A high intake of the omega-3 polynsaturated fatty acids (PUFA) docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) 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, DHA alone significantly altered the expression of 188 genes, down-regulating 92 and up-regulating 96. IL-1β significantly changed the expression of 2031 genes, down-regulating 997 and up-regulating 1034. Treatment with DHA, before IL-1 significantly affected the expression of 116 IL-1β -deregulated genes. Immunological, inflammatory and metabolic pathways were the most affected. In particular, newly identified DHA-regulated genes were involved in stemness, cellular growth and proliferation, cardiovascular system development and function, and cancer. These included cytochrome p450 4F2 (CYP4F2), transforming growth factor (TGF)-β2, caspase recruitment domain (CARD)11, and the junctional adhesion molecule (JAM)-A.
Conclusions: Endothelial exposure to DHA regulates several novel genes and related pathways. Such unbiased identification improves our understanding of mechanisms by which omega-3 PUFAs may affect human diseases.