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Series GSE223381 Query DataSets for GSE223381
Status Public on May 05, 2023
Title Shock drives a highly coordinated transcriptional and DNA methylation response in the endothelium_DNA methylation
Organism Homo sapiens
Experiment type Methylation profiling by genome tiling array
Summary Endothelial dysfunction is a critical factor in promoting organ failure during septic shock. Organ dysfunction during shock increases the risk of long-term sequelae in survivors through mechanisms that remain unknown. We postulated that vascular dysfunction during shock contributes to long-term morbidity post-shock through transcriptional and epigenetic changes within the endothelium. We performed cross-omics analyses on kidney endothelium from acute endotoxin-challenged mice lacking or not the JAK/STAT3 inhibitor SOCS3. This analysis revealed significant DNA methylation changes upon proinflammatory signaling associated with transcriptional activity through AP1, STAT, and IRF families, suggesting a mechanism driving transcription-induced gene-specific methylation changes. In vitro, we demonstrated that IL-6 induces similar changes in DNA methylation. Specific genes showed DNA methylation changes in response to an IL-6+R and consistently changes in their expression levels within 72 hours of IL-6+R treatment. Further, changes in the endothelial methylome remain in place for prolonged periods without IL-6, suggesting that this cytokine may elicit transcriptional changes long after the resolution of inflammation. Also, demonstrated that DNA methylation changes could directly alter the expression of these genes and that STAT3 activation had a causal role in this transcriptional response. Our findings provide evidence for a critical role of IL-6 signaling in regulating shock-induced epigenetic changes and sustained endothelial activation, offering a new therapeutic target to limit vascular dysfunction.
 
Overall design Human Umbilical Vein Endothelial Cells (HUVECs) were isolated in-house. The identity and purity of the HUVEC isolations were confirmed for each isolation by more than 99% positive immunostaining with endothelial cell markers (FITC-Ulex europaeus lectin, VE-cadherin) and more than 99.9% negative for α–smooth muscle actin. Cells were assayed between passages 3 and 8. To induce IL-6 signaling, cells were plated at full confluence at a density of 8 × 104 cells/cm2 on plates precoated for 30 min with 0.1% gelatin and incubated at least 48 h prior to the start of experiments. Cells were then treated with a combination of 200 ng/mL recombinant human IL-6 and 100 ng/mL sIL-6Rα (IL-6+R) or PBS (control) for 72 hours. After 72 hours, a subset of control and IL-6+R-treated cells were washed and incubated for 96 hours in EGM-2 Growth Medium. Treated HUVECs and controls were immediately centrifuged and stored until DNA extraction. DNA was isolated using the DNeasy Blood & Tissue Kit (Qiagen, USA) according to the manufacturer's instructions, including optional treatment with 100 mg/ml RNase A. The concentration of DNA was measured by the Qubit dsDNA BR Assay Kit (Molecular Probes). The DNA samples were stored at -80°C. Bisulfite conversion of 500 ng of genomic DNA was performed using an EZ DNA Methylation-Gold™ Kit (Zymo Research) following manufacturer’s instructions. Analysis of DNA methylation was carried out using Illumina Infinium MethylationEPIC BeadChip arrays33 for human genomes (>850K sites)
 
Contributor(s) Ramos RB, Adam AP
Citation(s) 37667913
Submission date Jan 20, 2023
Last update date Sep 12, 2023
Contact name Ramon Bossardi Ramos
E-mail(s) bossarr@amc.edu
Organization name Albany Medical College
Department Department of Molecular and Cellular Physiology
Lab Adam's Lab
Street address 43 New Scotland Avenue
City Albany
State/province Ny
ZIP/Postal code 12208
Country USA
 
Platforms (1)
GPL21145 Infinium MethylationEPIC
Samples (21)
GSM6946884 PBS_exp2_1_96
GSM6946885 PBS_exp3_1_96
GSM6946886 PBS_exp2_2_96
Relations
BioProject PRJNA925904

Download family Format
SOFT formatted family file(s) SOFTHelp
MINiML formatted family file(s) MINiMLHelp
Series Matrix File(s) TXTHelp

Supplementary file Size Download File type/resource
GSE223381_RAW.tar 460.8 Mb (http)(custom) TAR (of IDAT)
Processed data included within Sample table

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