Intermittent Hypoxia Alters Gene Expression in Peripheral Blood Mononuclear Cells (PBMC) of Healthy Volunteers

Intermittent hypoxia (IH) of obstructive sleep apnea (OSA) has been implicated in cardiovascular morbidity and mortality1. Although underlying mechanisms of the cardiovascular risk in OSA are not well defined, experiments in cell culture and animal models suggest that IH up-regulates pro-inflammatory genes that can promote atherosclerosis and insulin resistance. We hypothesized that short-term IH (5 h) will induce pro-inflammatory genes in healthy volunteers We performed a cross-over study of healthy volunteers (n = 8) who were exposed to IH or control conditions in a randomized fashion for 5 hours while awake as previously described4. IH was induced by inspiration of the hypoxic (95% N2 and 5% O2) gas until the oxyhemoglobin saturation dropped to 85% followed by reoxygenation to the baseline, resulting in ~25 hypoxic events/h. In control exposures hypoxic gas was substituted with air. Whole blood was collected before and after the exposures. Total RNA was isolated from PBMCs using the Trizol Reagent method (Invitrogen, Carlsbad, California 92008, cat. no. 15596-026) with subsequent RNEasy clean up (Qiagen, Valencia, CA 913555, cat. no. 74104). 0.5 µg of total RNA from each sample were labeled using the Illumina TotalPrep RNA Amplification Kit (Ambion, Austin, TX 78744-1832, cat. no. IL1791). mRNA was converted into double-stranded cDNA using an oligo-dT primer containing the T7 RNA polymerase promoter. Single stranded RNA (cRNA) was from double-stranded cDNA in an in vitro transcription reaction. cRNA was labeled by incorporating biotin-16-UTP. 0.85 ugs of biotin-labeled cRNA was hybridized (16 hours) to Illumina’s Sentrix HumanRef-8 Expression BeadChips (Illumina, San Diego, CA 92121-1975, cat.no. 11201828). The hybridized biotinylated cRNA was detected with streptavidin-Cy3 and quantified with Illumina’s BeadStation 500GX Genetic Analysis Systems scanners.