Microarray analysis of human umbilical vein endothelial cells highlights new anti-inflammatory and vasodilating properties of the omega-3 fatty acid docosahexaenoic acid

Abstract

High intake of ω-3 fatty acids has been associated with anti-inflammatory effects and cardiovascular protection, but the underlying molecular basis remains incompletely defined. Using a DNA microarray technology we investigated the early gene expression profile of human vascular endothelium conditioned by DHA under pro-inflammatory conditions. Methods: Human umbilical vein endothelial cells (HUVEC) were treated with 50 μmol/L DHA for 48 hours and then stimulated with 5 ng/mL IL-1β for 3 hours. Total RNA was extracted, and qualitatively/quantitatively analyzed with a NanoDrop spectrophotometer and an Agilent Bioanalyzer before RNA labeling and purification. Gene expression profile was performed with an Agilent Whole Human Genome Oligo Microarray covering 41 000 unique genes and transcripts. Slides were scanned with the Agilent's scanner and images processed using Agilent Feature Extraction software. The raw data were further processed with the GeneSpring® 10 software and differentially expressed RNA identified using Benjamini and Hochberg False Discovery Rate with a P-value <0.05. Functional and network analyses were identified by the Ingenuity Pathways version 8.0 Analysis. Results: Fixing a significance threshold at 1.5 fold of change (FC), IL-1 stimulation significantly changed the expression of 2031 genes, down-regulating 997 and up-regulating 1034. Treatment with DHA before IL-1 stimulation significantly affected the expression of 48 IL-1-deregulated genes. The application of the Ingenuity pathway analysis software allowed us to pinpoint immunological-, inflammatory- and metabolic-related pathways as the most affected. In particular, we identified new DHA regulated genes involved in cellular growth and proliferation, cardiovascular system development and function, and cancer. These included the programmed cell death ligand 1 (PD-L)1, apoliprotein (Apol)3, phosphodiesterase (PDE)5A, and transforming growth factor (TGF)-β2. Conclusions: Endothelial exposure to DHA before IL-1 stimulation modulates several IL-1 deregulated genes. The unbiased identification of novel genes regulated by DHA treatment improves our understanding of mechanisms by which fish oils may prevent or attenuate human chronic diseases, including atherosclerosis.