Abstract 483: Effect of Glucose on Endothelial Cell Expression of Inflammation-Related miRNAs

Abstract

Hyperglycemia is an independent risk factor for cardiovascular disease (CVD). A key factor underlying hyperglycemia-related CVD risk is endovascular inflammation. The mechanisms underlying hyperglycemia induced endovascular inflammation are not clear. It is now recognized that microRNAs (miRs) play a pivotal role in regulating cellular inflammation pathways, vascular health and, in-turn, cardiovascular disease (CVD). It is currently unknown whether high glucose concentrations adversely affect the expression of inflammation-related miRs in endothelial cells; if so, miR dysregulation may provide novel insight into the proinflammatory endothelial phenotype associated with hyperglycemia. The aim of this study was to determine the effect of high glucose concentrations on the expression of inflammation-related miRs (miR-34a, -92a, -126, -146a, -150, -155 and -181b). Cultured human umbilical vein endothelial cells were harvested on the 3rd passage and plated in 6-well plates at a density of 5.0x10 5 cells/condition. Cells were incubated with RPMI 1640 media containing 25mM D-glucose (concentration representing a hyperglycemic state) or 5mM D-glucose (control condition) for 48 hours. Thereafter, RNA was extracted from 1.0x10 5 cells and miRs were reverse transcribed and expression was determined by RT-PCR. Cellular expression was normalized to RNU6 and calculated as fold change in ΔΔCt from control (N=6, experimental units). There was no effect of high glucose concentrations on the expression of proinflammatory miRs: miR-34a (0.93±0.18 fold), -92a (0.93±0.07 fold) and -155 (0.85±0.10 fold). However, cellular expression of anti-inflammatory miRs: miR-146a (0.64±0.06 fold), -150 (0.69±0.12 fold), and -181b (0.73±0.07 fold), were significantly reduced (~60%, ~45%, and ~40% respectively; P<0.05) in response to the high glucose condition. Lower cellular expression of miR-146a, miR-150 and miR-181b is consistent with a pro-inflammatory endothelial phenotype. These miRs directly target and suppress NF-kB activation and immune cell activation, primary mediators of cellular inflammation. The seminal and novel finding of this study is that high glucose concentration compromises endothelial cell expression of key anti-inflammatory miRs.