Os riscos provocados pela Terapia de Reposição Hormonal, levaram à busca de novas terapias, como os fitoestrógenos. São substâncias com ação estrogênica e propriedades que podem retardar a formação de placas ateroscleróticas. Isoflavonas são os fitoestrógenos mais estudados e são encontradas na soja, no red clover e em outras plantas. Avaliamos os efeitos dos fitoestrógenos extraídos da soja Glycine max: genisteína, formononetina, biocanina A e daidzeína; a mistura entre eles (Mix1); o extrato padronizado de red clover (Menoflavon 40mg) e uma segunda mistura com os fitoestrógenos extraídos da Glycine max nas concentrações encontradas no Menoflavon (Mix2), na expressão de moléculas de adesão de leucócitos, VCAM-1, ICAM-1 e E-selectina, em cultura de células endoteliais de cordão umbilical humano (HUVEC), assim como na linhagem modificada de célula endotelial, ECV304, estimuladas com LPS. Resultados: foram padronizados os tempos e concentrações de exposição ao LPS no cultivo de HUVEC de 1ug durante 12 horas de exposição para as três moléculas de adesão; e no cultivo de ECV304 para a expressão de VCAM-1, de 500ng durante 12 horas, para ICAM-1 de 1ug durante 18 horas, para E-selectina 100ng durante 18 horas na superfície celular e 200ng durante 24 horas no sobrenadante de culturas de ECV304, permitindo que este tipo celular seja utilizado como modelo de inflamação. Os fitoestrógenos reduziram VCAM-1, ICAM-1 e E-selectina na superfície celular assim como as formas solúveis dessas moléculas, tanto em ECV304 como em HUVEC, sendo efetivos como agentes preventivos e também para tratamento da aterosclerose. A mistura entre os fitoestrógenos não apresentou maior eficiência na redução das moléculas de adesão na superfície celular, mas apresentou diferenças significativas na produção das formas solúveis. Tanto em ECV304, quanto em HUVEC os fitoestrógenos extraídos do red clover e os extraídos da soja Glycine max reduziram as moléculas de adesão na superfície celular e no sobrenadante, sendo que o Menoflavon, apresentou maior efetividade na redução das moléculas de adesão que os fitoestrógenos extraídos da soja Glycine max, em HUVEC. Ocorreram interações entre os fitoestrógenos e o 17 estradiol, tanto em ECV304 quanto em HUVEC, principalmente quando este se encontrava em baixas concentrações, sugerindo proteção para mulheres na menopausa. Esses efeitos dos fitoestrógenos ocorreram via receptor de estrógeno, como demonstrado pela inibição de suas ações por ICI. Conclusão: tanto os fitoestrógenos extraídos da soja Glycine max quanto os extraídos do red clover apresentaram efeitos anti-aterogênicos, podendo atuar como cardioprotetores para mulheres pós-menopausa. / The risks of hormone replacement therapy have led to a search for new alternatives such as the use of phytoestrogens, plant compounds with estrogen-like biological activity. Isoflavones are the phytoestrogens most extensively studied and can be found in soy, red clover and other plants. Due this estrogen-like activity phytoestrogens can have some effect on atherosclerosis. We evaluated the effects of the phytoestrogens extracts from Glycine max soy: genistein, formononetin, biocanin A and daidzein; a Mix between them (Mix1); a standardized red clover extracts (Menoflavon 40mg) and a second Mix using phytoestrogens from Glycine max with same Menoflavon concentrations (Mix2) on adhesion molecules expression, VCAM-1, ICAM-1 and E-selectin by endothelial cell HUVEC, and by endothelial cell line ECV304, stimulated with LPS. Results: were standardized time and concentration to LPS exposure, being 1ug during 12 hours for the three adhesion molecules expression on HUVEC, and 500ng during 12 hours for VCAM-1 expression, 1ug during 18 hours for ICAM-1 expression and 100ng during 18 hours for E-selectin expression on cell surface as well as 200ng during 24 hours to E-selectin increase on culture supernadant, on ECV 304 cell line. The phytoestrogens decreased VCAM-1, ICAM-1 and E-selectin levels on cell surface and on culture supernadant in HUVEC and ECV304, being useful as preventive agents as well as treatment agents. Mix1 were not most effective than isolated phytoestrogens on cell surface, but presented decreased results on soluble forms. Menoflavon presented more effectiveness than Glycine max on HUVEC. Phytoestrogens interacted with 17 oestradiol mainly, in low concentrations (10pg), showing protection for post menopausal women. These phytoestrogens effects happened by oestrogen receptor activation, this was demonstrated through phytoestrogens inhibition by ICI. Conclusions: the phytoestrogens from Glycine max as well as phytoestrogens from red clover presented antiatherogenic effects, mainly when 17 estradiol is low, being usefull for postmenopausal women.

Diabetic foot ulceration (DFU) is a known major complication of diabetes mellitus which contributes to lower extremities amputation. This study aimed to investigate the use of interventional devices either as a preventative or therapeutic strategy to improve clinical management of this pathology, as well as investigating the impaired function of endothelial progenitor cells in the diabetic condition. The first element targeted plantar callus formation among diabetic neuropathic (NRP) patients through the use of a SurroSenseRxTM biofeedback device. Reducing foot pressure with improved walking strategy in the 6 months study in diabetic neuropathy patients (n=20) appeared to minimise the size of non-ulcerative plantar callus (p < 0.05), potentially reducing future ulcer recurrence. The 2nd study focused on the use of a GekoTM electrical stimulation device to enhance DFU healing in 24 patients. Wounds were characterised as being neuroischaemic (NRI) or neuropathic (NRP) based on standard parameters adopted in the Manchester diabetes clinic. The device was worn by 11 intervention subjects and compared to 13 controls without any electrical stimulus. Results suggested healing and wound closure have potentially increased in participants with electrical stimulation. In addition, Neuropathy Disability Score (NDS) was improved among intervention patients compared to control (p < 0.0001). The 3rd, in vitro and mechanistic study focuses on the outgrowth of endothelial cells (OECs), abnormal angiogenic responses and inflammatory microenvironment which could contribute to impaired wound healing in diabetic patients. OECs were isolated from diabetic patients and healthy controls (HCs), characterised by immunohistochemistry and Polymerase Chain Reaction (PCR). The functions of the three OEC groups from NRI, NRP diabetic patients and healthy controls respectively were compared using in vitro proliferation, transwell migration and wound healing scratch assays, together with matrigel tube formation assays. Scratch assays showed 100% closure in HCs over 24 hours, while 86.6% closure was apparent in NRI vs 38.1% in NRP. Seahorse mitochondrial stress test was conducted and demonstrated mitochondrial dysfunction in NRP vs NRI vs HCs (p < 0.05). Western blot analysis showed a lack of ERK phosphorylation by NRP OECs and an up-regulation of plasma inflammatory cytokines (TNFa and IL-6) in diabetic samples vs HC (p < 0.0001), while the angiogenic factors ang-2, FGF-2, VEGF-D, HGF and IL-8, and nitric oxide bioavailability were all significantly reduced in diabetic samples vs HC (p < 0.05). The functional defects of the diabetic OECs were partially restored through glycomimetic (synthesis compounds for endothelial damage protection) treatment (p < 0.05). In summary, this study has highlighted areas worthy of future development both in terms of preventative and therapeutic strategies. With improvements in digital technology and the need to empower patients to take responsibility of their health and well-being as well as greater understanding of the cellular and molecular biological repair processes that may be exploited, there may be potentials to reduce the risk of future ulceration among patients using these novel approaches in the future.

Chronic kidney disease (CKD) is growing global public health problem affecting 1 in 10 adults in developed countries and recognised as an important risk factor for cardiovascular disease (CVD) development. CVD is the main cause of death among CKD patients. Endothelial injury and dysfunction are critical steps in atherosclerosis, a major CVD. Oxidative stress (increased level of reactive oxygen species, ROS) has been associated with CVD development. Intravenous (IV) iron preparations are widely used in the management of CKD mediated anaemia, and have been associated with increased oxidative stress and cellular dysfunction. This study examined the effect of pharmacologically-relevant concentrations of IV Venofer (iron sucrose) or IV Ferinject (Ferric carboxymaltose, FCM) on primary human umbilical vein endothelial cell (HUVEC) activation/damage and on intracellular ROS generation as well as studying the potential mechanisms responsible. Data from TUNEL assay and Annexin V-FITC/PI staining showed that, IV FCM had no effect, but IV iron sucrose increased HUVEC apoptosis at 24hr. IV iron sucrose inhibited cell proliferation and reduced cell viability. Both compounds induced EC activation through sustained activation of p38 MAPK and up-regulation of ICAM-1 and VCAM-1. Additionally, the compounds induced significant increase in total ROS and superoxide anion production, which was attenuated by the anti-oxidant N-acetylcysteine (NAC). P38 MAPK showed up-regulation of pro-apoptotic protein Bax and down-regulation of antiapoptotic Bcl-2 protein in HUVEC treated with IV iron sucrose and p38 inhibition reversed these effects. In summary, these results suggest that IV iron sucrose causes more severe EC injury than IV FCM. However, both IV iron preparations induced intracellular ROS and superoxide anion generation in HUVEC leading to EC activation/dysfunction, providing a potential explanation for vascular damage in CKD patients.

Five traditional Chinese medicines were screened for their antiangiogenic activities through zebrafish angiogenic assay. Two of them, Tripterygium wilfordii and Rheum palmatum showed potential in the primary screening. T. wilfordii was selected in further study. / In the further investigation of antiangiogenic activity of triptolide on mammal systems, triptolide showed potent activity in human umbilical vein endothelial cells (HUVECs) assays including proliferation, migration and tube formation assay. It inhibited HUVEC proliferation with IC50 as low as 34 nM. It also showed more potency in HUVEC migration and tube formation assay at as low concentration as nanomolar level than SU5416, a putative VEGFR-2 inhibitor currently in clinic trials. RT-PCR and western blotting analysis showed that the underlying mechanism of triptolide correlated with down-regulation of VEGFR-2 and Tie2 expression and production. Tie2 inhibition appeared to be a later event as compared with VEGFR-2. Tie2 overexpression in HUVEC could attenuate the inhibitory effect of triptolide on HUVEC proliferation. Tie2 knockdown mimicked the inhibition activity of triptolide in tube formation assay. These phenomemon revealed that Tie2 signaling pathway plays a crucial role in triptolide-mediated angiogenesis inhibition. In in vivo Matrigel Plug assay, triptolide showed inhibition effect at as low as 100 nM. / T. wilfordii is an immune-suppressive, anti-inflammatory herb used in China for centuries. Through bioassay-guided purification, three antiangiogenic terpenoids were isolated from the ethyl acetate fraction, namely, celastrol, cangoronine and triptolide. Among them, triptolide manifested the most potent antiangiogenic activities against vessel formation. As low as 0.31microM, triptolide inhibited 20% of vessel formation, and the inhibition reached a plateau of 50% at 1.2 microM. Celatrol reduced vessel formation by more than 30% at 0.62microM, but killed 50% of the embryos at higher concentrations. Cangoronine was much weaker, inhibiting vessel formation by 20% at 2.5microM. These three components all showed stronger antiangiogenic activities than 2-methoxyestradiol, a putative compound currently under clinical trials as an antiangiogenic agent for cancer treatment, as the latter inhibited angiogenesis in zebrafish embryos by 34% at 10microM. The loss of vessel formation in the embryos treated with triptolide was further confirmed using endogenous alkaline phosphatase staining. Semi-quantitative RT-PCR analysis revealed that triptolide dose- and time-dependently reduced the mRNA expression of angiopoietin (angpt2) and tie2 in zebrafish, indicating the involvement of angpt2/tie2 signaling pathway in the antiangiogenic action of triptolide. / This research revealed that zebrafish model is a promising antiangiogenic model for both the screening of antiangiogenic agents from Chinese herbal medicine and the subsequent discovery for the drug targets. Triptolide, an anti-inflammatory component from T. wilfordii, is a potent angiogenic inhibitor through targeting VEGFR-2 and Tie2 pathways in mammal models whereas targeting ang2-tie2 pathway in zebrafish model. The anti-tumor action of triptolide was demonstrated to be partly through inhibition of tumor angiogenesis. Moreover, the potent antiangiogenic action exerted by triptolide at nanomolar dosage level gives proof that it is a promising lead compound for the development of antiangiogenic drug for cancer treatment. (Abstract shortened by UMI.) / He, Mingfang. / Adviser: Paul Pui-Hey Bot. / Source: Dissertation Abstracts International, Volume: 71-01, Section: B, page: 0247. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 84-106). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / As doenças cardiovasculares são a principal causa de morte nos países ocidentais. Alguns estudos sugerem que o chá verde tem efeito benéfico sobre diferentes fatores de risco cardiovascular. No entanto, outros estudos não mostraram essa associação. Objetiva avaliar em mulheres pré-hipertensas obesas o efeito do consumo de chá verde sobre: a pressão arterial, a função endotelial, o perfil metabólico, a atividade inflamatória e a adiposidade corporal. Estudos clínico, randomizado, cruzado, duplo-cego e placebo-controlado. Durante 4 semanas as mulheres foram orientadas a ingerir 3 cápsulas de extrato de chá verde por dia (500mg extrato chá verde/cápsula) passando por 2 semanas de washout e posteriormente ingeriam por mais 4 semanas o placebo. As mulheres que iniciaram o estudo tomando placebo posteriormente utilizaram o chá verde. Ou seja, todas as pacientes receberam chá verde e placebo por um mesmo período. No início e final de cada tratamento foram analisadas as variáveis. Foram avaliadas 20 mulheres pré-hipertensas, obesidade grau I e II, idade entre 25 e 59 anos. O local do estudo foi o Laboratório da Disciplina de Fisiopatologia Clínica e Experimental Clinex. Universidade do Estado do Rio de Janeiro. As variáveis estudadas foram a pressão arterial, índice de hipertemia reativa (avaliada com Endo-PAT2000), proteína C reativa, interleucina-6, fator de necrose tumoral-&#945;, molécula de adesão intercelular e molécula de adesão vascular celular, inibidor de ativador do plasminogênio, fator de crescimento endotelial vascular, E-selectina, adiponectina, colesterol total, LDL-colesterol, HDL-colesterol, triglicérides, glicemia, insulina, HOMA, índice de massa corporal, circunferência de cintura, circunferência de quadril, relação cintura quadril e percentual de gordura corporal. Como resultados, na avaliação da pressão arterial pela monitorização ambulatorial da pressão arterial, observou-se redução significativa da pressão arterial sistólica de 24 horas (pré 130,31,7 mmHg vs. pós 127,02,0 mmHg; p= 0,02), pressão arterial sistólica diurna (pré 134,01,7 mmHg vs. pós 130,72,0 mmHg; p= 0,04) e pressão arterial sistólica noturna (pré 122,21,8 mmHg vs. pós 118,42,2 mmHg; p= 0,02), após o consumo do chá verde, em comparação ao uso do placebo. Após o consumo do chá verde foi observado aumento, embora estatisticamente não significativo, no índice de hiperemia reativa (pré 1,980,10 vs. pós 2,220,14), além de redução expressiva na concentração da molécula de adesão intercelular (pré 91,88,0 ng/ml vs. pós 85,85,6 ng/ml) e do fator de crescimento endotelial vascular (pré 195,846,2 pg/ml vs. pós 158,638,7 pg/ml), porém sem significância estatística. As demais variáveis avaliadas não se modificaram de forma significativa após o consumo do chá verde, em comparação ao placebo. Foi observada forte correlação entre redução de pressão arterial sistólica e diastólica de 24hs, avaliada pela monitorização ambulatorial da pressão arterial, e o aumento do índice de hipertemia reativa (r= -0,47; r= -0,50, respectivamente). Os resultados do presente estudo sugerem que o chá verde tem efeito benéfico sobre a pressão arterial e possivelmente sobre a função endotelial. / Cardiovascular diseases are the leading cause of mortality in Western countries. Some studies have suggested that green tea has beneficial effects on different cardiovascular risk factors. However, others have failed to show such an association. Objective to evaluate the effects of green tea on blood pressure, endothelial function, metabolic profile, inflammatory activity and body adiposity in obese pre-hypertensive women. Study clinical, randomized, crossover, double-blinded, placebo-controlled. During 4 weeks women were instructed to ingest 3 capsules of green tea extract per day (500mg green tea extract/capsule), after 2 weeks of washout and then ingested placebo for 4 weeks. The women who began the study with placebo later have used green tea. That is, all patients received placebo and green tea for the same period. At the beginning and end of each treatment the variables were analyzed. Study of 20 women with pre-hypertension, obesity grade I and II, aged between 25 and 59 years. Study site in Laboratory of the Discipline of Clinical and Experimental Pathophysiological Clinex. Rio de Janeiro State University. Variables studied was blood pressure, reactive hyperemia index (evaluated with Endo-PAT2000), C-reactive protein, interleukin 6, tumor necrosis factor alpha, vascular cell adhesion molecule, intercellular adhesion molecule, plasminogen activator inhibitor-1, vascular endothelial growth factor, E-selectin, adiponectin, total cholesterol, low density lipoprotein-cholesterol, high density lipoprotein-cholesterol, triglycerides, blood glucose, insulin, HOMA, body mass index, waist circumference, hip circumference, waist-to-hip ratio and body fat. As results, in the assessment of blood pressure by ambulatory blood pressure monitoring was observed significant reduction in 24 hours systolic blood pressure (pre 130.31.7 mmHg vs. post 127.02.0 mmHg; p= 0.02), daytime systolic blood pressure (pre 134.01.7 mmHg vs. post 130.72.0 mmHg; p= 0.04) and nighttime systolic blood pressure (pre 122.21.8 mmHg vs. post 118.42.2 mmHg; p= 0.02), after consumption of green tea compared with placebo. After consumption of green tea, there was an increase, although not statistically significant, in reactive hyperemia index (pre 1.980.10 vs. post 2.220.14), besides expressive reduction in the concentration of intercellular adhesion molecule (pre 91.88.0 ng/ml vs. post 85.85.6 ng/ml) and vascular endothelial growth factor (pre 195.846.2 pg/ml vs. post 158.638.7 pg/ml), however without statistical significance. The other variables evaluated did not change significantly after consumption of green tea. A strong correlation was observed between reduction in 24 hours systolic blood pressure and 24 hours diastolic blood pressure, assessed by ambulatory blood pressure monitoring, and the increase of reactive hyperthermia index (r= -0.47; r= -0.50, respectively). The results of this study suggest that green tea has a beneficial effect on blood pressure and possibly on endothelial function.

Oxisteróis (por exemplo, 7hidroxicolesterol) são gerados por modificações oxidativas que ocorrem na molécula de colesterol. Podem ser encontrados em elevados níveis plasmáticos em pacientes com aterosclerose e como componentes da placa aterosclerótica. Considerando que o colesterol é o principal componente da cavéola (domínios específicos da membrana plasmática que ancoram diversas proteínas de sinalização) formulamos a hipótese que os oxisteróis podem ser incorporados a estes domínios, interferindo com as vias de sinalização aí localizadas. Células endoteliais de veia umbilical humana (HUVECs) em cultura foram expostas a 7hidroxicolesterol (10g/mL) por diferentes tempos. Analisamos a incorporação desse oxisterol à cavéola utilizando espectrometria de massa e a atividade das proteínas de sinalização presentes neste domínio: óxido nítrico sintase endotelial (eNOS), CD40/CD40L, receptor do fator de crescimento de fibroblastos (rFGF), utilizando PCR quantitativo e imunoblots. Inicialmente mostramos que o 7hidroxycholesterol, em concentrações fisiológicas, foi incorporado às cavéolas mais acentuadamente que em outros domínios de membrana. Esse fenômeno impediu o desligamento entre eNOS e caveolina, prejudicando a função dessa enzima. Também mostramos que o receptor CD40 apresentou uma maior incorporação à cavéola e o rFGF manteve uma ativação mais longa quando células foram expostas ao 7hidroxicolesterol. Esses efeitos gerados pelo oxisterol não estavam relacionados à sua ação sobre mediadores inflamatórios ou receptores nucleares, desde que nenhuma diferença foi observada no perfil de citocinas ou na expressão de genes dependentes da ativação de LXR. Assim, concluímos que a incorporação de 7hidroxycholesterol nos domínios de cavéola pode interferir com vias de sinalização sabidamente envolvidas na aterogênese ou na ruptura da placa / Oxysterols (for example, 7hidroxycholesterol) are generated by oxidative modifications to cholesterol molecules. They have been described in high levels in patients with atherosclerosis and as components of the atherosclerotic plaque. Since cholesterol is the main component of caveolae (plasma membrane domains that anchor several signaling proteins), we hypothesized that oxysterol could be incorporated to these domains, interfering with the signaling networks that use this pathway. Human umbilical vein endothelial cells (HUVECs) in culture were exposed to 7hidroxycholesterol (10g/mL) for different times. We analyzed incorporation of this oxysterol to caveolae using mass spectroscopy and the activity of signaling pathways present in these domains: endothelial nitric oxide synthase (eNOS), CD40/CD40L, fibroblast growth factor receptor (FGFr), using quantitative PCR and immunoblots. Initially we showed that 7hidroxycholesterol, in physiological concentrations, was incorporated to caveolae more prominently than to other plasma membrane domains. This phenomenon caused a difficulty in eNOS release from caveolin, impairing its function. We also showed that the receptor CD40 presented a stronger incorporation to caveolae and FGFr maintained a longer activation when cells were exposed to 7hidroxycholesterol. These oxysterol effects were not related to its action in inflammatory mediators or nuclear receptors, since no difference could be observed in cytokine profiles or in the expression of genes dependent on LXR activation. Therefore we conclude that 7hidroxycholesterol incorporation in caveolae domains may interfere with signaling pathways known to be involved in atherogenesis or in plaque rupture

A relação entre risco cardiovascular e terapia hormonal na pós-menopausa é controversa. Ainda que o estrogênio endógeno possa estar associado ao menor risco cardiovascular observado em mulheres na pré-menopausa em relação às pós-menopáusicas, grandes ensaios clínicos, como o WHI, falharam em demonstrar efeito benéfico da terapia hormonal. Estes resultados podem ter sido influenciados por uma série de fatores, sendo os mais importantes: idade média das pacientes e tempo de menopausa superiores às candidatas usuais de terapia hormonal, tipo e dose dos hormônios utilizados. Desenvolvemos ensaio clínico randomizado, cross-over, com objetivo de avaliar os efeitos de dois tipos de tratamento hormonal na menopausa: tratamento oral baixa dose, associação de estradiol 17 β nasal 300 μcg e drospirenona 2 mg, diário e tratamento nâo oral, estradiol 17 β nasal diário e progesterona micronizada vaginal, 200 mg, 14 dias por mês , sobre variáveis relacionadas com inflamação e função endotelial, perfil antropométrico, metabólico e hormonal em mulheres na pós-menopausa recente e sem doença clínica evidente. Quarenta mulheres na pós-menopausa foram alocadas aleatoriamente para iniciar o tratamento hormonal por um dos dois grupos de tratamento: via oral baixa dose (n=20): ou via não oral (n=20). Ao final dos primeiros 2 meses do estudo, o grupo inicialmente tratado com terapia oral passou a receber tratamento não oral por mais 2 meses, e o grupo inicialmente tratado com terapia não oral passou a receber terapia oral também por mais 2 meses. A avaliação laboratorial foi realizada antes e ao final de 2 e 4 meses de tratamento hormonal. A amostra do estudo foi composta por mulheres com média etária de 51,2 ± 2,7 anos e tempo de amenorréia de 23,1 ± 10 meses. Após os primeiros 2 meses de tratamento, não houve diferença significativa entre os tratamentos sobre circunferência da cintura, relação cintura/quadril, índice de massa corporal e níveis de pressão arterial. Colesterol total diminuiu em ambos os tratamentos de forma semelhante. O tratamento oral teve um efeito maior em reduzir os níveis de LDL-C. HDL-C, triglicerídeos, glicemia e insulinemia de jejum, glicemia e insulinemia 2 horas após sobrecarga oral de glicose não se modificaram. PCR e FVWdiminuíram significativamente, e fibrinogênio permaneceu inalterado. Após o período de 4 meses de tratamento hormonal, não houve diferença significativa entre os tratamentos sobre circunferência da cintura, relação cintura/quadril, índice de massa corporal e níveis de pressão arterial. Durante o tratamento oral observou-se redução da circunferência da cintura e da relação cintura/ quadril em relação ao basal. Colesterol total diminuiu em ambos os grupos de tratamento, e HDL-C diminuiu discreta, mas significativamente após o tratamento oral, enquanto triglicerídeos diminuíram durante tratamento não oral. A glicemia 2 horas após sobrecarga oral de glicose apresentou valores mais elevados em relação ao basal após tratamento oral. Em contraste, glicemia e insulinemia em jejum e insulinemia 2 horas após sobrecarga oral de glicose não se modificaram. Níveis de FVW encontraram-se significativamente reduzidos após 4 meses de tratamento hormonal. Em conclusão, os resultados obtidos em nosso estudo sugerem que os tratamentos não induziram efeitos deletérios sobre variáveis relacionadas com risco cardiovascular, a curto prazo, em uma população de mulheres na pós-menopausa recente e aparentemente saudáveis. O tratamento hormonal baixa dose por via oral manteve os efeitos benéficos conhecidos do tratamento hormonal por via oral, a redução do colesterol total e do LDL-C, e evitou os efeitos nocivos tradicionalmente atribuídos à via oral: o aumento de marcadores próinflamatórios, relacionados à disfunção endotelial. O tratamento hormonal por via não oral mostrou-se também uma alternativa segura, não relacionado à modificações no perfil metabólico e nos marcadores de função endotelial. / The relationship between cardiovascular risk and hormone therapy (HT) for menopause is a contemporary and complex issue. While evidences suggest an association between endogenous estrogen and cardiovascular protection among premenopausal women, recent clinical trials have failed in demonstrate a benefic impact of HT on prevention of cardiovascular events. These results seem to be related by several factors, including selection biases like higher mean age of and time since menopause of participants, fixed type and dosages of hormones administered. A cross-over, randomized clinical trial was designed in order to evaluate the effects of two types of HT: low dose oral treatment, estradiol 17 β oral 1 mg and drospirenona 2 mg, by day and non-oral treatment, estradiol 17 β nasal 300 μcg by day and vaginal micronized progesterone, 200 mg/d, 14 days by month on variables associated with endothelial function, anthropometric, metabolic and hormonal variables on early and healthy postmenopausal women.Forty postmenopausal women were randomly allocated to start with one of the treatments: low dose oral treatment or non-oral treatment. At the end of two months, the group that started with low dose oral treatment passed to receive the non oral treatment for additional two months and vice-versa. Laboratory evaluations were performed before, at 1, 2 and 4 months of HT. The sample of the study included postmenopausal women presenting mean age of 51.2 ± 2.7 years and mean time since menopause of 23.1 ± 10 months. After 2 months, no significant differences were observed between treatments on waist circumference, waist to hip ratio, BMI and arterial pressure. Total cholesterol levels were reduced on both treatments. Low oral dose treatment had greater effect in reducing LDL cholesterol. HDL cholesterol, triglycerides, fast and 2 hours glucose and insulin levels did not change with either treatment. PCR and vW factor levels were reduced in both treatment groups and fibrinogen did not change. After 4 months of low oral dose treatment, a reduction on waist circumference and waist/circumference ratio was found. Total cholesterol was lower than basal levels on bothtreatment groups and while HDL cholesterol presented a slight but significant reduction on low oral dose treatment, triglycerides decreased significantly on non oral treatment. Two hours glucose was higher than basal levels but fast glucose and fast or 2 h insulin levels did not change after low oral dose therapy. After 4 months, vW factor decreased only on non oral treatment and PCR and fibrinogens were unchanged on both treatment groups. In conclusion, the present results suggest that the studied treatments did not induce deleterious effects on variables related to cardiovascular risk, at least at short period of time, in early postmenopausal and apparently healthy women. Low dose oral HT has maintained the well known beneficial effects on lipid profile (lower total and LDL cholesterol) and did not induced an increase on pro-inflammatory or endothelial function markers. On the other hand, non oral HT has shown to be a safe alternative, and was not related to changes on metabolic profile or markers of endothelial function.

O melanoma é uma neoplasia de pele invasivo, com maior taxa de morte, sem tratamento efetivo. Nanocápsulas poliméricas de núcleo lipídico (LNC) tem sido empregadas com sucesso como carreadores de fármacos hidrofóbicos. Como o eugenol é um composto hidrofóbico com atividades antiproliferativas e pró-apoptóticas em células cancerosas, visamos avaliar os efeitos dos tratamentos com acetileugenol (AC), LNC ou LNC contendo acetileugenol (LNC-AC) em modelo de melanoma in vivo em camundongos C57B6, e a citotoxicidade dos mesmos em células endoteliais (HUVEC) e de melanoma (SK-Mel-28) in vitro. Os resultados obtidos mostraram que: 1) tratamentos i.p. com as LNC ou com LNC-AC (50 mg/kg, 3-10 dia de indução do tumor) induziram toxicidade sistêmica e, somente o tratamento com LNC inibiu o desenvolvimento do melanoma. O tratamento com LNC, mas não com a mistura de triglicerídeos de cadeia média, por via oral, inibiu o desenvolvimento tumoral, sem toxicidade. Adicionalmente, os tratamentos com AC, LNC ou LNC-AC não foram eficazes quando administrados em fase tardia de evolução tumoral (50 mg/kg, 7-17 dia de indução do tumor, via oral); 2) os tratamentos agudos com AC, LNC ou LNC-AC (20 mg/kg, 200 &#181;L, e.v.) não alteraram o número de leucócitos circulantes, mas os tratamentos com LNC ou com LNC-AC reduziram o comportamento de rolling dos leucócitos em vênulas póscapilares do músculo cremaster e causaram hemólise, sendo que este último efeito também foi observado após tratamento in vitro em hemácias murinas; 3) Os estudos in vitro mostraram que as LNC e LNC-AC foram captadas pelas células HUVEC e SK-Mel-28 após 1 hora de incubação; que a incubação com LNC-AC induziu apoptose tardia e necrose com maior eficácia em SK-Mel-28 do que em HUVEC; que as incubações com LNC ou LNC-AC exerceram efeitos antiproliferativos, induzindo parada na fase G2/M do ciclo celular das duas linhagens de células avaliadas; que somente a incubação com AC ou LNC-AC inibiu a adesão ao Matrigel® com maior eficácia na linhagem SK-Mel-28 do que HUVEC; que somente a incubação com as LNC reduziram a expressão de VCAM-1 em HUVEC e que as incubações com LNC ou LNC-AC reduziram a expressão de &#946;3 integrina em SK-Mel- 28; que nenhum dos tratamentos alterou a migração celular das HUVEC ou SK-Mel- 28; que somente a incubação com LNC-AC reduziu os níveis de espécies reativas de oxigênio em HUVEC e SK-Mel-28; que a incubação com LNC ou LNC-AC aumentou a produção de óxido nítrico (NO) pelas duas linhagens de células avaliadas; que o tratamento com L-NAME reverteu os níveis de NO e a inibição sobre a proliferação celular induzida pela incubação com LNC ou LNC-AC e; que o tratamento de células de melanoma murino com LNC ou LNC-AC parece alterar a polarizar os neutrófilos para o fenótipo N1. Associados, os resultados obtidos mostram o tratamento oral com LNC inibe o crescimento do melanoma sem induzir efeitos tóxicos, e que este efeito benéfico pode ser dependente, pelo menos em parte, da nanoencapsulação dos triglicerídios de cadeia média e da supraestrutura da formulação, com toxicidade direta sobre as células de melanoma e possível modulação do microambiente tumoral. / Melanoma is the most invasive skin cancer, with high rates of death without effective treatment. Polymeric lipid-core nanocapsules (LNC) has been successfully used as carriers of hydrophobic drugs. As eugenol is an hydrophobic compound with antiproliferative and pro-apoptotic activity in cancer cells, here we aimed to evaluate the effects of treatments with acetyleugenol (AC), LNC or LNC containing acetyleugenol (LNC-AC) in an in vivo melanoma model in C57BL6 mice and the cytotoxicity of the treatments in vitro, using endothelial (HUVEC) and melanoma (SK-Mel- 28) cells. The results obtained showed that: 1) i.p. treatments with LNC or LNCAC (50 mg/kg, 3-10 days of tumor injection) induced systemic toxicity and, only the treatment with LNC inhibited the melanoma development. Treatment with LNC, but not with mix of triglycerides of medium chain, by oral route, inhibited the tumor development, without toxicity. In addition, the treatments with AC, LNC or LNC-AC were not effective when administered in the late stage of tumor evolution (50 mg/kg, 10-20 days of tumor induction, oral route); 2) the acute treatments with AC, LNC or LNC-AC (20 mg/kg, 200 &#181;L, intravenous route) did not altered the number of circulating leukocytes, but the treatments with LNC or LNC-AC reduced the rolling behavior of leukocytes in postcapillary venules of the cremaster muscle and induced hemolysis. The latter effect was also observed after in vitro treatment using murine erythrocytes; 3) In vitro studies showed that the LNC and LNC-AC suffered uptake by HUVEC and SK-Mel-28 cells after 1 hour of incubation; that the incubation with LNC-AC induced late apoptosis and necrosis more effectively in SK-Mel-28 than in HUVEC cells; that the incubation with LNC or LNC-AC presented antiproliferative effects, by inducing G2M arrest in cell cycle in both cells lines evaluated; that only the incubation with AC or LNC-AC inhibited the adhesion in Matrigel® with more efficaccy in SK-Mel-28 than in HUVEC cells; that only incubtion with LNC reduced the VCAM-1 expression in HUVEC and the incubation with LNC or LNC-AC reduced the &#946;3 integrin expression in SK-Mel-28 cells; that any treatment affected the HUVEC or SK-Mel- 28 migration; that only the incubation with LNC-AC reduced the levels of reactive species of oxygen in HUVEC and SK-Mel-28 cells; that the incubation with LNC or LNC-AC increased the nitric oxide (NO) production by both cell lines used; that the treatment with L-NAME reversed the NO levels and the inhibition on cell proliferation induced by incubation with LNC or LNC-AC and; that the in vitro treatment of murine with LNC or LNC-AC altered the neutrophil polarization to N1 phenotype. Together, results obtained show that the oral treatment with LNC inhibit the melanoma growth without any toxic effect, and that the beneficial effect could be dependent, at least in part, of nanoencapsulation of medium chain triglycerides and the supraestrucuture of the formulation, with direct toxicity on melanoma cells and possible modulation of tumor microenvironment.

The potential of embryonic stem (ES) cells to generate any developmental or adult cell type holds much promise for regenerative medicine and in vitro modelling of development and disease. Haematopoietic stem cells (HSCs) regenerate all lineages of the blood throughout adult life and are essential for the treatment of a vast number of haematalogic disorders. Current sources of HSCs for clinical use and research, including adult bone marrow, peripheral blood stem cells and umbilical cord blood, are limited by the number of HSCs they contain and by the availability of a suitable donor. A system that generates a reliable source of HSCs from ES cells would therefore be an ideal alternative. While much progress has been made in the generation of downstream lineages of the haematopoietic system, progress in the derivation of HSCs capable of long-term self-renewal and multilineage reconstitution from ES cells has been limited. Understanding of the developmental steps leading to HSC emergence in the embryo has been advancing in recent years. In particular, precursors of HSCs (preHSCs) have been isolated from the mouse embryo, characterised and matured into HSCs ex vivo using the specialised conditions of aggregate culture systems (Taoudi et al 2008, Rybtsov et al 2011). We hypothesised that application of the aggregate culture system in the differentiation of ES cells could provide a missing link in the in vitro generation of HSCs. Here I have developed a novel ES cell differentiation system that employs the specialised conditions of the aggregate culture system, after an initial stage of mesoderm differentiation. I show that this system creates an environment for efficient haematopoietic and endothelial progenitor formation and generates cells of a preHSC type I (VE-Cadherin+CD45-CD41lo) and preHSC type II (VE-Cadhein+CD45+) surface phenotype. Notably, the system gives rise to cells that achieve low-levels of haematopoietic repopulation in sublethally irradiated NSG mice. The low-level repopulating cells persist for over 4 months in animals and show both myeloid and lymphoid potential. I identify genes that are expressed in cells of a preHSC II surface marker-phenotype from the E11.5 dorsal aorta, but not in cells of this phenotype from the E11.5 Yolk sac or differentiated ES cells. I also show that enforced expression of Notch downstream target Hes1 in Flk1+ mesoderm during ES cell differentiation does not improve levels of ES-derived repopulation.

Made available in DSpace on 2017-07-10T14:17:10Z (GMT). No. of bitstreams: 1 STEFANY_ PEDROTTI 2015.pdf: 2292343 bytes, checksum: 2d00d0ff8c02256c8040bdbd293e6156 (MD5) Previous issue date: 2015-02-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Periodontal disease is characterized by a chronic infection, which may cause systemic inflammation due to high levels of inflammatory cytokines such as tumor necrosis factor-alpha (TNF-&#945;), interleukin-6 (IL-6), interleukin-8 (IL-8) and interleukin-18 (IL-18), contributing to the development of endothelial dysfunction in various organs. The impairment of endothelial function can lead to erectile dysfunction, because it is characterized by vascular changes such as occlusion of cavernous arteries by atherosclerosis and reduction vascular reactivity due to decreased release of nitric oxide. In this sense, inflammation associated with periodontal disease may damage the penile endothelial layer, leading to occurrence of erectile dysfunctional. However, the potential role of chronic periodontitis in the pathophysiology of erectile dysfunction remains scarce. Therefore, the aim of the study was to evaluate the tissue morphology of periodontal and penis rats associated or not with experimentally induced periodontitis. For this, 18 Wistar rats were obtained and induced periodontal disease in 9 rats, whereas the remaining 9 rats served as controls. After 30 days of induction of periodontal disease, the sexual behavior of rats was recorded and later, the animals were sacrificed to obtain gingival sample, hemimandible and penis to the morphological analysis, morphometric, immunological and radiographic. The results showed that experimental periodontitis contributed to the reduction in the sexual behavior of rats (p<0.05) and caused alveolar bone loss with a significant increase in osteoclastic activity and gingival volume (p<0.05). Moreover, the group with periodontitis had a higher concentration of inflammatory cytokines such as IL-6 in the gingiva and TNF-&#945; in the penis (p<0.05). There was no statistically significant difference in ventral-dorsal and side-to-side distance of the penis (p> 0.05), however there was a higher thickness and a shorter area statistically significant of penile dorsal artery in the group with experimental periodontitis, as well as a smaller area of vascular spaces of the penile cavernous tissue (p<0.05). Based on these results, it is concluded that the experimental periodontitis caused structural and functional changes in the penis Wistar rats and sexual behavior changes, therefore systemic inflammation caused by periodontal disease can be an important risk factor for erectile dysfunction, that is why is necessary the development of interventions involving many health professionals for prevention and treatment of these two diseases that endanger the health and general well-being of individuals / A doença periodontal caracteriza-se por ser uma infecção crônica, a qual pode causar uma inflamação sistêmica devido aos elevados níveis de citocinas inflamatórias envolvidas, tal como fator de necrose tumoral-alfa (TNF-&#945;), interleucina-6 (IL-6), interleucina-8 (IL-8) e interleucina-18 (IL-18), o que contribui para o desenvolvimento da disfunção endotelial em diferentes órgãos. O comprometimento da função endotelial pode levar à disfunção erétil, pois a mesma é caracterizada por alterações vasculares, como oclusão das artérias cavernosas por aterosclerose e redução da reatividade vascular devido à diminuição da liberação do óxido nítrico. Nesse sentido, a inflamação associada com a doença periodontal pode danificar as camadas endoteliais penianas, induzindo à ocorrência da ereção disfuncional. Contudo, o papel potencial da periodontite crônica na fisiopatologia da disfunção erétil permanece escasso. Portanto, o objetivo deste estudo foi avaliar a morfologia tecidual do periodonto e pênis de ratos associados ou não com a periodontite induzida experimentalmente. Para isso, foram obtidos 18 ratos Wistar e induzida a doença periodontal em 9 ratos, sendo que os 9 ratos restantes serviram como controle. Aos 30 dias de indução da doença periodontal, foi gravado o comportamento sexual dos ratos e após, os animais foram sacrificados para a obtenção de amostra gengival, hemimandíbula e pênis para as análises morfológicas, morfométricas, imunológicas e radiográficas. Os resultados mostraram que a periodontite experimental contribuiu para a redução do comportamento sexual dos ratos (p<0.05), bem como causou perda óssea alveolar, com significante atividade osteoclástica e aumento de volume gengival (p<0.05). Além disso, o grupo com periodontite apresentou uma maior concentração de citocinas inflamatórias, tal como IL-6 nas gengivas e TNF-&#945; no pênis (p<0.05). Não houve diferença estatisticamente significativa nas dimensões ventral-dorsal e látero-lateral do pênis (p>0.05), no entanto verificou-se uma maior espessura e menor área estatisticamente significante da artéria dorsal do pênis no grupo com periodontite experimental, assim como uma menor área dos espaços vasculares dos corpos cavernosos (p<0.05). Com base nesses resultados, conclui-se que a peridontite experimental provocou alterações estruturais e funcionais no pênis de ratos Wistar, bem como alterações de comportamento sexual, portanto a inflamação sistêmica causada pela doença periodontal pode ser um importante fator de risco para a disfunção erétil, por isso se faz necessário o desenvolvimento de intervenções envolvendo vários profissionais da saúde para prevenção e tratamento dessas duas patologias que comprometem a saúde e bem-estar geral dos indivíduos