Endothelial Nitric‐Oxide Blockade Influences Endothelial microRNA Expression

Abstract

Blunted endothelial nitric oxide synthase (eNOS) activity and lower nitric oxide (NO) bioavailability underlie endothelial cell inflammation, senescence, apoptosis and dysfunction and is etiologically involved in atherogenesis. However, the factors fundamental to the vascular consequences linked with reduced eNOS activity are not fully understood. MicroRNAs (miRs) are short single stranded noncoding RNAs that regulate gene expression by inhibiting target protein translation. It is well established that miRs are involved in the regulation of a number of cellular processes such as inflammation, senescence and apoptosis. In addition, altered endothelial cell expression of various miRs have been linked to the development and progression of atherosclerosis. Specifically, miR‐92a, miR‐126, miR‐146a, miR‐150 and miR‐181b are known to play regulatory roles in endothelial cell activation and inflammation; and miR‐217 and miR‐Let‐7a have been shown to modulate the cellular propensity towards senescence and apoptosis. The aim of this study was to determine the effect of eNOS inhibition on endothelial cell expression of miR‐92a, miR‐126, miR‐146a, miR‐150, miR‐155, miR‐181b, miR‐217 and miR‐Let‐7a. Cultured human umbilical vein endothelial cells were harvested after reaching ~90% confluence on the 3rd passage and plated at a density of 5.0×105 cells/condition. Cells were treated with media alone or media containing the eNOS inhibitor, L‐NG‐Nitroarginine methyl ester (L‐NAME; 300 mM) for 24 hours. To determine cellular miR expression, RNA was extracted from 1.0×105 cells and quantified using RT‐PCR. Cellular expression was normalized to RNU6 and calculated as fold change in ΔΔCt from the untreated cells (N=5, experimental units). Cellular expression of miR‐126 (0.80±0.04 fold), miR‐146a (0.75±0.09 fold) and miR‐150 (0.65±0.13 fold) was significantly decreased (25%, 35% and 55%, respectively) in response to L‐NAME treatment compared with untreated controls. In contrast, cellular expression of miR‐92a (0.92±0.07 fold), miR‐181b (0.99±0.16 fold), miR‐217 (1.1±0.13 fold) and miR‐Let‐7a (0.99±0.3 fold) was not significantly affected by L‐NAME. In summary, these data indicate that eNOS inhibition differentially effects endothelial cell miR expression. Cellular expression of anti‐inflammatory miRs (miR‐126, miR‐146a and miR‐150) were markedly reduced. Indeed, miR‐126, miR‐146a and miR‐150 are recognized key regulators of vascular inflammation as they target cell adhesion molecules and proteins involved in NF‐kB activation. Importantly, reduced expression of these miRs is associated with elevated vascular risk. Thus, the increase in endothelial inflammation associated with conditions characterized by diminished eNOS activity may be due, in part, to miR dysregulation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.