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Eine der häufigsten kardiovaskulären Erkrankungen ist die Atherosklerose. Bei der Entstehung einer Atherosklerose spielt eine Hyperlipoproteinämie eine entscheidende Rolle. Ein weiterer Faktor für die Entstehung kardiovaskulärer Erkrankungen ist ein hoher Blutdruck. In dieser Arbeit wurde eine mögliche Interaktion zwischen Lipoproteinen und den blutdruckregulierenden Endothelin- und Renin-Angiotensin-Systemen untersucht. Weiterführende Analysen erfolgten an Rezeptoren für die Aufnahme von nLDL und oxLDL. Abschließend wurden Signalwege untersucht, die durch nLDL und oxLDL aktiviert werden. Tierexperimentielle Untersuchungen in Aorten und Herzen fettreich gefütterter Wildtyp- Mäuse unterstützen die Zellkultur-Ergebnisse einer Induzierung des Endothelin-Systems durch erhöhte Lipoproteine. Zusammenfassend zeigt diese Arbeit neue Mechanismen der Interaktion von Lipoproteinen und blutdruckregulierenden Systemen in Endothelzellen. Die Rezeptoren scheinen dabei eine Schlüsselrolle zu spielen. Dies spricht für eine Potenzierung von Hyperlipoproteinämie und Hypertonie bei der Entstehung von Herz-Kreislauf-Erkrankungen.

Master of Science / Department of Chemistry / Duy H. Hua / (+)-myriceric acid A is known as a non-peptide ETA receptor antagonist. It is isolated from the natural plant Myrica cerfera with 0.01% yield which is very low. The total synthesis of (+)-myriceric acid A is being pursued in Hua’s lab. (+)-myriceric acid A specifically blocks the vasoconstriction caused by endothelin-1 (ET-1). Because some derivatives of (+)-myriceric acid A were shown to have ET-1 receptor antagonistic effect, the tetracyclic terpenoid intermediates toward the total synthesis of (+)-myriceric acid A are postulated to have the similar antagonistic activities. The objective of this project is to study the release of vasoconstriction of these synthetic intermediates and compare their antagonistic potency. The ET-1 receptor antagonistic bioactivity of six (+)-myriceric acid A intermediates as well as (+)-myriceric acid A were evaluated by the in vitro spiral modiolar artery (SMA) bioassay. The synthetic intermediates which have not been reported in the literature were previously synthesized in Hua’s laboratory by Dr. Angelo Aguilar and Dr. Aibin Shi. Their synthesis was described in Dr. Aguilar’s PhD thesis. All the antagonistic effect evaluations were based on the SMA’s diameter changes. SMA’s diameter changes were induced by the superfusion of different extracellular solutions. The dose-response curves and straight lines were plotted to compare the antagonistic potency of these compounds. Based on the EC50 value of (+)-myriceric acid A intermediates (0.090 µM ~ 0.582 µM for the curves and 0.095 µM ~ 0.385 µM for the straight lines), all of the compounds have ET-1 receptor antagonistic activity, therefore the synthesis and screening of (+)-myriceric acid A intermediates is probably a promising route to develop new non-peptide ETA receptor antagonists.

Exposure to air pollution, particularly fine and ultrafine particulate matter derived from combustion sources, has been consistently associated with increased cardiovascular morbidity and mortality. Recent controlled exposure studies demonstrate that short-term exposure to diesel exhaust, which can contribute up to 40% of urban particulate air pollution, results in impaired vascular endothelial and fibrinolytic function in healthy volunteers, and increased exercise-induced myocardial ischaemia in patients with coronary heart disease. These observations may, in part, explain the observed increase in cardiovascular events following exposure to air pollution. Despite these observations there remain uncertainties regarding the key constituents of the air pollution mixture that mediate these adverse effects, and the underlying physiological and biological pathways involved. In these studies, using two controlled exposure facilities, I explored the vascular effects of the most prevalent gaseous component of the air pollution mixture – nitrogen dioxide – and the mechanisms responsible for impaired vasomotor function following exposure to diesel exhaust. Furthermore, I investigated the effect of acute exposure to “real-world” urban air pollution in both healthy volunteers and patients with coronary heart disease, and the effect of reducing that exposure using a simple facemask. In total, 10 healthy volunteers were exposed to nitrogen dioxide, and 29 healthy volunteers exposed to dilute diesel exhaust in a series of doubleblind randomised crossover studies. Exposure to nitrogen dioxide had no effect on either vasomotor function or endogenous fibrinolysis, providing indirect evidence that the adverse vascular effects are predominantly driven by particulate components. Following exposure to diesel exhaust there was no up regulation of endothelin-1 production, although there was increased vasoconstriction to intra-arterial infusion of endothelin-1. Following endothelin A receptor antagonism, there was attenuated vasodilatation following exposure to diesel exhaust as compared to air, an effect abrogated by endothelin B receptor antagonism. My findings suggest that the endothelin system does not play a central role in the adverse vascular effects of air pollution, but given the tonic interaction between the endothelin and nitric oxide systems, these observations could be explained by reduced nitric oxide bioavailability. Following diesel exhaust inhalation, plasma nitrite concentrations (as a marker for nitric oxide generation) are markedly increased without changes in haemodynamics or basal blood flow consistent with increased nitric oxide consumption. In the presence of a nitric oxide clamp, and without endogenous nitric oxide release, the vascular responses to vasodilators are similar. This perturbation of nitric oxide consumption and release appears to underlie the observed vascular endothelial effects. Fifteen healthy volunteers and 98 patients with coronary artery disease were recruited in Beijing, China. Subjects walked along a predefined city centre route for 2 hours in the presence and absence of a highly efficient facemask to reduce personal particulate air pollution exposure in an open label randomised crossover study. When wearing a facemask, there was an attenuation of exercise-induced increases in blood pressure, an improvement in heart rate variability, reduced myocardial ischaemia and subjects reported fewer symptoms. My findings have identified the biological mechanisms underlying the adverse vascular effects of exposure to diesel exhaust, and have helped to clarify the components responsible for these effects. Moreover, I have identified important benefits of reducing personal exposure to particulate matter using a simple facemask that have the potential to reduce cardiovascular events in patients living in urban or industrialised areas. Ongoing research in this area will provide further insight into the underlying vascular mechanisms, and the potential benefits of reducing particulate air pollution exposure, and may result in important targeted interventions to reduce the impact of air pollution on cardiovascular health.

Introdução: A disfunção erétil (DE) é definida como a incapacidade de alcançar ou manter a ereção do pênis para um desempenho sexual satisfatório, contribuindo significativamente para a baixa qualidade de vida e morbidade psicossocial masculina. A endotelina-1 (ET-1), um potente peptídeo vasoconstritor que promove contração lenta e sustentada em células de músculo liso vascular, possui grande importância na fisiopatologia da DE. Diversos estudos mostram que o aumento da expressão de mediadores inflamatórios está intimamente ligado ao desenvolvimento da DE. O inflamassoma é um complexo multiprotéico do sistema imune inato que atua através da ativação da caspase-1 e resulta na maturação de citocinas pró- inflamatórias, tais como interleucina- IL (IL-l?). O receptor NLRP3 faz parte do inflamassoma e sua ativação leva a clivagem de caspase-1 e consequente secreção de IL-1?. A ET-1, também possui papel importante na inflamação crônica vascular, mediando a liberação de citocinas pró-inflamatórias. No entanto, ainda é desconhecido se a ação pró- inflamatória da ET-1 em células de músculo liso é mediada pela ativação da via do inflamassoma. Hipótese: A ET-1 ativa o NLRP3 em células do músculo liso do corpo cavernoso (CMLCC), promovendo alterações na reatividade do corpo cavernoso (CC). Objetivo: Avaliar o papel da endotelina-1 na ativação do NLRP3 em CMLCC de camundongos. Métodos: CMLCC de camundongos C578BL/6 (WT) e NLRP3-/- foram cultivadas em meio de cultura DMEM acrescido de soro fetal bovino (SFB), 10%, foram pré- incubadas com endotelina-1 nas concentrações de 10-9, 10-8 e 10-7 M, em presença de LPS ou veículo. Avaliamos o efeito da deleção do NLRP3 sobre a reatividade do CC (contratilidade e relaxamento mediante estímulos por campo elétrico e/ou farmacológico). Após, avaliamos o efeito da ET-1 na ativação do NLRP3, nas alterações sobre a reatividade do CC de camundongos WT, e se estas persistiriam nos camundongos NLRP3-/- e caspase1/11-/- . Resultados: As células apresentaram-se fluorescentes para marcação para ?-actina e não para Von Willebrand, caracterizando assim que não houve contaminação com células endoteliais. A incubação com a ET-1 10-7 M por 24 h na presença de LPS ou veículo aumentou a atividade da caspase-1 em CMLCC de camundongos WT e este efeito não ocorreu nas CMLCC de camundongos NLRP3-/-. Não se observou diferença com relação à massa corporal ou massa dos órgãos entre os animais WT e NLRP3-/-. O CC de animais NLRP3-/- apresenta prejuízo para o relaxamento mediado por nitroprussiato de sódio (NPS) quando comparado com as tiras de CC de camundongos WT. A incubação com ET-1 10-7 M por 4 horas promove aumento na contração para fenilefrina (PE) e prejuízo no relaxamento induzido por nitroprussiato de sódio (NPS), e o mesmo efeito não é observado nas tiras de CC de camundongos NLRP3-/- e caspase1/11-/-. Conclusão: O NLRP3 contribui para o aumento na contração e prejuízo no relaxamento produzido pela ET-1 em CC de camundongos, possivelmente através da ativação da caspase-1 / Introduction: Erectile dysfunction (ED) is defined as the inability to achieve or maintain penile erection to perform sexual intercourse, it contributes significantly to the low quality of life and male psychosocial morbidity. Endothelin-1 (ET-1), a potent vasoconstrictor peptide that promotes slow and sustained contraction of vascular smooth muscle cells, has great importance in the pathophysiology of ED. Several studies show that increased expression of inflammatory mediators is closely linked to the development of ED. The inflammasome is a multiproteic complex of the innate immune system that acts through activation of caspase-1, which leads to maturation of pro-inflammatory cytokines such as interleukin-1 beta (IL-l?). The activation of NLRP3 receptor, part of the inflammasome, leads to caspase-1 cleavage and subsequent secretion of IL-1?. ET-1 also plays an important role in chronic vascular inflammation by mediating the release of pro-inflammatory cytokines. However, it is still unknown whether pro-inflammatory actions of ET-1 on smooth muscle cells is mediated by the activation of the inflammasome. Hypothesis: ET-1 activates NLRP3 in smooth muscle cells of the corpora cavernosa (SMCCC), promoting changes in corpus cavernosum (CC) reactivity. Objective: To evaluate the role of endothelin-1 in the activation of the NLRP3 in SMCCC of mice. Methods: SMCCC of C57BL/6 (WT) and NLRP3-/- mice were grown in DMEM culture medium supplemented with bovine fetal serum (FBS) 10%, pre-incubated with endothelin-1 at concentrations of 10-9, 10- 8 and 10-7M, in the presence of LPS or vehicle. We evaluated the effect of the NLRP3 deletion on the reactivity of the CC (contractility and relaxation by electric field and/or pharmacological stimulation). After that, we evaluated the ET-1 effect on activation NLRP3, changes on the reactivity of the CC of WT, and if these alterations would persist NLRP3-/- and caspase1/11-/- mice. Results: The cells presented fluorescent labeling to ?-actin, but not for Von Willebrand factor, characterizing absence of endothelial cells contamination. The incubation with 10-7 M ET-1 for 24 h in the presence of LPS or vehicle increased caspase-1 activity in SMCCC from WT, but not from NLRP3-/- mice. No difference was observed in body mass or weight of the organs between WT and NLRP3-/- animals. The CC from NLRP3-/- animals displayed impaired relaxation mediated by sodium nitroprusside (SNP) when compared to WT CC. The incubation with ET-1 10-7 M for 4 hours promoted an increase in the contraction to phenylephrine (PE) and reduced relaxation induced by sodium nitroprusside (SNP). The same effect was not observed in CC strips from NLRP3-/- and caspase1/11-/- mice. Conclusion: NLRP3 contributes to the increase in contraction and impaired relaxation produced by ET-1 in mice CC, possibly by activation of caspase-1

Relógios biológicos são marcapassos endógenos presentes tanto em eucariotos quanto em procariotos. Relógios diferentes possuem períodos distintos, e aqueles que se aproximam de 24h de oscilação são chamados circadianos. Em mamíferos, o primeiro relógio circadiano identificado situa-se no núcleo supraquiasmático, localizado no hipotálamo. O funcionamento do relógio circadiano envolve mecanismos de retroalimentação positiva e negativa, em geral tendo início com a ativação dos genes Per e Cry por CLOCK e BMAL1. Atualmente sabe-se que os relógios estão presentes em áreas do cérebro fora do núcleo supraquiasmático e em muitos tecidos periféricos. Em Drosophila e Danio rerio, os osciladores periféricos podem ser sincronizados diretamente por luz, enquanto em mamíferos o reinício de fase dos mesmos parece ser controlado por sinais regulados pelo marcapasso do núcleo supraquiasmático. Uma nova opsina, denominada melanopsina, foi recentemente descoberta na retina de todos os vertebrados estudados, em uma subpopulação de células ganglionares intrinsecamente fotossensíveis. Ela é responsável pela captura de luz e envio dessa informação para o núcleo supraquiasmático. A endotelina (ET) é um peptídeo vasoconstritor composto por 21 resíduos de aminoácidos. Existem três isoformas endógenas de ETs, designadas ET-1, ET-2 e ET-3. Três tipos de receptores para endotelinas já foram clonados, sendo eles designados ETA, ETB e ETC. Todos pertencem à família dos receptores acoplados à proteína G. Órgãos, tecidos e células de Danio rerio constituem um excelente modelo para o estudo dos genes de relógio e de ritmos in vitro. Em células embrionárias ZEM 2S deste teleósteo, constatamos a presença de melanopsina, do receptor ETA para endotelina, e dos seis genes Cry através de PCR. A presença de melanopsina também foi confirmada por imunocitoquímica. Foram realizadas curvas de crescimento em células ZEM 2S previamente mantidas por cinco dias em regime de 14C:10E (luz acesa às 9:00h). No 6º. dia, as células foram transferidas para as seguintes condições: escuro constante; 14C:10E; 10C:14E e luz constante. Houve inibição da proliferação celular por luz. O padrão de expressão temporal dos genes Per1, Cry1b, Clock e da melanopsina foi estudado, assim como sua modulação por ET-1. Células ZEM 2S foram mantidas em fotoperíodo 12C:12E (luz acesa às 9:00h) durante cinco dias, após o que foram tratadas com ET-1 nas concentrações 10-11M, 10-10M, 10-9M e 10-8M, durante 24h. O RNA extraído a cada 3h foi submetido a RT-PCR para posterior análise por PCR quantitativo. RNA ribossômico 18S foi utilizado como normalizador do experimento. Melanopsina não apresentou ritmicidade de expressão em fotoperíodo 12C:12E. ET-1 exerceu efeito bifásico, aumentando a expressão nas menores concentrações de hormônio utilizadas e diminuindo nas maiores. Na concentração 10-10M, ET-1 aparentemente estabeleceu uma oscilação ao longo das 24 horas, com crescente expressão na fase de escuro, atingindo um pico em ZT21 e decrescente durante o período de luz, com o mínimo em ZTs 6 e 9. A expressão do gene Clock é rítmica em regime fotoperíodo 12C:12E, com valores significativamente maiores em ZT12 a ZT21 do que em ZT0, ZT3 e ZT9, indicando um aumento de expressão coincidente com o período de escuro. Foi observado um pico de expressão em ZT6, durante a fase de luz. ET-1 nas concentrações de 10-11 e 10-10M aboliu o ritmo de expressão de Clock, e inibiu o pico de expressão em ZT6. Expressão de Clock permaneceu elevada somente em ZT18. Nas maiores concentrações (10-9M e 10-8M), a inibição ocorreu em todos os ZTs, abolindo completamente o ritmo e atenuando qualquer variação previamente observada entre os ZTs. A expressão do gene Per1 é rítmica em regime fotoperíodo 12C:12E, com valores significativamente maiores nos ZTs 21, 0, 3, 6 e 9 do que nos ZTs 12, 15 e 18, indicando um aumento de expressão na fase de claro. Vale mencionar que já em ZT21, há um aumento significativo antecipatório da fase de luz. Nas concentrações de 10-11 e 10-10M, ET-1 não alterou o período ou a amplitude desse ritmo. A ação evidente de ET-1 foi a inibição da expressão de Per1 na fase de luz (ZT0, ZT3, ZT6 e ZT9), e também em ZT21 (fase de escuro) nas maiores concentrações (10-9M e 10-8M) não afetando o período da oscilação, mas diminuindo marcadamente sua amplitude. A expressão de Cry1b foi rítmica durante o ciclo claro:escuro, com aumento na fase de claro e diminuição na fase de escuro. Novamente a ET-1 apresentou um efeito bifásico sobre a expressão deste gene, aumentando a mesma durante a fase de luz na concentração de 10-11M, e em ZT6 e ZT9 na concentração 10-10M. No entanto, não alterou o período ou a amplitude do ritmo. Por outro lado, durante toda a fase de luz houve inibição deste gene na presença de ET-1 10-9 e 10-8M, diminuindo a amplitude observada nas células controle. / Biological clocks are endogenous timekeepers that are present both in eukaryotic as in prokaryotic organisms. Different clocks have different periods, and those that have about 24h of oscillation are called circadian clocks. In mammals, the first identified circadian clock is located in the suprachiasmatic nucleus, in the hipothalamus. It is now well known that clocks are present in brain regions other than the suprachiasmatic nucleus and in many peripheral tissues. In Drosophila and Danio rerio, peripheral oscillators can be synchronized directly by light, while in mammals the reset of the phase seems to be controlled by signals regulated by the suprachiasmatic timekeepers. The maintenance of the circadian clock is governed by positive and negative feedback loops, in general starting with the activation of Per and Cry genes by CLOCK and BMAL1. A new opsin called melanopsin, was recently discovered in the retina of all studied vertebrates, in a subset of intrinsically photosensitive ganglion cells. This photopigment is responsible for capturing light and sending this information to the suprachiasmatic nucleus. Endothelin (ET) is a 21-amino acid residue vasoconstrictor peptide. There are three endogenous isoforms of ETs, ET1, ET2 and ET3. Three subtypes of endothelin receptors have already been cloned: ETA, ETB and ETC, all members of the family of G protein -coupled receptors. Organs, tissues and cells of Danio rerio constitute an excellent model for the study of clock genes and rhythms in vitro. In ZEM 2S embryonic cells of this teleost, we demonstrated the presence of melanopsin, the endothelin receptor ETA, and the six Cry genes by PCR. The presence of melanopsin was also confirmed by immunohistochemistry. ZEM 2S cells previously kept for five days in 14L:10D (lights on 9:00am) were transferred in the sixth day to the following conditions: constant darkness, 14L:10D, 10L:14D and constant light, and growth curves were determined. ZEM 2S showed inhibition of proliferation by light. The temporal expression pattern of the genes Per1, Cry1b, Clock and of melanopsin and their modulation by ET-1 were studied. ZEM 2S cells were kept in 12D:12L photoperiod (lights on 9:00am) for five days, and then treated with 10-11M, 10-10M, 10-9M and 10-8M ET-1, for 24h. RNA extracted every 3 hours was submitted to RT-PCR for subsequent analysis by Real Time-PCR. 18S ribosomal RNA was used to normalize the results. Melanopsin did not show rhythmicity of expression in 12D:12L photoperiod. ET-1 exhibited a biphasic effect, increasing the expression in the lower concentrations, and reducing at the higher concentrations. At 10-10M, ET-1 apparently established an oscillation along the 24h-period, with increasing expression in the dark phase, reaching a peak at ZT2, and decreasing during the light phase, with the minimum at ZT6 and 9. The expression of Clock gene was rhythmic in 12D:12L photoperiod, with significant higher values in ZT12 to ZT21 than ZT0, ZT3 e ZT9, indicating an increase of expression coincident with the dark period. A peak of expression was observed at ZT6, during the light phase. At 10-11 and 10-10M, ET-1 abolished the rhythm of expression of Clock, and inhibited the peak of expression at ZT6. Expression of Clock remained high only at ZT18. At the higher concentrations (10-9M e 10-8M), the inhibition occurred at all ZTs, completely abolishing the rhythm and attenuating any variation previously observed among ZTs. The expression of Per1 gene was rhythmic in 12D:12L photoperiod, with significant higher values at ZTs 21, 0, 3, 6 and 9 than at ZTs 12, 15 and 18, indicating an increase of expression in the light phase. It is important to mention that at ZT21 there was already a significant increase, anticipatory of the light phase. At 10-11 e 10-10M, ET-1 did not alter neither the period nor the amplitude of this rhythm. The evident action of ET-1 was the inhibition of Per1 expression in the light phase (ZT0, ZT3, ZT6 e ZT9), and also at ZT21 (dark phase), at the higher concentrations (10-9M e 10-8M), with no change in the oscillation period, but markedly reducing its amplitude. The expression of Cry1b was rhythimic during the light:dark cycle, with increase in the light phase and reduction in the dark phase. Again, ET-1 showed a biphasic effect on this gene expression, increasing it during the light phase at the concentration of 10-11M, and at ZT6 and 9 at 10-10M. However, the hormone did not affect either the period or the amplitude of the rhythm. On the other hand, along the light phase, there was inhibition of Cry1b in the presence of ET-1 10-9 and 10-8M, reducing the amplitude observed in the control cells.

A endotelina-1 (ET-1) é um peptídeo vasoconstritor que exerce um papel importante no controle do tônus do corpo cavernoso. No entanto, tem sido demonstrado que esse peptídeo também está envolvido na disfunção erétil (DE) associada ao diabetes mellitus e hipertensão. O consumo de etanol aumenta os níveis plasmáticos de ET-1 e a resposta contrátil a esse peptídeo em tecidos vasculares. Os objetivos deste trabalho foram o de estudar as consequências funcionais e celulares do consumo crônico de etanol sobre o sistema endotelinérgico no corpo cavernoso e identificar os mediadores envolvidos nessa resposta. Ratos Wistar foram divididos em dois grupos, os quais receberam água (controle) ou solução de etanol a 20% (vol./vol.) por seis semanas. Nossos resultados mostram que em tiras de tecido cavernoso, não houve alteração da resposta de relaxamento induzida pela adrenomedulina e nitroprussiato de sódio após tratamento com etanol. Com relação à acetilcolina, o consumo crônico de etanol reduziu o relaxamento induzido pelo referido agonista. Além disso, observou-se redução dos níveis plasmáticos e teciduais de nitrato no grupo etanol. Em conjunto, esses resultados sugerem que o tratamento crônico com etanol reduz a síntese/liberação do NO tecidual sem prejuízo em sua via de sinalização. O tratamento com etanol aumentou os níveis plasmáticos de ET-1 e a resposta contrátil induzida por esse peptídeo em corpo cavernoso de ratos. A contração induzida pela fenilefrina ou KCl 120 mmol/L não foi afetada pelo tratamento com etanol, sugerindo que os efeitos do tratamento sobre a reatividade do corpo cavernoso não são inespecíficos. O antagonista dos receptores ETB, o BQ788, não alterou a resposta de contração induzida pela ET-1 em corpo cavernoso de animais do grupo controle ou etanol. Não houve alteração da resposta de relaxamento induzida pelo IRL1620, um agonista seletivo dos receptores ETB. O tratamento com etanol não alterou os níveis de RNAm assim como a expressão protéica dos receptores ETB. Esses resultados mostram que o aumento da contração induzida pela ET-1 após tratamento com etanol não está relacionado à redução do relaxamento mediado pelos receptores ETB. Em nosso estudo o BQ123, antagonista seletivo dos receptores ETA, deslocou a curva cumulativa para ET-1 para direita em músculo cavernoso de ratos do grupo controle com consequente redução do valor de pD2. O mesmo não foi observado no tecido de animais do grupo etanol, indicando que a resposta mediada pelos receptores ETA está favorecida após o tratamento. O consumo de etanol não afetou os níveis de RNAm dos componentes do sistema endotelinérgico (ET-1, ECE-1, receptores ETA e ETB) e das isoformas da enzima óxido nítrico sintase (NOS) (eNOS, nNOS e iNOS), porém aumentou a expressão protéica do receptor ETA, da ET-1 e da iNOS no músculo cavernoso. O tratamento com etanol induziu aumento do estresse oxidativo sistêmico assim como dos níveis de ânions superóxido (O2-) no corpo cavernoso. As espécies reativas de oxigênio (ERO), os metabólitos derivados da NOS e da ciclooxigenase (COX) modulam negativamente a contração induzida por ET-1 e mostraram-se importantes no aumento da contração à ET-1 observada no corpo cavernoso de animais tratados com etanol. O Y27632, um inibidor da Rho-cinase, reduziu a resposta contrátil da ET-1 em corpo cavernoso de animais de ambos os grupos. Portanto, os resultados mostram que o tratamento com etanol aumenta a resposta contrátil da ET-1 por mecanismos que envolvem o aumento da expressão dos receptores ETA e das ERO e a via da Rho-cinase. / Endothelin-1 (ET-1) is a vasoconstrictor peptide that plays an important role in controlling the tone of the cavernosal smooth muscle (CSM). ET-1 is also involved in erectile dysfunction (ED) associated with diabetes mellitus and hypertension. Ethanol consumption increases plasma levels of ET-1 and the contractile response to this peptide in vascular tissues. This study aimed to investigate the cellular and functional consequences of chronic ethanol consumption on the endothelinergic system in CSM as well as the mediators involved in this response. Male Wistar rats were treated with ethanol 20% (vol./vol.) for 6 weeks. Reactivity experiments were performed on isolated CSM. Our findings show that adrenomedullin and sodium nitroprusside-induced relaxation was not altered after treatment with ethanol. On the other hand, acetylcholine-induced relaxation was reduced in CSM from ethanol-treated rats. Moreover, chronic ethanol consumption reduced plasma and CSM nitrate levels. These observations suggest that chronic ethanol consumption reduces NO synthesis/release but does not alter NO signaling pathway. Ethanol consumption increases plasma levels of ET-1 and the contractile response to this peptide in isolated CSM. Chronic ethanol consumption did not alter the contraction induced by phenylephrine or KCl 120mmol/L in isolated CSM strips. These observations suggest that the effects of chronic ethanol consumption on the CSM reactivity are nonspecific. BQ788, a selective ETB receptor antagonist, did not alter ET-1-induced contraction in CSM from both control and ethanol-treated rats. The relaxation induced by IRL1620, a selective ETB receptors agonist, was not affected by ethanol consumption. mRNA levels and protein expression for ETB receptor were not affected by ethanol consumption. We concluded that CSM hyper-reactivity to ET-1 is not related to reduction of ETB receptor-mediated relaxation. BQ123, a selective ETA receptor antagonist, shifted the concentration-response curve for ET-1 to the right in CSM from control rats. However, this response was not observed in CSM from ethanol group, indicating that the response mediated by the ETA receptor is favored after ethanol treatment. It was found that chronic ethanol consumption did not alter mRNA levels for the components of the endothelinergic system (ET-1, ECE-1, ETA and ETB receptors) and the isoforms of nitric oxide synthase (NOS) (eNOS, nNOS and iNOS), but increased protein expression for ETA receptor, ET-1 and iNOS. Ethanol induced systemic and cavernosal oxidative stress. Reactive oxygen species (ROS), metabolites derived from cyclooxygenase (COX) and NOS, modulate negatively ET-1-induced contraction and appear to be important mediators of ethanol-induced ET-1 hyper-reactivity in the isolated CSM. Y27632, a Rho-kinase inhibitor, reduced ET-1-induced contraction in CSM from both control and ethanol-treated rats. Our results show that chronic ethanol consumption increases ET-1 induced contraction in isolated CSM and that this response is mediated by the Rho-kinase pathway and an increase in ROS generation and ETA receptor expression.

Introdução: Os fitoesteróis são indicados no tratamento da hipercolesterolemia. Embora sua ação sobre a redução do colesterol já tenha sido exaustivamente estudada, não se encontra bem elucidada na literatura, a forma como atua sobre biomarcadores inflamatórios e endoteliais em indivíduos hipercolesterolêmicos. Objetivo: Avaliar o efeito do fitoesterol adicionado ao leite de soja sobre as concentrações plasmáticas de lípides e biomarcadores, além de alterações em vias transcricionais envolvidos na patogênese da aterosclerose em células linfomononucleares em indivíduos moderadamente hipercolesterolêmicos. Metodologia: Foram estudados 38 pacientes, recrutados no Ambulatório do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo. Inicialmente, os pacientes foram submetidos a um período basal de três semanas para avaliar a aderência e tolerabilidade ao leite de soja padrão (Placebo). Foram orientados a manter os hábitos alimentares e a atividade física. Receberam 400 mL/dia de leite de soja padrão ou enriquecido com fitoesteróis (1,6 g/dia) por dois períodos de quatro semanas em que o estudo foi randomizado, cego e cruzado. Resultados: O peso corporal foi mantido durante o estudo. O tratamento com fitoesterol reduziu o colesterol total em 5,5 % (P<0,001), a lipoproteína de baixa densidade (LDL) em 6,4 % e os triglicérides em 8,3 % (P<0,05) e aumentaram as concentrações de campesterol e sitosterol no plasma em 18,9% e 21,6%, respectivamente. Não houve alteração nos valores de lipoproteína de alta densidade (HDL). Com relação aos biomarcadores de aterosclerose, os fitoesteróis provocaram redução apenas na endotelina-1 em 11 % (P<0,05). Esta ação foi independente da redução do LDL-C. Não houve alteração no RNAm para 3-hydroxy-3-methylglutaryl coenzyme A redutase e receptor de LDL. Conclusão: Os fitoesteróis são eficientes na redução da concentração plasmática do colesterol total, triglicérides, LDL-C e da endotelina-1, sendo esta última independente da redução do colesterol / Background: Phytosterols are recommended in the treatment of moderate hypercholesterolemia. Although their mechanisms of action on cholesterol reduction has been exhaustively studied, other activities, such as their effects on inflammatory arterial markers and on endothelial function, have not yet been fully investigated. Objective: To evaluate the efficacy of phytosterols added to soy milk on the reduction of plasma lipids, on biomarkers involved in the pathogenesis of atherosclerosis and on transcriptional changes in 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase and low density lipoprotein receptor in moderately hypercholesterolemic individuals. Design: Moderately hypercholesterolemic patients (n=38, mean age 58 y) were initially followed over a 3-wk baseline period to evaluate their adherence to the program and their tolerance to the added milk. Patients received soy milk (400 mL/d) or soy milk + phytosterol (1.6 g/d) for 2 periods of 4-wk each. The study was randomized, blinded and crossed. Results: Body weight was maintained during the study. Phytosterol treatment reduced the total cholesterol concentration 5.5 % (P < 0.001), low density lipoprotein (LDL) 6.4 % and triglycerides 8.3 % (P<0.05), without modifying high density lipoprotein (HDL). Phytosterol reduced plasma endothelin-1 by 11 % (P<0.05), independently of LDL-C lowering activity. No effects on LDL receptor or on HMGCoA reductase mRNA expression in mononuclear blood cells were found. Sitosterol and campesterol plasma concentrations increased with phytosterol consumption by 18.9% and 21.6%, respectively. Conclusions: Phytosterol effectively lowered plasma total cholesterol, LDL-C, triglycerides and endothelin-1 concentration. The reduction in endothelin-1 concentration was independent of the decrease in the LDL-C concentration

Pulmonary arterial hypertension is a rare, chronic disease characterized by progressive remodeling of the pulmonary vasculature. Historically, prognosis has been very poor with relatively low 3-year survival rates. Common symptoms include fatigue and shortness of breath upon exercise, chest pain, and syncope. Patients exhibit increased pressure and resistance in pulmonary arteries due to fibrosis, vessel narrowing, and elevated levels of vasoconstrictive agents; diagnosis is confirmed by right heart catheterization. Reduced blood flow through the pulmonary vasculature not only reduces the amount of oxygenated blood available for the systemic circulation, but increases afterload on the right ventricle and, if left untreated, ultimately causes right ventricular heart failure. In the past, few medications were available to pulmonary arterial hypertension patients. However, recent advancements in our molecular understanding of the disease have led to the development of new therapeutic options that show promise of slowing, or in some cases reversing, disease progression. Currently available treatments have been shown to significantly improve 3-year survival rates and help promote a better quality of life for patients. While an exact molecular or genetic mechanism of disease progression is not yet known, several studies have noted the presence of dysfunctional endothelial cells and an imbalance in molecular modulators of the pulmonary vasculature. Specifically, patients exhibit chronically low levels of vasodilating agents such as prostacyclin and nitric oxide. In addition, there is a heightened vasoconstrictive effect due to elevated endothelin-1 and thromboxane A2. Drugs have been developed to target these signaling pathways and show considerable promise and efficacy for managing pulmonary hypertension in patients. Although these therapeutics have been shown to significantly improve survival rates and symptoms, many have complex and inconvenient administration protocols and a host of adverse side effects. Moreover, many require monitoring or frequent follow up visits due to their off-target effects. Recent innovative advancements in pulmonary arterial hypertension pharmaceuticals hope to deliver safe, efficacious treatment options to patients debilitated by this chronic disease.

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.