Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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ACE-Scan of the thrombin DNA aptamer on 10^10 Gen1 DNA microarrays with sodium and potassium buffers in two colors.

ABSTRACT: The experiments were carried out to map the ligand binding landscape of various DNA and RNA duplexed aptamer families. Duplexed Aptamer (DA) constructs were engineered from (i) natural and synthetic DNA and RNA aptamers and (i) synthetic oligonucleotide aptamer-complementary elements synthesized on custom DNA microarrays. The aptamers tested consist of the ATP DNA aptamer, the ATP RNA aptamer, the cocaine DNA aptamer, the human alpha-thrombin DNA aptamer, and the natural add riboswitch aptamer from the pathogenic bacteria Vibrio vulnificus. Each duplexed aptamer family consists of 1000's of synthetic constructs, each formed by hybridizing the aptamer with an aptamer-complementary element (ACE) - here, ACEs consisted of various DNA oligonucleotides synthesized as a custom DNA microarray.

INSTRUMENT(S): Agilent Technologies G2565CA microarray scanner

ORGANISM(S): synthetic construct

SUBMITTER: Jeffrey Munzar

PROVIDER: E-MTAB-6378 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Cysteinyl leukotrienes (cysLT), i.e. LTC4, LTD4, and LTE4, are lipid mediators derived from the 5-lipoxygenase pathway. The cysLT receptors cysLT1-R and cysLT2-R are expressed on different target cells and mediate inflammatory reactions in tissue- and LT-R-specific ways. Though endothelial cells (ECs) predominantly express cysLT2-Rs, their role in vascular biology remains to be defined. To delineate cysLT2-RÂ´s action, we stimulated human umbilical vein EC with 100 nM LTD4 for 60 min, determined gene signatures by microarrays, and characterized the resulting EC phenotypes. As controls, we compared LTD4-induced genes with those induced by 10 nM thrombin, a prototype vasoactive activator of EC that binds to protease-activated receptor 1 (PAR-1). Following application of stringent filters 37 LTD4-upregulated genes were identified (> 2.5fold stimulation). Surprisingly, most of the LTD4-regulated genes were also induced by thrombin and expression of cysLT2-R- and PAR-1-regulated genes strongly correlated (Pearson correlation coefficient: r = 0.90). Moreover, LTD4 + thrombin, when added together, augmented expression of LTD4- or thrombin-stimulated genes (Wilcoxon signed rank test: p < 0.01). Prominently induced genes that may play roles in vascular injury were studied in detail: Early growth response (EGR) and nuclear receptor subfamily 4 group A; E-selectin; CXC ligand 2; interleukin 8 (IL-8); a disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif 1 (ADAMTS-1); and tissue factor (TF). Transcripts of these genes peaked at approximately 60 min, were unaffected by the cysLT1-R antagonist montelukast, and were superinduced by cycloheximide. The EC phenotype was markedly altered: LTD4 induced de novo synthesis of EGR1 protein and EGR1 localized in the nucleus in LTD4-stimulated cells; LTD4 upregulated IL-8 formation and secretion; and LTD4 raised TF protein and TF-dependent EC pro-coagulant activity. These data show that cysLT2-R activation results in a pro-inflammatory EC phenotype through activation of immediate-early genes that resemble those induced by PAR-1. As LTD4 and thrombin are formed concomitantly during vascular injury and pro-thrombotic states, cysLT2-R and PAR-1 may collaborate in vivo to mediate vascular injury and repair. Experiment Overall Design: HUVEC in passage 1 were cultured in serum-free EC medium until cells reached confluency. HUVEC were stimulated by 100 nM LTD4 (n = 4) or 10 nM thrombin (n = 4) or both agonists concomitantly (n = 3). The immediate-early gene programs of these experimental groups were identified at 60 minutes. In addition, one HUVEC preparation was stimulated with 100 nM LTD4 for 6 h and 24 h.

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ACE-Scan of the thrombin DNA aptamer on 10^10 Gen1 DNA microarrays with sodium and potassium buffers in two colors.

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