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Increased production of oxygen free radicals by phagocytes in hypertensive or coronary disease subjects

Muniz-Junqueira Imaculada; Mota Lícia; Aires Rodrigo; Junqueira Jr Luiz

University of Brasilia Department of Pathology and Department of Internal Medicine.
Laboratory of Cellular Immunology and Division of Cardiology
Brasilia. Brazil.

Abstract
Introduction
Objectives
Subjects and Methods
Results
Discussion and Conclusions

Abstract
Introduction: Complicated arterial hypertension and coronary disease are among the major causes of mortality in the world. These conditions can be linked to immunological mechanisms related to atherosclerotic damage of vascular endothelial cells dependent of altered production of cytokines by cells of the immune system, in association with others factors such as the oxygen free radicals.
Objective: To evaluate the capacity of phagocytes to produce oxygen free radicals in hypertensives and in subjects with ischemic cardiopathy as compared to individuals with others cardiopathies and health controls.
Subjects and Methods: It was evaluated 14 hypertensive patients, 8 with coronary disease, 11 with Chagas’ heart disease without cardiac failure, 4 with chronic rheumatic cardiopathy and 14 normal controls. Whole blood was placed on a slide and incubated in a wet chamber by 45 min at 37° C. The slides were washed and adhered monocytes and neutrophils were treated with nitrobluetetrazolium (NBT) solution during 20 minutes with and without stimulus of 5 Saccharomyces cerevisiae per phagocyte. The nuclei of phagocytes were dyed with safranin and the percentage of NBT reduction in cytoplasm were determined by light microscopy. The percentage of reduced NBT is directly proportional to the amount of superoxide (O2-) produced by the phagocytes. The means of the proportion presented by the groups were compared by analysis of variance.
Results: A statistically significant difference (p = 0.035) was observed between the mean ± sd percentage of reduced NBT for the patients with hypertension (88.9 ± 6.7%), coronary disease (91 ± 6.3%), rheumatic cardiopathy (85.2 ± 5.1%), Chagas’ heart disease (73.5 ± 17.6%) and normal control subjects (79 ± 17.7%).
Conclusion: The phagocytes of patients with hypertension and ischemic cardiopathy appears to have the more increased capacity to produce oxygen free radicals. These data suggest that the increased amount of superoxide produced in these patients may contribute to cause endothelial damage and consequently to induce the development of atherosclerosis. (PIBIC-UnB/CNPq)

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Introduction:

Complicated arterial hypertension and coronary disease are among the major causes of morbidity and mortality in the world. These conditions can be consequences of the atherosclerotic chronic damage of vascular endothelial cells. Atherosclerosis is the result of an intricate interplay between several factors as lipid metabolism, production of cytokines, haemodynamic vascular stress and behavioral risk factors. An inappropriate immune response to vascular injury can participate of initiation of atherosclerosis. Macrophages are present in high number in atherosclerotic plaques and these cells can produce a lot of cytokines and oxygen free radicals which can damage the cells and increase the lesion. It is not yet clear if an individual with cardiac damage due to atherosclerosis or other pathological process is able to respond to distinctive stimuli producing increased amount of oxygen free radicals that can potentially damage the endothelial cells.

Objectives: 

We aimed at evaluate the capacity of phagocytes to produce oxygen free radicals in hypertensives individuals and in subjects with ischemic cardiopathy as compared to individuals with other cardiopathies and health controls.

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Subjects and Methods: 

The ethical rules of Helsinki Declaration and those from the Health Ministry of Brazil for experimentation in human beings were strictly followed throughout this work, and the patients gave their informed consent before tested. It was evaluated 14 hypertensive patients, 8 with coronary disease, 11 with Chagas’ heart disease without heart failure, 4 with chronic rheumatic cardiopathy and 14 normal controls, of both sexes, between 18 to 60 years old . Peripheral blood was collected from individual volunteer, and 40 m l by well were placed on slides and incubated for 45 minutes in a wet chamber at 37° C. After rinsing the slides with 0.15M phosphate buffered saline (PBS), pH 7.2, the adherent cells (>98% neutrophils and monocytes) were incubated with 0.05% nitrobluetetrazolium (NBT) solution in Hanks triz with or without stimuli of 5 Saccharomyces cerevisiae in Hanks-triz, pH 7.2, per phagocyte during 20 min in a wet chamber, at 37oC. The mean number of adhered cells per well was previously assessed. The suspension of Saccharomyces cerevisiae was previously incubated with 20% fresh serum from the same individual for 30 minutes at 37oC. Slides were then rinsed with PBS, fixed with absolute methanol, and the nuclei of phagocytes were stained with safranin solution and the percentage of NBT reduction in cytoplasm of phagocytes were assessed in individual preparation by light microscopy. Microscopic fields distributed throughout the slides were randomly selected and all phagocyte in each particular field were examined. Superoxide (O2-) is detected by the reduction of the compound NBT to an insoluble form (formazan) which is observed as blue granules in the cytoplasm of phagocytes. The presence of reduced NBT in cytoplasm show the capacity of phagocyte to produce superoxide (O2-), and the percentage of cell reducing the dye is directly proportional to the amount of superoxide produced by the phagocytes. The means of proportion of cells reducing the NBT showed by the groups were compared by analysis of variance between independent samples with normal distribution. The differences between the groups were tested by analysis of variance, employing the SigmaStat Software Package (Jandel Scientific, USA). The data are expressed as mean ± sd, and a p value £ 0.05 was considered as significant.

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Results:

A statistically significant difference (p = 0.035) was observed between the mean percentage of reduction in NBT for the groups with hypertension (88.9 ± 6.7%), coronary disease (91 ± 6.3%), chronic rheumatic cardiopathy (85.2 ± 5.1), Chagas’ heart disease (73.5 ± 17.6%) and of normal control subjects (79 ± 17.7%). The Figure 1 illustrates the differences observed between the groups.

Figure 1


Percent reduction of nitrobluetetrazolium (NBT) in the normal control group and in groups of patients with Chagas’ heart disease (CHG), essential hypertension (HYP), coronary disease (COR) and chronic rheumatic cardiopathy (RHE). A statistically significant difference was detected between the groups by analysis of variance (p = 0.035). The data are shown as mean and range of 1 standard deviation

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Discussion and Conclusions: 

Phagocytes of patients with hypertension and ischemic cardiopathy showed increased capacity to produce oxygen free radicals as compared to rheumatic heart disease, Chagas’ heart disease and control individuals. In these two clinical situation (hypertension and ischemic cardiopathy) the inappropriate immune response to vascular injury can increase the possibility of initiate an plaque of atherosclerosis, and it is recognized that these free oxygen radicals may damage the endothelial cell. Our data suggest that the individuals with hypertension an coronary disease may produce an increase amount of these radicals than individuals without atherosclerotic disease, and it is possible that this inappropriate production of superoxide may play a role in physiopathogenesis of the disease facilitating the initial production of the plaque of atheroma.

 

Questions, contributions and commentaries to the Authors: send an e-mail message (up to 15 lines, without attachments) to hbp-pcvc@pcvc.sminter.com.ar , written either in English, Spanish, or Portuguese.

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© CETIFAC
Bioengineering
UNER

Update
Nov/14/1999


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