Comment[ArrayExpressAccession] E-GEOD-19593 Public Release Date 2010-12-18 Investigation Title Endothelial transcriptional profiles of porcine coronary and iliac arteries passages 1 through 4 Comment[Submitted Name] Endothelial transcriptional profiles of porcine coronary and iliac arteries passages 1 through 4 Experiment Description Atherosclerotic plaques tend to form in the major arteries at certain predictable locations. As these arteries vary in atherosusceptibility, inter-arterial differences in endothelial cell (EC) biology are of considerable interest. To explore the origin of differences observed between typical atheroprone and atheroresistant arteries, we used DNA microarrays to compare gene expression profiles of harvested porcine coronary (CECs) and iliac (IECs) artery ECs grown in static culture out to passage four. Fewer differences were observed between the transcriptional profiles of CECs and IECs in culture compared to in vivo, suggesting that most differences observed in vivo were due to distinct environmental cues in the two arteries. One-class significance of microarrays revealed that most in vivo interarterial differences disappeared in culture, as fold differences after passaging were not significant for 85% of genes identified as differentially expressed in vivo at a 5% false discovery rate. However, the three homeobox genes HOXA9, HOXA10, and HOXD3 remained under-expressed in coronary endothelium for all passages by at least 9, 8, and 2-fold, respectively. Continued differential expression despite removal from the in vivo environment suggests that primarily heritable or epigenetic mechanism(s) influence transcription of these three genes. Quantitative real-time polymerase chain reaction confirmed expression ratios for seven genes associated with atherogenesis and over- or under-expressed by 3-fold in CECs relative to IECs. The present study provides evidence that both local environment and vascular bed origin modulate gene expression in arterial endothelium. The transcriptional differences observed here may provide new insights into pathways responsible for coronary artery susceptibility. Endothelial cells were freshly harvested from right coronary and iliac arteries from four pigs. Cells were cultured out to passage four. RNA was isolated after each passage and expression profiles were obtained using oligo microarrays. Date of Experiment Term Source Name EFO Term Source Version Term Source File http://efo.svn.sourceforge.net/viewvc/efo/trunk/src/efoinowl/efo.owl Person Last Name Burridge Burridge Friedman Person First Name Kelley Kelley Morton Person Mid Initials A H Person Email burridge@duke.edu Person Affiliation Duke University Person Phone Person Fax Person Address Biomedical Engineering, Duke University, Rm 136 Hudson Hall, Durham, NC, USA Person Roles submitter Person Roles Term Source REF Person Roles Term Accession Number Normalization Type Normalization Term Accession Number Normalization Term Source REF Replicate Type Replicate Term Accession Number Replicate Term Source REF Experimental Design Experimental Design Term Accession Number Experimental Design Term Source REF Quality Control Type Quality Control Term Accession Number Quality Control Term Source REF Protocol Name P-GSE19593-1 P-GSE19593-6 P-GSE19593-3 P-GSE19593-12 P-GSE19593-8 P-GSE19593-7 P-GSE19593-9 P-GSE19593-2 P-GSE19593-10 P-GSE19593-11 P-GSE19593-4 P-GSE19593-5 Protocol Description ID_REF =
VALUE = Lowess normalized log2 ratio (Cy5/Cy3) representing test/reference • Make hybridization buffer fresh each time. • Add 38 ul hybridization buffer to 12 ul fragmented Cy Dye labeled aa-aRNA. • Add 1ul each poly dA and appropriate Cot-1 DNA. • Heat at 95ºC for 2 minutes and put on ice to cool down. • Mix well and load sample to the array slide with lifter cover slip. • Hybridize overnight in 42ºC water bath. Endothelial cells were grown in EGM-2MV media. LOWESS normalized. Agilent GeneSpring GX was used. LOWESS normalized. Agilent GeneSpring GX was used. Scanned by GenePix 4000B and quantified using GenePix software passage 3 passage 2 passage 4 passage 1 Qiagen RNeasy Micro kit following manufacturer's protocol Messenger RNA was amplified using a MessageAmp II aRNA amplification kit (Ambion). Two rounds of amplification were performed, and aminoallyl-UTP was used in the final round to provide reactive groups for dye incorporation. The RNA samples were labeled with Cy5 dyes and reference RNA sample was labeled with Cy3 dyes. Protocol Software Protocol Hardware Protocol Contact Protocol Type bioassay_data_transformation hybridization grow feature_extraction feature_extraction image_aquisition specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action nucleic_acid_extraction labeling Protocol Term Source REF Protocol Term Accession Number Experimental Factor Name CELL TYPE ANIMAL Experimental Factor Type cell type animal Experimental Factor Term Source REF Experimental Factor Term Accession Number Publication Title Publication Author List PubMed ID Publication DOI Publication Status Publication Status Term Source REF Publication Status Term Accession Number Comment[SecondaryAccession] GSE19593 Comment[GEOLastUpdateDate] 2010-12-18 Comment[AEExperimentType] transcription profiling by array Comment[GEOReleaseDate] 2010-12-18 Comment[ArrayExpressSubmissionDate] 2009-12-21 SDRF File E-GEOD-19593.sdrf.txt