ABSTRACT: Objective: To examine the effect of estradiol on gene expression in the monkey iliac arteries. Design: Eight ovariectomized cynomolgus monkeys were on a high fat diet for 6.5 years. The left iliac artery was biopsied before randomization to estradiol (1 mg Estrace® per day, n=4) or vehicle (n=4) for 8 months. The right iliac artery was obtained at necropsy. Gene expression in the iliac arteries in response to estradiol was determined by DNA microarray and confirmed by real time RT-PCR. Results: Atherosclerotic lesions in the iliac arteries ranged in size from 0-0.61 mm2 in estrogen-treated animals and from of 0-0.873 mm2 in controls. 106 genes were shown to be down-regulated and 26 genes were up-regulated in the monkey iliac arteries in response to estradiol. Among the genes shown to be up-regulated by DNA microarray and confirmed by real time RT-PCR were components of the insulin-like growth factor (IGF) pathway, IGF-1, IGF binding protein 4 (IGFBP4) and IGFBP5. Components of the Wnt signaling pathway were also up-regulated, including secreted frizzled-related protein 2 (SFRP2), SFRP4, low density lipoprotein receptor-related protein 6 (LRP6), and Wnt 1 inducible signaling pathway protein 2 (WISP2). Conclusions: The modest effect of estradiol on gene expression in monkey iliac arteries reported herein may reflect a reduction in response to estradiol as a result of the effect of the altered environment of the artery containing atherosclerotic plaque. Experiment Design: Goal of the experiment: To examine differential gene expression in the iliac arteries of ovariectomized cynomolgous monkeys on a high fat diet in response to treatment with estradiol. Brief description of the experiment: Objective: To examine the effect of estradiol on gene expression in the monkey iliac arteries. Design: Four ovariectomized cynomolgous monkeys were on a high fat diet for 6.5 years. The left iliac artery was biopsied before randomization to estradiol (1 mg Estrace® per day, n=4) for 8 months. The right iliac artery was obtained at necropsy after treatment. Gene expression in response to estradiol was determined by CodeLink Whole Human (Applied Microarrays, Tempe, AZ) DNA microarray and confirmed by real time RT-PCR. Results: Atherosclerotic lesions in the iliac arteries ranged in size from 0-0.61 mm2 (mean 0.34 ± 0.13 mm2) in pretreatment arteries and 0-1.411 (mean 0.789 ± 0.31mm2) in post-estrogen treated arteries. 106 genes were shown to be down-regulated and 26 genes were up-regulated in the monkey iliac arteries in response to estradiol. Among the genes shown to be up-regulated by DNA microarray and confirmed by real time RT-PCR were components of the insulin-like growth factor (IGF) pathway, IGF-1, IGF binding protein 4 (IGFBP4) and IGFBP5. Components of the Wnt signaling pathway were also up-regulated, including secreted frizzled-related protein 2 (SFRP2), SFRP4, low density lipoprotein receptor-related protein 6 (LRP6), and Wnt 1 inducible signaling pathway protein 2 (WISP2). Conclusions: The modest effect of estradiol on gene expression in monkey iliac arteries reported herein may reflect a reduction in response to estradiol as a result of the effect of the altered environment of the artery containing atherosclerotic plaque. Keywords: nonhuman primate, hormone replacement Experimental factors: hormone treatment Experimental design: Female cynomolgous monkeys (n=4) had been ovariectomized for 4 years and on a high fat diet for 6.5 years. The left iliac artery was removed at surgical biopsy. Animals were treated with estradiol for 8 months, then necropsied. The right iliac artery was obtained at necropsy. The presence and size of atherosclerotic plaque was quantified in the iliac arteries. 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 (Applied Microarrays, Tempe, AZ) 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 labeling: 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 6.5 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 before treatment with estradiol, the the right iliac artery was removed after the treatment period at necropsy. The presence and size of atherosclerotic plaque was quantified in the iliac arteries Experimental factor: hormone treatment 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. Labeled 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.
