Abstract 19506: The Role of Mir-16 on Endothelial Dysfunction in Rat with Atherosclerotic Diet with a Chronic Model of Femoral Artery Occlusion and Carotid Balloon Injury

Abstract

Background: Peripheral artery disease (PAD) is associated to coronary endothelial dysfunction and is an important predictor of future cardiovascular events. MicroRNAs play a critical role in modulating a variety of cellular processes by post-transcriptional regulation of their target genes. They are highly expressed in human endothelial cells (ECs), playing key role in essential biological processes. Thus, the aim of the present study was to evaluate the role, if any, of miRNAs in endothelial dysfunction in a model of PAD. Methods and Results: Male Wistar rats were provided of a standard atherogenic, high cholesterol diet and divided into 4 groups: no treatment (sham); rat subjected to vascular injury by balloon catheter (B.I.); rats underwent common femoral artery ligation (PAD); rats subjected to common femoral artery ligation and, after 21 days, subject to balloon injury (PAD+B.I.). RNA from intima was separately obtained from carotid arteries and evaluated for endothelial specific miRNAs using a stem-loop real-time-PCR. Interestingly, among the most important miRNAs expressed, real time RT-PCR showed an important up-regulation of miR-16 in ECs extracted by injured carotid artery of PAD+BI group respect the ECs of the others groups. A bioinformatics approach revealed highly conserved binding sites for miR-16 in the 3’UTR of SMAD7, an inhibitor of TGF-β1 signaling. The expression level of SMAD7 was measured in ECs treated with antagomiR-16 using immunoblotting analysis. Up-regulation of miR-16 revealed a reduction of SMAD7 levels. Also, we evaluated the effects of miR-16 inhibition in ECs on mRNA and protein levels of eNOS and NF-kB, regulators of endothelial dysfunction. Real-time PCR showed an increased expression of eNOS mRNA in ECs treated with antagomiR-16. Furthermore, immunoblotting revealed increased eNOS protein production as well as its activation in ECs by miR-16 inhibition. Finally, we showed an increased NF-kB activation in ECs after up-regulation of miR-16. Conclusions: Our data demonstrated that in rat with PAD and atherosclerotic diet, miR-16 is up-regulated in endothelial cell of carotid arteries after balloon injury. This findings might of importance to assess future strategies in patients with PAD.