Efeitos agudos da exposição “in vitro” ao ferro (ii) sobre a função endotelial de aorta de ratos

• Autor(a) principal: Carnelli, Anderson Ramiro Rangel
• Data de Publicação: 2023
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: Repositório Institucional da Universidade Federal do Espírito Santo (riUfes)
• Texto Completo: http://repositorio.ufes.br/handle/10/16937
• topic: Fisiologia; Hemocromatose; Anemia; Politransfusões; Ferro-heme; Estresse oxidativo; Radicais livres; Endotélio; Reatividade vascular; subject.br-rjbn

INTRODUCTION: Although iron is an essential mineral for homeostasis, excessive iron accumulation in our body can lead to intoxication or overload, since there are not regulated processes for its excretion. Oral intoxication, mainly by children due to accidental ingestion, or parenteral administration, due to high-dose infusions, exhibit high morbidity and mortality rates. In excess, free iron can damage organs and systems, especially the cardiovascular system. It is currently well known that not only chronic iron overload, but also acute incubation of rat myocardial tissue with ferrous ion (Fe2+) result in contractile dysfunction of the cardiac muscle. In addition, several studies also suggest significant vasculopathy in rats chronically overloaded with iron, related to intense oxidative stress. However, the “in vitro” effects of this metal on the vasculature have not been identified. Thus, due to the vasculopathy already described in chronic exposure "in vivo" models, and its oxidative potential, the hypothesis is that "in vitro" exposure of aortic segments to Fe2+ is capable of altering the endothelial structure and function, in its modulatory role on vascular tone. OBJECTIVE: To evaluate whether “in vitro” exposure to high concentrations of Fe2+ induce morphofunctional changes in the vascular endothelium of rat aortic segments. MATERIAL AND METHODS: Aortic rings isolated from male Wistar rats (250-350g) were used to evaluate the vascular reactivity to phenylephrine after incubation with a standard nutrient solution added with ferrous sulfate (FeSO4 10, 25, 100, 250, and 1000 µM) for 30 minutes. the vasodilatory responses to acetylcholine or a nitric oxide donor, sodium nitroprusside, were evaluated in rings previously pre-contracted with phenylephrine. In addition, the role of the endothelium in the effects of Fe2+ 100 and 1000 µM on vascular reactivity was analyzed by mechanical injury of the intimal layer. Furthermore, some segments with intact endothelium were pre-incubated with an inhibitor of nitric oxide synthase, a cyclooxygenase inhibitor, a hydroxyl radical scavenger, and a hydrogen peroxide inactivator (L-NAME, indomethacin, DMSO and catalase, respectively). Finally, samples of aortic segments were analyzed by scanning electron microscopy and energydispersive spectroscopy for evaluation of the morphology and elemental mapping of the endothelial surface; in addition to the extraction of vascular tissue for analysis of advanced protein oxidation products and the main product of lipid peroxidation, malondialdehyde. RESULTS: “In vitro” exposure to Fe2+ increased vascular reactivity to phenylephrine after 30 minutes, from 25 µM, with more significant effects observed after exposure to concentrations of 100, 250, and 1000 µM. In the acetylcholine curves, aortic rings exposed to high Fe2+ had a lower vasodilatory response, while the response sodium nitroprusside was preserved, suggesting an impairment in endothelial function. Removal of the endothelium and incubation with L-NAME increased vasoconstrictive response in all rings. However, the magnitude of this increase was lower in those arterial segments exposed to Fe2+ , indicating a reduction in endothelial modulation and participation of nitric oxide, which was confirmed with DAF fluorescence that evidenced reduced NO bioavailability in samples incubated with Fe2+ . In the presence of indomethacin, vasoconstriction of aortic rings exposed to Fe2+ 1000 µM decreased, suggesting a role of the AA-COX pathway in hypercontractility at higher concentration of Fe2+ . After incubation with catalase and DMSO, there was a decrease in the contractile response of segments exposed to Fe2+, indicating a role of reactive oxygen species (ROS) in this effect. Despite this, there was no significant difference in AOPP or MDA in the rings incubated with Fe2+, suggesting that, despite the effect on vascular reactivity, the increase in ROS at 30 minutes should occur at levels still undetectable by the techniques used and do not cause sufficient damage. Qualitative microanalysis spectroscopy showed significant variations in iron concentration among the studied groups. The control group had low or no presence of iron, while the Fe-incubated groups showed a considerable and even greater increase.. CONCLUSION: “In vitro” incubation with high concentrations of Fe2+ is sufficient to damage to endothelial cells, resulting in impaired endothelial modulation. The mechanisms appear to be related to the exacerbation of contractile pathways derived from COX and a decrease in the bioavailability of NO in association with the production of ROS.