Made available in DSpace on 2017-07-21T14:13:10Z (GMT). No. of bitstreams: 1 Willian Moreira Machado.pdf: 833860 bytes, checksum: b7acc75cbcbeaee87101553fc891d9af (MD5) Previous issue date: 2013-02-22 / Fundação Araucária de Apoio ao Desenvolvimento Científico e Tecnológico do Paraná / Periodontitis is a chronic inflammatory disease initiated and perpetuated by Gram-negative anaerobic bacteria that colonize the subgingival area. This disease is characterized by destruction of periodontal tissue insertion, bone resorption, leukocyte infiltration and formation of periodontal pockets. Studies show a significantly increased risk of cardiovascular disease, especially atherosclerosis and hypertension, among people with periodontitis. It is believed that systemic inflammation induced by periodontal disease and decreased nitric oxide bioavailability would cause endothelial dysfunction, which consequently leads to cardiovascular disease. Interestingly, studies show that statins that has been providing consistent and satisfactory results in reversal of endothelial dysfunction. Thus, the purpose of this study was to evaluate the effect of systemic administration of simvastatin on inflammatory parameters induced periodontitis and correlate their possible actions in the cardiovascular system and in alveolar bone loss. Wistar rats were subjected to induction of periodontitis by placement of ligatures or were subjected to false-surgery (the ligatures were immediately placed and removed). On day 8, the animals were randomized to treatment with simvastatin (10 mg/kg/day p.o.) or vehicle.During the experiment, on days 1, 7 and 14 the rats were prepared for analysis of systolic blood pressure and heart rate. And on day 14, the rats were prepared for analysis of cardiovascular parameters, lipid profile, systemic inflammatory markers and tissue injury. The maxilla and mandible were removed for analysis of bone loss. Our data show that animals with untreated periodontitis showed an endothelium-dependent vasodilator response (acetylcholine) reduced a characteristic of endothelial dysfunction in animals with periodontal disease. In contrast, we found an increased vasodilatory response in animals with periodontitis treated with simvastatin. The vascular changes induced by treatment with simvastatin were associated with a decrease in lipid profile, and lymphocytic inflammatory markers such as IL-6 and C-reactive protein, which are traditionally associated with cardiovascular risk. And interestingly, simvastatin decreased alveolar bone loss in animals with periodontitis. Our data show that simvastatin reduces alveolar bone loss, systemic inflammation and damage to the endothelium induced periodontitis. Thus, simvastatin can be considered a promising drug for the treatment of periodontal disease and prevention of cardiovascular complications. / A periodontite é uma doença inflamatória crônica iniciada e perpetuada por bactérias anaeróbicas Gram-negativas que colonizam a área subgengival. Esta doença é caracterizada pela destruição do tecido periodontal de inserção, reabsorção óssea, infiltração de leucócitos e formação de bolsa periodontal. Estudos mostram um aumento significativo do risco de doenças cardiovasculares, principalmente aterosclerose e hipertensão, entre pessoas com periodontite. Acredita-se que a inflamação sistêmica induzida pela doença periodontal e a diminuição da biodisponibilidade de óxido nítrico seriam a causa da disfunção endotelial, que consequentemente leva a doenças cardiovasculares. Interessantemente, estudos mostram que as estatinas vêm proporcionando resultados consistentes e satisfatórios na reversão da disfunção endotelial. Assim, a proposta deste trabalho foi avaliar o efeito da administração sistêmica da sinvastatina sobre parâmetros inflamatórios de periodontite induzida e correlacionar suas possíveis ações no sistema cardiovascular e na perda óssea alveolar. Ratos Wistar foram submetidos a indução da periodontite através da colocação de ligaduras ou eram submetidos a falsa-cirurgia (as ligaduras eram colocadas e imediatamente removidas). No dia 8, os animais foram novamente randomizados para receber o tratamento com a sinvastatina (10 mg/kg/dia v.o.) ou veículo. Durante o experimento, nos dias 1, 7 e 14 os ratos eram preparados para análise da pressão arterial sistólica e frequência cardíaca. E no dia 14, os ratos foram preparados para análise de parâmetros cardiovasculares, perfil lipídico, marcadores inflamatórios sistêmicos e de lesão tecidual. A maxila e a mandíbula eram retiradas para a análise da perda óssea. Nossos dados mostram que os animais com periodontite não tratados apresentaram uma resposta vasodilatadora endotélio-dependente (acetilcolina) reduzida, uma característica da disfunção endotelial em animais com doença periodontal. Em contrapartida, foi encontrada uma resposta vasodilatadora aumentada em animais com periodontite tratados com sinvastatina. As alterações vasculares induzidas pelo tratamento com sinvastatina foram associadas com uma diminuição do perfil lipídico, linfocitário e marcadores inflamatórios como IL-6 e proteína C-reativa, que são tradicionalmente associados com o risco cardiovascular. E interessantemente, a sinvastatina diminuiu a perda óssea alveolar nos animais com periodontite. Nossos dados mostram que a sinvastatina reduz a perda óssea alveolar, inflamação sistêmica e o dano ao endotélio induzidos pela periodontite. Desta forma, a sinvastatina pode ser considerada uma droga promissora para o tratamento da doença periodontal e prevenção de complicações cardiovasculares.

Made available in DSpace on 2017-07-21T14:13:13Z (GMT). No. of bitstreams: 1 Luiz Renato Olchanheski Junior.pdf: 1811415 bytes, checksum: 57ce362be4cfc124e9a1788156185688 (MD5) Previous issue date: 2014-02-18 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Periodontitis is defined as a disease characterized by the formation of a biofilm which promotes colonization of microorganisms responsible for the initiation of a local inflammatory process. Studies have shown that the inflammatory process is not restricted to the oral cavity, reaching the circulation and causing systemic effects, such as endothelial dysfunction. The dysfunction is mainly characterized by reduced nitric oxide production/ bioavailability which increased platelet aggregation, leukocyte adhesion and rolling, and result in a loss in the ability of vasodilation. Therefore predisposing to an increased risk of cardiovascular disease. According to research by our group, animals with experimental periodontitis showed a decrease in endothelium-dependent vasodilator response fourteen days after the induction of periodontitis, however, this reduced vasodilator response is not evident twenty one days. The purpose of this study was to evaluate a possible increase in compensatory mechanisms of vasodilation in animals with experimental periodontitis. For this, rats received ligatures for induction of periodontitis, or were sham. Twenty one days after the procedure, the animals were prepared for blood pressure recording. Two consecutive dose-response curves to acetylcholine and sodium nitroprusside were obtained before and 20 min after LNAME (NOS inhibitor) + indometacin (COX inhibitor) + TEA (selective inhibitor of potassium channels) or LNAME or indometacin, or TEA or indometacin + TEA or Apamin + TRAM-34 injection. Only the selective and simultaneous blockade of small and intermediate-conductance calcium-activated potassium channels by selective inhibitors (Apamin and TRAM-34, respectively) was able to reduce acethylcholine-induced reduction on blood pressure at periodontitis animals. The inhibition of NOS, COX and use of a non-selective inhibitor of potassium channels, did not change the endothelium-depended vasodilatation. Altogether, these results show that IKca and SKca may balance the endothelial function and therefore mask the impairment on NO production and endothelial dysfunction. / A periodontite é definida como uma doença caracterizada pela formação de um biofilme dental, que favorece a colonização de microrganismos que iniciam um processo inflamatório local. Este processo inflamatório não fica restrito a cavidade bucal, ele ganha a circulação causando efeitos sistêmicos, como a disfunção endotelial. A disfunção é caracterizada principalmente pela redução da produção e biodisponibilidade de óxido nítrico, predispondo o indivíduo a um aumento no risco de doenças cardiovasculares. Trabalhos recentes mostram que animais com periodontite experimental apresentam uma disfunção endotelial quatorze dias após a indução da periodontite, porém, esta redução da resposta vasodilatadora não é evidente vinte e um dias após o mesmo procedimento. A proposta do presente trabalho foi avaliar um possível aumento de mecanismos compensatórios de vasodilatação em animais com periodontite experimental. Para isso, os ratos receberam as ligaduras para indução da periodontite, ou passaram pela falsa-cirurgia. Vinte e um dias após o procedimento, todos os animais passaram pela administração intravenosa de três doses crescentes de acetilcolina e nitroprussiato de sódio. Em seguida, os animais receberam as seguintes administrações: LNAME (inibidor da NOS) + indometacina (inibidor da COX) + TEA (inibidor não seletivo dos canais de potássio), ou LNAME, ou indometacina, ou TEA, ou administração de indometacina + TEA ou Apamina (inibidor seletivo dos SKCa) + TRAM-34 (inibidor seletivo dos IKCa). Após um período de 20 minutos, todos os grupos passaram por uma nova administração dos dois agentes vasodilatadores. Vinte e um dias após a indução da periodontite, os animais, não apresentaram disfunção, e a inibição de NOS, COX e a utilização de um inibidor não seletivo dos canais de potássio, evidenciou uma alteração no tempo de vasodilatação do grupo ligadura. A combinação dos demais inibidores não resultou em alterações significativas. A administração de inibidores de SKCa e IK Ca resultou em uma diminuição do tempo da resposta vasodilatadora dependente de endotélio, demonstrando a importância destes canais no mecanismo de compensação vasodilatadora 21 dias após a indução da doença periodontal.

O lúpus eritematoso sistêmico (LES) é uma doença autoimune e protótipo das doenças por imunocomplexos (IC). A deposição de IC em tecidos ou órgãos leva a um processo inflamatório crônico, que resulta em lesão tecidual. A fisiopatologia deste processo envolve principalmente o sistema do complemento (SC), receptores de IgG (Fc?R), neutrófilos e moléculas de adesão. Os produtos de ativação SC atraem os neutrófilos para o foco inflamatório e sua interação com os IC depositados resulta na liberação de espécies reativas de oxigênio (ERO) e das enzimas dos neutrófilos sobre os tecidos. As ERO podem levar a danos nas estruturas celulares devido ao estresse oxidativo, resultando na exposição de autoantígenos e sustentação da autoimunidade. Várias anormalidades envolvendo neutrófilos, função e expressão dos Fc?R e CR foram descritas no LES. O objetivo deste estudo foi avaliar se essas alterações podem ter implicações para o dano tecidual através do depósito de IC. Devido à importância das interações neutrófilo-endotélio-SC para a inflamação e lesão tecidual por IC no LES, este estudo propôs avaliar o efeito da ativação de neutrófilos e produtos do SC sobre as células endoteliais in vitro. Os resultados mostram que a exposição das células endoteliais aos neutrófilos/LES ativo, sem estímulo, resulta em maior peroxidação lipídica comparada com o controle espontâneo, o que não foi observado com neutrófilos/saudáveis. Contudo, na presença de IC/SHN, a peroxidação lipídica foi maior quando as células endoteliais foram expostas aos neutrófilos/LES inativo comparada ao controle espontâneo. O efeito da ativação dos neutrófilos, em todos os grupos, sobre a peroxidação lipídica das células endoteliais foi dependente da opsonização dos IC pelo complemento (IC/SHN), uma vez que não foi observado quando as proteínas do complemento foram inativadas (IC/SHI). Não houve diferença na produção de ERO entre os neutrófilos dos grupos estudados. Observou-se menor expressão dos Fc?RIIa (CD32) e CR1 em neutrófilos/LES ativo, quando comparados com o grupo controle. Houve maior liberação de catalase pelos neutrófilos/LES, quando estes foram estimulados via Fc?R, e maior produção de glutationa por neutrófilos/LES inativo quando estas células foram estimuladas via Fc?R e CR. A expressão de ICAM-1 não foi diferente entre os grupos de neutrófilos, entretanto foi menor na ausência de complemento. Não houve diferença na medida da ativação da via clássica do complemento avaliada pelo fragmento C4d. Estes resultados mostram que as células endoteliais são mais suscetíveis à peroxidação lipídica na presença de neutrófilos/LES. Contudo, quando o complemento do soro é inativado, esta suscetibilidade desaparece, bem como há menor liberação de ICAM-1 por todos os grupos de neutrófilos e maior liberação de catalase por neutrófilos/LES. A interação dos neutrófilos/LES com células endoteliais pode ser deletéria para este último e depender da atividade das proteínas do sistema complemento. O modelo desenvolvido neste estudo pode contribuir para a compreensão do envolvimento dos neutrófilos e do SC nas lesões teciduais no LES. / Systemic lupus erythematosus (SLE) is an autoimmune disease and prototype of immune complex diseases (IC). The deposition of IC in tissues or organs leads to a chronic inflammatory process, which results in tissue injury. The pathophysiology of this process mainly involves the complement system (SC), IgG (Fc?R) receptors, neutrophils and adhesion molecules. SC activation products attract neutrophils to the inflammatory focus and their interaction with deposited IC results in the release of reactive oxygen species (ROS) and neutrophil enzymes into tissues. ROS can lead to damage to cell structures due to oxidative stress, resulting in the exposure of autoantigens and the support of autoimmunity. Several abnormalities involving neutrophils, function and expression of Fc?R and CR have been described in SLE. The aim of this study was to assess whether these changes may have implications for tissue damage through the deposition of IC. Due to the importance of neutrophil-endothelial-SC interactions for inflammation and tissue damage by IC in SLE, this study proposed to evaluate the effect of the activation of neutrophils and SC products on endothelial cells in vitro. The results show that exposure of endothelial cells to active neutrophils / SLE without stimulus results in higher lipid peroxidation compared to spontaneous control, which was not observed with neutrophils / healthy. However, in the presence of IC / complement from normal human serum (NHS), lipid peroxidation was greater when endothelial cells were exposed to inactive neutrophils / SLE compared to spontaneous control. The effect of neutrophil activation in all groups on endothelial cell lipid peroxidation was dependent on IC with complement (IC / NHS), as it was not observed when complement proteins were inactivated (IC / INHS). There was no difference in ROS production among the neutrophils of the studied groups. Lower expression of Fc?RIIa (CD32) and CR1 in active neutrophils / SLE, when compared with the control group, was observed. There was greater release of catalase by neutrophils / SLE, when they were stimulated via Fc?R, and increased production of glutathione by neutrophils / inactive SLE when these cells were stimulated via Fc?R and CR. There was no difference in expression of ICAM-1 between the neutrophil groups, however it was lower in the absence of complement. There was no difference in the extent of the activation of the classical complement pathway evaluated by the C4d fragment. These results show that endothelial cells are more susceptible to lipid peroxidation in the presence of neutrophils / SLE. However, when serum complement is inactivated, this susceptibility disappears, as well as there is a lower release of ICAM-1 by all neutrophil groups and greater release of catalase by neutrophils / SLE. The interaction of neutrophils / SLE with endothelial cells may be deleterious to the latter and depends on the activity of the complement system proteins. The model developed in this study may contribute to the understanding of neutrophil and SC involvement in tissue lesions in SLE.

5‐羟色胺（5-HT）是一种强有力的血管活性神经递质，被广泛的应用在调节血管张力。当5‐HT 被释放后，会被单胺氧化酶（MAOs）催化的酶促反应代谢，从而产生不同的代谢产物，比如5‐HIAA，5‐HTOL 和过氧化氢（H₂O₂）。然而，5‐HT对于内皮细胞活性氧物种（ROS）的产生作用以及5‐HT 转运体，5‐HT 受体，MAOs和ROS 的产生伴随着细胞内钙变化是否参与了其中的信号传导尚未被阐明。所以，这个研究最初的目的是考查外源性加入的5‐HT 对于脐静脉内皮细胞中ROS产生的影响以及其潜在的生物机理。 / 数据清楚的显示在没有L‐NAME（一种抑制一氧化氮（NO）产生的抑制剂）预处理的情况下，5‐HT 并不能在脐静脉内皮细胞内产生显著性的ROS。然而，在L‐NAME 预处理的情况下，NO 的产生被完全抑制，我们观察到明显的显著性的线粒体内的ROS 产生。5‐HT 产生的线粒体ROS 可以被clorgyline（一种MAO‐A 抑制剂），indatraline（一种5‐HT 转运体阻断剂），LY272015（一种5‐HT‐2B 受体拮抗剂），ketanserin（一种5‐HT2A 受体拮抗剂），XeC（一种IP3 受体拮抗剂），Gd³⁺（一种非选择性TRP 通道阻断剂），BAPTA（一种强效钙离子螯合剂），PEG‐Catalase，U73122（一种选择性PLC 抑制剂）以及没有钙离子的培养基所阻止。同时，5‐HT介导的胞内钙离子变化被XeC, Gd³⁺, BAPTA, U73122, ketanserin, LY272015 以及没有钙离子的培养基所阻止。另外，MAO‐A 基因敲除抑制了5‐HT 导致的线粒体ROS的产生却对5‐HT 介导的胞内钙离子变化没有影响。基于以上所述的结果，我们可以得出结论，通过5‐HT 转运体，5‐HT 被摄取入细胞内，然后通过MAO‐A 介导的酶促代谢反应，产生钙离子依赖性的线粒体内ROS 的产生，这一结论对于解释血小板聚集而引起的内皮细胞功能性障碍起到非常重要的作用。 / 根据前人所述，内皮细胞内产生的ROS 对于内皮细胞通透性变化有着重要的作用，但是5‐HT 诱导的脐静脉内皮细胞ROS 的增加是否会对内皮通透性有所影响并没有被说明。在这项研究中，我们设计了实验旨在测试平面细胞表面积（ PCSA ）， 跨内皮电阻（ TER ）， 细胞高度， 肌球蛋白轻链磷酸化（MLCphosphorylation）和肌动蛋白细胞骨架（F‐actin cytoskeleton）水平的变化。此外，b‐catenin 在ROS 引起的F‐actin cytoskeleton 重组中的作用也在我们的讨论范围之内。 / 数据表明，在L‐NAME 预处理的情况下，5-HT 降低了脐静脉内皮的PCSA，TER 以及细胞高度，却增加了MLCP 和与b‐catenin 表达负相关的F‐actincytoskeleton 的水平。这些作用明显被PEG‐Catalase 预处理和MAO‐A 基因敲除减弱，证明了5‐HT 通过MAO‐A 介导产生的H₂O₂ 可以增加内皮细胞的通透性。 / 据文献报道，不论内源性还是外源性的低浓度的H₂O₂ 都可以激活导致血管生成的信号通路。文献进一步表明5‐HT 可以通过特定的5‐HT 受体亚型促进各种类型的内皮细胞的血管生成。然而，5‐HT 诱导的H₂O₂ 对于脐静脉内皮的血管生成作用并没有被报道。我们通过最初的实验先验证5‐HT 对于内皮细胞增殖和迁移的影响，然后我们才去验证H₂O₂ 在其中的作用及其潜在的机理。 / 实验结果表明，在L‐NAME 预处理的情况下，不论是急性（30 分钟）还是慢性（24 小时）的5‐HT 的处理都可以导致脐静脉内皮细胞的迁移，而这个作用会被5‐HT‐2 受体拮抗剂ketanserin，LY272015，ROS 清除剂PEG‐Catalase 以及PI3K的抑制剂wortmannin 所抑制。同时，在L‐NAME 预处理下，5‐HT 增加了cortactin,p‐Akt 和 p‐eNOS 的蛋白表达量而并没有影响Akt, eNOS 和p‐cortactin 的蛋白表达量。而5‐HT 增加的p‐Akt 和p‐eNOS 的蛋白表达被wortmannin 和PEG‐Catalase所抑制。不论是在Cyuant 细胞增殖检测还是在BrdU 细胞增殖检测中，5‐HT 诱导了一种非显著性的DNA 合成的增加，并且再BrdU 细胞增殖检测中，增加了的DNA 合成被PEG‐Catalase 显著性降低。总结以上实验结果，我们可以得出结论，通过一种5‐HT‐2 受体介导的PI3K 依赖性通路，而不是cortactin 磷酸化依赖性的信号通，路5‐HT 可以引导内皮细胞迁移。 / 除此之外，ROS 也被印证可以加剧内皮细胞的炎症反应和加速内皮细胞的老化。因此，我们也观察了5‐HT 对于粘附蛋白比如ICAM‐1 和VCAM‐1 以及抗老化因子SIRT‐1 的表达是否有影响。数据表明，在L‐NAME 预处理的情况下，30 分钟的5‐HT 处理显著的增加了ICAM‐1，SIRT‐1 而不是VCAM‐1 的表达。同时，这些作用均可以被PEG‐Catalase 所抑制表明了5‐HT 通过诱导H₂O₂ 的产生来形式其促进炎症反应和抗衰老的作用。 / 最后，总结以上，通过抑制NO 的产生，5‐HT 可以通过MAO‐A 介导的酶促代谢反应在人体脐静脉内皮细胞线粒体诱导ROS 的产生。同时，5‐HT 诱导的H₂O₂参与了改变内皮细胞通透性，促进血管生成（内皮迁移）及炎症反应的过程。 / 5-Hydroxytryptamine (5-HT), a potent vasoactive neurotransmitter, is involved in the regulation of vascular tone. After its release, 5-HT is terminated at the nerve terminals via enzymatic metabolism catalyzed by monoamine oxidases (MAOs), resulting in the generation of different metabolites (e.g. 5-HIAA, 5-HTOL and H₂O₂). Our lab demonstrates for the first time that 5-HT-induced ROS production indeed occurs and therefore, the aim of this study is to investigate exogenously added 5-HT on ROS generation in human umbilical vein endothelial cells (HUVECs), in order to understand the mechanisms involved in 5-HT-induced ROS production. / Our results clearly demonstrated that in the absence of L-NAME(a NO production inhibitor), there wasno apparent ROS production induced by 5-HT. However, after the inhibition of NO synthesis by L-NAME, 5-HT caused a significant increase in mitochondrial H₂O₂ production. The 5-HT-induced mitochondrial H₂O₂ generation was sensitive to clorgyline (a MAO-A inhibitor), indatraline (a 5-HT transporter blocker), LY272015 (a 5-HT2B antagonist) and ketanserin (a 5-HT2A antagonist), Xextospongin C（XeC，a IP3 receptor antagonist), Gd³⁺ (a non-selective TRP channel blocker), BAPTA (a potent Ca²⁺ ions chelator), PEG-Catalase, U73122 (a selective PLC inhibitor), and in [Ca²⁺]o-free medium. Concurrently, 5-HT-mediated [Ca²⁺]i changes were sensitive to XeC, Gd³⁺, BAPTA, U73122, ketanserin, LY272015, and in [Ca²⁺]o-free conditions. In addition, gene knockdown of MAO-A suppressed 5-HT-elicited H₂O₂ production with no effects on [Ca²⁺]i changes. Based on all the results above, we can conclude that 5-HT caused a Ca²⁺-dependent mitochondrial H₂O₂ generation via MAO-A-mediated metabolism with the pre-requisite uptake of 5-HT into HUVECs through 5-HT transporter. / ROS derived from endothelial cells have been implicated in changes in endothelial permeability, but whether 5-HT-induced H₂O₂ generation could alter endothelial cells permeability has as yet not been demonstrated. Here, we measured the planar cell surface area (PCSA), transendothelial electrical resistance (TER), cell height, myosin light chain phosphorylation and F-actin cytoskeleton level in response to 5-HT challenge to investigate the change of endothelial permeability. Moreover, the participation of β-catenin in regulation of F-actin cytoskeleton remodeling in ROS-modulated alteration in endothelial permeability was also investigated. Results indicated that in the presence of L-NAME, 5-HT reduced the PCSA, TER and cell height in HUVECs. In contrast, 5-HT (with L-NAME) increased myosin light chain phosphorylation (MLCP) expression and F-actin cytoskeleton level, which are negatively associated with β-catenin expression. All of these effects were ameliorated by pre-treatment of PEG-Catalase or gene knockdown of MAO-A, implying 5-HT can consistently elicit the increase in endothelial permeability via MAO-A mediated H₂O₂ generation. / Low dose of ROS from exogenous or endogenous source can activate signaling pathway that lead to angiogenesis.5-HT can promote endothelial angiogenesis through specific 5-HT receptor subtype in various endothelial cell types, but the concomitant ROS generation had not previously been indicated to play a role in the process. In this study, we seek to test out the effects of 5-HT on endothelial cells migration, and should there be a functional role for ROS in the process. / Our results revealed that in the presence of L-NAME, both acute (30 min) and chronic (24 hr) treatment of 5-HT caused HUVECs migration, the effects of which were reversed by pre-incubation of 5-HT-2 receptor antagonists, ketanserin, LY272015, ROS scavenger PEG-Catalase or selective PI3K inhibitor wortmannin. With L-NAME, 5-HT consistently increased cortactin, p-Akt and p-eNOS expression without affecting total Akt, eNOS and p-cortactin protein expression whereas the increased p-Akt and p-eNOS expression are suppressed by pre-treatment of wortmanin or PEG-Catalase. Both in Cyuant cell proliferation assay and BrdU assay, 5-HT caused a trend but non-significant increase in DNA synthesis whereas the pre-treatment of PEG-Catalase significantly suppressed cell proliferation in the BrdU assay. Based on these results, we can conclude that 5-HT elicits endothelial migration via 5-HT-2 receptor-mediated H₂O₂ generation in a PI3K-dependent pathway. Under this circumstance, cortactin phosphorylation-dependent pathway was excluded. / Besides, ROS is notorious for effects like aggravation of inflammation and acceleration aging processes. The investigation extends to looking at alterations ofexpression of adhesion protein including ICAM-1 and VCAM-1 in the inflammatory response pathway and also to looking at the major aging parameter SIRT-1 in the presence of 5-HT in endothelium. Our data showed that in the presence of L-NAME, 30 min treatment of 5-HT significantly increased ICAM-1 and SIRT-1 expression without altering VCAM-1 expression and the up-regulation of ICAM-1 and SIRT-1 expression was prevented by PEG-Catalase. / In conclusion, with the eradication of the influence of NO, 5-HT induced mitochondrial H₂O₂ production via MAO-A-mediated metabolism in HUVECs. At the same time, 5-HT-induced H₂O₂ generation was involved in increasing endothelial permeability, inflammation and angiogenesis (cell migration). / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Zhang, Qian. / Thesis (Ph.D.) Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 326-450). / Abstracts also in Chinese. / Zhang, Qian.

Vitamin D deficiency in patients with chronic kidney disease, measured by reduced serum concentrations of 25 hydroxy vitamin D, is highly prevalent and associated with both endothelial dysfunction and an increased risk of cardiovascular disease. Observational studies in chronic kidney disease have demonstrated that vitamin D therapy reduces the risk of cardiovascular disease. In patients with chronic kidney disease and concomitant vitamin D deficiency, the effect of vitamin D therapy on endothelial function, which is associated with cardiovascular disease, is poorly understood. The mechanism by which vitamin D affects endothelial function is unclear. Methods Presented in this thesis, two studies have addressed these issues: 1. A double blind, randomized controlled trial evaluating the effect of ergocalciferol compared to placebo on microcirculatory endothelial function in patients with non-dialysis chronic kidney disease and concomitant vitamin D deficiency 2. In vitro and in vivo experiments to determine the mechanistic effect of ergocalciferol on endothelial function in an experimental model of uraemia. Results In the clinical study, ergocalciferol increased vitamin D serum concentrations and improved microcirculatory endothelial function measured by laser Doppler flowmetry after iontophoresis of acetylcholine. Oxidative stress measured by skin autofluorescence for advanced glycation end products did not change in the ergocalciferol group but increased significantly in the placebo group. Ergocalciferol increased endothelial nitric oxide synthase expression and activity in cultured human endothelial cells and improved endothelial function in an in vivo model of mild uraemia. The findings from the in vivo and clinical studies occurred independently of changes in blood pressure, conduit artery function, serum calcium, phosphate and parathyroid hormone supporting in vitro findings that ergocalciferol acts directly on the endothelium. Conclusion Ergocalciferol improved endothelial function in both rodent and human subjects with chronic kidney disease. Experimental evidence suggests this effect occurs through an endothelium dependent mechanism involving changes in the upregulation and function of endothelial nitric oxide synthase.

Ingestion of vegetables rich in inorganic nitrate (NO3-) content has emerged as an effective method, via the formation of a nitrite (NO2-) intermediate, for acutely elevating vascular nitric oxide (NO) levels. As such a number of beneficial effects of NO3- ingestion have been demonstrated including the suggestion that platelet reactivity is reduced. I initially investigated whether inorganic NO3- supplementation might also reduce platelet reactivity in healthy volunteers and have determined the mechanisms involved in the effects seen. I conducted a randomised crossover study in 24 (12 of each sex) healthy subjects assessing the acute effects of potassium nitrate capsules (KNO3, 8 mmol) vs placebo (KCl) control capsule ingestion on platelet reactivity. Inorganic NO3- ingested via supplementation raised circulating NO3- and NO2- levels in both sexes and attenuated ex vivo platelet aggregation responses to adenosine diphosphate (ADP) and, albeit to a lesser extent, collagen but not epinephrine in male but not female volunteers. These inhibitory effects were associated with a reduced platelet P-selectin expression and elevated platelet cyclic guanosine monophosphate (cGMP) levels. In addition, I have shown that NO2- reduction to NO occurs at the level of the erythrocyte and not the platelet. These results demonstrate that inorganic NO3- ingestion, whether via the diet or through supplementation, results in a modest decrease in platelet reactivity in healthy males. I then sought to examine the effects of 6 weeks daily intake of NO3--rich beetroot juice versus a placebo NO3--deplete juice on endothelial and platelet function in a cohort of otherwise healthy non-diabetic untreated hypercholesterolaemics. In this randomised double blind placebo controlled parallel study 69 subjects were recruited. The primary end point was change in endothelial function determined using ultrasound flow-mediated dilatation (FMD). Secondary endpoints included change in pulse wave analysis (PWA), aortic pulse wave velocity (aPWV), platelet P-selectin and platelet monocyte aggregate (PMA) expression and plasma, urine and salivary NO3- and NO2- levels. Baseline characteristics, including lipid levels, were similar between the groups. Dietary NO3- caused an improvement in FMD of ~24% from 4.6%±2.2% to 5.7%±2.6% in the treatment group (p<0.001) not seen in the placebo group (4.5%±1.9% versus 4.3%±1.8% p=0.07). This improvement in FMD was also noted following acute administration of dietary NO3-. Small but significant improvements also occurred in aPWV and PWA augmentation index (p=0.04). The % of platelet monocyte aggregates was significantly reduced in the NO3- limb by 7.6% versus an increase of 10.1% in the placebo group (p=0.004). No adverse effects of dietary NO3- were detected. In this study population, chronic dietary NO3- ingestion improves endothelial function, vascular stiffness and platelet markers of atherogenesis in a cohort of hypercholesterolaemics who are otherwise at increased risk of cardiovascular disease (CVD). This thesis provides strong support for assessment of the potential of dietary NO3- as a primary prevention strategy to prevent atherothrombotic and atherogenic complications in larger cohorts.

Cardiovascular disease is the leading cause of death in the developed world. The majority of cardiovascular diseases are characterized by disorders of the arteries, predominantly caused by endothelial dysfunction and arterial stiffening. Passive heat stress results in elevations in core temperature (inducing heat shock protein expression) and changes in cardiovascular hemodynamics, such as increased cardiac output and shear stress, that are similar to exercise. Thus, repeated passive heat stress (“heat therapy”) may provide an alternative means of improving cardiovascular health, particularly for patients with limited exercise tolerance and/or capabilities. Therefore, the goal of this dissertation was to perform integrative studies to determine the effects of heat therapy on vascular function and the associated cellular pathways in young, sedentary humans. Twenty subjects were assigned to participate in 8 weeks (4-5x/week) of heat therapy (N=10; immersion in a 40.5°C bath sufficient to maintain rectal temperature ≥38.5°C for 60 min/session) or thermoneutral water immersion (N=10; sham). As discussed in Chapter V, we found that heat therapy improved numerous well-established biomarkers of conduit vessel/macrovascular function, including flow-mediated dilation (a measure of endothelial function), arterial stiffness, intima media thickness, and blood pressure. Heat therapy also improved microvascular function, as discussed in Chapter VI, measured as improved cutaneous thermal hyperemia and nitric oxide-dependent dilation (the difference between microdialysis sites receiving Lactated Ringer’s [control] and nitric oxide synthase inhibition). No changes were observed in any variables in sham subjects. In Chapter VII, we showed that both direct cellular heating and serum collected from human subjects following heat therapy improved nitric oxide bioavailability and angiogenesis in cultured endothelial cells, providing potential mechanisms by which heat therapy improves vascular function in vivo. Therefore, the studies described herein provide comprehensive evidence that passive heat therapy improves vascular health and insight into the mechanisms involved. Our data presented in Chapters IV-VII, combined with pilot data we conducted in spinal cord injured individuals (Chapter VIII), strongly indicate that passive heat therapy could be used as a simple and effective tool to improve cardiovascular health in a variety of patient populations. This dissertation includes published and unpublished co-authored material.

Endothelial dysfunction is a broad term which implies alteration of the overall functions of endothelial cells, including impairment of the barrier functions, vasodilation, and disturbances in proliferative and angiogenic capacities, migratory as well as tube formation, and deterrence of leukocyte transmigration. Such a dysfunction is triggered by pro-inflammatory stimuli and has been associated to several pathological conditions including atherosclerosis. Myeloperoxidase is a heme peroxidase secreted by activated neutrophils at site of inflammation near blood vessels and plays an important role in the initiation of atherosclerotic plaque by interfering with endothelial function. ApoLs represent a family of newly discovered apolipoproteins with yet unrevealed function, but predicted to be involved in inflammatory processes and cell death mechanisms. We aimed to study the expression of ApoLs as well as Myeloperoxidase in endothelial cells and their possible contribution to endothelial dysfunction. We performed RNA sequencing on MPO-treated endothelial cells and found that most of the induced genes are related to angiogenesis and blood vessel morphogenesis mechanisms. MPO treatment resulted in intracellular MPO localization and mimicked the effects of VEGF on several signal transduction pathways, such as Akt, Erk and Fak involved in angiogenesis. Accordingly MPO, independently of its enzymatic activity, stimulated cellular proliferation, migration and tubules formation by endothelial cells. RNA interference also pointed at a role of endogenous MPO in tubulogenesis and endothelium wound repair in vitro.On the other hand, ApoL3 among other family members was shown to be a downstream responsive gene to MPO, VEGF and FGF treatment. ApoL3 invalidation reduces tubules formation in MPO and VEGF-induced angiogenesis and wound repair in vitro. Accordingly, pro-angiogenic signaling pathways (Erk1/2 and FAK but not Akt) and some pro-angiogenic genes were partially inhibited in ApoL3 Knock out cells. These findings uncover for the first time an important and unsuspected role for ApoL3 and MPO as drivers of angiogenesis. / Le dysfonctionnement endothélial est un terme qui désigne un dérèglement général de la fonction endothéliale, caractérisé par des perturbations de l’intégrité membranaire, de la croissance endothéliale, du rôle anti-inflammatoire ;anti-coagulant, ainsi que leur propriété angiogenique principalement la migration endothéliale et la formation des structures tubulaires. Cette condition patho-physiologique pourrait être déclenchée par des stimuli pro-inflammatoire et elle est souvent associée à l’athérosclérose. La myéloperoxydase est une enzyme secrétee par les neutrophiles et contribue à la formation de la plaque d’athérome. Une nouvelle famille de protéines, les apolipoprotéines L, susceptibles d'intervenir dans le processus inflammatoire est bien exprimée dans les cellules endothéliales. Néanmoins, aucune fonction ne lui a été attribuée jusqu’à présent dans ce type cellulaire.dans le cadre de ce travail, Nous nous sommes intéressés à étudier l’implication des ApoLs ainsi que la Myeloperoxydase dans la dysfonction endothéliale. L’analyse du transcriptome des cellules traitées avec la MPO a montré que lamajorité des génes induits contrôlent le processus angiogenique. La myeloperoxidase stimule la proliferation,migration et la tubulogenese des cellules endotheliales. Cet effet est médié par l’activation des cascades (ERK1/2, Akt et FAK) et des genes pro-angiogeniques. Tandis que la suppression de l’expression de la MPO endogène entraine l’inhibition de la capacité des cellules à migrer et de former des tubes.D’autre part, l’invalidation de l’ApoL3 inhibe la migration cellulaire et la tubulogenése dépendente de la MPO et le VEGF. Sur le plan mécanistique, ces altérations phénotypiques sont les conséquences d’une part, une baisse de phosphorylation des kinases Erk1/2 et FAk (mais pas Akt) et d’autre part de la réduction du taux d’expression des gènes pro-angiogeniques dans les cellules ApoL3 Knock out stimulées par la MPO et le VEGF. ce résultat nous permet de définir l’ApoL3 et la MPO en tant que nouvels acteurs dans le processus angiogenique. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

A privação do sono é um problema sério nos tempos atuais e pode ter graves conseqüências para a fisiologia humana. De fato, a redução no tempo de sono tem sido associada a um notável aumento na incidência de hipertensão arterial, diabetes mellitus, infarto do miocárdio, acidente vascular cerebral e estresse, porém os mecanismos envolvidos são pobremente compreendidos. Objetivos: Avaliar o impacto da privação parcial do sono na função endotelial venosa e no controle autonômico cardiovascular em homens saudáveis. Métodos: Treze voluntários do sexo masculino, saudáveis e com idade média de 31±2 anos, tiveram o sono monitorado por diário de sono e actigrafia de pulso durante 12 noites consecutivas, nas quais foram divididas em 2 dois períodos. Um período de 5 noites denominado de privação parcial do sono (dormir<5h por noite) e outro de 5 noites denominado de sono controle (dormir>7h por noite). Entre estes períodos, foi interposto por 2 noites de sono irrestrito (com pelo menos de 7 horas de sono por noite). A escolha do período inicial de sono foi randomizada. Ao término de cada período de 5 dias, foi analisada a reatividade vascular venosa (com a técnica da veia do dorso da mão, Dorsal Hand Vein), a sonolência diurna excessiva (através da Escala de Sonolência de Epworth), realizada avaliação hemodinâmica e autonômica (no momento em repouso e mediante o teste de inclinação postural tilt test), exames de sangue e dosagem de norepinefrina plasmática. A freqüência cardíaca e pressão arterial de batimento a batimento na posição supina e com a manobra de \"tilt test\" foi monitorado com intervalo (RR) e variabilidade de pressão arterial. Resultados: Os indivíduos dormiram em média 8.0 h durante o período de sono controle e 4.5 h no período de privação parcial do sono, sendo a diferença significativa entre os mesmos (p<0.01). O período privação de sono não mudou a frequência cardíaca e a pressão arterial basal significativamente, mas promoveu um aumento significante em baixas freqüências cardíacas e variabilidade da pressão arterial, como também na norepinefrina plasmática. O \"tilt test\" promoveu uma queda em PA sistólica depois da privação parcial do sono, que foi significativamente maior depois do período de sono controle (p<0.05). A privação parcial do sono causou uma redução significantiva na venodilatação endotélio-dependente e não mudou venodilatação endotélio-independente. Conclusão: Privação parcial do sono durante só 5 noites já é o suficiente para causar disfunção endotelial venosa, um aumento significantivo na atividade simpática e no prejuízo do controle da pressão arterial / Background: Sleep curtailment is a serious and common problem in western societies and can have significant consequences in the human physiology. In fact epidemiological studies showed that sleep deprivation (reduction in sleeping time) is associated with increased blood pressure, higher incidence of diabetes mellitus, myocardial heart attack, strokes in the brain, and stress, however the mechanisms are poorly understood. Objectives: Evaluate the impact of partial sleep deprivation in the venous endothelial function and the autonomic cardiovascular autonomic control in healthy men. Methods: Thirteen healthy male volunteers (average age: 31±2 years) had their sleep monitored by sleep diary and wrist actigraphy during 12 consecutive nights, these were divided into two periods. The subjects were randomized and crossed over to 5 nights of control sleep (> 7hs) and 5 nights of partial sleep deprivation (<5hs), interposed by 2 nights of unrestricted sleep (at least 7 hours sleep per night). The choice of the initial sleeping period was randomized. At the end of each period of 5 days heart rate and beat-to-beat blood pressure in the supine position and head up tilt test maneuver were monitored with off line determination of RR-interval and blood pressure variability. In addition, serum norepinephrine and venous endothelial functions were measured by dorsal hand vein technique; also we performed the evaluation of excessive day sleepiness (evaluated through the Epworth Sleepiness Scale), hemodynamic and autonomous evaluation (during sleep and through the tilt test). Results: The subjects slept 8.0 and 4.5 hs during control and partial sleep deprivation periods, respectively (p<0.01). Sleep deprivation did not change significantly the resting heart rate and blood pressure but promoted a significant increase in the low frequency bands of heart rate and blood pressure variability as well as serum norepinephrine. Tilt test promoted a significantly greater drop in systolic BP after partial sleep deprivation than after control sleep (p<0.05). Partial sleep deprivation caused a considerable reduction of acetylcholine induced venodilatation (endothelium dependent) and did not change sodium nitroprusside venodilatation (independent from the endothelium). Conclusion: Partial sleep deprivation for only 5 nights is sufficient to cause significant increase in sympathetic activity, impairment of blood pressure control and endothelial dysfunction

Introdução. A prática regular do exercício de força (EF) está associada a adaptações metabólicas, neuromusculares e cardiovasculares que repercutem de maneira positiva sobre a saúde e qualidade de vida de seus praticantes. No entanto, Indivíduos sedentários apresentam comprometimentos agudos na função endotelial após EF de alta intensidade. Objetivo. Avaliar a função endotelial de indivíduos sedentários de meia idade em resposta a diferentes intensidades do EF. Métodos. 11 indivíduos sedentários (40,1±3,9 anos; 27,3±1,4 kg/m2) realizaram EF em três condições experimentais: extensão de joelhos a 50% de 1RM (MI), 80% de 1RM (AI) e repouso na condição controle (CON). Foi realizada avaliação da vasodilatação mediada pelo fluxo (FMD) antes, 30 minutos após e 60 minutos após os protocolos. A quantificação das concentrações de NO2 e NO3 (NOx), endotelina-1 (ET-1) e TBARS foram realizadas antes, imediatamente após e 60 minutos após os protocolos. A pressão arterial foi mensurada antes e após os protocolos Resultados. A FMD aumentou significativamente 30 minutos após o exercício na condição MI (12,5± 4,10 para 17,2±3,9 %; p=0,01) bem como os níveis de NOx (6,8± 3,3 vs. 12,6± 4,2μM; p= 0,007). A concentração de ET-1 aumentou imediatamente após na condição AI (20,02±2,2 vs. 25,4± 2,1pg/ml; p= 0,004). A elevação da pressão arterial não diferiu entre as condições MI e AI. As concentrações de TBARS não se alteraram ao longo dos protocolos. Conclusão. O EF de moderada intensidade aumenta a FMD e os níveis NOx após uma sessão aguda de exercício em indivíduos sedentários de meia idade, estes resultados sugerem que menores intensidades do EF são mais seguras ao iniciar um programa de exercícios. / Regular resistance exercise (RE) is associated with metabolic, neuromuscular and cardiovascular adaptation that results in improvement of quality of life and health. However, sedentary subjects have been showing an acute impairment on endothelial function after high intensity resistance exercise. The aim of this study was to evaluate the endothelial function in sedentary middle age men after RE in different intensities. Methods. Eleven middle age sedentary men (40,1±3,9 years; 27,3±1,4 kg/m2) performed RE in three different conditions: knee extension at 50% of one 1RM (MI), at 80% of 1RM (HI) and rest in the control group (CON). Flow mediated dilation (FMD) was assessed before, 30 and 60 minutes of exercise. Venus plasma concentration of ET-1 NOx and TBARS were measured before, immediately after and 60 minutes after exercise. Blood pressure was evaluated before and after exercise. Results. There was a significant improvement in FMD 30 minutes after exercise in the MI condition (12,5± 4,10 vs 17,2±3,9%; p= 0,016; p=0,01). The plasma NOx concentration was significant higher immediately after MI (6,8± 3,3 vs. 12,6± 4,2μM; p= 0,007). There was a significant improvement in the plasma ET-1 concentration immediately after HI (20,02±2,2 vs. 25,4± 2,1pg/ml; p= 0,004). There was no significant difference in the BP between the experimental conditions (MI vs HI) and TBARS throughout the experimental conditions. Conclusions. Resistance exercise performed in moderate intensity improve endothelial function in sedentary middle aged men, there results suggest that lower intensities of RE could be safe for this population in the beginning of the exercise programs.