Atherosclerotic cardiovascular disease is the leading cause of death of adults in North America. Diabetes is a classical risk factor for atherosclerotic cardiovascular disease. Plasminogen activator inhibitor-1 (PAI-1) is the major physiological inhibitor of fibrinolysis. Elevated levels of PAI-1, oxidized low-density lipoprotein (oxLDL) and glycated LDL (glyLDL) were detected in patients with diabetes. Increased oxidative stress is associated with diabetic cardiovascular complications. Previous studies in our laboratory demonstrated that oxLDL or glyLDL increased the production of PAI-1 or reactive oxygen species (ROS) in vascular endothelial cells (EC). This study was undertaken to investigate transmembrane signaling mechanisms involved in oxLDL or glyLDL-induced upregulation of PAI-1 in cultured vascular EC. Further, we examined the mechanism for oxLDL or glyLDL-induced oxidative stress in EC. The results of the present studies demonstrated novel transmembrane signaling pathway for oxLDL-induced PAI-1 production in vascular EC. We demonstrated that lectin-like oxLDL receptor-1, H-Ras, a small G-protein and Raf-1/ERK-1/2 mediate oxLDL-induced PAI-1 expression in cultured EC. GlyLDL may activate EC via a distinct transmembrane signaling pathway. The results of the present study demonstrated that receptor for advanced glycation end products, NADPH oxidase and H-Ras/Raf-1 are implicated in the upregulation of heat shock factor-1 or PAI-1 in vascular EC under diabetes-associated metabolic stress. We investigated the effects of oxLDL or glyLDL on mitochondrial function in EC. Treatment with oxLDL or glyLDL significantly impaired the activities of electron transport chain (ETC) enzymes and also increased mitochondria-associated ROS in EC. The findings suggest that oxLDL or glyLDL attenuated activity of ETC and increased ROS generation in EC, which potentially contributes to oxidative stress in vasculature. In conclusion, diabetes-associated lipoproteins may upregulate stress response mediators and PAI-1 production via distinct transmembrane signaling pathways. OxLDL or glyLDL may increase ROS production via NOX activation and the impairment of mitochondrial ETC enzyme activity in EC. The understanding and identification of the regulatory mechanisms involved in diabetes-associated lipoprotein-induced signaling may help pharmacological design for the management of diabetic cardiovascular complications.

Made available in DSpace on 2015-07-22T20:50:56Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-07-27. Added 1 bitstream(s) on 2015-08-11T03:07:21Z : No. of bitstreams: 1 Publico-12807.pdf: 1925667 bytes, checksum: 40f94a2382fcb5e9e0c7155532f0079a (MD5) / Objetivo: Apresentar e avaliar os resultados histológicos, anatômicos e funcionais do procedimento de McIndoe modificado, utilizando a celulose oxidada (Surgicel®). Métodos: Onze mulheres com agenesia vaginal se submeteram à neovaginoplastia com ou sem canalização útero-vaginal utilizando um molde revestido com celulose oxidada. As cirurgias foram realizadas entre janeiro de 2009 e dezembro de 2010. Oito dessas pacientes tiveram o diagnóstico de Síndrome de Mayer-Rokitansky-Kuster-Hauser (MRKH), e as outras três tiveram o diagnóstico de Agenesia Cervico-vaginal (CVA). O tempo médio de seguimento foi 14 meses (variando de 6 a 24 meses), e incluiu exame físico e avaliação do questionário sexual (Female Sexual Function Index-FSFI). Amostras teciduais foram obtidas no momento da cirurgia e 1 a 12 meses após o procedimento. Biópsias vaginais para controle foram obtidas de um grupo de pacientes com doenças ginecológicas benignas. Avaliou-se a epitelização escamosa do tecido da neovagina, e a quantidade total de colágeno das neovaginas, e comparou-se com o grupo controle. Para análise estatística utilizou-se o teste ANOVA e o t-test. Resultados: Seis meses após a cirurgia, o sucesso anatômico foi encontrado em 100% das pacientes com síndrome MRKH (comprimento vaginal > 6 cm), e sucesso funcional em todas as pacientes que iniciaram atividade sexual (Score FSFI > 30). As biópsias mostraram epitelização da neovagina após 6 meses em todas as amostras, e o conteúdo total de colágeno foi comparável ao de uma vagina normal. Uma complicação maior ocorreu em uma paciente com CVA, que culminou em morte. O procedimento de canalização útero vaginal não foi bem sucedido em criar um pertuito para menstruações regulares mensais em nenhum dos casos. Nenhuma das pacientes com CVA obtiveram sucesso anatômico ou funcional. Conclusão: A neovaginoplastia com a técnica de McIndoe modificada, utilizando a celulose oxidada (Surgicel®), procedimento simples e de baixo custo, permite a formação de canal vaginal com epitelização e conteúdo de colágeno similar ao de uma vagina normal. É uma potencial alternativa terapêutica para síndrome de MRKH. / Objective: To present and evaluate the histological, anatomical and functional results of the McIndoe procedure, as modified by the application of oxidized cellulose (SurgicelTM) in women with vaginal agenesis. Study design: Eleven patients with vaginal agenesis underwent vaginoplasty with or without uterovaginal cannulation using a mold that had been wrapped with oxidized cellulose. The surgeries were performed between January 2009 and January 2010. Eight of the patients had been diagnosed with Mayer-Rokitansky- Kuster-Hauser (MRKH) syndrome, and the remaining three had been diagnosed with cervicovaginal agenesis (CVA). The mean follow-up time was 14 months (range, 6-24 months), and it included clinical examinations and evaluation of the Female Sexual Function Index (FSFI). Neovaginal biopsies were taken from the patients with MRKH syndrome at the time of surgery and 1-12 months after surgery. Control tissues were taken from a group of patients without vaginal disease. The histology of the samples was evaluated to determine squamous epithelialization of the neovaginal tissue over time, and the total collagen content of the neovaginas were compared with normal control subjects. For statistical analysis we employed the ANOVA test and the t-test. Results: At 6 months, anatomical success was achieved in 100% of the MRKH syndrome patients (neovaginal length . 6 cm), and functional success was achieved in 100% of the patients who started their sexual life (FSFI score . 30). Biopsy results showed complete epithelialization of the neovagina after 5 monthsin all samples, and the amount of collagen content in the neovaginas progressively increased. After 6 months, the collagen content was comparable to that of a normal vagina. One major postoperative complication occurred in a patient with CVA, which culminated in death. The uterovaginal canalization procedure was unsuccessful at creating an outflow tract for regular menses in all cases. None of patients with CVA achieved anatomical or functional success. Conclusions: The procedure described here offers patients a functional vagina by means of a simple and low-cost procedure that elicits squamous epithelialization of the neovaginal vault, with total collagen content similar to that of normal vaginal tissue. It is a potential alternative therapeutic approach for MRKH syndrome but not applicable to cases of CVA. / TEDE

Este trabalho apresenta as propriedades ópticas dos complexos Európio Tetraciclinas (EuTcs) na presença de LDL e de LDL oxidada com potenciais aplicações em análises clínicas. Foram escolhidos quatro elementos da família das Tetraciclinas: Tetraciclina (Tc), Clorotetraciclina (CTc), Metatetraciclina (MTc) e Oxitetraciclina (OTc) para fazerem parte dos complexos com o íon európio. As melhores condições para se formar os complexos eficientemente foram determinadas, através das medidas dos parâmetros ópticos como: absorção, emissão e de tempo de vida. As melhores concentrações de európio nos complexos EuTcs e possíveis influências de íons inorgânicos normalmente presentes no plasma sanguíneo também foram analisadas. As amostras foram preparadas em pH neutro e a luminescência visível do lantanídeo foi detectada após tempo de repouso das amostras de 15 minutos. Os resultados deste trabalho mostraram que as moléculas de LDL e de LDL oxidada apresentaram um importante papel no aumento da intensidade de emissão dos complexos das Tcs. As medidas realizadas com os complexos EuTcs não apresentaram deslocamentos nos comprimentos de onda dos espectros de absorção e de emissão na presença de LDL, o que demonstra a ausência de interação direta entre as moléculas de Tcs e as moléculas de LDL e LDL oxidada. No entanto, o íon európio pode interagir em diferentes sítios das moléculas de tetraciclinas o que diferenciou a intensidade de emissão de cada complexo. Comparando os resultados obtidos entre os complexos de EuTcs, o complexo EuTc foi o que apresentou perspectivas promissoras na quantificação de LDL e LDL oxidada. / This work presents the optical properties of europium complexes - Tetracyclines (EuTcs) in the presence of LDL and oxidized LDL with potential applications in clinical analysis. Four elements were chosen from the Tetracyclines family: Tetracycline (Tc), Chlortetracycline (CTc), Metatetraciclina (MTc) and Oxytetracycline (OTc) to be part of complexes with europium ion. The best conditions to form the complex efficiently were determined through measurements of optical parameters such as absorption, emission and lifetime. The best concentrations of europium complexes in EuTcs and possible influences of inorganic ions normally present in blood plasma were also analyzed. The samples were prepared at neutral pH and the visible luminescence of lanthanide was detected after resting time of the samples of 15 minutes. These results showed that the molecules of LDL and oxidized LDL have an important role in increase of the emission intensity for Tcs complexes . The measurements performed with the complex EuTcs showed no shifts in the wavelengths of the absorption and emission spectra in the presence of LDL, which demonstrates the absence of direct interaction between the molecules of Tcs and the molecules of LDL and oxidized LDL. However, the europium ion can interact at different sites of the tetracyclines molecules which differed the emission intensity of each complex. Comparing the results obtained between the complexes EuTcs, the complex EuTc is the one that presented the promising prospects in the quantification of LDL and oxidized LDL.

No description available.

This study investigated whether oxLDL and/or angiotensin (Ang) II signaling pathways mediate traffic-generated air pollution- exposure induced alterations in blood-brain barrier (BBB) integrity and permeability in a healthy wild type (C57Bl/6) mouse model; additionally, whether these outcomes are exacerbated by a high fat-diet investigated. An environmentally relevant concentration of a mixture of vehicle engine exhaust (MVE) was used. To investigate the hypotheses, 12 wk old male C57Bl/6 mice on either a high fat (HF) or low fat (LF) diet were randomly assigned to inhalational exposure of either filtered-air (FA) or 30 µg PM/m3 diesel exhaust + 70 µg PM/m3 gasoline exhaust (MVE) for 6 hr/day for 30 days. Additionally, we examined mechanisms involved in MVE-mediated alterations BBB integrity using a novel BBB co-culture in vitro model, consisting of mouse primary cerebral vascular endothelial cells on an apical transwell and astrocytes in the basal compartment, which was treated with plasma from the mice on our exposure study. Our in vivo exposure study results showed that MVE inhalation resulted in increased circulating plasma oxLDL and Ang II, compared to FA controls. Additionally, we observed increased cerebral microvascular expression of oxLDL receptors, LOX-1 and CD-36, and Ang II receptor subtype 1 (AT1) in MVE-exposed C57Bl/6 mice, which was further exacerbated with consumption of an HF diet. Increased signaling of both Ang II and oxLDL was associated with decreased BBB integrity, as evidenced by the concurrent reduction in expression of tight junction (TJ) protein claudin-5 and increased permeability of sodium fluorescein (Na-F) from the blood into the cerebral parenchyma. Our results suggest that possible mechanisms involved in oxLDL and/or Ang II-mediated alterations in BBB integrity include oxidative stress and upregulated expression and activity of matrix metalloproteinase (MMP)-9, which is associated with degradation of TJ proteins in the BBB. Our in vitro BBB co-culture results confirm our in vivo findings, as we observe increased BBB permeability (TEER) and decreased integrity (decreased expression of TJ proteins) in the endothelial (apical) layer when treated with plasma from MVE-exposed mice, which was further exacerbated when treated with plasma from MVE-exposed mice on an HF diet. Pre-treatment of the endothelial cells with the AT1 receptor antagonist, Losartan, prior to applying plasma, resulted in attenuation of the alterations observed in endothelial integrity in the BBB co-culture treated with plasma from either MVE+LF or MVE+HF animals. These results suggest Ang II – AT1 signaling mediate, at least in part, the alterations in the BBB integrity observed after exposure to MVE. Moreover, we observed that treatment of the endothelial (apical) layer with plasma from MVE-exposed animals resulted in increased production of inflammatory mediators interleukin-6 (IL-6) and transforming growth factor-β in the astrocyte media (basal compartment). Additionally, these same astrocytes also displayed increased production of angiotensin-converting enzyme (ACE) and also AT1 receptor mRNA expression, while showing decreased expression of the aryl hydrocarbon receptor (AhR) and glutathione peroxidase (GPx). Collectively, these results suggest that exposure to the ubiquitous environmental air pollutant, vehicle engine emissions, results in increased oxLDL and Ang II signaling in the cerebral microvasculature, which is associated with decreased vessel integrity and increased oxidative stress and inflammatory signaling in the CNS. The observed detrimental outcomes are even further exacerbated when coupled with the consumption of an HF diet.

No description available.

No description available.

Atherosclerosis is a disease characterized by cholesterol-rich plaques within the intima of medium and large arteries. Cholesterol deposition is thought to occur by infiltration of low-density lipoprotein (LDL) into lesions followed by uptake into macrophages, generating lipid-loaded “foam cells.” Foam cells can also be generated in vitro by treatment of macrophages with LDL or oxidized LDL (oxLDL). The purpose of the current investigation was to determine the contribution of selective cholesteryl ester (CE) uptake versus whole-particle uptake during LDL-induced foam cell formation in cultured macrophages. Murine bone marrow-derived macrophages (BMMs) exhibited significant cholesterol accumulation when treated with LDL as indicated by quantification of cellular cholesterol and visualization of Oil Red-O-stained neutral lipid droplets. Uptake of LDL cholesterol was determined by measuring uptake of 3H and 125I into BMMs during treatment with [3H]CE/125I-LDL. [3H]CE uptake was linearly related to the LDL concentration at the concentrations used and was much larger than 125I uptake, indicating that the majority of LDL-cholesterol was acquired by nonsaturable, selective CE uptake. This pathway was demonstrated to be independent of whole-particle uptake by showing that inhibition of actin polymerization blocked LDL particle uptake but not selective CE uptake. Analysis by thin-layer chromatography (TLC) indicated that following uptake, [3H]CE was rapidly hydrolyzed into [3H]cholesterol by cells and largely effluxed into the culture medium. In contrast to LDL, studies of [3H]CE/125I-oxLDL uptake demonstrated that CE was acquired from oxLDL by whole-particle uptake with little or no selective CE uptake. Using a series of ten different [3H]CE/125I-oxLDLs oxidized for 0-24 hours, selective [3H]CE uptake was shown to be progressively impaired by LDL oxidation, while 125I-LDL particle uptake was increased as expected. Interestingly, the impairment of selective CE uptake occurred very early in LDL oxidation and this minimally oxidized LDL induced significantly less cholesterol accumulation in BMMs compared to native LDL. Together, these results demonstrate that selective CE uptake is the primary mode of cholesterol uptake from LDL but not oxidized LDL, a finding that has important implications for cholesterol metabolism in atherosclerotic lesions. Future studies seek to identify the molecular components that participate in the macrophage selective CE uptake mechanism.

O objetivo deste trabalho foi avaliar se as enzimas secretadas pelo fungo de decomposição parda Gloeophyllum trabeum podem atuar como auxiliares às celulases comerciais na hidrólise de substratos celulósicos oxidados. Para isto foi realizada a oxidação de celulose microcristalina Avicel mediante reação de Fenton (Fe+2, H2O2) e tratamento com CuSO4. O substrato oxidado foi caracterizado e posteriormente foi determinado o efeito desta modificação sobre a eficiência hidrolítica de enzimas comerciais (Celluclast/Novozym) e sobre misturas destas enzimas comerciais suplementadas com extrato enzimático obtido de G. trabeum. As reações realizadas com Avicel indicaram que ambos tratamentos químicos foram efetivos para modificar grupos funcionais presentes na celulose. A caracterização fisicoquímica dos substratos oxidados permitiu concluir que o tratamento Fenton gerou transformações mais intensas na celulose do que o tratamento com CuSO4. O análise dos hidrolisados ácidos das amostras oxidadas por Fenton mediante espectrometria de massas (GC-MS) confirmou que a oxidação ocorre principalmente no C1. As hidrólises enzimáticas utilizando preparações com diferentes cargas das enzimas comerciais apresentaram uma diminuição significativa da conversão de celulose quando as amostras são tratadas com o reagente Fenton. Por outro lado, a celulose oxidada gerada pelo tratamento com CuSO4 ainda é eficientemente hidrolisada pelas enzimas. As duas amostras de celulose Avicel oxidadas também foi hidrolisada utilizando misturas de enzimas comerciais e extratos enzimáticos de G. trabeum. Em todas as condições ensaiadas a suplementação da mistura reacional com extratos enzimáticos do fungo de decomposição parda G. trabeum diminuíram a eficiência de hidrólise enzimática da celulose oxidada. O emprego do dobro de carga enzimática de enzimas comerciais permitiu recuperar os níveis de hidrólise observados na celulose in natura. / The goal of this work was to evaluate whether enzymes produced by the brown-rot fungus Gleophyllum trabeum would be able to act as auxiliary enzymes of commercial cellulases during the enzymatic hydrolysis of oxidized cellulose. To reach this goal an oxidation of microcrystalline-cellulose (Avicel) was performed by two differente reaction systems: Fenton reaction (Fe2+, H2O2) or CuSO4 treatments. The oxidized subtracts were characterized and the effect of cellulose modification on the hydrolytic efficiency of commercial enzymes, supplemented or not with the enzymes recovered from a G. trabeum culture, was determined. The Avicel treatment by the oxidative systems resulted in cellulose structural changes as evidenced by infrared spectroscopy, viscosity measurements and chemical characterization by HPLC and GC/MS chromatography of the monomers recovered after acid hydrolysis. The physicochemical characterization of the oxidized celluloses allowed to conclude that the transformation of the cellulose was more intense when Fenton treatment was used as compared with the CuSO4 treatment. The analysis of the acid hydrolysates by GC-MS indicated that the oxidation by Fenton reaction occurred mainly at the C1 of the cellulose chain, resulting in gluconic acid in the acid hydrolysates. A meaningful decrease of the cellulose conversion was observed when Fetnon-treated samples were submitted to enzymatic hydrolysis using different amounts of commercial enzymes. On the other hand, the CuSO4-oxidized cellulose was still efficiently hydrolyzed by high enzyme dosages. The oxidized Avicel substrates were also hydrolyzed using a mixture of the commercial enzymes with enzymes recovered from G. trabeum cultures. At all tested conditions, the supplementation of the reaction mixture with enzymatic extracts from G. trabeum decreased the enzymatic hydrolysis efficiency. Nevertheless, the normal levels of enzymatic hydrolysis were recovered when the commercial enzymes dosage two-fold increased.

A obesidade é considerada uma doença crônica e multifatorial, onde eventos como a inflamação de baixa intensidade e as modificações oxidativas estão presentes. A elevada prevalência de obesidade tem impacto direto no desenvolvimento precoce de diabetes mellitus, hipertensão e outros fatores de risco cardiovasculares. Esse perfil tem motivado a identificação de biomarcadores precoces, sendo o monitoramento da lipoproteína de baixa densidade oxidada (LDLox) e lipoproteína de baixa densidade eletronegativa [LDL(-)] potenciais candidatos. Diante do exposto, o objetivo deste estudo foi realizar a análise comparativa e de correlação entre o conteúdo de LDLox e [LDL(-)] em adolescentes. Foram selecionados 137 adolescentes de ambos sexos, com faixa etária de 10 a 19 anos e regularmente em matriculados em escolas públicas da cidade de São Paulo. O peso, altura e circunferência da cintura (CC) foram avaliados. Após jejum (12h-15h) foi coletada uma amostra de sangue e, a partir do plasma, foram realizadas as seguintes análises: glicose, insulina, perfil lipídico, apolipoproteína (AI e B), ácidos graxos não esterificados, tamanho de HDL, atividade da CETP e LDL(-) e LDLox. Os resultados encontrados foram analisados por meio do programa SPSS 15.0, considerando valor de significância de p< 0,05. Os 137 adolescentes foram distribuídos em dois grupos: 71 no Eutrófico (51,82%) e 66 no Obeso (48,18%), segundo a classificação do IMC. 48 (35,04%) dos adolescentes eram do sexo masculino e 89 (64,96%) do sexo feminino, com idade média de 14,2 (2,3) anos. Em relação à CC, observou-se que essa confirmou a classificação feita pelo IMC. Observou-se também uma maior prevalência de hipertensão (65% p = 0,011) e obesidade (64,7% p=0,041) nos antecedentes familiares do grupo Obeso quando comparado ao grupo Eutrófico. Os adolescentes obesos apresentaram maiores valores de triglicerídeos, HDL, APO B, CETP, insulina e LDL(-) e LDLox, quando comparados aos eutróficos. Perfil inverso foi observado para Apo AI. O conteúdo de LDLox e LD(-) variou significativamente em função do IMC. Entretanto, essas partículas de LDLs não se correlacionaram entre si, embora tenham apresentado associação com outros parâmetros cardiometabólicos. Os resultados obtidos confirmam o impacto negativo da obesidade sobre os parâmetros cardiometabólicos de adolescentes e, apesar do conteúdo de LDLox e LDL(-) ter aumentado em função do IMC, essas partículas parecem ser estruturalmente distintas. Essa possibilidade foi reforçada pelas diferentes associações dessas partículas com outros marcadores bioquímicos. / Obesity is considered a chronic and multifactorial disease, where events such as low intensity inflammation and oxidative modifications are present. The high prevalence of obesity has a direct impact on the early development of diabetes mellitus, hypertension and other cardiovascular risk factors. This profile has motivated the identification of early biomarkers, and monitoring the oxidized low density lipoprotein (oxLDL) and electronegative low-density lipoprotein [LDL (-)] are potential markers. The aim of this study was to conduct a comparative analysis and correlation between the content of oxLDL and [LDL (-)] in adolescents. We selected 137 adolescents of both sexes, aged 10-19 years, enrolled in public schools in the city of São Paulo. The weight, height, waist circumference (WC) were assessed. After fasting (12-15h) samples of blood were collected and from plasma were performed the following analyzes: glucose, insulin, lipid profile, apolipoprotein (AI and B), NEFA, HDL size, CETP activity and LDL (-) and oxLDL. The results were analyzed by using SPSS 15.0 considering significant value of p <0.05. The 137 adolescents were divided into two groups: 71 Normal Weight (51.82%) and 66 Obese (48.18%), according to BMI classification. 48 (35.04%) of the adolescents were male and 89 (64.96%) females with a mean age of 14.2 (2.3) years. Regarding the CC observed, it confirmed the classification made by BMI. There was a higher prevalence of hypertension (65% p = 0.011) and obesity (64.7% p = 0.041) in the family history group Obese when compared to normal weight. Obese adolescents had higher triglyceride, HDL, APO B, CETP, insulin and LDL (-) and oxLDL, compared to normal weight. Reverse profile was observed for Apo AI. The content of oxLDL and LDL (-) varied significantly according to BMI. However, these LDL particles were not correlated with each other, although they showed cardiometabolic combination with other parameters. The results confirm the negative impact of obesity on cardiometabolic parameters of teenagers and although the content of oxLDL and LDL (-) increased as a function of BMI, these particles appear to be structurally distinct. This possibility was reinforced by different associations of these particles with other biochemical markers.