O hormônio melatonina produzido pela glândula pineal no período de escuro, participa na regulação circadiana de processos, fisiológicos e fisiopatológicos envolvendo vasos sanguíneos. Alguns destes estudos sugerem que as células endoteliais, que revestem os vasos sanguíneos são alvo para a melatonina circulante e medeiam a regulação do tônus vascular, em condições fisiológicas, e da interação neutrófilo-endotélio, em resposta a um estímulo injuriante. O óxido nítrico produzido pelas células endoteliais é um dos responsáveis por grande parte dos eventos vasculares, e a melatonina inibe a produção de óxido nítrico em diversos modelos. O objetivo deste estudo foi verificar o efeito da melatonina na produção de óxido nítrico induzido por bradicinina em células endoteliais em cultura. Para tanto, utilizamos uma técnica de cultura primária de células endoteliais de rato e através de um marcador fluorescente de óxido nítrico intracelular, mensuramos a fluorescência em microscópio confocal. Foi verificado que a melatonina e seu precursor N-acetilserotonina inibem a produção de óxido nítrico induzido por bradicinina e este efeito não ocorre pela inibição do aumento de cálcio que induz a produção de óxido nítrico. O análogo de receptores MT2 (4P-PDOT) e MT3 (5-MCA-NAT) não provocaram qualquer alteração sobre o aumento de óxido nítrico induzido por bradicinina, e a utilização do antagonista de receptores MT1 e MT2 (luzindol) não reverteu o efeito inibitório da melatonina. Portanto, nossos dados indicam que o efeito da melatonina sobre a atividade da NOS constitutiva não é mediado por receptores de membrana. Considerando que a melatonina é capaz de ligar-se à calmodulina, inibindo desta maneira a atividade da NOS endotelial constitutiva, poderíamos sugerir que este seria o mecanismo de ação. No entanto, é preciso ressaltar que tal atividade não é comprovada para a N-acetilserotonina, assim, apesar de ser este um possível mecanismo de ação, há a necessidade de demonstrar que a N-acetilserotonina está se ligando a calmodulina extraída de células endoteliais. Em resumo, neste trabalho mostramos que a melatonina em concentrações compatíveis com o pico noturno encontrado na circulação, pode modular eventos vasculares no organismo, através da inibição da produção de óxido nítrico em células endoteliais induzida por bradicinina. / Melatonin, the hormone synthesized by the pineal gland at night, signalizes darkness and modulates, in a circadian basis, blood vessels activity. Previous studies suggest that endothelial cells are the target for circulating melatonin and mediate changes in vascular tone and leukocyte-endothelial adherence properties. Melatonin effects can be mediated by several pathways, such as G protein-coupled receptors (MT1 and MT2 receptors), a putative membrane receptor, most probably an enzyme-binding site (MT3 receptor), and several intracellular mechanisms, including calmodulin binding and inhibition of constitutive and induced nitric oxide synthase. The aim of the present study was to characterize melatonin effect on the production of nitric oxide by bradykinin-stimulated endothelial cells in culture. Nitric oxide production was measured in real time at cellular level by detecting fluorescent stimulation of the probe DAF by confocal microscopy. After determining the ideal conditions for recording cumulative dose-response curves for bradykinin (1 – 100 nM) the effect of pre-incubated (1 min) melatonin and analogs was evaluated. Melatonin and its precursor, N-acetylserotonin, but not the selective ligands for receptors MT2 (4P-PDOT) and MT3 (5-MCA-NAT) receptors inhibited bradykinin-stimulated nitric oxide production. This effect was not blocked by the classical antagonist of MT1 and MT2 receptors, luzindol; excluding therefore the participation of membrane receptors. Taking into account that melatonin inhibits calmodulin activation of several enzymes, including constitutive nitric oxide synthase in brain and cerebellum, it could be suggested a similar mechanism for endothelial cells. However, this hypothesis is discussed taking into account that N-acetylserotonin was shown to do not bind neural cells calmodulin. In addition, here we discuss the relevance of the present finding according to physiological and physiopathological roles of endothelial nitridergic system. This analysis point melatonin modulation of constitutive nitric oxide synthase activity as a putative mechanism for explaining melatonin control of vascular tone.

A melatonina é produzida pela glândula pineal somente durante o escuro e atinge rapidamente a circulação, além disso, outros tecidos e células são capazes de produzir melatonina. As células endoteliais, devido a sua localização, são excelentes alvos para as ações da melatonina. O entendimento dos mecanismos de ação pelos quais a melatonina desenvolve seus efeitos sobre as células endoteliais, possibilitaria o uso desta indolamina e de seus análogos como uma importante ferramenta farmacológica. No presente trabalho, demonstramos que a melatonina em concentrações compatíveis com as encontradas na circulação durante o pico noturno de produção pela pineal, atua sobre as células endoteliais inibindo a produção de NO proveniente da enzima constitutiva (eNOS), enquanto altas concentrações de melatonina, que podem ser atingidas por exemplo pela produção por células imunocompetentes ativadas, inibem a produção induzida de NO mediada pela iNOS. A melatonina (1 nM) inibe a produção constitutiva de NO induzida por agonistas que atuam através da ativação de receptores acoplados à proteína G (histamina, carbacol e ATP/P2Y), e este efeito deve-se à inibição do aumento de [Ca2+]i por liberação de estoques intracelulares, sendo independente da ativação de receptores de melatonina. A melatonina inibe os efeitos decorrentes da produção de NO induzida por bradicinina como a produção de GMPc por células endoteliais e a vasodilatação de arteríolas \"in vivo\". A melatonina inibe a produção de NO induzida por LPS também de maneira independente da ativação de seus receptores, porém, em concentrações muito maiores (1-10 µM) do que a necessária para inibir a produção constitutiva. Estes efeitos devem-se à inibição da expressão da enzima iNOS por impedir a translocação do NF-kB ao núcleo. A vasodilatação de aortas induzida por LPS também é inibida por melatonina. Podemos concluir até o momento que as células endoteliais, devido a sua localização, são excelentes sensores para as ações da melatonina e podem auxiliar no melhor entendimento do conceito \"eixo imune-pineal\". Os estudos sobre os mecanismos pelos quais a melatonina atua em condições fisiológicas e fisiopatológicas são essenciais para se conhecer o potencial terapêutico da melatonina. / Melatonin, the darkness hormone, produced at night by the pineal gland, is also synthesized in a non-rhythmic manner by other cells. Pineal and extra-pineal melatonin reaches endothelial layer, and the understanding of its mechanism of action will improve the possibilities of using this indolamine and derivates as pharmacological tools. Here we showed that melatonin, in concentrations compatible to nocturnal melatonin surge impairs the activity of eNOS, while much higher concentrations, which can be attained by activated immune competent cells, impair the induction of iNOS synthesis. As a consequence of inhibiting eNOS we showed that melatonin inhibits vasodilation of the microcirculation induced by bradykinin. The inhibitory effect of melatonin is observed only when eNOS is activated by triggering G protein-coupled receptors (bradykinin B2, muscarinic and P2Y purine receptors). Activation of eNOS by calcium-channel operated receptors (P2X) is not blocked by melatonin. Inhibition of the transcription of iNOS results in inhibition of the LPS-induced vasodilation of rat aorta. As a matter of fact, here we show that LPS effect is dependent on the endothelial layer. The mechanism of action of melatonin in inhibiting iNOS transcription is due to block of the NF-kB pathway. Our work contributed to unravel the role of endothelium cells as targets for melatonin and as a key player in the \"immune-pineal axis\". The understanding of the concentrations ranges reached by endogenous production, i.e., the discrimination between the levels achieved during physiological and physiopathological responses, are essential for using these substances as analogous therapeutical tools.

Tumour angiogenesis is a hallmark of cancer. Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase involved in endothelial cells (ECs) survival, proliferation and migration. FAK has several tyrosine phosphorylation sites thought to be involved in FAK function but the requirement of phosphorylation of these residues in vivo is unknown. We have generated mice where endogenous FAK is deleted simultaneously with the expression of nonphosphorylatable FAK-Y397F or FAK-Y861F mutated or wild type forms of FAK in adult endothelium in order to test this. My data show that EC-FAK-Y397FKI mice present with decreased tumour angiogenesis (in sygeneic B16F0, CMT19T and LLC) but impaired B16F0 and CMT19T tumour growth only, with increased tumour hypoxia. FAK-Y397F tumour endothelium is not perfusion, leakage or vascular maturation defective. This mutation affects VEGF-, PlGF- and bFGF-driven angiogenesis in vivo and VEGF+Ang2 administration is able to partially rescue this phenotype ex vivo. In contrast, endothelial FAK-Y861F mutation leads to an initial delay in B16F0 tumour angiogenesis, that subsequently resolves, and does not affect B16F0 tumour growth. LLC and CMT19T tumour growth and angiogenesis are not affected by the endothelial FAK-Y861F mutation; neither are tumour blood vessel perfusion, leakage, vascular maturation or tumour hypoxia. VEGF-, PlGFand bFGF-driven angiogenesis in vivo and ex vivo was not affected by the endothelial FAK-Y861F mutation, whereas increased in vivo angiogenesis was triggered by Ang2 administration. Lastly, to understand whether cytokine profiles that might affect angiocrine signalling are affected differentially in FAK-Y397F vs FAK-Y861F endothelial cells, I show that CCL1 and CCL2 are increased in FAK-Y397F but IL- 13, IL-1F3, CCL4, IL-1F1, CCL2 and others are increased in FAK-Y861F endothelial cells. Overall my data indicates that endothelial-specific FAK mutations on two phosphorylation sites has different effects on tumour angiogenesis, tumour growth, growth factor stimulated angiogenesis in vivo and ex-vivo and cytokine production.

Junctional adhesion molecule C (JAM-C) is a component of endothelial cell (EC) tight junctions that has been implicated in a number of endothelial functions, such as angiogenesis and trafficking of leukocytes through the endothelium during inflammation. Work within our lab has identified that loss of JAM-C at EC junctions results in increased reverse transendothelial migration (rTEM) of neutrophils back into the circulation, a response that has been associated with the dissemination of inflammation to distant organs. Whilst the mechanism by which JAM-C is lost or redistributed away from EC junctions has begun to be elucidated, little is known about how loss of endothelial JAM-C impacts the functions of ECs. As such, this thesis aimed to investigate the effect of JAM-C deficiency on EC functions to unravel possible molecular and cellular mechanisms of mediating neutrophil rTEM. To address the effect of JAM-C deficiency on EC functions, an in vitro RNA interference (RNAi) approach was used to efficiently knock-down (KD) JAM-C in human umbilical vein ECs (HUVECs). Importantly, KD of JAM-C did not affect expression of other key EC junctional markers such as JAM-A and VE-Cadherin and cell proliferation and apoptosis were similarly unaffected. Gene expression profiling using microarrays revealed that JAM-C depleted HUVECs exhibited a pro-inflammatory phenotype under basal conditions that was characterised by increased expression of pro-inflammatory genes such as ICAM1 and IL8. Following IL-1β-induced inflammation, no difference in expression of pro-inflammatory genes was detected between control and JAM-C KD HUVECs. However, protein levels of secreted chemokines such as IL-8 were reduced in JAM-C KD HUVECs following stimulation with IL-1β. This was corroborated by in vivo studies demonstrating reduced levels of secreted chemokines in the plasma of mice where JAM-C was conditionally deleted from ECs. A novel finding of this work is the demonstration that JAM-C KD HUVECs exhibit increased autophagy under basal conditions. This might provide a potential mechanism for the reduced chemokine secretion that is observed in this system, whereby chemokines are preferentially trafficked for autophagosome-mediated degradation. Taken together, these findings indicate a multi-functional role for JAM-C in regulating EC homeostasis under basal conditions. JAM-C KD ECs respond aberrantly to inflammatory stimuli by secreting reduced chemokine levels, a consequence that could provide novel insights into the mechanisms of neutrophil rTEM under conditions of endothelial JAM-C loss.

Glucagon-like peptide-1 (GLP-1) synthetic analog therapies are prescribed for type 2 diabetes due to their effects on insulin and glucagon secretion, and glycemic control. Recent studies also suggest that they may have cardiovascular benefits; however, the mechanism responsible for this is unknown. To examine this question, we evaluated the effects of GLP-1 and the GLP-1 synthetic analog, liraglutide on cell signaling and function in human aortic endothelial cells (HAECs). The results indicate that both agents inhibit TNFα and LPS induced cellular adhesion molecule expression and monocyte adhesion. They also show that incubation with 30pM GLP-1 and 100nM liraglutide stimulates an immediate increase in intracellular calcium, which activates calcium/calmodulin-dependent protein kinase kinase β (CaMKKβ). This in turn led to a 2.5 fold increase in the phosphorylation of both AMP-activated protein kinase (AMPK) and calcium/calmodulin-dependent protein kinase 1 (CaMK1) within 5 minutes. In addition both GLP-1 and liraglutide caused a 2-fold increase in the phosphorylation of the downstream AMPK/CaMK1 targets: endothelial nitric oxide synthase (eNOS) and cAMP response element-binding protein (CREB). Inhibition of CaMKKβ with STO-609 (0.5ug/mL) blocked the phosphorylation of both AMPK and CaMK1, confirming its pivotal role. Incubation of the HAECs for three hours with lipopolysaccharide (LPS, 2ug/mL) and TNFα (10ng/mL) increased the expression of vascular cell adhesion molecule-1 (VCAM-1) and E-selectin by 5 and 2 fold, respectively. Comparable increases in THP-1 monocyte adhesion to the HAECs, a putative initiating event in atherogenesis, also occurred. Pre-incubation for one hour with either GLP-1 or liraglutide inhibited these events. Likewise, pre-incubation with the CaMKK inhibitor STO-609, or use of lentivirus shRNA to knock down AMPK, blocked the inhibitory effects of both GLP-1 and liraglutide on monocyte adhesion. These results suggest that the recently observed cardiovascular benefits of GLP-1 and liraglutide could be mediated by their effects on CaMKKβ, AMPK and CaMK1 activation, which lead to decreased adhesion molecule expression and monocyte adhesion in endothelial cells. The finding that these effects occur at concentrations of GLP-1 (30pM) and liraglutide (100nM) observed in vivo also suggests they are physiologically relevant.

Chloride intracellular channels (CLICs), 1 and 4 are expressed in endothelial cells where they promote cell proliferation, migration and vessel morphogenesis in vitro. Clic4-/- mice exhibit defects in retinal angiogenesis suggesting CLIC4 functions as an angiogenic regulator. S1P signaling, through S1P receptors S1P1 and S1P2, is essential for endothelial cell functions during vascular development. S1P treatment promotes CLIC4 localization to cell surface suggesting a link between CLICs and S1P pathways. Here we demonstrate that CLICs function in embryonic development, retinal angiogenesis and vascular permeability regulation. Clic1-/-;Clic4-/- embryos die in utero and exhibit severe growth restriction with vascular defects prior to death. Loss of Clic4 in murine endothelium (Clic4ECKO) caused aberrant retinal angiogenesis characterized by reduced vascular outgrowth and increased vessel sprouting. Clic4ECKO mice exhibited increased vessel leakiness as assessed by a lung permeability assay. We establish that CLIC1 and CLIC4 function in distinct branches of the S1P pathway to promote angiogenesis. Knockdown of CLIC1 or CLIC4 in endothelial cells impeded S1P1-mediated induction of AKT and Rac1 and reduced endothelial cell migration and adherence junctions formation. CLIC1 knockdown alone inhibited RhoA activation and actin stress fibers downstream of S1P2. Using pharmacological perturbation of S1P signaling in Clic knockout mice we established that Clic4 is essential for S1P1-mediated regulation of retinal angiogenesis and vascular permeability. We conclude that CLIC1 and CLIC4 function as effectors in the S1P pathway, where they have overlapping functions in S1P1-PI3K signaling and CLIC1 uniquely acts as an effector in S1P2-RhoA signaling cascade. Through these findings, our work defines a molecular mechanism through which CLICs function in endothelium.

Yeung Lam. / Thesis submitted in: December 2003. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 134-150). / Abstracts in English and Chinese. / ABSTRACT --- p.I / 摘要 --- p.III / ACKNOWLEDGMENT --- p.V / Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- General review of angiogenesis --- p.1 / Chapter 1.2 --- Vascular endothelial growth factors (VEGFs) --- p.2 / Chapter 1.2.1 --- VEGF-A --- p.2 / Chapter 1.2.2 --- P1GF --- p.4 / Chapter 1.2.3 --- VEGF-B --- p.5 / Chapter 1.2.4 --- VEGF-C and VEGF-D --- p.6 / Chapter 1.3 --- VEGF receptors (VEGFRs) --- p.9 / Chapter 1.3.1 --- VEGFR-1 (or flt-1) --- p.9 / Chapter 1.3.2 --- VEGFR-2 ( or flk-1) --- p.10 / Chapter 1.3.3 --- VEGFR-3 ( or flt-4) --- p.11 / Chapter 1.4 --- Hormonal regulation of VEGFs by LH/hCG --- p.14 / Chapter 1.5 --- General review of the testis --- p.17 / Chapter 1.5.1 --- Structure and function of the testis --- p.17 / Chapter 1.5.2 --- Testicular vasculature --- p.18 / Chapter 1.5.3 --- Testicular angiogenesis --- p.19 / Chapter 1.6 --- Localization of VEGF and VEGF receptors in the testis --- p.20 / Chapter 1.7 --- Aims of the present study --- p.21 / Chapter 2. --- Materials and methods --- p.23 / Chapter 2.1 --- Animals --- p.23 / Chapter 2.1.1 --- Depletion of Leydig cell --- p.23 / Chapter 2.1.2 --- Suppression of Leydig cell and stimulation by hCG --- p.24 / Chapter 2.1.3 --- Collection of tissue --- p.25 / Chapter 2.2 --- Preparation of primary cells from rat testes --- p.27 / Chapter 2.2.1 --- Sertoli cell preparation --- p.27 / Chapter 2.2.2 --- Germ cell preparation --- p.29 / Chapter 2.2.3 --- Interstitial cell and Leydig cell preparation --- p.30 / Chapter 2.3 --- Cell cultures --- p.32 / Chapter 2.3.1 --- Reagents and cell lines --- p.32 / Chapter 2.3.2 --- "Mouse Leydig cell line, TM3 and Sertoli cell line, TM4" --- p.33 / Chapter 2.3.3 --- "Mouse tumor Leydig cell line, MLTC-1" --- p.34 / Chapter 2.3.4 --- "Rat tumor Leydig cell line, R2C" --- p.34 / Chapter 2.3.5 --- "Rat tumor Leydig cell line, LC540" --- p.35 / Chapter 2.4 --- Reverse-transcription polymerase chain reaction (RT-PCR) and semi-quantitative RT-PCR --- p.35 / Chapter 2.4.1 --- Extraction of total RNA --- p.35 / Chapter 2.4.2 --- Quantitation of total RNA --- p.37 / Chapter 2.4.3 --- RT-PCR --- p.37 / Chapter 2.4.4 --- Purification and authentication of PCR products --- p.47 / Chapter 2.5 --- Immunohistochemical staining --- p.48 / Chapter 2.5.1 --- Perfusion and processing of testes for histological sections --- p.48 / Chapter 2.5.2 --- Immunohistochemical staining of tissue sections --- p.50 / Chapter 2.6 --- Western immunoblotting --- p.52 / Chapter 2.6.1 --- Extraction and quantitation of total protein --- p.52 / Chapter 2.6.2 --- SDS-PAGE --- p.53 / Chapter 2.6.3 --- Immunoblotting --- p.55 / Chapter 2.7 --- Statistical analyses --- p.57 / Chapter 3. --- Results --- p.58 / Chapter 3.1 --- Expression and localization of VEGFs in the rat testis --- p.58 / Chapter 3.1.1 --- VEGF-A --- p.58 / Chapter 3.1.2 --- VEGF-B --- p.64 / Chapter 3.1.3 --- VEGF-C --- p.69 / Chapter 3.1.4 --- VEGF-D --- p.73 / Chapter 3.1.5 --- P1GF --- p.77 / Chapter 3.2 --- Effect of Leydig cell depletion on VEGFs expression in the rat testis --- p.81 / Chapter 3.2.1 --- Effect on VEGF-A --- p.81 / Chapter 3.2.2 --- Effect on VEGF-B --- p.82 / Chapter 3.2.3 --- Effect on VEGF-C --- p.88 / Chapter 3.2.4 --- Effect on VEGF-D --- p.91 / Chapter 3.2.5 --- Effect on P1GF --- p.94 / Chapter 3.3 --- Effect of Leydig cell suppression and hCG stimulation on VEGFs expression in the rat testis --- p.97 / Chapter 3.3.1 --- Effect on VEGF-A --- p.97 / Chapter 3.3.2 --- Effect on VEGF-B --- p.107 / Chapter 3.3.3 --- Effect on VEGF-C --- p.113 / Chapter 3.3.4 --- Effect on VEGF-D --- p.119 / Chapter 4. --- Discussion --- p.126 / Chapter 5. --- References --- p.134

ANCA-associated vasculitis is a group of devastating autoimmune diseases that predominantly target and destroy small blood vessels. The interaction of neutrophils and monocytes with the endothelial cell lining of blood vessels is imperative to understanding the pathophysiology of the disease. The nature and temporal dynamics of these interactions are mostly unknown and could provide a currently unmet clinical need for more reliable biomarkers of disease activity. This study describes the development of a 4-dimensional in vitro live cell imaging model allowing the interactions of leukocytes with endothelial cells to be analysed in the context of health and disease. Monocytes and neutrophils were isolated from peripheral venous blood of AAV patients and healthy donors. Cells were fluorescently labelled and imaged on a monolayer of human umbilical vein endothelial cells (HUVEC) using a spinning disc confocal microscope. Leukocyte migration, partial and full transmigration, route of transmigration, degranulation and the presence of leukocyte-derived particles inside endothelial cells were measured and the influence of ANCA or BVAS status considered. Following a series of preliminary experiments, it was determined that neutrophil degranulation and partial transmigration indicated early promise as potential biomarkers of disease activity. Several circulating serum analytes correlated with in vitro leukocyte functions, complementing these findings but also highlighting the prevalent immune dysfunction in the pathophysiology and development of the disease. Fatigue is a common symptom within the AAV community and the complex relationship between autoimmune fatigue and leukocyte functions was examined. The data in this thesis describes the development of a novel in vitro live cell imaging platform which can be used to investigate leukocyte functions as potential markers of disease activity as well as understanding their role in the pathophysiology of AAV.

O endotélio é a camada celular interna dos vasos sanguíneos, responsável pela homeostase vascular. Além disso, é a porta de entrada para as células de defesa frente a um quadro de inflamação. A camada endotelial é alvo de diversos estudos devido ao seu caráter de fácil expansão em cultura, porém sua biologia ainda não é completamente compreendida. Devido à sua localização privilegiada, o endotélio está susceptível a alterações da composição da corrente sanguínea. A melatonina, hormônio produzido ritmicamente pela glândula pineal e de forma não rítmica em diversos outros tecidos, tem propriedade citoprotetora. Diversos estudos já demonstraram que ela atua, por diferentes mecanismos de ação e faixas de concentração, como um mediador anti-inflamatório sobre o endotélio. Segundo a hipótese de trabalho de nosso grupo, o eixo imune-pineal, a glândula pineal e o sistema imunológico se interligam em uma comunicação bidirecional, na qual a produção rítmica de melatonina é inibida frente a um quadro de inflamação para que ocorra a montagem da resposta inflamatória independente da hora do dia. Assim sendo, esta dissertação se baseou na hipótese de que as células endoteliais apresentam um ritmo em sua maquinaria que altere a intensidade de resposta frente a um estímulo inflamatório, e propôs avaliar como a melatonina agiria na regulação desse ritmo. Dessa forma, avaliamos como o tratamento sistêmico com LPS afetaria a produção noturna de melatonina, modulando a reatividade de células endoteliais da microcirculação. Demonstramos que o tratamento com LPS diminui os níveis circulantes deste hormônio e inibe a transcrição gênica da enzima chave, AA-NAT. Na periferia, o tratamento com LPS aumenta a reatividade de células endoteliais independente da hora do dia de administração mesmo após 18 dias em cultura. Este efeito é transiente, pois quando o tratamento é realizado seis horas antes da morte, os parâmetros analisados retornam aos níveis basais. A melatonina, administrada juntamente com LPS, reverte o efeito do LPS sobre as células endoteliais, além de reduzir a concentração plasmática de TNF. Além disso, células endoteliais obtidas de animais mortos à noite possuem menor estado de ativação que células provenientes de animais mortos de dia. De maneira geral, o efeito observado sobre as células endoteliais é inversamente correlacionado com a concentração plasmática de melatonina. Esses dados sugerem que as células endoteliais possuem uma espécie de \"memória celular\", pois armazenam as informações do estado do animal doador mesmo após todos os procedimentos em cultura. Adicionalmente, demonstramos a dinâmica do eixo imune-pineal in vivo. Juntamente, nossos dados permitem concluir que a melatonina prima as células endoteliais, modulando sua reatividade de acordo com a hora do dia e o estado de saúde do animal. / The endothelium is the vascular internal cellular layer, responsible for vascular homeostasis. Additionally, it regulates immune cells entrance during an inflammatory response. The endothelial layer is the focus of many studies due to its facility of culture expansion, but its biology is not yet totally understood. Because of its privileged localization, the endothelium is susceptible to plasma compounds changes. Melatonin, rhythmically produced by pineal gland and in a non rhythm way in others tissues, has citoprotector properties. Many studies have already shown that melatonin acts on endothelium as an anti-inflammatory mediator, through different mechanisms of action and concentrations ranges. Considering our work hypothesis, the immune-pineal axis, that suggests that the pineal gland and immune system are integrated through a bidirectional communication, melatonin rhythm production is inhibited during an injury, permitting the mounting of immune response independently of the hour of the day. This dissertation is based on the hypothesis that endothelial cells presents a rhythm in its machinery that alters the response intensity due to an inflammatory stimuli. We analyzed how LPS systemic treatment affects the melatonin nocturnal production, modulating the endothelial cells reactivity of microcirculation. We demonstrated that LPS treatment reduced plasma melatonin level and inhibited gene transcription of key enzyme, AA-NAT. On the periphery, LPS treatment increased endothelial cells reactivity independently of the hour of the day even after 18 days in culture. This effect was transient, once the parameters analyzed returned to basal levels when the treatment was done six hours before the death. Melatonin administrated together with LPS, reverted LPS effects on the endothelial cells, and also reduced plasma TNF concentration. Endothelial cells obtained from animal killed at nighttime are more activated than cells obtained from animals killed at daytime. Generally, the endothelial cells effects are inversely correlated with plasma melatonin level. These data suggests that endothelial cells have a \"cellular memory\", because they are capable to retain the information of donor animal state even after all culture proceedings. Additionally, we demonstrated the immune-pineal axis dynamics in vivo. All together, our results permit to conclude that melatonin primes the endothelial cells, modulating their reactivity according to the hour of the day and donor animal health.

archives@tulane.edu / Experimental stroke in endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) knockout mice showed diverse effects on brain injury. nNOS and eNOS have been shown to uncouple in pathological conditions to produce superoxide. Oxidative stress is believed to be the underlying cause of several cardiovascular diseases including ischemic stroke. However, the role of eNOS and nNOS uncoupling in ischemic stroke is not well studied. Our objective of the study was to determine the effect of eNOS and nNOS inhibition on reactive oxygen species (ROS), NO, viability and mitochondrial bioenergetics in rat brain microvascular endothelial cells (BMECs) and rat cortical neurons following oxygen-glucose deprivation-reoxygenation (OGD/R). We found that non-specific inhibition of NOS in endothelial cells reduced ROS levels in BMECs but increased ROS levels in neurons under normoxia. This suggests that a pool of uncoupled NOS exists in the BMECs whereas the dominant functional NOS in neurons produces NO. We observed increased levels of ROS following OGD/R that is sensitive to NOS inhibition in both BMECs and neurons indicating eNOS and nNOS uncoupling during OGD/R. Furthermore, NOS inhibition reduced mitochondrial respiration while it improved cell survival rate in both BMECs and neurons following OGD/R. Thus, it is possible that decreased mitochondrial respiration in the immediate aftermath (4 hours) of OGD/R could be protective against reoxygenation injury. Moreover, we identified the expression of nNOS in BMECs from rat, human, and mouse. We observed that the nNOS in the BMECs constitutively produces superoxide under physiological conditions instead of NO. In contrast, nNOS in the neurons produces NO and doesn’t contribute to ROS. We also confirmed the nNOS expression and its function in freshly isolated rat brain microvessels. In addition, we developed a novel method to measure mitochondrial respiration in freshly isolated mouse brain microvessels using Seahorse XFe24 Analyzer. We validated the method by demonstrating impaired mitochondrial respiration in cerebral microvessels isolated from old mice compared to young mice. In summary, the present doctoral research investigated the distinct role of NOS isoforms in BMECs and Neurons leading to the identification of novel functional variant of nNOS in BMECs and brain microvessels. / 1 / RAMARAO SVNL