Abstract 12680: Silencing miR-145 Prevents Potentiating Effects of Human Electronegative Low-Density Lipoprotein on Platelet Activation
Background: During ST-elevation myocardial infarction (STEMI) and ischemic stroke, platelet activation triggers thrombosis. Low-density lipoprotein in human plasma can be chromatographically resolved into 5 subfractions (L1-L5); L5, the most electronegative and atherogenic subfraction, is significantly elevated in patients with these acute ischemic events. Whereas L5 potentiates platelet activation and aggregation by activating nuclear factor kappa B (NF-κB), L1 does not. Because NF-κB can be negatively regulated by ubiquitin-specific peptidase 31 (USP31), we examined whether L5-mediated thrombosis can be prevented by silencing microRNA (miR)-145, an upstream modulator of USP31.
Methods and Results: Cultivated human platelets were treated with L5 or L1 from STEMI or stroke patients. L5, but not L1, induced phosphorylation of IκB kinase β (IKKβ) and degradation of its inhibitor IκBα, thereby activating NF-κB. These effects were prevented by neutralizing LOX-1, the lectin-like receptor that mediates L5 signaling. Activated platelets rapidly formed aggregates bridged by the stimulated GPIIb/IIIa receptors. L5-induced degradation of IκBα was inhibited via deubiquitination by USP31, which was in turn inhibited by miR-145. Quantitative analysis by stem-loop real-time PCR showed overexpression of miR-145 after pre-miR-145 overproduction in L5-treated platelets. Specific antisense oligodeoxynucleotide (ODN) against miR-145, but not scrambled ODN, prevented USP31 inhibition, thus preventing NF-κB activation. We injected 5 mg/kg of L5 or L1 into C57/BL6 mice. After 30 min, the tail-bleeding time was 44% shorter in the L5 mice than the L1 mice (n=12; P≤0.05); this reduction was prevented by coadministering miR-145 antisense ODN, but not scrambled ODN.
Conclusions: miR-145 plays a role in L5-mediated platelet aggregation. Further investigation is warranted to examine whether silencing miR-145 reduces the risk of myocardial infarction and stroke.
- © 2013 by American Heart Association, Inc.