The doctoral dissertacion is focused on the role of inflammation in the pathogenesis of preterm labor. In the first part, we describe the current view on pathophysiology of preterm labor. In the second part, we evaluated the relationship of specific markers of inflammation and endothelial dysfunction to preterm birth and fetal inflammatory response. The most important findings of our study was that we found decreased levels of MMP-2 and decreased levels of sRAGE in women with preterm labor in comparison with the control group of pregnant women. Similarly, we found decreased levels of MMP-2 in women with subsequent diagnosed fetal inflammatory response. sRAGE is currently ranked among patttern recognition receptors. In the case of sRAGE we followed the results of our pilot project, it can be assumed that the its low level are connected with tissue damage. We confirmed that it can play an important role in the pathogenesis of preterm labor. We assume abnormal regulatory mechanisms of the production of MMP-2. In both cases, however, further studies are required to elucidate the functional significance of our results.

Contexto: A suplementação de ácido fólico (AF) melhora a função endotelial de indivíduos infectados pelo HIV em uso contínuo de terapia antirretroviral (TARV). A literatura não demonstra com clareza esse benefício em indivíduos coinfectados HIV-HCV. Introdução: Indivíduos infectados pelo HIV ou em coinfecção pelo HIV-HCV apresentam um conjunto de fatores de risco que podem levar a disfunção endotelial. Estudos demonstram que a administração de AF possui efeitos benéficos sobre a função endotelial de diferentes populações com risco cardiovascular, inclusive em HIV monoinfectados. Objetivo: Determinar o efeito da suplementação de AF por quatro semanas sobre a dilatação mediada (FMD) pelo fluxo da artéria braquial em indivíduos infectados pelo HIV ou HIV-HCV em uso contínuo de TARV. Delineamento: Ensaio clínico randomizado (ECR), controlado por placebo. Local: Ambulatório de doenças infecciosas do Hospital Universitário de Santa Maria. População: Foram avaliados 69 indivíduos com idade entre 18-50 anos, de ambos os sexos, infectados pelo HIV com ou sem coinfecção pelo HCV, em TARV e com carga viral indetectável há mais de seis meses. Excluíram-se participantes com diabetes mellitus, infarto agudo do miocárdio, revascularização miocárdica, ou acidente vascular encefálico prévios, com creatinina >1,5 mg/dL, diagnóstico clínico, ecográfico, endoscópico ou laboratorial de cirrose hepática, em uso de estatinas, fibratos, terapia de reposição hormonal, sulfonamidas, suplementos vitamínicos, ou AF nos últimos 30 dias. Adicionalmente foram excluídas mulheres grávidas, e participantes de outro ECR. Intervenção: Indivíduos alocados para o grupo intervenção receberam AF, 5 mg, via oral, em dose única diária, pela manhã, durante quatro semanas. Os participantes alocados para grupo placebo receberam orientação para seguir a mesma posologia, sendo os comprimidos indistinguíveis em cor, aroma, sabor, forma e tamanho. Desfechos: Utilizaram-se as variações nos níveis de homocisteína, vitamina B12 e na FMD, aferida por Doppler, na artéria braquial, obtidos na randomização e ao final do seguimento. Resultados: Realizou-se o rastreamento de 239 participantes, sendo 72 elegíveis, 69 randomizados e 68 acompanhados, entre outubro de 2012 e julho de 2013. Em indivíduos infectados pelo HIV houve aumento significativo nos níveis de AF (12,8 ng/ml) no grupo intervenção em comparação ao placebo (P<0,001), acompanhada de variação negativa de homocisteína (-1,9 umol/l) no grupo intervenção e aumento mínimo no grupo placebo (P<0,001). Não houve variação significativa na FMD (P=0,9). Entre indivíduos coinfectados por HIV-HCV, a variação nos níveis de AF foi decorrente da elevação no grupo intervenção (12,6 ng/ml) e redução no grupo placebo (-0,7 ng/ml) (P<0,001). Os níveis de homocisteína aumentaram no grupo placebo (4,6 umol/l) e diminuíram no grupo intervenção (-1,0 umol/l) (P<0,0003). Em relação ao FMD, houve tendência à redução percentual no grupo intervenção e aumento no grupo placebo (P=0,007). As variações de vitamina B12 não foram significativas, independente do status de infecção para HCV. Conclusão: Esse estudo demonstrou que a suplementação de AF por um curto período de tempo, esteve associada com redução de homocisteína sérica, mas não modificou a FMD da artéria braquial, aferida por Doppler, em indivíduos adultos infectados pelo HIV ou HIV-HCV em uso de TARV. / Context: The supplementation of folic acid (FA) improves endothelial function in HIV-infected individuals in continuous use of highly active antiretroviral therapy (HAART). The literature does not clearly show this benefit in coinfected HIV-HCV. Introduction: Individuals infected with HIV or HIV-HCV coinfected presents a set of risk factors that can lead to endothelial dysfunction. Studies show that FA management has beneficial effects on endothelial function in different populations with cardiovascular risk, including HIV monoinfected. Objective: To determine the effect of FA supplementation for four weeks on the mediated dilation (FMD) by brachial artery flow in patients infected with HIV or HIV-HCV continuous HAART. Design: Randomized clinical trial (RCT), placebo-controlled study. Location: Division of Infectious Diseases, University Hospital of Santa Maria. Population: A total of 69 subjects aged 18-50 years, of both sex, HIV or HIV-HCV infected, on HAART, with undetectable viral load for more than six months. Patients presenting with diabetes mellitus, acute myocardial infarction, coronary revascularization, or stroke prior, with creatinine >1,5 mg/dL, clinical diagnosis, ultrasound, endoscopic or laboratory evidence of liver cirrhosis, on statins, fibrates, hormone replacement therapy, sulfonamides, vitamin supplements, or FA in the last 30 days. In addition pregnant women were excluded, and participants in a RCT. Intervention: Subjects allocated to the intervention group received FA, 5 mg orally once daily in the morning for four weeks. Participants allocated to placebo group were instructed to follow the same dosage, being indistinguishable tablets in color, aroma, taste, shape and size. Outcomes: changes were used in the levels of homocysteine, vitamin B12 and FMD, measured by Doppler, in the brachial artery obtained at randomization and at the end of follow-up. Results: We carried out the screening of 239 participants, 72 eligible, 69 randomized and 68 accompanied, between October 2012 and July 2013. In HIV-infected patients there was a significant increase in the levels of FA (12.8 ng/ml) in the intervention group compared to placebo (P <0.001), accompanied by negative variation of homocysteine (1.9 umol/L) in the group intervention and minimal increase in the placebo group (P <0.001). There was no significant change in FMD (P = 0.9). Between individuals coinfected with HIV-HCV, the variation in FA levels was due to the increase in the intervention group (12.6 ng/ml) and reduction in the placebo group (-0.7 ng / ml) (P <0.001). Homocysteine levels increased in the placebo group (4.6 umol/L) and decreased in the intervention group (-1.0 umol/L) (P <0.0003). Regarding the FMD, there was a tendency to percentage reduction in the intervention group and increased in the placebo group (P = 0.007). Variations of B12 were not significant, independent of HCV infection status. Conclusion: This study showed that AF supplementation for a short term, was associated with reduced serum homocysteine, but did not change the FMD of the brachial artery, measured by Doppler in adults infected with HIV or HIV-HCV in HAART.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2018-09-11T10:46:39Z No. of bitstreams: 2 Tese - Thiago Sardinha de Oliveira - 2018.pdf: 4687551 bytes, checksum: 1e4f2d0e9cb8438779749f49756c0b0b (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2018-09-11T11:12:23Z (GMT) No. of bitstreams: 2 Tese - Thiago Sardinha de Oliveira - 2018.pdf: 4687551 bytes, checksum: 1e4f2d0e9cb8438779749f49756c0b0b (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-09-11T11:12:24Z (GMT). No. of bitstreams: 2 Tese - Thiago Sardinha de Oliveira - 2018.pdf: 4687551 bytes, checksum: 1e4f2d0e9cb8438779749f49756c0b0b (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-08-31 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Estrone (E1), the major component of Premarin® and predominant estrogen in postmenopausal women, does not have its vascular effects involving its vasodilatory mechanism of action completely elucidated. Therefore, the aim of this study was to evaluate the effects of E1 treatment on the vascular reactivity of isolated aortic rings and blood pressure in ovariectomized Wistar rats (OVX). For this purpose, 12 week-old Wistar rats were divided into four experimental groups, Sham (physiological estrous rats treated with vehicle), OVX (OVX rats treated with vehicle), OVX + E1 (OVX rats treated with 825μg / kg of E1) and OVX + 17β-estradiol (E2) (OVX rats treated with 15μg / kg of E2). The treatments started after the 8 weeks of surgery trough subcutaneous pathway for 30 days. At the end of treatment, blood pressure measurement by tail plethysmograph was performed. In addition, aortic rings were isolated to evaluate contractile response to phenylephrine (Phe), relaxation to acetylcholine (ACh) or sodium nitroprusside (NPS) by means of concentration curves. The response to ACh in rings previously incubated with superoxide dismutase (SOD), catalase (CAT) or apocynin (NADPH oxidase inhibitor) was also evaluated. The protein expression of SOD, CAT, NOX 1, NOX 2 and NOX 4 were quantified by Western blotting. Follow-up of the weight gain after the OVX or Sham procedure was also performed. After euthanasia, the weight of retroperitoneal fat, uterus and heart were evaluated. E1 treatment decreased body weight and retroperitoneal fat, increased uterine weight, and corrected both the increased blood pressure, and the decreased hyperreactivity to Phe vasoconstrictor agent and also increased endothelium-dependent vasodilatory response to ACh. The effects presented by this hormone are related to compensatory mechanisms in the activity of antioxidant enzymes such as SOD and catalase besides the reduction in the expression of the NADPH oxidase NOX 4 isoform. In addition, E1 reverses the increase in total and LDL cholesterol in the OVX group. Our study confirms the role of oxidative stress in endothelial dysfunction of OVX rats and further demonstrates that E1 reverses elevation of blood pressure and restores endothelial function in OVX rats. / A estrona (E1), componente majoritário do Premarin® e estrogênio predominante na circulação feminina na pós-menopausa, não possui seus efeitos vasculares e mecanismos de ação completamente elucidados. Diante disso, o objetivo do presente trabalho foi avaliar os efeitos do tratamento com E1 em ratas Wistar ovariectomizadas (OVX) sobre a reatividade de anéis de aorta e pressão arterial. Para este estudo, ratas com 12 semanas de idade foram divididas em quatro grupos experimentais, Sham (ratas em estro fisiológico tratadas com veículo), OVX (ratas OVX tratadas com veículo), OVX + E1 (ratas OVX tratadas com 825μg/Kg de E1) e OVX + 17β-estradiol (E2) (ratas OVX tratadas com 15μg/Kg de E2). Os tratamentos foram iniciados após 8 semanas da cirurgia pela via subcutânea pelo período de 30 dias. Ao final do tratamento, foi realizada a medida de pressão arterial por pletismografia de cauda.Além disso, anéis de aorta foram isolados para avaliar resposta contrátil à fenilefrina (Phe), relaxamento à acetilcolina (ACh) ou ao nitroprussiato de sódio (NPS) por meio de curvas de concentração efeito cumulativas. Foi ainda avaliada a resposta à ACh em anéis incubados previamente com superóxido dismutase (SOD), catalase (CAT) ou apocinina (inibidor da NADPH-oxidase (NOX)). A expressão protéica da SOD, CAT, NOX 1, NOX 2 e NOX 4 foi quantificada por Western-blot. Foi realizado o acompanhamento do ganho de peso após o procedimento de ovariectomia nos grupos OVX ou Sham e, após a eutanásia, foi avaliado o peso da gordura retroperitoneal, útero e coração. O tratamento com E1 diminuiu o peso corporal e a gordura retroperitoneal, aumentou o peso uterino, normalizou os níveis de pressão arterial, o aumento da resposta contrátil à Phe e a resposta vasodilatadora dependente de endotélio à ACh. Os efeitos apresentados por este hormônio estão relacionados com mecanismos compensatórios na expressão e atividade de enzimas antioxidantes como SOD e CAT, além da redução na expressão da isoforma NOX 4. Além disso, a E1 reverteu o aumento do colesterol total e LDL observados nas ratas OVX. Os dados apresentados demonstram o papel benéfico da E1 frente ao estresse oxidativo na disfunção vascular, restabelecendo a função endotelial e os níveis fisiológicos da pressão arterial.

Contexto: A suplementação de ácido fólico (AF) melhora a função endotelial de indivíduos infectados pelo HIV em uso contínuo de terapia antirretroviral (TARV). A literatura não demonstra com clareza esse benefício em indivíduos coinfectados HIV-HCV. Introdução: Indivíduos infectados pelo HIV ou em coinfecção pelo HIV-HCV apresentam um conjunto de fatores de risco que podem levar a disfunção endotelial. Estudos demonstram que a administração de AF possui efeitos benéficos sobre a função endotelial de diferentes populações com risco cardiovascular, inclusive em HIV monoinfectados. Objetivo: Determinar o efeito da suplementação de AF por quatro semanas sobre a dilatação mediada (FMD) pelo fluxo da artéria braquial em indivíduos infectados pelo HIV ou HIV-HCV em uso contínuo de TARV. Delineamento: Ensaio clínico randomizado (ECR), controlado por placebo. Local: Ambulatório de doenças infecciosas do Hospital Universitário de Santa Maria. População: Foram avaliados 69 indivíduos com idade entre 18-50 anos, de ambos os sexos, infectados pelo HIV com ou sem coinfecção pelo HCV, em TARV e com carga viral indetectável há mais de seis meses. Excluíram-se participantes com diabetes mellitus, infarto agudo do miocárdio, revascularização miocárdica, ou acidente vascular encefálico prévios, com creatinina >1,5 mg/dL, diagnóstico clínico, ecográfico, endoscópico ou laboratorial de cirrose hepática, em uso de estatinas, fibratos, terapia de reposição hormonal, sulfonamidas, suplementos vitamínicos, ou AF nos últimos 30 dias. Adicionalmente foram excluídas mulheres grávidas, e participantes de outro ECR. Intervenção: Indivíduos alocados para o grupo intervenção receberam AF, 5 mg, via oral, em dose única diária, pela manhã, durante quatro semanas. Os participantes alocados para grupo placebo receberam orientação para seguir a mesma posologia, sendo os comprimidos indistinguíveis em cor, aroma, sabor, forma e tamanho. Desfechos: Utilizaram-se as variações nos níveis de homocisteína, vitamina B12 e na FMD, aferida por Doppler, na artéria braquial, obtidos na randomização e ao final do seguimento. Resultados: Realizou-se o rastreamento de 239 participantes, sendo 72 elegíveis, 69 randomizados e 68 acompanhados, entre outubro de 2012 e julho de 2013. Em indivíduos infectados pelo HIV houve aumento significativo nos níveis de AF (12,8 ng/ml) no grupo intervenção em comparação ao placebo (P<0,001), acompanhada de variação negativa de homocisteína (-1,9 umol/l) no grupo intervenção e aumento mínimo no grupo placebo (P<0,001). Não houve variação significativa na FMD (P=0,9). Entre indivíduos coinfectados por HIV-HCV, a variação nos níveis de AF foi decorrente da elevação no grupo intervenção (12,6 ng/ml) e redução no grupo placebo (-0,7 ng/ml) (P<0,001). Os níveis de homocisteína aumentaram no grupo placebo (4,6 umol/l) e diminuíram no grupo intervenção (-1,0 umol/l) (P<0,0003). Em relação ao FMD, houve tendência à redução percentual no grupo intervenção e aumento no grupo placebo (P=0,007). As variações de vitamina B12 não foram significativas, independente do status de infecção para HCV. Conclusão: Esse estudo demonstrou que a suplementação de AF por um curto período de tempo, esteve associada com redução de homocisteína sérica, mas não modificou a FMD da artéria braquial, aferida por Doppler, em indivíduos adultos infectados pelo HIV ou HIV-HCV em uso de TARV. / Context: The supplementation of folic acid (FA) improves endothelial function in HIV-infected individuals in continuous use of highly active antiretroviral therapy (HAART). The literature does not clearly show this benefit in coinfected HIV-HCV. Introduction: Individuals infected with HIV or HIV-HCV coinfected presents a set of risk factors that can lead to endothelial dysfunction. Studies show that FA management has beneficial effects on endothelial function in different populations with cardiovascular risk, including HIV monoinfected. Objective: To determine the effect of FA supplementation for four weeks on the mediated dilation (FMD) by brachial artery flow in patients infected with HIV or HIV-HCV continuous HAART. Design: Randomized clinical trial (RCT), placebo-controlled study. Location: Division of Infectious Diseases, University Hospital of Santa Maria. Population: A total of 69 subjects aged 18-50 years, of both sex, HIV or HIV-HCV infected, on HAART, with undetectable viral load for more than six months. Patients presenting with diabetes mellitus, acute myocardial infarction, coronary revascularization, or stroke prior, with creatinine >1,5 mg/dL, clinical diagnosis, ultrasound, endoscopic or laboratory evidence of liver cirrhosis, on statins, fibrates, hormone replacement therapy, sulfonamides, vitamin supplements, or FA in the last 30 days. In addition pregnant women were excluded, and participants in a RCT. Intervention: Subjects allocated to the intervention group received FA, 5 mg orally once daily in the morning for four weeks. Participants allocated to placebo group were instructed to follow the same dosage, being indistinguishable tablets in color, aroma, taste, shape and size. Outcomes: changes were used in the levels of homocysteine, vitamin B12 and FMD, measured by Doppler, in the brachial artery obtained at randomization and at the end of follow-up. Results: We carried out the screening of 239 participants, 72 eligible, 69 randomized and 68 accompanied, between October 2012 and July 2013. In HIV-infected patients there was a significant increase in the levels of FA (12.8 ng/ml) in the intervention group compared to placebo (P <0.001), accompanied by negative variation of homocysteine (1.9 umol/L) in the group intervention and minimal increase in the placebo group (P <0.001). There was no significant change in FMD (P = 0.9). Between individuals coinfected with HIV-HCV, the variation in FA levels was due to the increase in the intervention group (12.6 ng/ml) and reduction in the placebo group (-0.7 ng / ml) (P <0.001). Homocysteine levels increased in the placebo group (4.6 umol/L) and decreased in the intervention group (-1.0 umol/L) (P <0.0003). Regarding the FMD, there was a tendency to percentage reduction in the intervention group and increased in the placebo group (P = 0.007). Variations of B12 were not significant, independent of HCV infection status. Conclusion: This study showed that AF supplementation for a short term, was associated with reduced serum homocysteine, but did not change the FMD of the brachial artery, measured by Doppler in adults infected with HIV or HIV-HCV in HAART.

O endotélio vascular desempenha um papel central no controle do tônus vascular pela liberação de fatores relaxantes derivados do endotélio (EDRFs) e fatores contráteis derivados do endotélio (EDCFs). O óxido nítrico (NO) é um dos mais importantes mediadores da vasodilatação, sua produção é catalisada pelas enzimas NO-sintases (NOS). A eNOS é constitutiva e depende do Ca2+ para ser ativada. A fosfolipase A2 (cPLA2), também é dependente de Ca2+, esta enzima é responsável pela conversão de fosfolipídeos de membrana a ácido araquidônico, o precursor de prostanóides, tais como prostaciclina (PGI2) e tromboxano (TXA2). A disfunção endotelial está relacionada com uma menor biodisponibilidade de NO e maior produção de EDCFs e está presente em várias doenças cardiovasculares, como hipertensão arterial. Embora esta disfunção seja multifatorial, o aumento da produção de espécies reativas de oxigênio (EROs) parece contribuir de forma considerável. O aumento na [Ca2+]c está relacionado com o aumento na produção de EROs e liberação de EDCFs. Em nosso estudo, utilizamos o ionóforo de cálcio A23187 para avaliar as alterações na sinalização celular decorrentes da mobilização de cálcio, independente da ativação de receptores, que ocorrem na hipertensão arterial. O objetivo deste trabalho foi estudar as respostas vasculares desencadeadas pelo aumento da [Ca2+]c promovido pelo A23187 em aorta de ratos normotensos (2R) e hipertensos renais (2R-1C). Verificamos que o A23187 induz efeito vasodilatador dependente da produção de NO em aortas de ratos 2R e 2R-1C, pois o relaxamento foi abolido na presença do inibidor da NOS (L-NAME), bem como em aortas sem endotélio. Em aortas de ratos 2R-1C, mas não em aortas de ratos 2R, verificamos a participação da PGI2 no efeito vasodilatador induzido pelo A23187. A produção de PGI2 está aumentada em aortas de ratos 2R-1C em comparação com aortas de ratos 2R, o que indica que esse prostanóide está ativando receptores TP, induzindo contração. O A23187 induziu efeito contrátil de forma independente do endotélio em aorta de ratos 2R. Entretanto, em aortas com endotélio, de ratos 2R-1C o efeito contrátil está prejudicado. O efeito anti-contrátil em aortas de ratos 2R-1C é devido à produção de NO, que está aumentada a ponto de impedir a contração induzida pelo A23187. Verificamos que a contração induzida pelo A23187 é dependente da produção dos prostanóides contráteis, TXA2 e PGI2, que ativam os receptores TP, pois quando utilizamos o inibidor da ciclooxigenase (ibuprofeno) o efeito contrátil foi atenuado. Além disso, quando utilizamos o antagonista dos receptores TP (SQ29548) o efeito foi completamente abolido. O estímulo com A23187 aumentou a produção de TXA2 em aorta de ratos 2R e 2R-1C. Porém, a produção em aorta de ratos 2R-1C foi maior do que aquela observada em aorta de ratos 2R. Observamos que a catalase atenuou a resposta contrátil induzida pelo A23187, demonstrando que o peróxido de hidrogênio modula positivamente a contração induzida pelo A23187. / The vascular endothelium plays a pivotal role in the vascular tone due to the release of relaxing factors (EDRFs) and contractile factors (EDCFs). Nitric oxide (NO) is one of the most important EDRFs involved in the vasodilation and it is produced by the NO-synthases (NOS). eNOS is a constitutive isoform and its activity is dependent of the transient calcium. Besides eNOS, other important enzymes are modulated by Ca2+ such as phospholipase A2 (cPLA2). This enzyme converts membrane phospholipids to araquidonic acid, responsible for the formation of the prostanoids prostaciclin (PGI2) and thromboxane (TXA2). Endothelial dysfunction is related to the decreased NO bioavailability and increased production of EDCFs. It is present in several cardiovascular disorders like hypertension. Endothelial dysfunction is a multifactorial proccess that is also caused by the increased production of reactive oxygen species (ROS). Increased cytosolic calcium concentration ([Ca2+]c) is related to augmented ROS and EDCFs production. In the present study, we have used the calcium ionophore A23187 in order to evaluate the altered cellular signaling caused by [Ca2+]c in a receptor activation-independent way that occurs in hypertension. This work aimed to study the vascular responses stimulated by A23187 in normotensive rat (2K) aorta and in renal hypertensive (2K-1C) rat aorta. We have verified that A23187 induces vasodilator effect dependent on the production of NO in 2K and 2K- 1C rat aortas. The vascular relaxation was abolished by the non-selective NOS inhibitor (L-NAME) and by the endothelium removal. In 2K-1C but not in 2K rat aortas, PGI2 contributes to He vasodilator effect induced by A23187. PGI2 production is greater in 2K-1C than in 2K rat aortas, which suggests that PGI2 activates TP receptors inducing contraction. The contractile effect of A23187 is endotheliumdependent in 2K rat aorta. However, in 2K-1C intact-endothelium aortas, the contractile effect of A23187 is impaired. The anti-contractile effect is due to increased NO production that inhibits the contractile response to A23187. The contractile response induced by A23187 is dependent of the prostanoid production like TXA2 and PGI2 that activate TP receptors because this response is inhibited by the cyclooxygenase inhibitor (ibuprofen). In addition, this effect was abolished by the TP receptor antagonist (SQ29548). TXA2 production was stimulated with A23187 in 2K and 2K-1C rat aorta, which was greater in 2K-1C than in 2K rat aorta. We have also observed that catalase blunted the contractile response induced by A23187. These results suggest that hydrogen peroxide positively modulates A23187-induced contractile response.

O endotélio vascular é responsável por várias funções como o controle do tônus vascular pela produção e/ou liberação de substâncias vasoconstritoras (EDCFs) e relaxantes (EDRFs). Na hipertensão arterial ocorre disfunção endotelial caracterizada pelo desequilíbrio entre EDCFs e EDRFs. Vários autores sugerem que essas alterações são decorrentes do aumento nas concentrações de espécies reativas de oxigênio (EROs). As EROs podem afetar a sinalização, produção e/ou biodisponibilidade do óxido nítrico (NO), principal EDRF, assim como aumentar os níveis de prostanóides como prostaglandinas e tromboxanos, EDCFs produtos da COX. O principal alvo para o NO é a ativação da guanilil ciclase solúvel (GCs) no musculo liso vascular causando vasorelaxamento. No modelo de hipertensão arterial dois rins um clipe (2R-1C), ocorre aumento nos níveis de EROs e ativação da NADH/NADPH-oxidase, principal enzima produtora de EROs em células endoteliais. Em nosso estudo, utilizamos o composto NCX2121, que é estruturalmente formado por um doador de NO e inibidor da COX (indometacina). O estudo teve por objetivo caracterizar farmacologicamente a resposta relaxante do NCX2121 na aorta de ratos hipertensos 2R-1C e investigar a contribuição do endotélio vascular e das EROs para essa resposta. Verificamos que o composto NCX 2121 produz relaxamento da aorta de ratos 2R e 2R-1C, que é reduzido pela remoção do endotélio e inibição da enzima NO-Sintase (NOS). O relaxamento do composto NCX 2121 deve ser promovido pelo NO, uma vez que em aortas sem endotélio esse relaxamento foi abolido pelo ODQ. Porém, foi apenas reduzido em aortas com endotélio, isoladas de ratos normotensos (2R) e não foi alterado nas aortas com endotélio, isoladas de ratos 2R-1C. O NCX 2121 não alterou a fosforilação dos sítios de ativação ou inibição da eNOS. O NO não foi detectado em solução por análise amperométrica. O composto NCX2121 aumentou a concentração citosólica de NO, medida pela sonda fluorescente sensível a NO (DAF-2DA), por microscopia confocal. Na aorta de ratos 2R-1C, o relaxamento estimulado com o composto NCX2121 foi inibido pelas EROs e os níveis de EROs em células endoteliais isoladas, foi reduzido pelo composto NCX2121. O composto NCX 2121 reduziu os níveis de tromboxano na aorta de ratos 2R e 2R-1C. Os nossos resultados demonstram que o composto NCX2121 promove relaxamento pela liberação intracelular de NO e inibição da COX por reduzir a produção de prostanóides vasoconstrictores como o tromboxano. O composto NCX2121 não interfere com a ativação da NOS, mas reduz as EROs nas células endoteliais. / The vascular endothelium plays multiple roles on the tone control by the production and/or release of contractile factors (EDCFs) and relaxing factors (EDRFs). There is an imbalance between EDCFs and EDRFs in hypertension that is defined by endothelial dysfunction. In accordance to several authors, these alterations are due to increased production of reactive oxygen species (ROS). The ROS can affect the nitric oxide (NO) signaling, production and bioavailability that is the major EDRF. ROS can also increase the levels of prostaglandins and thromboxane (TX) that are EDCFs products of COX. The main target for NO is the activation of soluble guanylyl-cyclase (sGC) in the vascular smooth muscle cells causing vasorelaxation. In renal hypertensive rats (2K-1C), there is an increased production of ROS by NADH-NADPH-oxidase in the rat aorta endothelial cells. In the present study we used the compound NCX2121, in which chemical structure there is a NO donor and a non-selective COX inhibitor indomethacin. This study aimed to pharmacologically characterize the NCX2121 relaxing effect in 2K-1C rat aorta, and to investigate the contribution of the endothelial factors and ROS for this response. We verified that the relaxation-induced by NCX2121 was impaired by the endothelium removal and NO-synthase (NOS) inhibition. The relaxation induced by NCX2121 is due to NO, since sGC inhibition by ODQ completely abolished its effect in denuded endothelium 2K-1C rat aorta. However, in intact endothelium normotensive 2K rat aorta, the relaxing effect of NCX2121 was only partially inhibited whereas in 2K-1C it was not changed. NCX2121 did not change the phosphorylation sites of activation or inhibition of NOS. NO was not detected by amperometry in the organ bath during the relaxation induced by NCX2121, but it was measured in the cell cytoplasm by confocal microscopy. The vasorelaxation was inhibited by ROS, and NCX2121 decreased the ROS in isolated endothelial cells. NCX2121 reduced TX in 2K and 2K-1C rat aortas. Therefore, our results indicate that the compound NCX2121 induces relaxation by intracellular NO release and COX inhibition by the reduced production of contractile prostanoids such as TX. The compound NCX2121 does not modulate NOS, but it decreases ROS in the endothelial cells.

L'endothélium artériel pulmonaire joue un rôle déterminant dans la régulation du tonus vasomoteur en libérant des substances vasodilatatrices et vasoconstrictrices. Toute perturbation des fonctions endothéliales entraîne une perte de l'équilibre physiologique très finement régulé entre réponse vasoconstrictrice et vasodilatatrice, avec perte conjointe du contrôle de la prolifération des cellules musculaires lisses vasculaires et de l'effet antiagrégant plaquettaire. Ces modifications conduisent à un remodelage du lit vasculaire pulmonaire avec une augmentation des résistances vasculaires parfois à l'origine d'une hypertension pulmonaire (HTP) irréversible. Le rôle de l'hypoxémie n'y est pas exclusif. Le réseau bronchique est soumis quant à lui à des agressions physiques par les particules inhalées. Celles-ci peuvent entraîner un remodelage bronchique et une perturbation du tonus bronchique et de la broncho-réactivité, dont les médiateurs peuvent être en partie partagés par la dysfonction vasculaire.Le but de ce travail était d'évaluer dans un premier temps l'existence d'une dysfonction endothéliale, en tant que précurseur potentiel d'une HTP, dans deux modèles de bronchopathies chroniques à des stades opposés de la sévérité de l'atteinte respiratoire : la mucoviscidose terminale et le tabagisme avec peu ou pas d'impact sur la fonction respiratoire.Nous avons ainsi montré à partir d'explants pulmonaires qu'une dysfonction endothéliale était fréquente au cours de la mucoviscidose au stade pré-greffe et qu'elle était au-moins en partie liée à une surexpression vasculaire pulmonaire de l'ET-1 et de ses récepteurs ET-A.Nous avons montré par ailleurs que 25% environ des sujets fumeurs à fonction respiratoire normale ou peu altérée présentaient également une telle dysfonction. Nous en avons étudié ici deux voies physiopathologiques potentielles, celle de l'ET-1 et celle qui gouverne la synthèse du NO : la balance NO Synthases (NOS)/arginases. Nous avons mis en évidence une surexpression des récepteurs ET-A de l'ET-1 chez les sujets avec dysfonction ainsi qu'une corrélation inverse entre cette expression et la réponse vasoactive à l'Acétylcholine (Ach). Par ailleurs, si le NO a entre autres un effet anti-prolifératif sur les cellules musculaires lisses, la voie des arginases - système enzymatique compétitif avec les NOS - mène quant à elle à la réparation et au remodelage. Nous avons étudié l'expression vasculaire de ces différentes enzymes ainsi que l'effet pharmacologique d'inhibiteurs des NOS ou des arginases sur la réponse vasoactive à l'Ach. Nous avons montré qu'il n'existait pas de déficit d'expression des NOS et qu'il ne semblait pas y avoir d'effet délétère des arginases dans la dysfonction endothéliale dans ce modèle.Parallèlement aux mécanismes sous-tendant le remodelage vasculaire au cours du tabagisme nous avons recherché l'existence d'une « dysfonction » bronchique chez ces fumeurs, qui pourrait précéder le remodelage bronchique qui les caractérise. La présence d'une hyperréactivité bronchique est un marqueur prédictif de remodelage des voies aériennes et de l'obstruction bronchique qui en découle. Le rôle de la balance NOS/arginases dans le contrôle du tonus bronchique est encore méconnu. Notre premier objectif était d'évaluer l'expression de cette balance enzymatique dans le tissu bronchique de ces patients et le second d'étudier les effets de l'inhibition des NOS et des arginases sur la réponse bronchoconstrictrice à l'Ach. Nous avons montré qu'une augmentation d'expression bronchique de la NOS 2 chez les fumeurs était impliquée dans la régulation du tonus bronchique et dans l'obstruction bronchique, tandis qu'une augmentation de l'activité des arginases était impliquée dans la sensibilité bronchique. / Pulmonary arteriel endothelium has a key role in the regulation of vascular tone by the release of dilating and constrictive mediators. Impairment of endothlium functions leads to a loss of the physiological equilibrium between vasoconstriction and vasodilation, together with the loss of vascular smooth muscle cells (SMC) proliferation. These alterations induce pulmonary vascular remodeling and elevation of vascular resistance which can lead to an irreversible pulmonary hypertension (PH). The role of hypoxemia is not exclusive. Airways are exposed to physical aggressions by inhaled particles, which can lead to bronchial remodeling and impaired bronchial tone and reactivity, the mediators of which can be partly shared by endothelial dysfunction.Our goal was to evaluate the existence of endothelial dysfunction as a precursor of PH in 2 models of chronic bronchopathy of opposite stages of disease severity: end-stage cystic fibrosis (CF) and tobacco smoking with or without impaired lung function. We showed that in end-stage CF pulmonary explants, endothelial dysfunction is frequent and that it was at least partly due to a vascular upregulation of the endothelin (ET)-1 pathway.Furthermore, approximately ¼ of smokers with normal or poorly impaired lung function also displayed a similar endothelial dysfunction. We studied therein 2 potential physiopathological pathways, that of ET-1 and that which governs nitric oxide (NO) synthesis: the NO synthases (NOS)/arginases balance. We showed an upregulation of ET-A receptor expression and an inverse correlation between this expression and the vasoactive response to acetylcholine (Ach). If NO has an anti mitogenic effect on SMC, the arginases pathway-competitive with the NOS- leads to tissue repair and remodeling.We studied the vascular expression of these enzymes and the pharmacological effect of NOS and arginases inhibitors on response to Ach. We showed that the expression of NOS was not deficient and that arginases did not seem to have a deleterious effect on endothelial function in this model.Concomitantly to these mechanisms leading to vascular remodeling, wr searched for a bronchial dysfunction in smokers which could antecede bronchial remodeling, a well known feature of tobacco smoking. Bronchial hyperresponsiveness is a predictive marker of airway remodeling and subsequent bronchial obstruction. The role of the NOS/arginases pathway in the control of bronchial tone is still unknown. We evaluated the bronchial expression of the NOS/arginases balance in smokers and the effects of NOS and arginases inhibitors on bronchoconstrictive response to Ach. We found that an upregulation of NOS2 expression in COPD patients is involved in airway tone regulation and functional airflow limitation, whereas increased arginase activity is involved in airway sensitivity.

Introduction : Chez les malades porteurs d’une broncho-pneumopathie chronique obstructive (BPCO) modérée à sévère, l’hypoxie induit à la fois une augmentation du tonus vasculaire pulmonaire et la survenue d’un remodelage tissulaire touchant les trois tuniques des vaisseaux pulmonaires. Des données récentes montraient que la voie RhoA/Rho-kinase était impliquée dans le dysfonctionnement endothélial et dans la vasoconstriction induite par l’endothéline-1 (ET-1) chez les sujets tabagiques et les patients porteurs d’une hyperension artérielle pulmonaire (HTAP). Objectifs : Le but de ce travail était de clarifier le rôle de la voie RhoA/Rho-kinase dans la vasodilatation pulmonaire dépendante de l’endothélium et la vasoconstriction pulmonaire induite par l’ET-1, ainsi que les différents niveaux d’interaction entre la voie RhoA/Rho-kinase et celle de la NO synthase endothéliale (NOS-3) chez les patients BPCO post-tabagiques. Matériels et Méthodes : Les prélèvements d’artères pulmonaires et de tissus pulmonaire provenaient des patients porteurs d’un cancer broncho-pulmonaire, opérés pour une lobectomie ou pneumonectomie. L’expression protéique de la NOS-3, des récepteurs de l’ET-1 (ET-A et ET-B), des Rho-kinases (ROCK- 1 et ROCK-2), de la petite protéine G RhoA (sous sa forme activée, liée au GTP, ou sous sa forme inactivée), et de la forme phosphorylée de la sous-unité pMYPT-1 ont été évaluées par immunohistochimie, western blot, et immunoprécipitation. Les activités enzymatiques de la NO synthase et de la Rho-kinase, ainsi que la concentration du GMPc ont été mesurées par la méthode Elisa. L’expression des ARNm de l’ET-1, ET-A, et ET-B a été quantifiée par RT-PCR. Résultats : La relaxation dépendante de l’endothélium des vaisseaux pulmonaires était nettement réduite chez les BPCO par rapport aux témoins (P < 0,05). De façon plus surprenante, nous avions retrouvé la même différence significative entre le groupe témoin et le groupe des patients tabagiques sans TVO. La vasoconstriction pulmonaire en réponse à l’ET-1 était augmentée chez les patients BPCO par rapport aux deux autres groupes (P < 0,05). L’expression protéique de la NOS-3 ainsi que les expressions en ARNm de l’ET-1 et des récepteurs ET-A et ET-B n’étaient pas significativement différentes entre les trois groupes (P > 0,05). L’expression de la RhoA-GTP, ROCK-1, ROCK-2, et pMYPT-1 était augmentée chez les patients BPCO mais également chez les fumeurs sans TVO (P < 0,01 et P < 0,05). L’activité de la NOS-3 était diminuée chez les BPCO avec et sans hypoxémie (P < 0,01, P < 0,05). La concentration du GMPc dans les tissus pulmonaires et dans les artères pulmonaires était diminuée de façon parallèle chez les patients BPCO hypoxémiques par rapport aux BPCO non-hypoxémiques. En revanche, l’activité des Rho-kinases n’était augmentée que chez les patients BPCO (P < 0,001) et ne semblait pas être augmentée chez les fumeurs sans TVO par rapport aux témoins. Conclusion : Notre étude a montré que la voie de signalisation RhoA/Rho-kinase module la fonction endothéliale en diminuant l’expression et l’activité de la NO synthase et en favorisant l’effet de l’ET-1, ce qui aboutit à une augmentation du tonus vasculaire pulmonaire des patients porteurs d’une BPCO post-tabagique mais également chez des fumeurs sans TVO / Background : In patients with moderate to severe chronic obstructive pulmonary disease (COPD), hypoxia induces an increase of pulmonary vascular tonus and vascular remodeling. Recent data showed that RhoA/Rho-kinase pathway had been involved in endothelial dysfunction and vasoconstriction induced by endothelin-1 (ET-1) in smokers and in patients with pulmonary hypertension (PA). Objective : The aims of study were to clarify the role of RhoA/Rho-kinase pathway in pulmonary vasodilatation endothelium dependent and the interaction between Rho-A/Rho-kinase and endothelial NOS (NOS-3) signalization in cigarette smoke-induced COPD patients. Material and Methods : Lung tissues and pulmonary arteries from patients with broncho-pulmonary cancer, operated for lobectomy or pneumonectomy. Protein expression of NOS-3, ET-1 receptors (ET-A and ET-B), Rho-kinase (ROCK-1 and ROCK-2), a small protein G RhoA (active form combined to GTP or inactive forme), and phosphorylated form of pMYPT-1 subunit had been evaluated by immunohistochemistry, western blot, and pull-down assay. Enzyme activity of NO synthase, Rho-kinase, and the concentration of cGMP had been measured by Elisa. mRNA expression had been quantified by RT-PCR. Results : Endothelium-dependent relaxation of pulmonary vessels was clearly reduced in COPD patients in comparison with control subjects (P < 0.05). Surprisingly, we founded the significant difference of relaxation between smokers without airflow obstruction and control subjects. Pulmonary vasoconstriction in response to ET-1 was increased in COPD patients as compared to two other groups (P < 0.05). Protein expression of NOS-3 and mRNA expression of ET-1 and ET-A/ET-B receptors were not significant different between three groups (P > 0.05). Expression of GTP-Rho-A, ROCK-1, ROCK-2, and pMYPT-1 were increased in COPD and in smokers without airway obstruction (P < 0.01 and P < 0.05). NOS-3 activity was reduced in COPD patients with and without hypoxemia (P < 0.01 and P < 0.05). The concentration of cGMP in lung tissues and pulmonary arteries were reduced in hypoxemic COPD patients in comparison with non-hypoxemic COPD patients. Whereas, Rho-kinase activity had been increased predominantly in COPD patients (P < 0.001) than in smokers without COPD as compared to control subjects. Conclusion : The present study showed that RhoA/Rho-kinase pathway modulated the endothelial function by decreasing the expression and activity of NO synthase. It facilitated the effect of ET-1 in pulmonary vasoconstriction. These phenomena produced an increase of pulmonary vascular tonus in cigarette smoke-induced COPD and in smokers without airway obstruction

The development of tissue engineered blood vessel mimics for the testing of intravascular devices in vitro has been established in the Cal Poly tissue engineering lab. Due to the prevalence of cardiovascular disease in diabetic patients and minimal accessible studies regarding the interactions between diabetes and intravascular devices used to treat vascular disease, there is a need for the development of diabetic models that more accurately represents diabetic processes occurring in the blood vessels, primarily endothelial dysfunction. This thesis aimed to create a diabetic blood vessel mimic by implementing a high glucose environment for culturing human endothelial cells from healthy umbilical veins (HUVECs) and from diabetic coronary arteries (DHCAECs). The characterization of these BVMs was achieved using immunofluorescence, scanning electron microscopy (SEM), and qPCR gene expression analysis. From this study, it was determined that HUVECs and DHCAECs are robust enough to be cultured in a high glucose environment – analogous to hyperglycemia – and these cells exhibited different characteristics when evaluated under microscopy and qPCR gene expression. The immunofluorescence and SEM imaging showed presence of cells within each blood vessel mimic. The qPCR gene expression analysis demonstrated that mRNA expression of endothelial nitric oxide synthase (eNOS), platelet endothelial cell adhesion molecule (PECAM), and receptor for advanced glycation end products (RAGE) differs between HUVECs and DHCAECs, as well as between cells cultured in v normal and elevated glucose concentrations. These differences in gene regulation indicate the potential of the diabetic BVM to more accurately represent the endothelial response to diabetes and to the implementation of intravascular devices in the future. It was determined that culturing DHCAECs in a high glucose cell media for use in blood vessel mimics results in a model that differs considerably from HUVECs grown in normal glucose media. It was also determined that there was a difference between DHCAECs cultured in high glucose media and normal glucose media, as well as HUVECs cultured in high glucose media and normal glucose media. This study aided the development of a diabetic BVM; however, there are still improvements to be made, namely the inclusion of vascular smooth muscle cells in the model and improving the confluency of the BVM.

Die chronische Herzinsuffizienz gehört zu den häufigsten internistischen Krankheitsbildern in Europa. Eine wichtige Rolle in der Therapie der chronischen Herzinsuffizienz spielt das moderate körperliche Ausdauertraining. HDL ist als Vasoprotektor bekannt und ist in der Lage, über die Regulation der endothelialen Stickstoffmonoxidsynthase (eNOS) die Dilatationsfähigkeit von Gefäßen zu regulieren. Da eine gestörte Endothelfunktion verbunden mit einer geringeren eNOS-Expression einen wichtigen Aspekt in der Pathophysiologie der Herzinsuffizienz darstellt, war das Ziel dieser Arbeit zunächst, die HDL-induzierte eNOS-Aktivierung und NO-Produktion in Endothelzellen bei chronisch Herzinsuffizienten mit der von Gesunden zu vergleichen. Des Weiteren wurde der Einfluss körperlichen Ausdauertrainings auf die HDL-Funktion bei chronischer Herzinsuffizienz untersucht. Dafür wurde HDL jeweils aus Blutserum von herzgesunden Probanden und Herzinsuffizienten vor und nach körperlichem Ausdauertraining isoliert. Damit wurden humane aortale Endothelzellen inkubiert und anschließend mittels Western Blot die HDL-induzierte Phosphorylierung der endothelialen Stickstoffmonoxidsynthase (Regulation der eNOS-Aktivierung), der Proteinkinase C-βII sowie der p70S6K ermittelt. Des Weiteren wurde ESR-spektroskopisch die HDL-induzierte NO-Produktion in Endothelzellen gemessen. Letztendlich bestand die Frage, worin der Unterschied zwischen HDL von Gesunden und HDL von Herzinsuffizienten besteht, der die funktionalen Differenzen erklären kann. Dazu wurde die Menge des HDL-gebundenen Malondialdehyds ermittelt. Die Endothelfunktion wurde sonographisch als Fluss-vermittelte Vasodilatation bestimmt. Die Ergebnisse der Untersuchungen belegen, dass die HDL-induzierte eNOS-Aktivierung bei Patienten mit chronischer Herzinsuffizienz im Vergleich zu Gesunden vermindert ist. Des Weiteren kann der Einfluss von HDL auf die eNOS-Aktivierung durch körperliches Ausdauertraining bei Patienten mit chronischer Herzinsuffizienz verbessert werden. Die Verbesserung der HDL-induzierten NO-Produktion korreliert dabei mit der verbesserten Fluss-vermittelten Vasodilatation. Als Unterschied zwischen HDL von Gesunden und dem von chronisch Herzinsuffizienten konnte bei den Letztgenannten eine höhere Menge von gebundenem Malondialdehyd nachgewiesen werden.