Name: ANDERSON RAMIRO RANGEL CARNELLI
Publication date: 06/04/2023
Advisor:
Name |
Role |
|---|---|
| LEONARDO DOS SANTOS | Advisor * |
Examining board:
| Name |
Role |
|---|---|
| ALESSANDRA SIMAO PADILHA | Internal Examiner * |
| LEONARDO DOS SANTOS | Advisor * |
| VINÍCIUS BERMOND MARQUES | External Examiner * |
Summary: 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 energy-
dispersive 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
