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- Vascular mechanisms of testosterone: the non-genomic point of viewPublication . Lorigo, Margarida; Mariana, Melissa; Lemos, Manuel C.; Cairrão, ElisaTestosterone (T) is the predominant endogenous androgen in the bloodstream. At the vascular level, T presents genomic and non-genomic effects, and both effects may overlap. The genomic actions assume that androgens can freely cross the plasma membrane of target cells and bind to nuclear androgen receptors, inducing gene transcription and protein synthesis. The non-genomic effects have a more rapid onset and may be related to the interaction with protein/receptor/ion channels of the plasma membrane. The key T effect at the vascular level is vasorelaxation, which is primarily due to its rapid effect. Thus, the main purpose of this review is to discuss the T non-genomic effects at the vascular level and the molecular pathways involved in its vasodilator effect observed in in vivo and in vitro studies. In this sense, the nuclear receptor activation, the influence of vascular endothelium and the activation or inhibition of ion channels (potassium and calcium channels, respectively) will be reviewed regarding all the data that corroborated or not. Moreover, this review also provides a brief update on the association of T with the risk factors for cardiovascular diseases, namely metabolic syndrome, type 2 diabetes mellitus, obesity, atherosclerosis, dyslipidaemia, and hypertension. In summary, in this paper we consider the non-genomic vascular mode of action of androgen in physiological conditions and the main risk factors for cardiovascular diseases.
- Adverse vascular effects of phthalates: Possible contribution to hypertension in pregnancyPublication . Mariana, Melissa Rodrigues; Oliveira, Maria Elisa Cairrão Rodrigues; Soares, Amadeu Mortágua Velho da Maia; Sousa, Miguel Castelo Branco Craveiro dePhthalates are chemical compounds used as plasticizers to increase flexibility and elasticity of rigid polymers and as non-plasticizers as solvents and additives in cosmetics and personal care products. These compounds have low water solubility and since they are not covalently bound to the polymer they are easily released into the environment and absorbed by the human body. Afterwards, phthalates are rapidly metabolized into their respective monoesters being already detected in different biological samples, including blood, urine, saliva, amniotic fluid, umbilical cord blood and breast milk. In addition, phthalates can disrupt the endocrine system by binding to molecular targets and interfering with hormonal homeostasis, thus being classified as endocrine-disrupting compounds (EDCs). Due to these properties and their adverse effects, phthalates were classified as priority environmental pollutants by the United States Environmental Protection Agency (US EPA). Alongside this increase in exposure to phthalates, the incidence of cardiovascular diseases (CVD) has also increased over the years, with the occurrence of hypertensive disorders in pregnancy (HDP) being one of the main causes of maternal and foetal morbidity and mortality. There are several risk factors that influence the development of HDP, including exposure to EDCs, but the mechanisms involved are not yet known. Several studies have also suggested an association with phthalates, in rats they seem to promote changes in the vascular function, to affect the expression of proteins involved in cardiac function, to contribute to increased blood pressure, changes in lipid metabolism, and atherosclerosis. Thus, the aim of this thesis was to investigate how phthalates’ exposure affects the cardiovascular health, focusing on HDP. For that, studies were carried out on rat aorta and the human umbilical artery to evaluate the effects of di(2-ethylhexyl) phthalate (DEHP) and diethyl phthalate (DEP) in the vascular function. In the first study, rat aorta devoid of endothelium was contracted with noradrenaline and potassium chloride (KCl) and then subjected to different concentrations of DEHP (0.001-100 μM) to assess the vascular reactivity. Besides, since Ca2+ levels are the main determinant of contraction and relaxation, DEHP effects on Ca2+ currents were also analysed using the A7r5 cells. The data obtained showed that DEHP caused a concentration-dependent vasorelaxation after contraction with KCl, and, at the highest concentrations, it inhibited both the basal and the BAY K8644-stimulated calcium currents. These results suggest that DEHP induces vasorelaxation of the rat aorta by inhibiting the L-type Ca2+ channels (LTCC). Then, the genomic and non-genomic effects of DEP on the rat aorta were analysed. Using the organ bath technique, DEP (0.001-1000 μM) was analysed after contraction of the arteries with noradrenaline and KCl. Similarly, to understand the possible mechanisms involved, the effects of DEP were analysed on the relaxing effect of nifedipine (NIF - specific calcium channel blocker) and sodium nitroprusside (SNP - soluble guanylate cyclase activator). Besides, through electrophysiology studies the non-genomic (occurring within a few minutes) and genomic (occurring after 24h of exposure) effects of DEP on the Ca2+ currents of the A7r5 cells were investigated. These showed that both the rapid and 24h effects of DEP exposure led to a decrease in LTCC currents, being in agreement with the vasorelaxation obtained through the organ bath experiments. For the first time, the vascular electrophysiological properties of an EDC were analysed after long-term exposure, concluding that DEP promotes an inhibition of the calcium current in the rat aorta, which may be responsible for electrical disturbances such as arrhythmias. To evaluate the effect of phthalates on maternal health, the last experimental work of this thesis analysed the contribution of DEP (0.001-1000 μM) in the development of gestational hypertension. Therefore, human umbilical arteries (HUA) were collected from normotensive and hypertensive pregnant women. To assess the non-genomic vascular effects, the HUA were pre-contracted with serotonin, histamine and KCl and then subjected to DEP, resulting in a vasorelaxation by interfering with Ca2+ channels and serotonin and histamine receptors. On the other hand, to assess the genomic effects, the arteries were incubated with DEP for 24h, and then the effects of NIF and SNP were analysed. The results showed that DEP seemed to act through the NO/sGC/cGMP/PKG signalling pathway, and to interfere with LTCC. In addition, primary culture of HUA smooth muscle cells was also performed for cell viability essays, showing that DEP has no toxic effect on these cells. Overall, this experimental work showed that DEP affects the HUA vascular resistance through disturbances in hormone and calcium homeostasis, suggesting that the development of hypertension in pregnancy may be a consequence of exposure to phthalates. In general, the results obtained show that phthalates modify the vascular homeostasis in both animals and humans, promoting vasorelaxation mainly through the inhibition of Ca2+ channels. This is the first time that an experimental study has been able to establish the link between exposure to DEP and gestational hypertension. We are beginning to unravel the mechanistic pathways involved in the vascular effects of phthalates that explain the pathological functional alterations of hypertension in pregnancy. However, more experimental studies are needed to understand how different classes and mixtures of phthalates affect the cardiovascular health during pregnancy and in future generations.
- Efeito dos macrocíclos na regulação de funções vascularesPublication . Mariana, Melissa Rodrigues; Verde Lusquiños, José Ignacio; Rodrigues, Maria Elisa CairrãoAs células do músculo liso (SMC) vascular são fundamentais para a regulação do tónus vascular e têm capacidade de responder a diversos estímulos hormonais e hemodinâmicos. São células altamente especializadas que podem contribuir para o estudo de vários processos de sinalização envolvidos no controlo do tónus vascular, tais como o metabolismo do cálcio e diferentes vias envolvidas na modulação da reatividade vascular. A regulação do fluxo de Ca2+ é fundamental para a contração e a sua desregulação pode estar envolvida na patofisiologia de diversas doenças cardiovasculares. A entrada de cálcio nas células do músculo liso vascular através de canais de Ca2+ tipo L (LTCC) pode ser desencadeada pela ativação de recetores presentes na membrana plasmática ou por despolarização da membrana. A formação do complexo Ca2+-calmodulina leva à ativação da cadeia leve de miosina (MLC), e é o estado de fosforilação desta que determina a atividade contráctil do músculo liso. Os compostos macrocíclicos são macromoléculas cíclicas que podem ser usadas como transportadores moleculares, uma vez que, para além de terem a capacidade de atravessar a membrana celular sem alterar a sua integridade, não são tóxicos nas concentrações usadas e podem transportar outras moléculas. Os macrocíclos são considerados como importantes hospedeiros seletivos em algumas aplicações biológicas, nomeadamente em solubilizadores e estabilizadores de fármacos e agentes anticancerígenos, formando complexos estáveis e específicos. O principal objetivo deste trabalho é analisar se os macrocíclos têm atividade biológica. Assim, foi estudado o efeito de diferentes compostos macrocíclicos (MaC) sobre a atividade dos canais de Ca2+ tipo L e sobre a contractilidade das células A7r5. Utilizou-se a técnica Patch Clamp na configuração whole cell para medir a atividade dos LTCC nas células A7r5. O efeito de quatro macrocíclos (Me2[28]py2N6, [16]phenN2, [32]phen2N4, [30]phen2N6) foi analisado a diferentes concentrações (0,01μM, 0,1μM, 1μM e 10μM) sobre a corrente basal de Ca2+ e estimulada por Bay K (Bay K8644 – ativador específico dos LTCC). Foi também analisado o efeito destes MaC, nas mesmas concentrações, sobre a contractilidade das SMC vasculares através da técnica de PCSA (Planar Cell Surface Area), usando um microscópio de fluorescência. Este efeito foi analisado após contração das células com serotonina e noradrenalina. Pelos resultados obtidos através do Patch clamp e do PCSA pode-se observar que, nas concentrações mais baixas, o [32]phen2N4 inibe ligeiramente os LTCC, tanto a corrente basal como a corrente estimulada por Bay K. Relativamente à contractilidade, este composto não tem qualquer efeito sobre a contração induzida por serotonina ou por noradrenalina. No que diz respeito ao [16]phenN2 e ao [30]phen2N6, verifica-se que estes MaC não ativam nem inibem os LTCC mas, por outro lado, induzem redução da área celular. No caso do Me2[28]py2N6, este ativa os LTCC, de forma não significativa, mas este efeito parece não estar concordante com o efeito a nível da contractilidade, pois este MaC não tem efeito significativo sobre a redução da área celular. Assim, dos macrocíclos estudados, o [32]phen2N4 na concentração mais baixa (0,01μM) pode ser considerado como um bom transportador de fármacos, uma vez que não apresenta um efeito prejudicial sobre o sistema cardiovascular.
- Vascular Pathways of Testosterone: Clinical ImplicationsPublication . Lorigo, Margarida; Mariana, Melissa; Oliveira, Nelson; Lemos, Manuel C.; Cairrão, ElisaCardiovascular diseases (CVD) are one of the leading causes of death worldwide. Testosterone (T) is an important sex hormone that triggers several genomic and non-genomic pathways, leading to improvements of several cardiovascular risk factors and quality of life in men. At the vascular level, the key effect of T is the vasorelaxation. This review discusses the molecular pathways and clinical implications of T in the vascular system. Firstly, the mechanisms involved in the T vasodilator effect will be presented. Then, it will be discussed the association of T with the main risks for CVD, namely metabolic syndrome, type 2 diabetes mellitus, obesity, atherosclerosis, dyslipidaemia and hypertension. Several studies have shown a correlation between low T levels and an increased prevalence of several CVD. These observations suggest that T has beneficial effects on the cardiovascular system and that testosterone replacement therapy may become a therapeutic reality for some of these disorders.