Mestrado em Ciências Fisiológicas

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Navegando Mestrado em Ciências Fisiológicas por Assunto "2R1C"

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    Efeito da hipertensão renovascular 2R1C sobre as células tronco da medula óssea de camundongos
    (Universidade Federal do Espírito Santo, 2008-08-11) Campagnaro, Bianca Prandi; Meyrelles, Silvana dos Santos; Vasquez, Elisardo Corral; Gouvêa, Sônia Alves
    Angiotensin II has been recognized for a long time as a powerful vasoconstrictor. In addition, several studies have attributed a variety of other biological activities to this peptide, such as, cellular growth, proinflammatory and immunomodulator effects. Moreover, high angiotensin II levels increase reactive oxygen species production. Although, some reports show that different cardiovascular diseases affect the number of several bone marrow cell populations, the effect of this peptide on these cells and DNA remains unclear. The objective of this study was to evaluate the effects of 2K1C renovascular hypertension on the number and DNA damage of bone marrow stem cells in mice. Experiments were conducted on male C57 mice (averaging 23 g), which were randomly separated in two groups: Sham (n=10) and two-kidney one-clip (2K1C, n=10). The renovascular 2K1C hypertension was induced by placing a stainless clip around the left renal artery. The Sham group was subjected to the same surgical procedure, without clip placement. Animals were studied 14 days later, when a catheter was inserted into the right carotid artery for direct arterial pressure measurements. Then, the animals were euthanized, bone marrow was flushed out of the tibiae and femurs and the mononuclear cells isolated by density-gradient centrifugation. Cells were counted using a Neubauer chamber. The identification and quantification of different bone marrow cell population were determined by immunofluorescence detection using a mixture of antibodies. Mononuclear cells were stained with CD117-FITC and CD90.2-PE (5μl/106 cells). The hematopoietic and mesenchymal stem cells were quantified by flow cytometry. The level of DNA damage was determined by the Comet Assay. Cell samples were mixed with low melting point agarose, spread on slides precoated with normal melting point agarose and submerged in lysis solution. Then, comet slides were placed on an electrophoresis chamber filled with unwinding alkali buffer electrophoresed, neutralized, fixed, stained with ethidium bromide and visualized in a fluorescence microscope. Data are expressed as means±SEM. Statistical analysis was performed with Student´s t test. *p<0.05. As expected, blood pressure was higher in 2K1C than in Sham mice (Sham: 133±1,5 mmHg vs. 2K1C: 182±12,5 mmHg). Renovascular hypertension did not affect cell viability (Sham: 97%±0.54 vs. 2K1C: 96%±0.54) and monocyte cell number (Sham: 2.81±0.46 vs. 2K1C: 3.32±0.34 cells/ml x 106 ). However, 2K1C mice presented a significant decrease in stem cell number (2.26±0.13 cells/ml x107 ) when compared with Sham (2.66±0.11 cells/ml x107 ) and a simultaneous increase in lymphocyte number (1.98±0.15 vs. 1.22±0.25 cells/ml x106 ), compared with Sham mice. The flow cytometry analysis showed a significant increase in hematopoietic stem cell number in hypertensive mice (0,41±0,16%) when compared with Sham mice (1,75±0,18%). The mesenchymal stem cell number did not show difference between the groups (Sham: 2,36±0,61% vs. 2K1C: 1,48±0,22%). The comet assay showed that 2K1C mice presented high to severe DNA damage, while Sham mice presented none to moderate DNA damage. Our data suggest that angiotensin II-dependent renovascular hypertension reduce stem cell number by the augmentation of asymmetric cell division rate, which leads to an increase of hematopoietic stem cells differentiation. The elevated differentiation rate could be confirmed by the augment of inflammatory cells number produced in bone marrow. In addition, this model of experimental hypertension leads to DNA damage which could be due to augmented reactive oxygen species produced by angiotensin II high levels, which is known to cause genotoxicity by DNA degradation.
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    Efeitos do tratamento com sildenafil sobre a função vasoconstritora na hipertensão experimental
    (Universidade Federal do Espírito Santo, 2014-12-18) Fahning, Bernah Mathias; Vasquez, Elisardo Corral; Stefanon, Ivanita; Meyrelles, Silvana dos Santos; Pereira, Thiago de Mello Costa
    Cardiovascular diseases account for the majority of pre-mature deaths in thee world. Hypertension is a disease and a risk factor for CVD, and is considered one of the major challenges in public health. Hypertension is a multifactorial condition characterized by high and sustained levels of blood pressure often associated with functional and/or structural changes in target organs. The renovascular hypertension is defined as hypertension due to renal ischemia, usually caused by a partial or complete obstructive lesion of one or both renal arteries. The first experimental studies dating back to 1934 by Goldblatt and colleagues. The model was initially used in dogs which was partially blocked one of the renal arteries in order to study their effects. It was later adapted to rats and mice and such a model used worldwide was known for 2K1C. The mechanism involved in the development and maintenance of 2K1C hypertension is the continuous activation of the renin-angiotensin-system. Previous studies have shown that this model develops endothelial dysfunction and increased oxidative stress, which damages your vascular function. Within this context, sildenafil appears as an alternative treatment, due to the fact that an inhibitor of PDE5 enzyme, increasing the supply of cGMP and therefore the NO. In addition, other studies have shown that sildenafil decreases the activity of NADPH oxidase is a key enzyme production of ROS. The objective of this study was to evaluate the effects of sildenafil on the contractile function, oxidative stress and production of angiotensin fractions. Male mice were C57BL/6 mice with approximately 23 grams. The animals were divided into three groups: Sham, 2K1C 2K1C and treated with sildenafil (40 mg/kg/day). Treatment started 14 days after the induction of hypertension. 28 days after implantation of the dummy clip or surgery some animals were cannulated for blood pressure measurement and cardíacada frequency, other animals were anesthetized, LVM was cannulated and isolated for evaluation of contractile function through the construction of dose-response curves to norepinephrine (NOR). The kidneys were removed and some were stored in liquid nitrogen for determination of thiobarbituric acid (TBARS) which reflects lipid peroxidation consequence of oxidative stress present in this model of hypertension. The plasma was stored for checking the levels of angiotensin fractions. The maximal response (Rmax) and the negative logarithm of drug concentration which caused halfmaximal response (pEC 50) was calculated. Results of pharmacological maneuvers were expressed as the difference in area under the curve (ΔAUC). Results were expressed as mean ± SEM. Statistical comparisons of dose-response curves were made by 2-way ANOVA followed by post hoc Tukey test. Comparisons of Rmax and pEC50, biological parameters, hemodynamic measurements, angiotensin dosage and TBARS were made by ANOVA 1 way followed by post hoc Bonferroni. A value of p <0.05 and p <0.01 were considered statistically significant. The 2K1C animals showed an increase in mean arterial pressure and heart rate (127.9 ± 3.8 and 514.2 ± 7.4, respectively) when compared to the sham group (105.2 ± 2.4 and 441 ± 9.7) and treatment with sildenafil decreased MAP and HR (114.7 ± 2.4 and 471.4 ± 12) compared to 2K1C animals. The fractions of plasma angiotensin I and II did not show significant changes between the groups and the fraction of angiotensin-(1-7) proved to be higher in animals treated with sildenafil compared to 2K1C and sham groups (146 ± 13.3 vs. 102 ± 10.4 and 100, respectively). Likewise the TBARS 2K1C animals show increased if compared to the sham group being restored after treatment with sildenafil (101.5 ± 9.1 vs 63.7 ± 7.0 vs. 64.4 ± 12.5, respectively). The 2K1C animals showed marked hyperresponsiveness when compared to the sham NOR (Rmax 162 ± 14 vs. 118 ± 12, respectively) and the treatment with sildenafil was able to revert the displayed hyperreactivity (116 ± 9). The ΔAUC after removal of endothelium demonstrates that the endothelial function is impaired in 2K1C animals compared to sham (86 ± 7 vs 201 ± 27, respectively), while treatment with sildenafil provides an improvement in this function (134 ± 13). The ΔAUC before and after the addition of apocynin (blocker of NADPH oxidase) shows that the increased contractility 2K1C animals is associated with increased oxidative stress when compared to Sham (101 ± 16 vs 15 ± 6, respectively) and sildenafil deletes this increase in NADPH oxidase ativadade (4 ± 2). Thus it can be concluded that the treatment with sildenafil improves vascular function in experimental hypertension. The mechanisms involved involve increased levels of Ang-(1-7), decreased oxidative stress and improved endothelial function, reflecting also on the improvement of MAP and HR.