Caracterização por citometria de fluxo das células de sangue, medula óssea e aorta de camundongos hipertensos
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Data
2012-12-07
Autores
Campagnaro, Bianca Prandi
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Universidade Federal do Espírito Santo
Resumo
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. However, some reports show that high angiotensin II levels increase reactive oxygen species (ROS) production and, consequently, apoptosis in target organs of the disease, the analysis of this peptide effect by flow cytometry remains unclear in the 2K1C model. The objective of this study was to evaluate the effects of 2K1C renovascular hypertension on blood, endothelium and bone marrow cells in mice. Experiments were conducted on male C57 mice (averaging 23 g), which were randomly separated in two groups: Sham (n=25) and two-kidney one-clip (2K1C, n=25). 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, and blood collected by heart puncture. After perfusion, endothelial cells were mechanically isolated from thoracic aorta and placed in warmed HBSS. Bone marrow cells were flushed out of tibias and femurs and placed in ice-cold PBS. Cells were counted using a Neubauer chamber. The identification of endothelial cells was determined by immunofluorescence detection after incubation with CD31-APC (5μl/106 cells). Oxidative stress was determined by H2O2 and O2•- production using the probes DHE and DCFH-DA, respectively. 106 cells were diluted with 1mL PBS and incubated with 20mM DCFHDA and 160µM DHE for 30 min at 37ºC in the dark, then washed and resuspended in 0.5ml of PBS. In order to analyze apoptosis, 106 cells were resuspended in Binding Buffer (BB) and incubated with 5µl of Annexin V-FITC and 5µl of propidium iodide (PI) at room temperature for 15 min in the dark and resuspended in 0.5ml of BB. To assay DNA content, 106 cells were fixed in ice-cold 70% ethanol and fixed at -20ºC. Cells were washed with ice-cold PBS and resuspended in 200μl of staining solution (20mg/ml RNAse, 500μg/ml PI, 10% Triton X-100). All cell preparations were stored on ice, and flow cytometric measurements were terminated within 4 hr. A FACSCanto II cytometer was used for the flow cytometric analysis. FSC and SSC were used to establish size gates and exclude cellular debris from the analysis. In each measurement, 30000 events were analyzed. Data were acquired and analyzed using the BD FACSDiva and FCS Express softwares. Data are means±SEM. Statistical analysis was performed with Student’s t test and Wilcoxon’s. As expected, blood pressure was higher in 2K1C than in Sham mice (Sham: 103±0.8 vs. 2K1C: 144±5.6 mmHg). Flow cytometric analysis showed that 2K1C mice presented a significant increase in O2• - production was higher in blood (Sham: 941±63 vs. 2K1C: 2155±289 a.u.), endothelial (Sham: 432±51 vs. 2K1C: 2630±184 a.u.) and bone marrow (Sham: 1309±175 vs. 2K1C: 12036±2205 a.u.) cells of 2K1C mice compared to Sham. Simultaneously, H2O2 production was also augmented in blood (Sham: 252±23 vs. 2K1C: 531±48 a.u.), endothelial (Sham: 300±30 vs. 2K1C: 1049±112 a.u.) and bone marrow (Sham: 2107±222 vs. 2K1C: 7517±1067 a.u.) cells of 2K1C mice compared to Sham. Apoptosis analysis by Annexin V-FITC/PI demonstrated that hypertensive mice presented higher percentages of apoptotic cells in blood (Q2Sham: 1.5±0.1 vs. Q22K1C: 15.3±3.8; Q4Sham: 1.6±0.2 vs. Q42K1C: 18.3±4.3 %), endothelial (Q2Sham: 1.2±0.2 vs. Q22K1C: 21.1±3.0; Q4Sham: 12.5±2.6 vs. Q42K1C: 31.2±2.4 %) and bone marrow (Q2Sham: 5.7±0.6 vs. Q22K1C: 22.5±2.5; Q4Sham: 12.9±1.1 vs. Q42K1C: 27.2±2.5 %) cells compared to Sham. DNA content analysis showed augmented DNA fragmentation in bone marrow cells of 2K1C (1.54±0.26 %) mice compared to Sham (0.50±0.09 %). Our data suggest that renovascular hypertension increases reactive oxygen species production leading to oxidative stress and, consequently, apoptosis in blood, endothelial and bone marrow cells of hypertensive mice. In addition, this model of experimental hypertension leads to DNA damage which could be due to augmented reactive oxygen species which is known to cause DNA fragmentation.
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Palavras-chave
Renovascular hypertension , Angiotensin II , Flow cytometry , ROS , Apoptosis , DNA fragmentation , Hipertensão renovascular , Angiotensina II , Citometria de fluxo , Apoptose , Fragmentação do DNA
Citação
CAMPANARO, Bianca Prandi. Caracterização por citometria de fluxo das células de sangue, medula óssea e aorta de camundongos hipertensos. Tese (Doutorado em Ciências Fisiológicas) - Universidade Federal do Espírito Santo, Centro de Ciências da Saúde, Vitória, 2012.