema secondary to breakdown of the inner blood-retinal barrier is the most common cause of vision impairment in diabetic retinopathy. Müller cells play a critical role in the trophic support of retinal neurons and blood vessels. In chronic diabetes, Müller cells are increasingly unable to maintain their supportive functions and may themselves undergo changes that exacerbate the retinal pathology. The consequences of early diabetic changes in retinal cells are primarily considered in this thesis. Aims: This thesis aims to investigate the effect of perivascular cells (Müller cells, RPE, pericytes) on retinal endothelial cell permeability using an established in vitro model. Methods: Immunohistochemistry, cell morphology and cell growth patterns were used to characterise primary bovine retinal cells (Müller cells, RPE, pericytes and endothelial cells). An in vitro model of the blood-retinal barrier was refined by coculturing retinal endothelial cells with perivascular cells (Müller cells or pericytes) on opposite sides of a permeable Transwell filter. The integrity of the barrier formed by endothelial cells was assessed by transendothelial electrical resistance (TEER) measurements. Functional characteristics of endothelial cells were compared with ultrastructural morphology to determine if different cell types have barrier-enhancing effects on endothelial cell cultures. Once the co-culture model was established, retinal endothelial cells and Müller cells were exposed to different environmental conditions (20% oxygen, normoxia; 1% oxygen, hypoxia) to examine the effect of perivascular cells on endothelial cell permeability under reduced oxygen conditions. Barrier integrity was assessed by TEER measurements and permeability was measured by passive diffusion of radiolabelled tracers from the luminal to the abluminal side of the endothelial cell barrier. A further study investigated the mechanism of laser therapy on re-establishment of retinal endothelial cell barrier integrity. Müller cells and RPE, that comprise the scar formed after laser photocoagulation, and control cells (Müller cells and pericytes, RPE cells and ECV304, an epithelial cell line) were grown in long-term culture and treated with blue-green argon laser. Lasered cells were placed underneath confluent retinal endothelial cells growing on a permeable filter, providing conditioned medium to the basal surface of endothelial cells. The effect of conditioned medium on endothelial cell permeability was determined, as above. Results: Co-cultures of retinal endothelial cells and Müller cells on opposite sides of a permeable filter showed that Müller cells can enhance the integrity of the endothelial cell barrier, most likely through soluble factors. Low basal resistances generated by endothelial cells from different retinal isolations may be the result of erratic growth characteristics (determined by ultrastructural studies) or the selection of vessel fragments without true â barrier characteristicsâ in the isolation step. When Müller cells were co-cultured in close apposition to endothelial cells under normoxic conditions, the barrier integrity was enhanced and permeability was reduced. Under hypoxic conditions, Müller cells had a detrimental effect on the integrity of the endothelial cell barrier and permeability was increased in closely apposed cells. Conditioned medium from long-term cultured Müller cells and RPE that typically comprise the scar formed after lasering, enhanced TEER and reduced permeability of cultured endothelial cells. Conclusions: These studies confirm that bovine tissues can be used as a suitable model to investigate the role of perivascular cells on the permeability of retinal endothelial cells. The dual effect of Müller cells on the retinal endothelial cell barrier under different environmental conditions, underscores the critical role of Müller cells in regulating the blood-retinal barrier in health and disease. These studies also raise the possibility that soluble factor(s) secreted by Müller cells and RPE subsequent to laser treatment reduce the permeability of retinal vascular endothelium. Future studies to identify these factor(s) may have implications for the clinical treatment of macular oedema secondary to diseases including diabetic retinopathy.

Doctor of Philosophy (Medicine)
Background: Diabetic vascular complications affect both the macro- and microvasculature. Microvascular pathology in diabetes may be mediated by biochemical factors that precipitate cellular changes at both the gene and protein levels. In the diabetic retina, vascular pathology is found mainly in microvessels, including the retinal precapillary arterioles, capillaries and venules. Macular oedema secondary to breakdown of the inner blood-retinal barrier is the most common cause of vision impairment in diabetic retinopathy. Müller cells play a critical role in the trophic support of retinal neurons and blood vessels. In chronic diabetes, Müller cells are increasingly unable to maintain their supportive functions and may themselves undergo changes that exacerbate the retinal pathology. The consequences of early diabetic changes in retinal cells are primarily considered in this thesis. Aims: This thesis aims to investigate the effect of perivascular cells (Müller cells, RPE, pericytes) on retinal endothelial cell permeability using an established in vitro model. Methods: Immunohistochemistry, cell morphology and cell growth patterns were used to characterise primary bovine retinal cells (Müller cells, RPE, pericytes and endothelial cells). An in vitro model of the blood-retinal barrier was refined by coculturing retinal endothelial cells with perivascular cells (Müller cells or pericytes) on opposite sides of a permeable Transwell filter. The integrity of the barrier formed by endothelial cells was assessed by transendothelial electrical resistance (TEER) measurements. Functional characteristics of endothelial cells were compared with ultrastructural morphology to determine if different cell types have barrier-enhancing effects on endothelial cell cultures. Once the co-culture model was established, retinal endothelial cells and Müller cells were exposed to different environmental conditions (20% oxygen, normoxia; 1% oxygen, hypoxia) to examine the effect of perivascular cells on endothelial cell permeability under reduced oxygen conditions. Barrier integrity was assessed by TEER measurements and permeability was measured by passive diffusion of radiolabelled tracers from the luminal to the abluminal side of the endothelial cell barrier. A further study investigated the mechanism of laser therapy on re-establishment of retinal endothelial cell barrier integrity. Müller cells and RPE, that comprise the scar formed after laser photocoagulation, and control cells (Müller cells and pericytes, RPE cells and ECV304, an epithelial cell line) were grown in long-term culture and treated with blue-green argon laser. Lasered cells were placed underneath confluent retinal endothelial cells growing on a permeable filter, providing conditioned medium to the basal surface of endothelial cells. The effect of conditioned medium on endothelial cell permeability was determined, as above. Results: Co-cultures of retinal endothelial cells and Müller cells on opposite sides of a permeable filter showed that Müller cells can enhance the integrity of the endothelial cell barrier, most likely through soluble factors. Low basal resistances generated by endothelial cells from different retinal isolations may be the result of erratic growth characteristics (determined by ultrastructural studies) or the selection of vessel fragments without true ‘barrier characteristics’ in the isolation step. When Müller cells were co-cultured in close apposition to endothelial cells under normoxic conditions, the barrier integrity was enhanced and permeability was reduced. Under hypoxic conditions, Müller cells had a detrimental effect on the integrity of the endothelial cell barrier and permeability was increased in closely apposed cells. Conditioned medium from long-term cultured Müller cells and RPE that typically comprise the scar formed after lasering, enhanced TEER and reduced permeability of cultured endothelial cells. Conclusions: These studies confirm that bovine tissues can be used as a suitable model to investigate the role of perivascular cells on the permeability of retinal endothelial cells. The dual effect of Müller cells on the retinal endothelial cell barrier under different environmental conditions, underscores the critical role of Müller cells in regulating the blood-retinal barrier in health and disease. These studies also raise the possibility that soluble factor(s) secreted by Müller cells and RPE subsequent to laser treatment reduce the permeability of retinal vascular endothelium. Future studies to identify these factor(s) may have implications for the clinical treatment of macular oedema secondary to diseases including diabetic retinopathy.

Le développement de l’uvéite auto-immune expérimentale (UAE) se fait en plusieurs étapes, commençant par l’activation en périphérie de lymphocytes T auxiliaires auto-réactifs spécifiques d’antigènes rétiniens, leur migration vers l’œil, où ils sont réactivés de façon antigène-spécifique et CMH II (complexe majeur d’histocompatibilité de classe II)-dépendante pour enfin induire la rupture de la barrière hémato-rétinienne (BHR), permettant le recrutement aspécifique de cellules inflammatoires responsables des dommages tissulaires. Tous ces processus représentent des cibles potentielles pour des thérapies biologiques ciblées. Dans cette perspective, notre travail a pour but d’approfondir la compréhension des mécanismes impliqués dans le recrutement de cellules inflammatoires, la présentation locale d’antigènes rétiniens et la rupture de la BHR interne lors de l’induction de l’UAE par transfert adoptif.L’expression de molécules d’adhésion par les cellules de la BHR joue un rôle central dans l’infiltration de cellules inflammatoires dans l’œil. Dans ce contexte, nous avons d’abord montré qu’à l’instar de ce que nous avions démontré pour VCAM-1, l’expression d’ICAM-1 est fortement induite dans la rétine durant l’UAE, avec une intensité et une extension corrélées à la sévérité de la maladie. Cependant, alors que VCAM-1 est uniquement inductible, une expression basale d’ICAM-1 est détectée dans la rétine naïve. Le ligand d’ICAM-1, LFA-1, est exprimé de façon ubiquitaire par les cellules immunes circulantes, contrairement au ligand de VCAM-1, VLA-4, qui n’est exprimé que par une minorité de cellules. Par ailleurs, nous avons observé une répartition tissulaire différente de ces deux molécules d’adhésion dans la rétine. En effet, si ICAM-1 prédomine dans l’épithélium pigmentaire rétinien, VCAM-1 est fortement exprimé au niveau des lésions de vasculite, à la fois sur les cellules endothéliales et gliales péri- vasculaires. Ces 2 sites correspondent respectivement à la BHR externe et interne. Ces différences majeures en termes de distribution rétinienne des molécules d’adhésion pourraient refléter des voies d’entrée distinctes pour les cellules inflammatoires lors de leur pénétration dans l’œil.Comme les lymphocytes T auto-réactifs n’induisent la maladie qu’après avoir localement reconnu leur antigène, nous nous sommes ensuite intéressés à identifier les cellules présentatrices d’antigène (CPA) potentielles exprimant du CMH II dans la rétine lors de l’UAE. Nous avons tout d’abord observé une forte induction de l’expression de molécules du CMH II dans la rétine lors de l’inflammation intraoculaire, corrélée avec la sévérité de la maladie. Celle-ci est associée avec l’induction de l’expression de molécules de co-stimulation, particulièrement sur les cellules exprimant fortement le CMH II. L’expression la plus forte de CMH II se retrouve dans la rétine interne, au niveau des vaisseaux enflammés et s’étend vers les couches externes de la rétine et l’espace sous-rétinien dans les uvéites sévères. Nous avons identifié 3 populations de CPA potentielles exprimant le CMH II dans la rétine :des cellules CD45-CD11b- non-hématopoïétiques exprimant faiblement le CMH II et des cellules CD45+CD11b+ hématopoïétiques exprimant plus fortement le CMH II, pouvant être subdivisées en cellules Ly6C+ et Ly6C-. L’analyse bio-informatique à l’aveugle du transcriptome de ces 3 populations mène à une ségrégation claire des échantillons, avec un enrichissement en marqueurs de macrophages et de microglie dans les cellules Ly6C+ et Ly6C-, respectivement. Cependant, l’expression de Ly6C ne permet pas une ségrégation absolue entre macrophages infiltrants et microglie résidente. L’analyse fonctionnelle à l’aide de DAVID (Database for Annotation, Visualization and Integrated Discovery) révèle que les 2 populations de cellules hématopoïétiques sont plus compétentes dans la présentation d’antigène associée au CMH II et l’activation des lymphocytes T que les cellules non-hématopoïétiques.Paradoxalement, nos données n’ont pas mis en évidence d’expression de CMH II par les principales cellules de la BHR que sont les cellules endothéliales et les cellules de l’épithélium pigmentaire rétinien. Cependant, il est bien établi que les cellules endothéliales rétiniennes subissent un changement majeur de phénotype lors du développement d’une UAE. Afin d’investiguer de façon globale les mécanismes sous-jacents à la rupture de la BHR interne, nous avons étudié la régulation de l’expression génique des cellules endothéliales rétiniennes lors de l’uvéite non-infectieuse. En accord avec les données de nos travaux précédents, l’analyse du transcriptome des cellules endothéliales rétiniennes n’a pas mis en évidence d’expression de CMH II lors de l’UAE. En revanche, cette approche nous a permis d’identifier 65 gènes modulés dans les cellules endothéliales rétiniennes lors du développement d’une UAE, confirmant non seulement l’implication de certaines molécules dont le rôle pathogénique est déjà connu, mais procurant également une liste de nouveaux gènes candidats et de voies fonctionnelles potentiellement associées à la rupture de la BHR lors d’une uvéite non- infectieuse.
Doctorat en Sciences médicales (Médecine)
info:eu-repo/semantics/nonPublished

Orientadores: Jacqueline Mendonça Lópes de Faria, José Butori Lopes de Faria
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: A patogênese da retinopatia diabética (RD) está associada ao estresse nitrosativo. Alterações na barreira hemato-retiniana (BHR) externa, formada pelas células do epitélio pigmentar da retina (EPR), estão associadas às fases precoces da RD e podem acarretar no desequilíbrio da manutenção dos fotorreceptores e consequentemente promoverem mudanças nas células neuronais da retina. O estresse nitrosativo como conseqüência do aumento da produção de óxido nítrico (NO¿) produzido pela super expressão da óxido nítrico sintetase induzida (iNOS) esteve presente em todas as camadas da retina, inclusive no EPR em condições de RD experimental in vivo precoce ou na linhagem celular humana do EPR (ARPE-19) expostas à alta concentração de glicose. O tratamento com agentes químicos como a S-nitrosoglutationa (GSNO), ou naturais (cacau enriquecido com polifenol) atuaram em diferentes vias de inibição da iNOS, prevenindo o estresse nitrosativo. Para o estudo in vivo com o colírio de GSNO (artigo I) foram utilizados animais espontaneamente hipertensos (SHR) com 4 semanas de idade. O diabetes (DM) foi induzido por STZ. Após a confirmação do DM (48 horas), os animais foram divididos em 6 grupos: controles (CTs) veículo; GSNO 900nm e GSNO 10?m ou DMs veículo; GSNO 900nm e GSNO 10?m. O efeito do tratamento com colírio de GSNO foi dependente da presença ou ausência da condição do DM. Nos animais CT, o GSNO atuou como um agente nitrosativo e nos animais DM preveniu o aumento da expressão da iNOS, preservando a retina funcional. Os estudos in vitro, demonstraram que o efeito do GSNO foi deletério ou protetor dependente da concentração de glicose. Nas células ARPE-19 expostas a condições normais de glicose, o tratamento promoveu um aumento na produção de NO¿ sem aumentar a expressão de iNOS e nas células sob alta glicose induziu uma modificação pós-translacional de proteína, a S-glutationilação da iNOS prevenindo o estresse nitrosativo. No estudo do cacau (artigo II), foi avaliado in vitro (ARPE-19 exposta a alta concentração de glicose) o seu efeito protetor dependente da concentração de polifenóis. Para isso foram testadas duas formulações de cacau que diferiram somente na concentração de polifenol: 0,5% para o cacau com baixo teor de polifenol e 60,5% para o cacau com alto teor de polifenol. A epicatequina (EC), encontrada na concentração de 12% no cacau com alto teor de polifenol foi tão eficaz quanto o próprio e esteve envolvida no controle da expressão da iNOS através da estimulação do receptor ?-opióide (DOR) diminuindo os níveis de TNF-?. A modulação da iNOS, preveniu a S-nitrosilação da caveolina-1 (CAV-1) e diminuição da expressão das junções intercelulares claudina-1 e ocludina através da prevenção da interação CAV-1?junções. Em ambos os estudos, o alvo terapêutico foi a iNOS em duas diferentes modalidades: modificação pós-translacional de proteína e modulação do TNF-? via DOR no EPR em modelos experimentais de RD. Os tratamentos apresentados neste trabalho demonstraram a iNOS como alvo terapêutico e mostraram-se eficaz em conter danos funcionais e morfológicos promovidas pela situação de mimetismo do DM no EPR demonstrando o importante papel da iNOS no desenvolvimento da RD
Abstract: The pathogenesis of diabetic retinopathy (DR) is associated with nitrosative stress. Changes in outer blood-retinal barrier (BRB), formed by retinal pigment epithelium cells (RPE) are associated in the early stages of DR and can cause imbalance in the maintenance of photoreceptors and thereby cause changes on retinal neuronal cells. The nitrosative stress as a result of increased production of nitric oxide (NO) produced by overexpression of nitric oxide synthase (iNOS) was present in all layers of the retina and mainly in RPE cells in early in vivo experimental DR or in human RPE cell line (ARPE-19) exposed to high glucose condition. Therapy with chemical agents such as S-Nitrosoglutathione (GSNO) or natural compounds (enriched cocoa polyphenol) acted in different pathways of iNOS inhibition, preventing nitrosative stress. For the in vivo study with GSNO eye drops (article I), it were used spontaneously hypertensive rats (SHR) rats with 4 week old. Diabetes (DM) was induced by streptozotocin (STZ). After DM confirmation (48 hours), the animals were divided into 6 groups: controls (CTs) vehicle; GSNO 900nm and GSNO 10?m or DMs vehicle; GSNO 900nm e GSNO 10?m. The effects of treatments were dependent on glucose concentration. In CT animals, GSNO acted as a nitrosative agent and in DM rats prevented iNOS overexpression, preserving the retina function. In vitro study showed that GSNO protective or deleterious effects were dependent on the glucose concentration. In ARPE-19 cells exposed to normal glucose, the treatment promoted an increase of NO¿ production without increase iNOS expression and in cells under high glucose (HG) condition induced post-translational protein modification, S-glutationylation of iNOS, preventing nitrosative stress. In the study with cocoa (article II), it was evaluated its protective effect dependent on concentration of polyphenols in ARPE-19 cells under HG condition. For this study, the composition of cocoa was the same in both preparations with the only difference in the amounts of polyphenol, 0.5% for low polyphenol cocoa (LPC) and 60.5% for high polyphenol cocoa (HPC). Epicatechin (EC), found in 12% of HPC was similarly protective compare to HPC and it was involved in controlling iNOS expression by stimulation of the delta opioid receptor decreasing TNF- ? levels. The modulation of iNOS prevented S-nitrosylation of caveolin-1 (CAV-1) and decreased expression of claudin-1 and occluding tight junctions by preventing CAV-1/junctions interactions. The treatments presented here showed iNOS as a therapeutic target containing functional and morphological changes promoted by DM milieu in RPE showing the important role of iNOS in the development of DR
Doutorado
Clinica Medica
Doutora em Clínica Médica

Tese de Doutoramento em Ciências Veterinárias, na especialidade de Ciências Biológicas e Biomédicas
Careful control of iron availability in the retina is central to maintenance of iron homeostasis, as its imbalance is associated with oxidative stress and progress of several retinopathies, such as diabetic retinopathy. Ferritin, known for its role in iron storage and detoxification, has also been proposed as an iron-transporter and can be regarded as a potential deliverer of a considerable large amount of iron to the retina compared to transferrin, the classical ironcarrier protein. Ferritin can bind to scavenger receptor class A member 5 (Scara5) and T-cell immunoglobulin and mucin-domain 2 (TIM2) receptors and is likely endocytosed. In this study, the presence of TIM2, which remained unknown in the retina, was investigated. Although no human ortholog for mouse TIM2 has been identified, human TIM1 and mouse TIM2 have similar functions. Our results revealed for the first time the presence of TIM2 receptors in the mouse retina, mainly expressed in Müller cells, unveiling new aspects of retinal iron metabolism regarding the putative role of TIM2 in this tissue. A knockout mouse for this membrane receptor was generated in order to better understand TIM2 functions in the retina. TIM2 deficiency affected retinal iron metabolism. Iron-loaded ferritin accumulation, probably due to increased ferritin uptake mediated by Scara5, and increased iron uptake by transferrin receptor 1 (TfR1)- transferrin binding led to retinal iron overload. Consequently, increased vascular permeability and blood-retinal barrier (BRB) breakdown were observed, inducing edema of the central retina. Paracellular and transcellular transports were impaired with tight junction integrity loss and increased caveolae number. Two mechanisms seem to be involved in this process: association of iron and ferritin overload with vascular endothelial growth factor (VEGF) overexpression and oxidative stress triggered by reactive oxygen species (ROS) overproduction generated by retinal iron overload. Altogether, these results point to TIM2 as a new key player in iron homeostasis in the mouse retina, possibly modulating cellular iron levels, and a potential target for the treatment of diabetic macular edema.
RESUMO - Análise da deficiência de TIM2 na retina de murganho - A retina necessita especificamente de ferro, devido a este ser um co-factor essencial da enzima guanilato ciclase que assegura a síntese de monofosfato de guanosina cíclico, segundo mensageiro na cascata de fototransdução. Para além disso, a retina é particularmente dependente de ferro devido à contínua necessidade de síntese de membranas, para suprir a constante renovação dos segmentos externos dos fotorrecetores, que requer como co-factor este elemento. Porém, o desequilíbrio da homeostasia do ferro está associado ao dano oxidativo e ao desenvolvimento de várias situações de retinopatia, como por exemplo a retinopatia diabética. A retina é particularmente propensa a stress oxidativo e o excesso de ferro exacerba potencialmente esta situação, devido à participação do ferro na reação de Fenton, que gera a superprodução de espécies reativas de oxigénio que, por sua vez, desencadeiam stress oxidativo. Por conseguinte, a manutenção da homeostasia do ferro é crucial neste tecido. Contudo, mecanismos de regulação do ferro na retina ainda não são completamente conhecidos. A retina obtém ferro a partir da circulação sanguínea. No entanto, a barreira hemato-retiana isola a retina da circulação sanguínea, protegendo-a de potenciais estímulos nocivos. Assim, são necessários mecanismos específicos e rigorosamente regulados de absorção de ferro para atravessar esta barreira e importar a quantidade de ferro estritamente essencial para o normal funcionamento da retina. Classicamente, a transferrina foi estabelecida como a proteína transportadora de ferro na retina, sendo aceite que a transferrina sérica se liga ao seu recetor de membrana, recetor da transferrina 1, na superfície das células endoteliais e do epitélio pigmentar da retina. Após a endocitose deste complexo, o ferro é libertado no parênquima retiniano. Mais recentemente, a ferritina, considerada classicamente como uma proteína de armazenamento de ferro e destoxificação, foi também proposta como uma proteína transportadora deste elemento. A vantagem da ferritina sérica em relação à transferrina no transporte de ferro prende-se na capacidade da ferritina de incorporar ~ 4,500 átomos de ferro, ao passo que a transferrina apenas transporta 2 átomos de ferro, constituindo, assim, a ferritina uma fonte muito eficiente de ferro para os tecidos. A molécula da ferritina é composta por 24 subunidades de dois tipos: cadeia leve (L) e cadeia pesada (H) que se unem aos recetores Scara5 (scavenger receptor class A member 5) e TIM2 (T-cell immunoglobulin and mucin-domain 2), respetivamente. O nosso grupo identificou pela primeira vez a presença de recetores Scara5 na retina humana e do murganho. No entanto, até à data, a presença de recetores TIM2 na retina não foi reportada na bibliografia. O TIM2, uma proteína transmembranar do tipo 1, é um membro da família de genes portadores dos domínios mucina e imunoglobulina de células T e, para além de ser um recetor para a ferritina-H, está envolvido na regulação da resposta imunitária...
N/A

Le système nerveux central est un organe privilégié et protégé, notamment grâce à l’existence des barrières histologiques entre le sang et les tissus nerveux. La barrière-hémato encéphalique (BHE) et la barrière hémato-rétinienne (BHR) séparent respectivement le parenchyme cérébral et la rétine des composés contenus dans l’espace vasculaire, grâce à l’expression de jonctions serrées et de transporteurs membranaires permettant une régulation spécifique des échanges entre le sang et le parenchyme nerveux. Ce travail a porté sur l’étude d’un nouveau transporteur de cations organiques mis en évidence fonctionnellement à la BHE de la souris. Ce transporteur appartenant très probablement à la superfamille des solute carrier (SLC), fonctionne comme un antiport proton. Actuellement, sa présence ne peut être démontrée que de façon fonctionnelle car son identité moléculaire est encore inconnue. Cet antiport proton constitue un nouvel acteur de la perméabilité cérébrale et ouvre une nouvelle voie d’accès au cerveau. Nous nous sommes tout d’abord attachés à approfondir les connaissances fonctionnelles de ce transporteur en étudiant de nouveaux substrats et tissus d’expression. Le transport cérébral de psychotropes a été étudié in vivo par la technique de perfusion carotidienne in situ chez la souris et in vitro grâce à une lignée de cellules endothéliales cérébrales humaines immortalisées (hCMEC/D3). Nous avons démontré que la haute perméabilité cérébrale de la cocaïne fait intervenir à la fois une diffusion passive et surtout une diffusion médiée par un antiport proton. La vitesse d’entrée des substances d’abus dans le cerveau est associée à un plus fort risque d’addiction et fait de ce transporteur un nouvel acteur critique de la régulation du passage cérébral. En effet, d’autres substances comme la nicotine et certaines amphétamines comme le MDPV et l'ecstasy sont également des substrats de cet antiport. Ce transporteur apparaît comme une cible pharmacologique potentielle dans la prise en charge de toxicomanies. Malgré la diversité chimique et pharmacologique d’interactions des composés avec cet antiport, les concentrations nécessaires pour l’inhiber dépassent celles retrouvées dans le sang. Pour aider l’identification d’inhibiteurs sélectifs et efficaces nous avons développé un modèle pharmacophorique d’inhibiteurs du transporteur à partir de données générées in vitro et de l’approche FLAPpharm. Ce modèle semble prédictif de nouveaux composés pouvant constituer de meilleurs inhibiteurs de ce transporteur. L’étude des échanges in vivo au niveau du tissu nerveux nous a menés à étudier l’impact de transporteurs ABC et de l’antiport-proton au niveau cérébral et rétinien à l’aide de substances spécifiques ou de substrats mixtes comme le vérapamil. L’antiport proton est fonctionnel au niveau de la BHR et transporte notamment la clonidine, le DPH et le vérapamil. Cependant, dans le cas d’un substrat mixte P-gp et SLC (ex : vérapamil), ce transport d’influx n’est visible à la BHE que lorsque la P-gp est neutralisée. Au contraire, à la BHR l’influx lié à cet SLC est visible naturellement. L’impact de la P-gp à la BHR étant 6.3-fois plus faible ce processus est probablement moins masqué. Cette étude illustre la difficulté actuelle de prédire l’impact fonctionnel d’un transporteur pour des substrats multi-spécifiques et l’existence d’une priorisation du transport. Enfin, nous avons essayé d’identifier l’antiport proton au niveau moléculaire par une méthode de photo-activation à l’aide d’un composé adapté. Cette méthode s’est avérée efficace pour fixer une molécule sur le transporteur, permettant par la suite de l’isoler plus facilement. En conclusion, ce travail a permis de mettre en évidence l’importance de l’antiport proton dans la distribution cérébrale de psychotropes et d’ouvrir de nouvelles perspectives dans l’addiction et la compréhension du transport de substrats multi-spécifiques
The central nervous system is a privilege organ protected by histological barriers between the blood and the nervous tissue. The blood-brain barrier (BBB) and the blood-retinal barrier (BRB) separate cerebral parenchyma and retina from the circulating blood and both express tight junctions and membrane transporters, allowing a precise regulation of the exchanges between the blood and nervous tissues. We studied a new cationic transporter functionally evidenced at the mouse BBB. This molecularly unknown transporter belong to the solute carrier super family (SLC) and is a proton antiporter. It could constitute a new actor in the cerebral permeability and may be a new brain access pathway. First, we worked on the functional identification studying new substrates and new localization. Psychotropic brain transport was studied in vivo by brain in situ perfusion on mouse and in vitro with human immortalized endothelial cells (hCMEC/D3). We showed that cocaine brain entry depends on passive diffusion but also mainly on a proton antiporter. Brain entry rate of drugs of abuse is associated with modulation of addiction liability, making this transporter a new component of brain entry of cocaine, and also nicotine and some amphetamines such as ecstasy and MDPV. This proton antiporter appears to be a new potential target in addiction. Various chemical entities interact with this transporter; however concentrations used to inhibit the transporter are much higher than the one possibly found in the blood. In order to help find or design new selective and potent inhibitors, we developed a pharmacophore model of the proton antiporter inhibitors using in vitro data and the FLAPpharm approach. The model predicts well new possible inhibitors of this transporter. We also studied the impact of the ABC transporters and the proton antiporter at the BBB and the BRB using specific or multi-specific substrates such as verapamil. The proton antiporter is functionally expressed at the BRB and transports clonidine, DPH and verapamil. However, for the multi-specific (P-gp and SLC) compound verapamil, influx transport by the proton antiporter is visible at the BBB only when P-gp efflux is neutralized. On the contrary, at the BRB, the proton antiporter influx is always visible. This is certainly due to the lower impact (by 6.3 fold) of P-gp at the BRB compared to the BBB. These results show the difficulty to predict the functional impact of a transporter for multi-specific compounds and a probable transport prioritization. Finally we worked on the molecular identification of the proton antiporter using a photolabeling method. This work evidenced the importance of the proton antiporter in the brain distribution of psychotropic and drugs of abuse and opened toward new perspectives in addiction and transport comprehension

La rupture de la barrière hémato-rétinienne interne (iBRB) se produit dans de nombreux troubles de la rétine et peut provoquer un œdème rétinien souvent responsable de la perte de vision. Le but de cette étude était de caractériser l'impact d'une rupture de l’iBRB sur les changements homéostatiques rétiniens de la dystrophine Dp71, AQP4 et Kir4.1 provoqués par les altérations les cellules gliales de Müller CGM. L'effet protecteur de la Dex a été étudié dans ce modèle. Par ailleurs, les explants rétiniens ont été utilisé pour étudier la formation et la résolution de l'œdème de CGM sans l'influence de l'inflammation du cristallin ainsi que l’effet de différentes doses de glucocorticoïdes (Dex, triamcinolone et fluocinolone) et des inhibiteurs de la voie de l'acide arachidonique. Nous avons observé que la chirurgie partielle du cristallin induit une rupture de l'iBRB et des changements moléculaires dans le CGM, une diminution de l’expression de la Dp71 et d’AQP4 et la délocalisation de Kir4.1. La Dex semble protéger la rétine par l’augmentation de l’expression du HSF1. Nous avons également observé que même si les glucocorticoides étudié ont des effets différents sur l’expression de la Dp71, AQP4 et Kir4.1 les trois sont capables de prévenir la formation de l’œdème de CGM. Nos résultats suggèrent que la formations d'œdème semblent être régulée par la voie des leucotriènes. Nous avons étudié le rôle des isoformes de la dystrophine Dp71 dans les processus d'adhésion intercellulaire des cellules PC12. Nos résultats suggèrent l’existence d’au moins deux mécanismes différents seraient impliqués dans l'adhésion intercellulaire associée à la Dp71, l'une impliquant Dp71dΔ71 et Cx43
The breakdown of the internal blood-retinal barrier (iBRB) occurs in many retinal disorders and may cause retinal edema, often responsible for vision loss. The aim of this study was to characterize the impact of iBRB disruption on retinal homeostatic changes in Dp71 dystrophin, AQP4 and Kir4.1 caused by Müller glial cells (MGC) alterations. The protective effect of Dex has been studied in this model. In addition, retinal explants were used to study the formation and resolution of CGM edema without the influence of lens inflammation and the effect of different doses of glucocorticoids (Dex, triamcinolone and fluocinolone) and inhibitors of the arachidonic acid pathway. We observed that partial lens surgery induced iBRB breakdown and molecular changes in MGC, decreased expression of Dp71 and AQP4, and miss localization of Kir4.1. Dex seems to protect the retina by increasing the expression of HSF1. We also observed that although the glucocorticoids studied have different effects on the expression of Dp71, AQP4 and Kir4.1 all three can prevent the formation of MGC edema. Our results suggest that edema formation appears to be regulated by leukotrienes. We have studied the role of isoforms of dystrophin Dp71 in intercellular adhesion processes of PC12 cells. Our results suggest the existence of at least two different mechanisms involved in intercellular adhesion associated with Dp71, one involving Dp71dΔ71 and Cx43

Diabetic retinopathy (DR) is a microvascular complication of diabetes leading to blindness. Hyperglycemic/hypoxic microenvironment concurs to mechanical breakdown of blood-retinal barrier (BRB) as well as the aberrant angiogenesis activating many downstream target genes including inflammatory cytokines and vasoactive peptides, such as interleukin-1beta and vascular endothelial growth factor (VEGF). To date, intravitreal injection of agents directed against VEGF represents the elective DR therapy. However, they counteract efficaciently ocular neovascularization only in 50% of treated patients, suggesting that other pathogenic events may play an important role in non-responders. Therefore, there is an increased need for new molecule able to counteract microenvironmental alterations of DR. It has been largely demonstrated that pituitary adenylate cyclase-activating peptide (PACAP) and vasoactive intestinal peptide (VIP) exert a protective effect against retinal injuries, including DR. However, until now, no study has investigated their protective role against the harmful combined effect of both hyperglycemia and hypoxia. The main objectives of this PhD thesis have been evaluate whether PACAP protects retinal tissue of diabetic rats through modulation of hypoxia and inflammation, both converging on angiogenic process. Moreover, we have investigated the protective effects of PACAP and VIP on BRB integrity whose breakdown due to hypoxic and inflammatory event, leads to diabetic macular edema (DME). The results have demonstrated that a single intravitreal administration of PACAP in streptozotocin-injected rats modulated the expression of hypoxic, inflammatory and angiogenic factors. Moreover, PACAP and VIP restored the integrity of BRB through the activation of phosphoinositide 3 kinase (PI3K)/Akt and mammalian mitogen activated protein kinase/Erk kinase (MAPK/ERK) survival signaling pathways. Overall, these data indicate that these peptides, both in vivo and in vitro, perform a beneficial effect to attenuate some pathologic events occuring in DR.

La rétinopathie diabétique (RD) se compose d’une part d’une ischémie rétinienne périphérique et d’autre part d’une exsudation rétinienne responsable d’un œdème maculaire diabétique, première cause de cécité chez les moins 55 ans. Les traitements utilisés actuellement sont non spécifiques et traitent les complications tardives de la RD. Les phases précoces de la RD ne sont donc pas ciblées. L’hyperglycémie chronique entraine un stress oxydant et une activation des PKC qui participent à l’altération des BHR. L’objectif de ce travail a été 1°) d’étudier l’implication de la PKCζ et de la voie Rho/ROCK/Myosine II sur la physiopathogénie de la RD et 2°) de montrer l’effet bénéfique de leurs inhibiteur sur les BHR et sur la reperfusion des capillaires rétiniens. Nous avons confirmé l’hyperactivation de la PKCζ et de la voie Rho/ROCK/Myosine II chez les rats diabétiques et leur participation à la rupture de la BHR externe. Le traitement par leurs inhibiteurs respectifs normalise l’activation des deux enzymes et restaure l’intégrité anatomique et fonctionnelle de la BHR externe. De plus l'hyperactivation de ROCK altère la perfusion rétinienne par 1) constriction focale artériolaire, 2) protrusions membranaires endoluminales des cellules endothéliales (blebbing) et 3) vasoconstriction capillaire diffuse. Nous avons montré que l'ensemble de ces phénomènes étaient réversibles par traitement intravitréen de son inhibiteur le Fasudil. De manière importante le traitement par Fasudil induit également une diminution du VEGF rétinien responsable de la perméabilité des barrières et témoin indirect de l’ischémie rétinienne. Ces travaux éclairent la physiopathogénie de la RD et ouvre des perspectives thérapeutiques permettant de cibler les événements précoces de la RD
Diabetic retinopathy (DR) mainly results from peripheral retinal ischemia and exudation leading to sight threatening complications such as retinal neovascularization or macular edema. This latter represents the main cause of visual loss among working age individuals. Current treatments address late complications of DR and are non-specific. Therefore, early events are currently not addressed. Chronic hyperglycemia increases oxidative stress and activates PKC leading to blood retinal barrier (BRB) breakdown. The aims of the present work were two fold. First, to assess the implication of PKCζ and the Rho/ROCK/Myosin II pathway on the pathogenesis of DR and second, to assess whether their specific inhibitors have the potential to restore the phenotype. Herein we have demonstrated the pathogenic role of PCKζ and ROCK hyperactivation on the development of diabetes induced external BRB breakdown. Furthermore their inhibitors restored the morphologic and functional aspect of the external BRB. We also found that ROCK hyperactivation was responsible for altered retinal perfusion through several mechanism namely 1) focal constriction of retinal arterioles; 2) endoluminal protrusions of the endothelial cell membrane (blebs) and 3) capillary diffuse vasoconstriction. We were able to demonstrate that all this aspects were reversible by Fasudil, a ROCK inhibitor, administrated into the vitreous. Of importance this treatment decreased also retinal VEGF that is a well-known factor responsible for barrier breakdown and a surrogate marker for retinal ischemia. To conclude the present findings not only shed light on the mechanisms of DR but also open new therapeutic avenues addressing the early events of DR a current unmet medical need

Tese de Doutoramento em Ciências Veterinárias
A retina contém diferentes populações celulares derivadas dos monócitos: macrófagos perivasculares e células da micróglia. Neste trabalho, descreve-se a presença de um novo subtipo de macrófagos residentes, distintos das células da micróglia, localizados no espaço perivascular, em retinas humanas e de murganho. Estas células emitem uma fluorescência específica e expressam constitutivamente receptores scavenger da classe A. Exibem um movimento alternado constante, ao longo dos vasos sanguíneos, providenciando um revestimento adicional a áreas mais delgadas da parede vascular. Para além disso, acumulam peroxidase de rábano e lipoproteínas de baixa densidade acetiladas em circulação, sem rotura da barreira hemato-retiniana. No conjunto, estes resultados sugerem que estas células, com função scavenger, contribuem para o funcionamento da barreira hemato-retiniana. Acumulam, também, ferro, em lisossomas, provavelmente por um mecanismo de endocitose de ferritina, mediada por receptores TIM-2, sugerindo o seu envolvimento na homeostasia do ferro na retina e em situações de alteração desta. Em fases iniciais de degenerescência dos fotorreceptores, estas células migram para o local das lesões, sugerindo a sua participação nos processos de retinopatia. Todos estes aspectos são semelhantes aos descritos para as células Mato cerebrais. Por conseguinte, este estudo estabelece, pela primeira vez, a presença de células Mato na retina.
ABSTRACT - Retinal Mato cells: morphologic and functional characterization of a new autofluorescent perivascular cell population in physiological conditions and during retinopathy -- The retina contains distinct populations of monocyte-derived cells: perivascular macrophages and microglia. In this work we describe the presence in mouse and human healthy retinas of a new subtype of resident macrophages, present in the perivascular space, different from microglia. These cells emitted specific autofluorescence and constitutively expressed the scavenger receptor class A. They were seen to move in an oscillatory manner along blood vessels, providing an additional coating to thinner areas of the vascular wall. Furthermore, these macrophages also accumulated blood-borne horseradish peroxidase and acetylated low-density lipoprotein without blood-retinal barrier pathological disruption. Taken together these findings suggest that these cells fulfil a major role in the scavenging of blood-borne molecules contributing to blood-retinal barrier function. In addition, they stored iron in lysosomes, probably by the TIM-2 mediated binding of ferritin, indicating they probably are involved in retinal iron homeostasis and related disorders. These cells were also early detected at lesions during photoreceptor degeneration, suggesting a possible role in the onset and development of retinopathy. All these features are consistent with those described for brain Mato cells. Thus, this study shows, for the first time, the presence of autofluorescent perivascular Mato cells in the retina.
CIISA\72.retina, financiado pelo CIISA - Centro de Investigação Interdisciplinar em Sanidade Animal, Faculdade de Medicina Veterinária, Universidade Técnica de Lisboa Investigador principal: Luísa Mendes-Jorge. FIS PI061837, financiado pelo Instituto de Salud Carlos III e CIBER de Diabetes y Enfermedades Metabolicas Asociadas Investigador principal: Jesús Ruberte París

The BRB consists of both capillary endothelial cells (REC) and retinal pigment epithelial cells (RPE). Both cell types have been suggested as potential activators of circulating T cells. In this study, an in vitro model using cultured rat REC and syngeneic T cells was developed. Furthermore RPE and for the purpose of comparative studies, AEC were also successfully cultured. It was demonstrated that activation of T lymphocyte LFA-1 is a critical event governing the adhesion of T cells to RPE and REC as IFN-γ induced up-regulation of RPE and REC ICAM-1 expression did not increase binding of resting T lymphocytes. The enhanced adhesion of activated lymphocytes (but not resting lymphocytes) to normal and IFN-γ treated RPE and REC was inhibited by LFA-1 mAb and to a lesser extent by ICAM-1 mAb but not OX34 (CD2). Treatment of lymphocytes with the anti-VLA-4 mAb resulted in differential effects on binding to AEC and REC. MAb to VLA-4 significantly blocked enhanced adhesion of activated T cells to AEC but not to REC. The results also demonstrated that VLA-4 mAb significantly inhibited unactivated T cell binding to IFNγ+TNFα+LPS stimulated AEC but not REC, suggesting that VLA-4 may also function in an activation-independent manner. It was shown that activation of T cells can enhance their migratory activity across cultured REC monolayers. Migration was decreased by both adhesion receptor-dependent mechanisms i.e., mAb to LFA-1 (but not ICAM-1) and adhesion receptor-independent mechanisms by means of PGE₂. The results of this thesis have shown that activation of LFA-1 is required for functioning of the LFA-1/ICAM-1-mediated lymphocyte adhesion and migration. In addition to the role of adhesion molecules, inflammatory mediator PGE₂, but not NO^o, was found to be important in regulation of T cell adhesion and migration across REC.

Lymphocyte-endothelial interactions play a central role in the progression of T-cell mediated autoimmune disease. In the eye the mechanisms by which lymphocytes adhere to the vascular endothelium and migrate to cause areas of retinal and choroidal inflammation are not completely understood. To understand these mechanisms a method was developed to track lymphocyte circulation in vivo. By combining the use of fluorescent dyes to label lymphocytes ex vivo and the scanning laser ophthalmoscope (SLO), the lymphocyte circulation in both the retinal and choroidal circulation was detected and quantified. In the normal retinal and choroidal circulation leukocyte velocity was found to be 10.4 (5.3)(SD) mm/s in the retinal arteries, 5.1 (2.5) in retinal veins mm/sec, 1.1 (1.2)mm/sec in retinal capillaries and 2.1 (2.1) mm/sec in the choroidal vessels. A relatively small fraction of circulating lymphocytes was found to adhere to the retinal capillaries and the choroid. In the retinal capillaries adhering cells were found to stop abruptly without prior rolling. The imaging method was applied to investigate an animal model of endogenous posterior uveitis, experimental autoimmune uveoretinitis (EAU). Increased lymphocyte adhesion to both the retinal capillary and choroidal circulations was detected before the onset of clinical inflammation in the posterior segment. The increased adhesion to the retinal capillaries appeared to be dependent on the activity of leukocyte-function antigen-1 (LFA-1) on lymphocytes. However, the increased lymphocyte adhesion to the choroid could not be reduced by initiation of LFA-1 activity.

Norrin is a secreted cystine-knot growth factor that plays critical roles in vascular development in the brain, retina, and cochlea, as well as the uterus. Although Norrin is unrelated to the lipid-modified morphogens Wnts, Norrin activates the canonical Wnt/β-catenin pathway by binding to receptor Frizzled4 cysteine-rich domain (Fz4-CRD) and co-receptors of low density lipoprotein receptor related protein 5/6 ectodomain (Lrp5/6-ECD) in conjunction with Tetraspanin-12 (Tspan-12). Like Wnts, Norrin has limited extracellular diffusion properties as a result of associating with heparan sulfate proteoglycans (HSPGs). Mutations lead to inherited disordered retinal vascularization diseases such as Norrie disease, familial exudative vitreoretinopathy and coats' disease. However, the molecular mechanism of how Norrin initiates signalling by engagement with Fz4, Lrp5/6, and HSPGs has remained unresolved. Here, novel strategies for protein production of recombinant human Norrin and Fz4-CRD as well as the complex are developed. The crystal structures of Norrin and its complex with Fz4-CRD, plus complex bound with the heparin mimic sucrose octasulphate, and unliganded structures of Fz4-CRD are presented. These structural data together with biophysical and cellular assays not only reveal the Fz4 and Lrp5/6 binding sites on distinct patches of the Norrin surface, but also indicate the HSPGs binding site on Norrin and Fz4-CRD as well as providing a framework to explain numerous disease-related mutations. Structural comparison with Xenopus Wnt8 in complex with mouse Fz8-CRD provides molecular insights for our understanding of ligand-receptor binding specificity and promiscuity, which has important implications for developing therapeutic strategies against Norrin dependent retinal disorders, and cancers caused by abnormal Wnt signaling.

The presence of blood-brain barrier (BBB) at the level of endothelial cells (ECs) that line the capillaries in the brain is a challenge while treating intracranial tumors or many neurological disorders since BBB could block the passage of many solvents or drugs entering from systemic circulation to brain parenchyma. Researchers are trying to understand how BBB is induced during development and maintained in adult life in order to develop new tools to enhance drug delivery across BBB. Many signaling pathway that are active during BBB development are unraveled through mainly reverse genetic approach and gene knock out studies. Wnt/β-catenin signaling well known for its role in organ development, morphogenesis and in cancer causation is also reported to be essential for BBB induction and maintenance in most vertebrates. However many target molecules or effectors of this pathway remain to be identified. Previously we have identified Sox17 a SoxF family transcription factor, as a downstream molecule of Wnt/β-catenin signaling. It plays a key role in arterial differentiation of the vasculature of different organs. We found that Sox17 is also expressed at high levels in brain ECs throughout embryo development and in the adult vasculature. EC-specific gain of function of β-catenin (GOF) increases Sox17 expression in BBB ECs and may induce ectopic expression of Sox17 also in the choroid plexus vasculature that lacks the BBB. We hypothesized therefore that Sox17 might be involved in BBB development and maintenance downstream to the Wnt pathway. In Sox17 null mice we analyzed different functional characteristics of BBB such as permeability control and specific markers expression. The absence of this transcription factor leads to increase in BBB permeability to high molecular weight molecules and marked increase in PLVAP, a protein inversely related to maturation of BBB. We also observed significant reduction in the β-catenin signaling itself by employing BAT-gal reporter in Sox17 15 null background. In addition, many direct β-catenin targets like Axin2 and LEF1 were decreased upon Sox17 abrogation. These data suggested that Sox17 is not only a target of β-catenin signaling but also could maintain steady state, detectable levels of β-catenin signaling. We could rescue β-catenin signaling and correct BBB defects by either inhibiting the β-catenin destruction complex or by employing GOF β- catenin in Sox17 null background. Our study shows that Sox17 is an important regulator of BBB and it partially acts by sustaining β-catenin signaling. Sox17 expression and signaling may be important in pathological conditions like intracranial tumors and modulation of its activity could have clinical implications and therapeutic benefits.

El edema macular diabético (DME) es una de las primeras causas de disminución de la agudeza visual en pacientes con retinopatía diabética (DR). La rotura de la barrera hematorretiniana (BHR) debido a la disrupción de las tight junctions (TJ) es el principal factor causante del DME. Además, el incremento de las citoquinas proinflamatorias como la IL-1β juega un papel importante en la patogénesis del DME y la retinopatía diabética proliferativa (PDR). Mientras que la alteración de las proteínas implicadas en la disrupción de las TJ de la BHR interna ha sido ampliamente estudiada, existe poca información sobre este proceso en la BHR externa. En el estudio FIELD sobre DR, el tratamiento con fenofibrato redujo en un 30% la necesidad de tratamiento con láser en pacientes diabéticos. Además, en el estudio ACCORD-Eye se observó una reducción del 40% en la progresión de la DR en el grupo de pacientes diabéticos a los que se les administró fenofibrato. Sin embargo, los mecanismos a través de los cuales el fenofibrato ejerce sus efectos beneficiosos en pacientes con DR se desconocen. En base a lo explicado anteriormente, los objetivos de esta tesis han sido: 1. Estudiar el efecto de la hiperglicemia sobre la funcionalidad de la BHR externa y la expresión de las proteínas de TJ en una línea celular humana de RPE (ARPE-19). 2. Estudiar el efecto protector del ácido fenofíbrico (el metabolito activo del fenofibrato) sobre la funcionalidad y la expresión de las proteínas de TJ en células ARPE-19 cultivadas bajo diferentes concentraciones de glucosa, con o sin IL-1β. Además se evaluó la implicación de la vía de la AMPK en la hiperpermeabilidad inducida por la IL-1β y el efecto del ácido fenofíbrico sobre la activación de la AMPK. En nuestro primer trabajo observamos una disminución de la permeabilidad y un aumento de los niveles de claudina-1 en condiciones de hiperglicemia. Sin embargo, después de bloquear la expresión de claudina-1 no se detectaron cambios en la resistencia transepitelial (TER) ni en la permeabilidad. Estos resultados sugieren que la sobreexpresión de claudina-1 inducida por la hiperglicemia no está relacionada con los mecanismos a través de los cuales la glucosa aumenta la función oclusiva de las TJ en las células ARPE-19. Se demuestra que es la combinación de los diferentes factores presentes en el medio diabético, y no la hiperglicemia por sí sola, los que producen una alteración de la funcionalidad de la BHR externa y la disrupción de las TJ. En el segundo trabajo utilizamos una combinación de IL-1β + hiperglicemia con el fin de simular en las células ARPE-19 la lesión producida por el medio diabético. El tratamiento con ácido fenofíbrico redujo (a 25 μmol/l) o previno (a 100 μmol/l) de manera dosis dependiente, el aumento de permeabilidad y la desorganización de las TJ inducida por la IL-1β + 25 mmol/l D-Glucosa, hecho que se asoció a una mayor preservación de la función de sellado de las células del RPE. Este efecto no está relacionado con cambios en la expresión de las proteínas de TJ y sugiere que para el correcto funcionamiento de la BHR externa es más importante una adecuada distribución y estructura de las TJ que un aumento en el contenido neto de éstas. Además, demostramos que el efecto protector del ácido fenofíbrico se debe a su capacidad para suprimir la activación de la AMPK inducida por la IL-1β, previniendo así la hiperpermeabilidad inducida por el medio diabético. Nuestros resultados contribuyen significativamente a aumentar el conocimiento sobre los mecanismos de acción a través de los cuales el fenofibrato ejerce sus efectos beneficiosos en el desarrollo y la progresión del DME.
Diabetic macular edema (DME) is one of the primary causes of poor visual acuity in patients with diabetic retinopathy (DR). The breakdown of the blood-retinal barrier (BRB) due to the disruption of tight junctions (TJ) is the main factor accounting for DME. In addition, the increase of proinflammatory cytokines such as IL-1β plays a key role in the pathogenesis of DME and proliferative diabetic retinopathy (PDR). While extensive work has been carried out to identify the factors involved in the disruption of TJ of the inner BRB, the mechanisms implicated in the outer BRB regulation have been poorly explored. In the FIELD study on DR, treatment with fenofibrate reduced the need for laser treatment for DME and PDR by 30% in diabetic patients. In addition, the ACCORD Eye Study showed a 40% reduction in DR progression in the group of patients receiving fenofibrate. However, the mechanisms by which fenofibrate exerts its beneficial effects in DR remain to be elucidated. With all this background, the main points of this thesis were: 1. To study the effects of high glucose concentration on the outer BRB function and the expression of TJ proteins in a human RPE cell line (ARPE-19). 2. To study the effect of fenofibric acid (the active metabolite of fenofibrate) on the barrier function and the levels of TJ proteins in ARPE-19 cells cultured under different glucose concentrations with and without IL-1β. In addition, we evaluated the role of AMPK in mediating the hyperpermeability induced by IL-1β and the effect of fenofibric acid on AMPK activation. Our first study showed a reduction of permeability and an increment of claudin-1 levels under hyperglycemic conditions. However, after blocking claudin-1 expression no changes in transepithelial electrical resistance (TER) and permeability were observed. These results suggest that the overexpression of claudin-1 induced by high glucose concentration is not involved in the mechanisms by which glucose increases the TJ sealing function in ARPE-19 cells. In addition, our findings demonstrate that hyperglycemia per se is not the only cause responsible for the breakdown of the outer BRB in patients with DR. It is the combination of different factors present in the diabetic milieu with hyperglycemia, the one responsible for the impairment of the outer BRB and TJ disruption. In our second study we used a combination of IL-1β + hyperglycemia to mimic the diabetic milieu and induce the breakdown of ARPE-19 monolayer. Treatment with fenofibric acid was able to reduce (at 25 μmol/l) or prevent (at 100 μmol/l) in a dose-dependent manner, the increment of permeability and the disorganization of TJ induced by IL-1β + 25 mmol/l D-Glucose, which was associated with the preservation of the sealing function of RPE cells. This effect was unrelated to changes in the content of TJ proteins, suggesting that an ordered distribution, rather than a crude assortment, of TJ proteins is essential for the efficient functioning of the outer BRB barrier. In addition, we demonstrated that the protective effect of fenofibric acid was mainly mediated by its ability to lower AMPK activation induced by IL-1β, which prevented the hypermermeability induced by the diabetic milieu. These findings contribute significantly to increasing our knowledge about the mechanisms involved in the beneficial effects of fenofibrate on the development and progression of DME.

Relatório de Estágio do Mestrado Integrado em Ciências Farmacêuticas apresentado à Faculdade de Farmácia
O aumento da prevalência de doenças relacionadas com o envelhecimento da população, tal como doenças neurodegenerativas e doenças degenerativas da retina levou a um crescente interesse em otimizar o tratamento destas.No entanto, a entrega de fármacos no cérebro e no olho não é assim tão linear, devido à presença de barreiras que protegem estes órgãos.Tornou-se, portanto, necessário desenvolver estratégias para a formulação de fármacos eficazes no tratamento destas doenças.A utilização de nanotecnologia para o tratamento de doenças tem crescido nos últimos anos. O uso de nanopartículas tem provado um enorme potencial em melhorar a farmacocinética do fármaco e a sua veiculação.Devido às suas características, o uso de nanopartículas para o tratamento de doenças do sistema nervoso central e da retina tem sido imensamente estudado. Esta revisão pretende abordar as dificuldades em ultrapassar as barreiras que protegem o cérebro e o olho, bem como os vários estudos de desenvolvimento de nanopartículas com o objetivo de formular estratégias terapêuticas para o tratamento de doenças neurodegenerativas e doenças degenerativas da retina. Ao longo desta revisão é feita menção a varios estudos publicados em que são abordadas diferentes nanopartículas com potencial para atuar nos alvos destas doenças. No presente trabalho encontra-se os relatórios referentes aos estágios realizados em farmácia comunitária e indústria farmacêutica, onde é feita uma abordagem SWOT (Strengths, Weaknesses, Opportunities and Threats) para discutir os aspetos mais pertinentes dos estágios.
The increasing prevalence of diseases related to an aging population, such as neurodegenerative diseases and retinal degenerative diseases, has led to a growing interest in optimizing their treatment.However, drug delivery to the brain and eye is not that easy, due to the presence of barriers that protect these organs. Therefore, it became necessary to develop strategies for the formulation of effective drugs in the treatment of these diseases.The use of nanotechnology to treat diseases has grown interest in recent years. The use of nanoparticles has proven an enormous potential in improving drug pharmacokinetics and its delivery.Due to their characteristics, the use of nanoparticles for the treatment of diseases of the central nervous system and the retina has been extensively studied.This review intends to address the difficulties in overcoming the barriers that protect the brain and the eye, as well as the various studies on the development of nanoparticles with the aim to formulate therapeutic strategies for the treatment of neurodegenerative diseases and retinal degenerative diseases.Throughout this review, mention is made of several published studies in which different nanoparticles with potential to act on the targets of these diseases are addressed.In this work, the reports refering to the internships in communitary pharmacy and pharmaceutical industry are discussed, to evaluate the relevant aspects refering to the internships is used a SWOT (Strengths, Weaknesses, Opportunities and Threats) approach.

Relatório de Estágio do Mestrado Integrado em Ciências Farmacêuticas apresentado à Faculdade de Farmácia
O aumento da prevalência de doenças relacionadas com o envelhecimento da população, tal como doenças neurodegenerativas e doenças degenerativas da retina levou a um crescente interesse em otimizar o tratamento destas.No entanto, a entrega de fármacos no cérebro e no olho não é assim tão linear, devido à presença de barreiras que protegem estes órgãos.Tornou-se, portanto, necessário desenvolver estratégias para a formulação de fármacos eficazes no tratamento destas doenças.A utilização de nanotecnologia para o tratamento de doenças tem crescido nos últimos anos. O uso de nanopartículas tem provado um enorme potencial em melhorar a farmacocinética do fármaco e a sua veiculação.Devido às suas características, o uso de nanopartículas para o tratamento de doenças do sistema nervoso central e da retina tem sido imensamente estudado. Esta revisão pretende abordar as dificuldades em ultrapassar as barreiras que protegem o cérebro e o olho, bem como os vários estudos de desenvolvimento de nanopartículas com o objetivo de formular estratégias terapêuticas para o tratamento de doenças neurodegenerativas e doenças degenerativas da retina. Ao longo desta revisão é feita menção a varios estudos publicados em que são abordadas diferentes nanopartículas com potencial para atuar nos alvos destas doenças. No presente trabalho encontra-se os relatórios referentes aos estágios realizados em farmácia comunitária e indústria farmacêutica, onde é feita uma abordagem SWOT (Strengths, Weaknesses, Opportunities and Threats) para discutir os aspetos mais pertinentes dos estágios.
The increasing prevalence of diseases related to an aging population, such as neurodegenerative diseases and retinal degenerative diseases, has led to a growing interest in optimizing their treatment.However, drug delivery to the brain and eye is not that easy, due to the presence of barriers that protect these organs. Therefore, it became necessary to develop strategies for the formulation of effective drugs in the treatment of these diseases.The use of nanotechnology to treat diseases has grown interest in recent years. The use of nanoparticles has proven an enormous potential in improving drug pharmacokinetics and its delivery.Due to their characteristics, the use of nanoparticles for the treatment of diseases of the central nervous system and the retina has been extensively studied.This review intends to address the difficulties in overcoming the barriers that protect the brain and the eye, as well as the various studies on the development of nanoparticles with the aim to formulate therapeutic strategies for the treatment of neurodegenerative diseases and retinal degenerative diseases.Throughout this review, mention is made of several published studies in which different nanoparticles with potential to act on the targets of these diseases are addressed.In this work, the reports refering to the internships in communitary pharmacy and pharmaceutical industry are discussed, to evaluate the relevant aspects refering to the internships is used a SWOT (Strengths, Weaknesses, Opportunities and Threats) approach.

Tese de doutoramento em Engenharia Biomédica, apresentada à Faculdade de Ciências e Tecnologia da Universidade de Coimbra
A retinopatia diabética é uma das complicações associadas à diabetes e uma das principais causas de perda de visão na população em idade ativa em todo o mundo. No entanto, os tratamentos disponíveis para esta doença são escassos, invasivos, não eficazes para todos os doentes, apresentam alguns riscos adversos e são normalmente administrados nas fases tardias da retinopatia diabética, quando a visão já se encontra significativamente comprometida. Desta forma, novas estratégias terapêuticas instituídas nos estadios iniciais da retinopatia diabética poderão ser efetivas para retardar ou atenuar a progressão da doença, prevenindo a perda de visão. Terapias antidiabéticas baseadas nas incretinas, os inibidores da dipeptidil-peptidase-IV (DPP-IV) e os agonistas do recetor do peptídeo semelhante ao glucagão (GLP-1), melhoram o controlo glicémico em doentes com diabetes do tipo 2 através do aumento da secreção de insulina. Estudos anteriores do nosso laboratório demonstraram que a sitagliptina (um inibidor da DPP-IV) previne o stresse nitrosativo, a inflamação e a apoptose das células da retina, exercendo ainda efeitos protetores ao nível da barreira hemato-retiniana (BHR) e melhorando o controlo glicémico num modelo animal de diabetes do tipo 2. Até ao início deste trabalho a informação existente sobre os efeitos diretos das terapias baseadas na modulação das incretinas na retina diabética era escassa. Nesse contexto, o principal objetivo da primeira parte deste trabalho foi investigar os efeitos da sitagliptina, independentes do seu efeito insulinotrópico, na retina diabética. Para tal foi usado um modelo animal de diabetes do tipo 1 induzida por estreptozotocina. O tratamento com sitagliptina (5 mg/kg/dia) foi iniciado após duas semanas de diabetes e prolongou-se por mais duas semanas, não se tendo observado qualquer efeito nos níveis de glicemia ou de insulina nos animais diabéticos. A sitagliptina preveniu o aumento da atividade e dos níveis da enzima DPP-IV no soro e na retina, induzido pela diabetes. A sitagliptina preveniu o aumento da permeabilidade da BHR, bem como alterações das proteínas das junções oclusivas induzidas pela diabetes. Além disso, a sitagliptina preveniu o aumento dos níveis da citocina pró-inflamatória interleucina-1β e da molécula de adesão intercelular-1 nas retinas diabéticas. A sitagliptina preveniu ainda o aumento dos níveis da proteína pró-apoptótica Bax, o aumento do número de células positivas a TUNEL e a degenerescência de células neuronais nas retinas diabéticas. Os resultados obtidos demonstram que a sitagliptina previne eficazmente a rutura da BHR e algumas das alterações que contribuem para a disfunção da BHR nas fases iniciais da retinopatia diabética, tais como a inflamação e a morte celular. Uma vez que o mecanismo de ação proposto para a sitagliptina baseia-se no aumento dos níveis endógenos do GLP-1, pretendeu-se esclarecer se os efeitos benéficos da sitagliptina nas retinas diabéticas foram, de alguma forma, mediados pelo GLP-1. Com a finalidade de esclarecer essa questão, na segunda parte deste estudo, foram avaliados os potenciais efeitos benéficos de um agonista do recetor do GLP-1, a Exendina-4 (Ex-4), contra a rutura da BHR e inflamação. Foi usado um modelo animal de isquemia-reperfusão (IR) que apresenta alterações semelhantes às observadas na retinopatia diabética. A IR ocular foi induzida em ratos através do aumento da pressão intra-ocular durante 45 min seguido de 48 h de reperfusão. O tratamento com Ex-4 (10 µg/kg) (duas administrações iniciais antes da isquemia e depois a cada 12 h durante o período de reperfusão) preveniu o aumento da permeabilidade vascular da retina induzido por IR. No entanto, estudos in vitro utilizando culturas primárias de células endoteliais da retina mostraram que o Ex-4 não preveniu o aumento da permeabilidade induzido por agentes permeabilizantes. O Ex-4 inibiu ainda a expressão de vários genes inflamatórios nas retinas de ratos sujeitos a IR. Em cultura de células da microglia, o Ex-4 reduziu a resposta inflamatória ao lipopolissacarídeo, muito provavelmente devido à inibição do fator nuclear-kB e à ativação do CREB. Além disso, a inibição da ativação das células da microglia foi capaz de proteger contra a disfunção da barreira de células endoteliais. Este estudo sugere que o Ex-4 previne a rutura da BHR e a inflamação induzidas por IR através da inibição da produção de citocinas inflamatórias pelas células da microglia. Em resumo, estes resultados oferecem uma nova perspetiva sobre os efeitos protetores das terapias baseadas nas incretinas contra a disfunção da BHR, inflamação e apoptose das células da retina. Com este trabalho foi ainda possível desvendar um novo mecanismo que ajuda a explicar os benefícios destas terapias na retinopatia diabética, isto é, a modulação da resposta pró-inflamatória nas células da microglia. Este trabalho contribui ainda para ajudar a criar condições para a utilização destas terapias não só para a retinopatia diabética, mas como para outras patologias oculares caraterizadas por um aumento da permeabilidade vascular e inflamação.
Diabetic retinopathy, a major complication of diabetes, is a leading cause of vision loss and blindness in working-age population worldwide. However, the treatments available are scarce, invasive, inefficient to a high percentage of patients, present some adverse risks, and are typically targeted for the later stages of diabetic retinopathy, when vision has already been significantly affected. Therefore, novel therapeutic approaches targeting the early stages of diabetic retinopathy could slow down the progression of the disease, being protective against vision loss. Incretin-based antidiabetic agents, the dipeptidyl peptidase-IV (DPP-IV) inhibitors and glucagon-like peptide-1 (GLP-1) receptor agonists, improve glycemic control by increasing insulin secretion in patients with type 2 diabetes. We have previously demonstrated that the DPP-IV inhibitor sitagliptin prevents nitrosative stress, inflammation and apoptosis in retinal cells and exerts beneficial effects on the blood-retinal barrier (BRB), while improving glycemic control in an animal model of type 2 diabetes. When this work started, the available information about the direct effects of incretin-based therapies in the diabetic retina was scarce. In this context, the main purpose of the first part of this work was to investigate the insulinotropic-independent effects of sitagliptin in the diabetic retina, by using an experimental model of streptozotocin-induced type 1 diabetes. Treatment with sitagliptin (5 mg/kg/day) started after two weeks of diabetes and lasted for two additional weeks. Sitagliptin treatment did not affect glycemia or insulin levels in diabetic animals. Sitagliptin prevented the diabetes-induced increase in both DPP-IV activity and DPP-IV levels in serum and retina. Sitagliptin prevented the increase of BRB permeability as well as alterations in tight junction proteins induced by diabetes. Furthermore, sitagliptin was able to prevent the upregulation of the pro-inflammatory cytokine interleukin-1β and the intercellular adhesion molecule-1 in the diabetic retinas. Sitagliptin also prevented the upregulation of the pro-apoptotic protein Bax, the increase in the number of TUNEL-positive cells and degenerating neuronal cells in the retina of diabetic animals. Altogether, these data show that sitagliptin is efficient in preventing BRB breakdown and associated features, such as inflammation and cell death, which are known to underlie and contribute to BRB dysfunction in the early stages of diabetic retinopathy. Since the proposed mechanism of action of sitagliptin lies in increasing the endogenous levels of GLP-1, we further aimed to elucidate whether the beneficial effects of sitagliptin in the diabetic retinas were somehow mediated by GLP-1. To clarify this issue, in the second part of this study, the potential beneficial effects of a GLP-1 receptor agonist, Exendin-4 (Ex-4), against BRB breakdown and inflammation were evaluated. For this purpose, an animal model of ischemia-reperfusion (IR) injury, presenting alterations known to occur in diabetic retinopathy, was used. Ocular IR injury was induced in rats by increasing the intraocular pressure for 45 min followed by 48 h of reperfusion. Ex-4 (10 µg/kg) treatment (two initial administrations prior to ischemia and then every 12 h for the next 48 h during the reperfusion period) was able to prevent the increase in retinal vascular permeability triggered by IR injury. However, in vitro studies, using primary cultures of retinal endothelial cells, showed that Ex-4 fails to prevent the increase in permeability triggered by permeabilizing agents. Ex-4 also inhibited the expression of IR-responsive inflammatory genes in the rat retinas. In cultured microglial cells, Ex-4 decreased the inflammatory response to lipopolysaccharide, most likely due to inhibition of nuclear factor-κB and CREB activation. Furthermore, inhibition of microglia activation was able to protect against endothelial cell barrier dysfunction in vitro. This study suggests that Ex-4 can prevent IR injury induced-BRB breakdown and inflammation through inhibition of inflammatory cytokine production by activated microglia. The results presented herein provide a new insight into the protective effects of incretin-based therapies against BRB breakdown, inflammation and retinal cell apoptosis. Additionally, this work reveals a new mechanism that helps explaining the benefits of the incretin-based therapies in diabetic retinopathy, i.e., : the modulation of the pro-inflammatory response in microglial cells. Moreover, this work helps to envisage these therapies for diabetic retinopathy, as well as for other ocular pathologies characterized by increased vascular permeability and inflammation.
FCT - SFRH/BD/103936/2014

Dissertação de Mestrado em Bioquímica apresentada à Faculdade de Ciências e Tecnologia
A retinopatia diabética é uma das principais causas de perda de visão, incluindo cegueira, em indivíduos diabéticos em idade ativa, nos países desenvolvidos. Atualmente, os tratamentos disponíveis para a retinopatia diabética são direcionados para as fases mais avançadas da doença, em que a visão já se encontra comprometida. Estes tratamentos são invasivos, não sendo eficazes para todos os doentes, e possuem vários riscos associados, nomeadamente o de infeções como a endoftalmite infeciosa e o risco de descolamento da retina. Além disso, a adoção destes tratamentos permite apenas retardar a progressão da doença e não a sua cura. Neste sentido é importante que sejam desenvolvidas e implementadas estratégias terapêuticas direcionadas para as fases mais iniciais da retinopatia diabética, em que os processos sejam reversíveis e que seja possível prevenir a perda de visão. As terapias antidiabéticas baseadas nas incretinas são divididas em duas classes: os inibidores da enzima dipeptidil-peptidase-4 (DPP-4) e os agonistas do recetor do peptídeo semelhante ao glucagão (GLP-1R). Ambas visam o aumento do tempo de meia-vida da hormona incretina GLP-1 em circulação (composto importante para a manutenção da homeostase da glicose). A administração destes fármacos melhora o controlo glicémico através do aumento de produção e secreção de insulina em doentes com diabetes tipo 2. Estudos anteriores do nosso grupo mostraram que a sitagliptina, que é um inibidor da DPP-4, tem efeitos pleiotrópicos, prevenindo a inflamação e a apoptose das células da retina e exercendo um efeito benéfico na integridade da barreira hemato-retiniana (BHR) em modelos animais de diabetes, tanto do tipo 2 (DMT2) como do tipo 1 (DMT1). Ainda, estudos mais recentes do grupo mostraram que a Exendina-4 (Ex-4), um agonista do GLP-1R, previne a rutura da BHR e a inflamação na retina, através da inibição de citocinas inflamatórias pelas células da microglia, num modelo animal de lesão retiniana por isquémia-reperfusão (IR), que mimetiza as principais características da retinopatia diabética (neurodegenerescência, neuroinflamação e aumento da permeabilidade da BHR). Este trabalho teve como objetivo avaliar os potenciais efeitos benéficos dos agonistas do GLP-1R na retina diabética, por um mecanismo independente do aumento da produção e secreção de insulina. Para tal utilizou-se um modelo animal de diabetes do tipo 1, com diabetes induzida por streptozotocina e estudou-se o efeito de dois agonistas do GLP-1R, o liraglutido e a Ex-4. Mais especificamente, avaliou-se o potencial efeito protetor destes agonistas na rutura da BHR, bem como de que forma estes compostos modulam a reatividade das células da microglia. Este aspeto foi avaliado em termos da sua capacidade migratória nas camadas da retina e das suas alterações morfológicas.O tratamento com liraglutido (200 µg/Kg/2x dia) foi iniciado duas semanas após a indução da diabetes e prolongou-se por mais duas semanas, não se tendo verificado nenhuma alteração no peso e nos níveis de glicémica e hemoglobina glicosilada dos animais diabéticos tratados com o fármaco, comparativamente com os animais diabéticos. O liraglutido preveniu o aumento da permeabilidade da BHR, induzido pela diabetes, bem como as alterações nos níveis das proteínas das junções oclusivas (claudina-5 e ocludina). Além disso, verificou-se que este agonista do GLP-1R modula a capacidade migratória das células da microglia na retina, induzida pela diabetes. O tratamento com a Ex-4 (10 µg/Kg/2x dia) foi iniciado uma semana após a indução da diabetes, tendo-se prolongado por mais três semanas. A Ex-4 não teve qualquer efeito nos níveis de insulina dos animais diabéticos. Os resultados obtidos mostraram que a Ex-4 previne a rutura da BHR e que este fármaco tem um efeito anti-inflamatório nas retinas de ratos diabéticos, através da inibição de citocinas pró-inflamatórias, prevenindo o aumento dos níveis da molécula de adesão molecular (ICAM-1). A Ex-4 mostrou, ainda, que tem a capacidade não só de prevenir a alteração da distribuição das células da microglia, nas diferentes camadas da retina, como também previne as alterações morfológicas destas células, induzidas pela diabetes.Na sequência de um estudo anterior do grupo, este trabalho vem reforçar a ideia de que os agonistas do GLP-1R, liraglutido e Ex-4, conseguem prevenir a rutura da BHR por diminuição do estado pró-inflamatório via inibição da reatividade das células da microglia da retina.
Diabetic retinopathy is one of the leading causes of vision loss, in developing-age diabetic individuals, in developed countries. Currently, the treatments available for diabetic retinopathy are against to the advanced stages of the disease, where vision is already compromised. These treatments are invasive and not effective for all patients and have several associated risks, namely infections such as endophthalmitis, and the risk of retinal detachment. In addition, they only delay the progression of the disease, being not able to healing it. In this sense, it is important to develop new therapeutic strategies directed to the earliest stages of diabetic retinopathy, in which the processes are reversible, and could prevent the loss of vision.Incretin-based antidiabetic therapies are divided into two classes: dipeptidyl peptidase-4 inhibitors (DPP-4) and glucagon-like peptide receptor agonists (GLP-1R). Both aim to increase the half-life of the circulating incretin hormone GLP-1 (which is an important compound for the maintenance of glucose homeostasis). Administration of these drugs improves glycemic control by increasing insulin production and secretion in patients with type 2 diabetes. Previous studies in our group have shown that sitagliptin, which is a DPP-4 inhibitor, has pleiotropic effects, preventing inflammation and apoptosis of retinal cells and exerting a beneficial effect on the integrity of the blood-retinal barrier (BRB) in animal models type 2 (T2DM) and type 1 (T1DM) of diabetes. Furthermore, recent studies of the group have shown that Exendin-4 (Ex-4), a GLP-1R agonist, prevents BRB rupture and inflammation in the retina by inhibiting inflammatory cytokines by microglial cells in an ischemia-reperfusion animal model (IR), which mimics the main features of diabetic retinopathy (neurodegeneration, neuroinflammation and increased permeability of BRB).This work aimed to evaluate the potential beneficial effects of GLP-1R agonists on the diabetic retina, by an independent mechanism of increased insulin production and secretion. For this purpose, an animal model of type 1 diabetes with streptozotocin-induced diabetes was used and the effect of two GLP-1R agonists, liraglutide and Ex-4, was evaluated. More specifically, the potential protective effect of these agonists on the breakdown of BHR was evaluated, as well as how these compounds modulate the reactivity of microglial cells. This aspect was evaluated in terms of its migratory capacity in the different layers of the retina as well as morphological changes.Treatment with liraglutide (200 μg/kg/2x day) started two weeks after the induction of diabetes and lasted for two more weeks. No changes were observed between diabetic and diabetic treated animals, in terms of body weight, glycemic levels and glycosylated hemoglobin levels. Liraglutide prevented the increase of BHR permeability and changes in occlusive junction protein levels (claudin-5 and occludin), in diabetic animals. In addition, it has been found that this GLP-1R agonist modulates the migratory capacity of the microglial cells in the retina, induced by diabetes.Treatment with Ex-4 (10 μg/kg/ 2x day) was started one week after the induction of diabetes and lasted for another three weeks. Ex-4 had no effect on the insulin levels of diabetic animals. The results showed that Ex-4 prevents the breakdown of BRB and has an anti-inflammatory effect in the retinas of diabetic rats, by inhibiting pro-inflammatory cytokines and preventing the increase of levels of the molecular adhesion molecule (ICAM-1). Ex-4 also has the ability not only to prevent alteration of the distribution of microglia cells in the different layers of the retina, but also to prevent the morphological alterations of these cells, induced by diabetes.Following an earlier study by the group, this work reinforces the idea that GLP-1R agonists, liraglutide and Ex-4, can prevent BRB rupture by decreasing the pro-inflammatory state via inhibition of cell reactivity microglia of the retina.

Diabetic Retinopathy (DR), characterized by retinal microvasculature impairment, is frequent a consequence of Diabetes mellitus (DM). DR is the leading cause of blindness among working-age adults worldwide, and its prevalence is expected to rise along with DM’s number of cases. This disease affects the patients’ life and represents a major public health burden. Current therapies are very invasive, only useful in advanced stages of DR and effective only in half of the cases. Therefore, new therapies that can overcome these limitations, must be developed. The key factor for DR development is DM-related chronic inflammation, whose hallmarks include increased recruitment and transmigration of leukocytes into the affected tissues. In early-stage DR, these events cause loss of integrity of the Blood-Retinal Barrier, and consequent ophthalmoscopically detected hallmarks. Thus, retinal inflammation appears to be a potential target for early-stage DR treatment. Previous studies with Kyotorphin (KTP) and its derivatives, revealed that this endogenous dipeptide presents anti-inflammatory activity when administered topically to mice. The aim of this work was to determine if the anti-inflammatory activity of KTP persisted with local (intravitreal injection) and systemic (intraperitoneal injection) administration, while reverting the retinal vascular lesions of DR. The experimental model used in this work (DM development for 3 months), did not allow the observation of significant differences between the diabetic and control mice, concerning inflammatory biomarkers (leukocytes’ dynamics using intravital microscopy and IL-6 and TNF- quantification using ELISA). Consequently, the effect of KTP in the systemic inflammation could not be successfully tested. Nonetheless, according to the retinal ex-vivo analysis, the formation of DR’s hallmarks in the diabetic mice (Acellular Capillaries and Pericyte Loss), was reverted by KTP. For future work, optimization of the DM-development period and the increase of sample size are the priority.