Normalization of vascular intussusception is essential for promoting lung regeneration over fibrosis (MeRIP-Seq)

Proliferation of vascular endothelial cells (ECs) is critical for providing sufficient cells lining the neo-vascular lumen after injury. As compared with parenchymal cells, vascular ECs normally do not develop genetic mutation during injury. Therefore, epigenetic changes such as RNA modification in ECs might be essential for modulating vascular regeneration in damaged lungs. N6-methyladenosine (m6A) modification has been shown as a major mammalian internal mRNA modification. However, the roles of RNA modification in the regulation of N6-methyladenosine (m6A) modification remain elusive. Here we examined m6A modification landscape in transcriptome-wide, and find that m6A is dynamically regulated during ECs proliferation after pneumonectomy (PNX). The result showed the number of m6A peaks in lung ECs 7 days post PNX was higher than that in sham group, and m6A-RIP sequencing revealed that the m6A peaks in Foxo1 exon 1 and exon 2 were increased on day 7 by PNX, suggesting that m6A methylation of these two exons in Foxo1 mRNA is increased in regenerating lung ECs after PNX. CD31+CD45- ECs were isolated from lung 0 day (Sham) and 7 days after PNX. The lung tissues were cut off and digested to get cell suspension. Immunomagnetic bead cell sorting was used to isolated mouse lung vascular ECs and magnetic beads were pre-coated with CD31 and CD45 antibody respectively. The endothelial cells were obtained as CD45 negative and CD31 positive cells.