Disfunção endotelial pela inibição da síntese de óxido nítrico: proposta e caracterização de um modelo celular in vitro

Abstract

Vascular endothelium is a type of epithelial tissue, formed by a monolayer of cells, which internally covers blood vessels and is essential for the maintenance of vascular homeostasis, guaranteed by the production of vasoactive biomolecules. An imbalance in the bioavailability of these biomolecules may trigger a pathological state, such as endothelial dysfunction (ED), which is mainly characterized by a reduction in the availability of nitric oxide (NO) and an increase in the production of reactive oxygen species (ROS). Considering the importance of endothelial dysfunction for the progress of pathologies such as atherosclerosis, for example, it is necessary to develop experimental models that simulate such a condition. This study was then carried out to develop and characterize an in vitro cellular model of ED by inhibiting NO synthesis. Initially, the thymus-derived endothelial cells (tEnd.1 line) were treated with the NO synthesis inhibitor (L-NAME) at 1μM, 10 μM, 100 μM and 1mM for 12, 24, 48, 72 , 96 and 120 hours, with and without retreatment every 24 hours. Subsequently, the best treatment conditions for inducing an ED state in the cells were determined based on the evaluation of cell viability and nitrite concentration. Treatments with 10 μM and 100 μM of L-NAME for 72 hours without retreatment and 96 hours with retreatment were selected. Real-time PCR was performed for p22phox, p47phox and eNOS genes. Results were significant for p22phox, in which the relative levels of gene mRNA were reduced by 65% in response to 100 μM L-NAME for 96 hours with retreatment. Western blotting analysis of eNOS protein expression was not successful. Finally, results of the evaluation of lipid peroxidation using the Buege and Aust technique were not statistically significant. Together, the results show that treatment with 100 μM L-NAME for 96 hours with retreatment was able to induce ED, decreasing the availability of NO, which was verified by the reduction in the concentration of nitrite and by the reduction in the expression of p22phox. However, additional studies aiming to determine the role of ROS in cells submitted to treatment with L-NAME are necessary for the improvement and characterization of the proposed experimental model.