Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Expression of human aortic endothelial cells treated with or without oxidized phospholipids (II)

ABSTRACT: Oxidized phospoholipids are a pro-inflammatory component of minimally modified lipoproteins that get trapped in the subendothelial space of atherosclerotic plaques of large arteries. To model the response of endothelial cells in a pro-atherosclerotic enviroment we measured the expression in primary endothelial cells with and without treatment with oxidized phsopolipids from 96 genetically identical donors of anonymous origin. These samples are an extention of samples of similar origin deposited in GSE20060 Primary human aortic endothelial cells were collected from aortic heart explants of heart transplant donors and cultured in standard cell culture condiditions. At confluence, cells were treated with or without 40 ug/ml oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphatidylcholine (Ox-PAPC) for 4 hours in media 199 containing 1% fetal bovine serum. After 4 hours, the cells were collected for RNA extraction and quantification of transcripts via microarrays. Application of electronic probe mask (HT_U133Amsk.cdf) on raw data.

ORGANISM(S): Homo sapiens

SUBMITTER: Casey Romanoski

PROVIDER: E-GEOD-30169 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Oxidized phospholipids are thought to promote atherogenesis by stimulating endothelial cells (ECs) to produce inflammatory cytokines, such as IL-8. In studies with mouse models, we previously demonstrated that genetic variation in inflammatory responses of endothelial cells to oxidized lipids contributes importantly to atherosclerosis susceptibility. We now show that similar variations occur in cultured aortic ECs derived from multiple heart transplant donors. These variations were stably maintained between passages and, thus, reflect either genetic or epigenetic regulatory differences. Expression array analysis of aortic EC cultures derived from 12 individuals revealed that >1,000 genes were regulated by oxidized phospholipids. We have used the observed variations in the sampled population to construct a gene coexpression network comprised of 15 modules of highly connected genes. We show that several identified modules are significantly enriched in genes for known pathways and confirm a module enriched for unfolded protein response (UPR) genes using siRNA and the UPR inducer tunicamycin. On the basis of the constructed network, we predicted that a gene of unknown function (MGC4504) present in the UPR module is a target for UPR transcriptional activator ATF4. Our data also indicate that IL-8 is present in the UPR module and is regulated, in part, by the UPR. We validate these by using siRNA. In conclusion, we show that interindividual variability can be used to group genes into pathways and predict gene-gene regulatory relationships, thus identifying targets potentially involved in susceptibility to common diseases such as atherosclerosis. Twelve individual HAEC lines were treated in duplicate (in 35-mm dishes) for 4 h in media containing PAPC (40 μg/ml) or oxidized PAPC (OxPAPC) at 20 ug/ml or 40 μg/ml. Cytoplasmic RNA was isolated by using an RNAeasy kit (Qiagen) and analyzed on an Agilent 2100 Bioanalyzer (Agilent, Palo Alto, CA) to assess RNA integrity. Double-stranded cDNAs were synthesized from total RNA by using the cDNA Synthesis System (Invitrogen, Carlsbad, CA). Biotin-labeled cRNA was generated and used to probe Affymetrix (Santa Clara, CA) Human Genome U133 Plus 2.0 arrays according to the manufacturer’s recommendations (Affymetrix). The Microarray Suite 5.0 software (Affymetrix) was used to analyze image data and make the absolute call for each measurement. The array data were normalized with the median invariant method (31).

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Expression of human aortic endothelial cells treated with or without oxidized phospholipids (II)

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