Otenaproxesul previne o remodelamento vascular induzido por estenose da artéria carótida de camundongos

Abstract

Neointimal hyperplasia is a critical event involved in the pathophysiology of arterial occlusive diseases, such as restenosis after balloon angioplasty. Increased cyclooxygenases (COXs) expression and reduced level of hydrogen sulfide (H2S), an endogenous gasotransmitter, play a role in vascular remodeling after vascular injury. Therefore, some non-steroidal anti-inflammatory drugs (NSAID), as well as inorganic H2S donors have been shown promising effects in preventing restenosis. We hypothesized that Otenaproxesul, which is a hybrid molecule of Naproxen coupled to an H2S-realising group, prevents vascular injury in mice submitted to occlusion of the carotid artery. The aim was to investigate the effect of Otenaproxesul versus its prototype drug, Naproxen, on arterial remodeling induced by carotid stenosis. All the procedures were approved by the Ethics Committee on the Use of Animals (CEUA/UFMG), under protocol number 13/2019. Male C57BL/6 mice (10 weeks) were submitted to total occlusion of the left common carotid artery to promote the cessation of local blood flow. The animals were divided into four experimental groups (N= 5-7): sham (false operated), stenosis, stenosis + Naproxen (10 mg/kg) and stenosis + Otenaproxesul (16 mg/kg). For local drug delivery in the external carotid artery, drug-loaded hydrogel formulations were made using a commercial polymer hydrogel (Pluronic®). After 21 days, non-invasive systolic blood pressure (SBP) was measured, and blood perfusion in the carotid arteries was analyzed by single-point laser doppler. Thereafter, the animals were euthanized, the tissues were collected and processed for morphometric analysis on sectioned arteries stained with hematoxylin-eosin; immunostaining for COX-1, COX-2, and MMP-2; determination of reactive species of oxygen (ROS), nitric oxide (NO) levels, and gelatinolytic activity (in situ zymography) by fluorescent probes. There was no change in SBP between groups. Carotid artery ligation resulted in a reduction of doppler flow compared to the sham group, which was fully restored by Otenaproxesul. Neointimal thickening and luminal narrowing induced by carotid artery stenosis were partially reduced by Naproxen and abolished by Otenaproxesul. Stenosis was accompanied by stronger immunostaining for both COX-1 and -2. Naproxen and Otenaproxesul reduced COX-1 levels, whereas only Otenaproxesul reversed the increase in COX-2 fluorescence staining. Stenotic arteries presented increased ROS generation and reduced NO levels. Both drugs exhibited antioxidant effect. Importantly, only Otenaproxesul restored NO levels. The expression and activity of MMP-2 were increased by occlusion of the carotid artery, which was reduced only by Otenaproxesul. In conclusion, neointima formation is correlated to the positive regulation of COX enzymes. Otenaproxesul was superior to Naproxen in improving the morphometric and perfusion parameters, as well as in regulating the levels of mediators of intimal hyperplasia of injured arteries. These data suggest that H2S and COX inhibition have synergistic effects on the amelioration of vascular remodeling and that Otenaproxesul is a therapeutic alternative for preventing restenosis.