Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of mouse aorta smooth muscle cells reveals they differentiate into lymphoid tissue organizers upon combined TNFR1/LTBR NF-kB signaling

ABSTRACT: Cultured mouse aorta endothelial cells (from 8-12 weeks old C57BL/6J mice, passage 2-3) were exposed to phosphate buffered saline (control) or a combination of TNFalpha plus agonistic alpha-LTÃ?R antibody for 24 hours as described in LÃ¶tzer et al. 2009. Arterioscler. Thromb. Vasc. Biol., in press. Total RNA was extracted and microarrays were prepared. Experiment Overall Design: Mouse aorta endothelial cells in tissue culture were stimulated with agonists and subsequently microarray, RT-PCR, and protein assays / analyses were performed.

ORGANISM(S): Mus musculus

SUBMITTER: Andreas Johann Richard Habenicht

PROVIDER: E-GEOD-19139 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Mouse aorta smooth muscle cells (SMCs) express TNF receptor superfamily member 1A (TNFR1) and lymphotoxin Ã receptor (LTÃR). Circumstantial evidence has linked the SMC LTÃR to tertiary lymphoid organogenesis in diseased aortae of hyperlipidemic mice. Here, we explored potential roles of TNFR1 and LTÃR activation in cultured SMCs. TNFR1 signaling by TNF activated the classical RelA NF-ÎºB pathway, whereas LTÃR signaling by agonistic anti LTÃR antibody activated both the classical RelA and alternative RelB NF-ÎºB pathways. Addition of both agonists synergized to enhance p100 inhibitor processing to the p52 subunit of NF-ÎºB and promoted its nuclear translocation suggesting RelA-RelB cross-talk in transcription regulation. Correspondingly, microarrays showed that simultaneous TNFR1 and LTÃR activation when compared to activation of single receptors was followed by markedly elevated levels of mRNAs encoding leukocyte homeostatic chemokines CCL2, CCL5, CXCL1, and CX3CL1. Furthermore, SMCs acquired prototypical features of mesenchymal cells known as lymphoid tissue organizers (LTOs), which control tertiary lymphoid organogenesis in autoimmune diseases, through hyperinduction of CCL7, CCL9, CXCL13, CCL19, CXCL16, VCAM-1, and ICAM-1. Experiments with ltbr-/- SMCs suggested that the LTÃR-RelB activation component of NF-ÎºB signaling was obligatory to generate the LTO phenotype. TNFR1-LTÃR crosstalk also resulted in augmented synthesis and prolonged secretion of lymphorganogenic chemokine proteins into the culture medium. Thus, combined TNFR1-LTÃR signaling triggers SMC transdifferentiation into a phenotype that strikingly resembles LTOs. LTO-like SMCs may adopt a thus far unrecognized role in diseased arteries, i.e. to coordinate tertiary lymphoid organogenesis in atherosclerosis, aortic aneurysm, and transplant vasculopathy. Experiment Overall Design: Smooth muscle cells in tissue culture were stimulated with agonists and subsequently microarray, RT-PCR, and protein assays / analyses were performed

Project description:Activation of the vascular endothelium with cytokines such as TNF is widely used to study the role of the vasculature in proinflammatory disease. To gain insight into mechanisms of prolonged vascular endothelial activation we compared changes in gene expression induced by continuous activation in stable tmTNFalpha expressing cells with changes due to acute TNFalpha challenge in vitro. Affymetrix GenechipÂ® analysis was performed on RNA from control, acute and continuous TNFalpha-activated endothelial cells. Only 36% of the significant changes in gene expression were convergent between the acute and continuously activated endothelial cells in comparison to the control. From the divergently regulated genes, for example the cytokine ENA-78 was specifically induced in chronically activated cells, while E-Selectin, a cell adhesion molecule, was upregulated only in acutely activated endothelial cells. Antioxidant SOD gene induction was noted in acute activation while NADPH oxidase was selectively upregulated in continuous activated endothelium in accordance with significant ROS induction occurred only in these cells. In addition, the anti-angiogenic genes PAI-1 and thrombospondin were upregulated only in acutely activated endothelium, consistent with reduced angiogenic activity observed in acute versus chronically activated endothelial cells in vitro. These data suggest that continuous activation of endothelial cells leads to specific expression and functional changes, consistent with alterations observed in dysfunctional endothelium exposed to or involved in chronic inflammation. Experiment Overall Design: This experiment compares the response of endothelial cells from newborn mice that were either stably transfected with a non-cleavable transmembrane mutant form of murine TNFalpha, or treated with soluble TNFalpha (20ng/ml) for 4 hours. Each treatment was compared to mock-transfected control cells. Four replicates for each condition were used. Five micrograms of total RNA was assayed per Genechip using standard Affymetrix protocols.

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Transcription profiling of mouse aorta smooth muscle cells reveals they differentiate into lymphoid tissue organizers upon combined TNFR1/LTBR NF-kB signaling

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