Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Identification of inflammatory gene modules based on variations of human endothelial cell responses to oxidized lipids

ABSTRACT: 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).

ORGANISM(S): Homo sapiens

SUBMITTER: Juyong Kim

PROVIDER: E-GEOD-29903 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Long wavelength Ultraviolet (UVA-1) radiation causes oxidative stress that leads to the formation of noxious substances within the skin. As a defensive mechanism skin cells produce detoxifying enzymes and antioxidants when they detect modified molecules. We have recently shown that UVA-1 irradiation oxidizes the abundant membrane phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (PAPC), which then induced the synthesis of the stress response protein heme oxygenase 1 (HO-1) in dermal fibroblasts. Here we examined the effects of UVA-1 and (UV-) oxidized phospholipids on the global gene expression in human dermal fibroblasts. We identified a cluster of genes that were co-induced by UVA-1-oxidized PAPC and UVA-1 radiation. The cluster included HO-1, glutamate-cysteine ligase modifier subunit (GCLM), aldo-keto reductases-1-C1 and -C2 (AKR1C1, AKR1C2), and interleukin 8 (IL8). These genes are members of the cellular stress response system termed âantioxidant responseâ or âPhase II detoxificationâ. Accordingly, the regulatory regions of all these genes contain binding sites for NF-E2-related factor 2 (Nrf2), a major regulator of the antioxidant response. Both UVA-1 irradiation and treatment with oxidized lipids led to increased nuclear accumulation of Nrf2. Silencing expression of Nrf2 using siRNA or using cells and tissue from Nrf2-deficient mice, we show that the induction of the co-regulated genes was suppressed. Expression of other canonical UVA-1-induced genes, including cyclooxygenase 2 (Cox2) and interleukin 6 (IL6) was unaltered in the absence of Nrf2. Together, our data show that UVA-1-mediated lipid oxidation induces induction of antioxidant response genes, which is dependent on the redox-regulated transcription factor Nrf2. To activate Nrf2 is a major strategy for novel antioxidant drugs, the skin photo-adaptation (SPA) inducers. Our finding that specific uv-oxidized lipids act similar sheds a new (ultraviolet) light on the usually detrimental âimageâ of UV generated lipid mediators. Experiment Overall Design: we profiled global mRNA expression levels in human dermal fibroblasts that had been treated with either UVA-1 or oxidized lipids. To investigate the effect of oxidized phospholipids on gene regulation, we used two preparations, which differed in their degree of oxidation; the minimally oxidized UV-PAPC resulting from UVA-1 irradiation of PAPC, and air-oxidized PAPC (OxPAPC), which represents the full spectrum of oxidation products (Gruber 07) (Reis et al., 2005). We irradiated dermal fibroblasts with UVA-1 (40J/cmÂ²) or treated them with UV-PAPC, OxPAPC or native PAPC (100Âµg/ml each). We analyzed global gene expression four hours after stimulation with gene arrays (Affymetrix U133A Plus 2.0 Gene Chips).

Project description:The transition of the endothelium to a pro-inflammatory state is key to progression of chronic inflammatory diseases including rheumatoid arthritis, chronic bowel disease and atherosclerosis. In atherosclerosis it is hypothesized that low density lipoproteins (LDL) that become trapped in the intima of the blood vessels are oxidized to minimally modified LDL (mmLDL) and that these serve as an important contributing factors to endothelial dysfunction. Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphatidylcholine (OX-PAPC), a model of the active phospholipid components of mmLDL affects the expression of hundreds of genes involved in inflammatory and other biological processes in human aortic endothelial cells (HAECs). We hypothesized that microRNAs (miRNAs) partially regulate this response. Using next generation sequencing, we identified miR-21-3p and miR-27a-5p to be induced 4-fold and 3-fold, respectively in response to OX-PAPC treatment compared to control treatment in HAECs. To identify the targets, we performed whole genome transcript profiling following transient over-expression of these two miRNAs followed by. In total, 1254 genes were down-regulated with 925 of them overlapping between the two miRNAs. Functional enrichment analysis using Gene Ontology predicted that the two miRNAs were involved in the regulation of NF-κB signaling. We characterized the Toll/interleukin-1 receptor (TIR) domain-containing adaptor protein TICAM2 as a direct target of miR-21-3p and miR-27a-5p. Furthermore, we showed that over-expression of miR-21-3p and miR-27a-5p lead to decreased p65 translocation to the nucleus and decreased the expression of known NF-κB downstream target genes confirming both miRNAs’ role in negatively regulating NF-κB signaling in endothelial cells. mRNA expression profiling of human aortic endothelial cells from two separate donors that were transfected with 1 nM microRNA mimics and negative control. The miRIDIAN mimics used were miR-21-3p (Catalog Number:C-301023-01-0005), miR-27a-5p (Catalog No: C-301028-01-0005), negative control (Catalog No: CN-001000-01-05)

Project description:Long wavelength Ultraviolet (UVA-1) radiation causes oxidative stress that leads to the formation of noxious substances within the skin. As a defensive mechanism skin cells produce detoxifying enzymes and antioxidants when they detect modified molecules. We have recently shown that UVA-1 irradiation oxidizes the abundant membrane phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (PAPC), which then induced the synthesis of the stress response protein heme oxygenase 1 (HO-1) in dermal fibroblasts. Here we examined the effects of UVA-1 and (UV-) oxidized phospholipids on the global gene expression in human dermal fibroblasts. We identified a cluster of genes that were co-induced by UVA-1-oxidized PAPC and UVA-1 radiation. The cluster included HO-1, glutamate-cysteine ligase modifier subunit (GCLM), aldo-keto reductases-1-C1 and -C2 (AKR1C1, AKR1C2), and interleukin 8 (IL8). These genes are members of the cellular stress response system termed “antioxidant response” or “Phase II detoxification”. Accordingly, the regulatory regions of all these genes contain binding sites for NF-E2-related factor 2 (Nrf2), a major regulator of the antioxidant response. Both UVA-1 irradiation and treatment with oxidized lipids led to increased nuclear accumulation of Nrf2. Silencing expression of Nrf2 using siRNA or using cells and tissue from Nrf2-deficient mice, we show that the induction of the co-regulated genes was suppressed. Expression of other canonical UVA-1-induced genes, including cyclooxygenase 2 (Cox2) and interleukin 6 (IL6) was unaltered in the absence of Nrf2. Together, our data show that UVA-1-mediated lipid oxidation induces induction of antioxidant response genes, which is dependent on the redox-regulated transcription factor Nrf2. To activate Nrf2 is a major strategy for novel antioxidant drugs, the skin photo-adaptation (SPA) inducers. Our finding that specific uv-oxidized lipids act similar sheds a new (ultraviolet) light on the usually detrimental “image” of UV generated lipid mediators.

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Identification of inflammatory gene modules based on variations of human endothelial cell responses to oxidized lipids

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