Aorta macrophage inflammatory and epigenetic changes in a murine model of obstructive sleep apnea: Potential role of CD36

Obstructive sleep apnea (OSA) affects 8–10% of the population, is characterized by chronic intermittent hypoxia (CIH), and causally associates with cardiovascular morbidities. In CIH-exposed mice, closely mimicking the chronicity of human OSA, increased accumulation and proliferation of pro-inflammatory metabolic M1-like macrophages highly expressing CD36, emerged in aorta. Transcriptomic and MeDIP-seq approaches identified activation of pro-atherogenic pathways involving a complex interplay of histone modifications in functionally-relevant biological pathways, such as inflammation and oxidative stress in aorta macrophages. Discontinuation of CIH did not elicit significant improvements in aorta wall macrophage phenotype. However, CIH-induced aorta changes were absent in CD36 knockout mice, Our results provide mechanistic insights showing that CIH exposures during sleep in absence of concurrent pro-atherogenic settings (i.e., genetic propensity or dietary manipulation) lead to the recruitment of CD36(+)high macrophages to the aortic wall and trigger atherogenesis. Furthermore, long-term CIH-induced changes may not be reversible with usual OSA treatment.

USA). Briefly, purified total RNAs were processed for labeling using the Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies, Santa Clara, CA) and hybridized with whole-genome mouse Agilent microarrays (8x60K). Equal quantities of total RNA were labeled with each reaction containing 25 ng of total RNA and 2 µl (34 pg) of RNA spike-in control. The quality of each cRNA sample was evaluated using 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Each sample was hybridized to an Agilent oligonucleotide microarray for all of the 32 independent experiments. The microarray slides were scanned using Agilent dual-laser Microarray Scanner and the digitized images were acquired and processed using Agilent Feature Extraction (FE) software v.12.0.
Background-subtracted intensities were normalized using the quantile method across all microarray experiments. We first corrected microarray expression intensity background using thr norm-exp algorithm with offset value of 50 1 . The background corrected data were normalized between arrays using cyclic loess method 2 . We applied limma moderated t-test to detect differentially expressed genes, considering the batch effect caused by different chips as covariates in the linear model. P-values were adjusted by Benjamini-Hochberg method 3 . Differentially expressed genes (DEGs) were identified either using false discovery rate (FDR) of 0.05 or log-fold change of 1.5 and FDR of 0.05.

qRT-PCR Validation
qPCR analysis was performed for selected mRNAs using ABI PRISM 7500 System (Applied Biosystems, Foster City, CA). To confirm candidate mRNAs, cDNA was synthesized using 250 ηg of total RNA using a High-Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA). Ribosomal 18S rRNA, was used as a reference gene to normalize the expression ratios. All experiments were performed in triplicates. The cycle number (C t ) values were averaged and the difference between the 18S C t and the gene of interest C t were calculated to calculate the relative expression of the gene of interest using the 2 -ΔΔCt method 4 . The results are presented as fold change.

Epigenomics profiling
Chromatin Immunoprecipitation: Macrophages were isolated from aortas of mice exposed to CIH (n=20) or RA (n=20) conditions for 20 weeks, as described above. Immediately after isolation, cells were crosslinked and chromatin immunoprecipitated using the True MicroChip kit (Diagenode, Denville NJ) and antibodies specific for H3K9ac and Many of the remaining DB regions were observed to coincide with repetitive sequences. Therefore, a further filtration was applied based on the 50-mer mappability score track obtained from the UCSC genome browser. All positions in DB regions were required to have a mappability score of 1 which implies that all 50-mer sequences found in the DB region are uniquely found in the known genome sequence. After filtering, peaks were annotated with the nearest genes using bedops package (Supplementary Tables S2 and S3). attach and spread on the electrode surface, impedance is altered, and serves as a measure for disruption of the endothelial cellular junction [7][8][9] . This method is based on measuring non-invasively the frequency-dependent electrical impedance of cell-covered gold-film electrodes along the time course of the experiment. The baseline was established using culture medium (300 µl/well-1) alone and compared with values obtained using electrodes covered with a monolayer of cells in 500 µL medium. Monolayer resistance was recorded at 4,000 Hz for 24 hours after application of 20,000 monocytes per well. A change in the resistance across the cell monolayer indicated increased or decreased tran-cellular permeability. Data were normalized to the average of five recordings prior to application of monocytes and each sample was run in duplicates.

IHC and Image Processing
Carotid arteries were harvested from several mice in each treatment group and embedded in paraffin. Sections from each carotid artery were designated for H&E staining (at 1,000, 3,000, and 5,000 μm), and other staining protocols. The carotid arteries were submitted on numbered cassettes, and their identity was kept on a separate document (blinded). Slides were labeled according to the cassettes to allow for blinding at the time of interpretation and reading. Slides were read using 60× magnification on a Nikon Eclipse TE 2000-U microscope (Nikon Instruments Inc., Melville, NY, USA) and interpreted with calibrated Image-Pro Plus software (version 6.1.0.346, Media Cybernetics Inc., Bethesda, MD, USA).
The mean of the measurements done at the 1,000, 3,000, and 5000 μm mark distal to the bifurcation were used in the statistical analyses, with each mouse's CIMT was then averaged to represent the final CIMT measurement from the 2 treatment groups 10 .