GSE169532
Transcriptomic analysis of lung endothelial subsets in experimental sepsis
Purpose: Sepsis affects almost all aspects of endothelial cell function. Lung endothelial subsets, capillaries (capEC)  and post capillary venules (PCV), are known to play a pivotal role in maintaining normal homeostasis. The aim of the study is to reveal transcriptional changes in lung endothelial subsets during sepsis. Methods: Sepsis was induced in male C57BL/6 mice by cecal ligation and puncture (CLP). Lung tissues were collected 4 hour after induction of sepsis. Single cell suspension was prepared by enzymatic digestion. Blood endothelial cells (BECs) were enriched by depletion of hematolymphoid cells and epithelial cells by using anti-CD45 and CD326 microbeads. CapEC and PCV were sorted from enriched BECs using fluorescent-labeled antibodies. capEC subset was identified as CD31+Icam1+Vcam1- and PCV subset was identified as CD31+Icam1+Vcam1+. EC subsets were directly sorted into RLT buffer. Total RNA was isolated from the sorted cells with RNeasy Plus Micro kit (Qiagen) and the quality of RNA was checked by Bioanalyzer RNA 6000 pico assay. Sequence library was prepared using SMARTer® Stranded Total RNA-Seq Kit v2-Pico Input Mammalian (Takara Bio USA, Inc.). After library preparation and quality check, the double-stranded-cDNA was sequenced on NextSeq 500 (Illumina) using (read one-index reads-read two, bp): 75-8-8-75 setup. Differentially expression analysis was performed using DESeq2 in R program. Results: Differential expression analysis revealed that lung capEC is transcriptionally different than PCV. capEC and PCV responded differently after induction of sepsis. Enrichment analysis revealed that capEC are more enriched with genes related to regulation of coagulation, vascular permeability, wound healing and lipid metabolic process. On the other hand, PCV are more enriched with genes related to cell chemotaxis, cell-cell adhesion by integrins, chemokine biosynthesis process, regulation of actin filament process, neutrophil homeostasis. Overall design: Abdominal sepsis was induced in C57BL/6 mice by CLP. Lung endothelial cells were sorted for total RNA extraction and transcriptomic analysis.