Abstract 18577: Oxidative Stress-Induced Mir-200c Disrupts Sirt1, Foxo1 and Enos Regulatory Loop, Causing Endothelial Dysfunction and Oxidative Stress Increase
Introduction: Reactive oxygen species (ROS) play a causal role in different pathophisyologic conditions. We previously showed that hydrogen peroxide (H2O2) up-regulated miR-200c in endothelial cells (ECs) causing apoptosis and senescence and that acute hindlimb ischemia enhanced miR-200c in skeletal muscle of wild-type mice, whereas in p66ShcA -/- mice, which display lower ROS after ischemia, miR-200c up-regulation was markedly inhibited. The loop among Sirtuin 1 (SIRT1), endothelial nitric oxide synthase (eNOS) and forkhead box O1 (FOXO1) plays a pivotal role in ECs. SIRT1 enhances eNOS-derived nitric oxide (NO) bioavailability. NO stabilizes SIRT1 mRNA and protein. p53 and FOXO1 transcription factor are SIRT1 deacetylation targets; acetylated p53 increases ROS production and apoptosis; FOXO1 acetylation inhibits its transcriptional activity on SIRT1 and ROS scavengers promoters.
Hypothesis: We hypothesized that miR-200c is involved in the ROS-dependent disruption of SIRT1/eNOS/FOXO1 regulatory loop.
Methods: Human umbilical ECs (HUVEC) were treated with 200 μM H2O2 from 8 to 24 hours. miR-200c over-expression was obtained by lentiviral transduction by premiR-200c encoding vectors; miR-200c inhibition was achieved by Locked Nucleic Acid oligonucleotides against miR-200c. Hindlimb ischemia was induced by left femoral artery dissection in mice. Human fibroblasts were established from 5 healthy young donors (<10 years) and 6 healthy old donors (>65 years).
Results: We found that miR-200c decreased SIRT1, eNOS and FOXO1 protein expression, targeting their 3’UTRs directly. Anti-miR-200c treatment partially rescued SIRT1, eNOS and FOXO1 protein down-modulation caused by H2O2. miR-200c inhibited NO production and increased FOXO1 and p53 acetylation. Accordingly, miR-200c inhibited FOXO1 transcriptional activity down-modulating ROS scavengers as well as increasing ROS production and p66SchA phosphorylation. In two in vivo models of oxidative stress i.e. skin fibroblasts from old donors and a mouse model of hindlimb ischemia, miR-200c was higher vs control and its targets were down-modulated.
Conclusions: miR-200c disrupts SIRT1/FOXO1/eNOS regulatory loop, causing senescence, apoptosis and ROS production.
Author Disclosures: M. D’Agostino: None. F. Carlomosti: None. S. Beji: None. G. Zaccagnini: None. B. Maimone: None. V. Di Stefano: None. F. De Santa: None. S. Cordisco: None. A. Antonini: None. R. Ciarapica: None. E. Dellambra: None. F. Martelli: None. D. Avitabile: None. M.C. Capogrossi: None. A. Magenta: None.
- © 2016 by American Heart Association, Inc.