Induction of microRNA-199 by Nitric Oxide in Endothelial Cells is Required for Nitrovasodilator Resistance via Targeting of Prostaglandin I2 Synthase
Background—Nitrates are widely used to treat coronary artery disease, but their therapeutic value is compromised by nitrate tolerance, owing to the dysfunction of prostaglandin I2 synthase (PTGIS). Micro RNAs (miRNAs) repress target gene expression and are recognized as important epigenetic regulators of endothelial function. The aim of this study was to determine whether nitrates induce nitrovasodilator resistance via microRNA-dependent repression of PTGIS gene expression.
Methods—Nitrovasodilator resistance was induced by nitroglycerin (GTN; 100 mg/kg/day, 3 days) infusion in Apoe-/- mice. The responses of aortic arteries to nitric oxide (NO) donors were assessed in organ chamber. The expressional levels of miR-199a/b were assayed by RT-qPCR or FISH.
Results—In cultured HUVECs, NO donors induced miR-199a/b endogenous expression of and downregulated PTGIS gene expression, both of which were reversed by carboxyl-PTIO or silence of serum response factor. Evidence from computational and luciferase reporter gene analyses indicates that the seed sequence of 976-982 in 3'-UTR of PTGIS mRNA is a target of miR-199a/b. Gain-functions of miR-199a/b resulting from chemical mimics or adenovirus-mediated overexpression increased PTGIS mRNA degradation in HEK293 cells and HUVECs. Furthermore, GTN-decreased PTGIS gene expression was prevented by miR-199a/b antagomirs or was mirrored by the enforced expression of miR-199a/b in HUVECs. In Apoe-/- mice, GTN induced the ectopic expression of miR-199a/b in the carotid arterial endothelium, decreased PTGIS gene expression, and instigated nitrovasodilator resistance, all of which were abrogated by miR-199a/b antagomirs or LNA-anti-miR-199. Importantly, the effects of miR-199a/b inhibitions were abolished by adenovirus-mediated PTGIS deficiency. Moreover, the enforced expression of miR-199a/b in vivo repressed PTGIS gene expression and impaired the responses of aortic arteries to GTN/sodium nitroprusside/acetylcholine/cinaciguat/riociguat, whereas the exogenous expression of PTGIS gene prevented nitrovasodilator resistance in Apoe-/- mice subjected to GTN infusion or miR-199a/b overexpression. Finally, indomethacin, iloprost, and SQ29548 improved vasorelaxation in GTN-infused Apoe-/- mice, while U51605 induced nitrovasodilator resistance. In humans, the increased expressions of miR-199a/b were closely associated with nitrate tolerance.
Conclusions—NO-induced ectopic expression of miR-199a/b in endothelial cells is required for nitrovasodilator resistance via the repression of PTGIS gene expression. Clinically, miR-199a/b is a novel target for the treatment of nitrate tolerance.
- Received April 29, 2017.
- Revision received January 18, 2018.
- Accepted January 24, 2018.