ABSTRACT: Goal of the experiment: To examine differential gene expression in the iliac arteries of cynomolgous monkeys in the presence of small, medium, or large atherosclerotic plaque. Brief description of the experiment: Objective: To examine global gene expression patterns in the iliac arteries of monkeys containing small, medium, or large atherosclerotic plaque. Design: The left iliac artery of 12 ovariectomized cynomolgous monkeys on a high fat diet for 8 years was biopsied. Gene expression was analyzed by DNA microarray and real time RT-PCR. Results: Significant up- or down-regulation of 986 genes was observed in monkey iliac arteries in the presence of atherosclerotic plaque. Changes in gene expression with atherosclerosis ranged from 0.1-151.9-fold. Differentially expressed genes included many cytokines, chemokines, components of signal transduction pathways, and transcriptional activators and repressors, among others. Real time RT-PCR confirmed down-regulation of estrogen receptor 1 (ESR1), claudin 11, BH protocadherin 7 (PCDH7), and the up-regulation of apolipoprotein E (ApoE), growth differentiation factor 15 (GDF15), superoxide dismutase 2 (SOD2), SET domain, bifurcated 2 (SETDB2), phospholipase A2 group IIA (PLA2IIA), phospholipase A2 group VII (PLA2VII), and ring finger protein 149 (RNF149). Conclusions: The gene expression environment in arteries containing atherosclerotic plaque is profoundly different from that of arteries without atherosclerosis. The data suggest that the changes in gene expression contribute to the disease process in diseased arteries. Experimental factors: presence of disease (atherosclerosis) Experimental design: Female cynomolgous monkeys had been ovariectomized for 4 years and on a high fat diet for 8 years. The left iliac artery was removed at surgical biopsy. The presence and size of atherosclerotic plaque was quantified in the iliac arteries. Samples were divided categories based on plaque size; categories were small (0-0.113mm2, n=4), medium (0.30-0.542 mm2, n=5), and large (0.61-1.003 mm2, n=3) atherosclerotic plaques. Arterial tissue from the iliac arteries was used for DNA microarray analysis of gene expression. Quality control steps: The cRNA that was synthesized from each iliac artery was used for hybridization to a single CodeLink (Amersham/GE) whole human microarray. Only one sample was hybridized with each slide and only one dye (Alexa 647) was used so no dye swaps were necessary. Bacterial control spikes were used as per manufacturer's instructions. Samples used, extract preparation and labelling: The origin of each biological sample: The samples were iliac arterial tissue from cynomolgous monkeys. Manipulations of biological samples and protocols used: Cynomolgous monkeys were placed on a high fat diet 8 years before the experiment and ovariectomized 4 years prior to the experiment to induce a surgical menopause. The left iliac artery was surgically removed from each animal in the study. The presence and size of atherosclerotic plaque was quantified in the iliac arteries Experimental factor: size of atherosclerotic plaque Technical protocols: The iliac arteries were homogenized in TRI reagent, bromochloropropane and sodium acetate were added, and the samples were centrifuged to separate the phases. The RNA-containing layer was removed and the RNA purified on an RNeasy extraction column (Qiagen). The sample was treated with an on-column DNase treatment (RNase-free DNase, Qiagen). The purity and quantity of RNA were evaluated by an Agilent Bioanalyzer using the RNA 6000 Nanoassay LabChip. Labelled cRNA was prepared using the MessageAmp II-Biotin enhanced kit (Ambion). 0.275 microgram of total iliac artery RNA was mixed with bacterial control RNA spikes and primed with T7 oligo(dT) primer for 10 min at 70C. (The bacterial control spikes included araB, entF, fixB, gnd, hisB, and leuB.) The first strand of cDNA was synthesized with first strand buffer, dNTP mix, RNase inhibitor, and reverse transcriptase for 2 h at 42C. The second strand cDNA synthesis reaction was prepared using second strand buffer, dNTP mix, DNA polymerase mix, and RNase H; the reaction was carried out for 2h at 16C. The double-stranded cDNA was purified on QIAquick columns (Qiagen) and the eluent was dried down in a SpeedVac concentrator. The double-stranded cDNA was resuspended in a mixture containing T7 reaction buffer, T7 ATP, T7 GTP, T7 UTP, T7 CTP, biotin-11-UTP, and T7 enzyme mix for the synthesis of cRNA. The cRNA synthesis reaction was terminated after 14h at 37C by purifying the cRNA on RNeasy columns (Qiagen). The concentration of cRNA was determined by spectrophotometry. Hybridization procedures and parameters: 10 micrograms of cRNA was mixed with fragmentation buffer and heated to 94C for 20 min. The fragmented cRNA was mixed with CodeLink hybridization buffer, loaded on the microarray slides, and hybridized for 18 hours at 37C. The slides were washed in 0.75x TNT (Tris-HCl, NaCl, Tween-20) at 46C for 1h then incubated with streptavidin-Alexa 647 fluorescent dye for 30 min at room temperature. The Alexa fluor was prepared in TNB blocking buffer (0.1M Tris-HCl, 0.15M NaCL, 0.5% NEN Blocking Reagent-PerkinElmer) The slides were then washed 4 times for 5 min each in 1x TNT and twice in 0.05% Tween 20 for 5 sec each. The slides were dried by centrifugation and scanned in an Axon GenePix 4000B scanner. Keywords: nonhuman primate, disease state analysis
