Inhibiting endothelial <i>Rhoj</i> blocks profibrotic vascular intussusception and angiocrine factors to sustain lung regeneration.

Abstract

Lung regeneration after fibrosis requires formation of functional new vasculature, which is essential for gas exchange and cellular cross-talk with other lung cells. It remains unknown how the lung vasculature can be regenerated without fibrosis. Here, we tested the role of N6-methyladenosine (m6A) modification of <i>forkhead box protein O1</i> (<i>Foxo1</i>) mRNA in lung regeneration after pneumonectomy (PNX) in mice, a model for lung regrowth after surgical resection. Endothelial cell (EC)-specific knockout of <i>methyltransferase-like 3</i> (<i>Mettl3</i>) and <i>Foxo1</i> caused nonproductive intussusceptive angiogenesis (IA), which impaired regeneration and enhanced fibrosis. This nonproductive IA was characterized by enhanced endothelial proliferation and increased vascular splitting with increased numbers of pillar ECs. Endothelial-selective knockout of <i>Mettl3</i> in mice stimulated nonproductive IA and up-regulation of profibrotic factors after PNX, promoting regeneration to fibrotic transition. EC-specific mutation of m6A modification sites in the <i>Foxo1</i> gene in mice revealed that endothelial <i>Mettl3</i> modified A504 and A2035 sites in the <i>Foxo1</i> mRNA to maintain pro-regenerative endothelial glycolysis, ensuring productive IA and lung regeneration without fibrosis. Suppression of <i>Mettl3-Foxo1</i> signaling stimulated a subset of hyperglycolytic and hyperproliferative <i>6-phosphofructo-2-kinase/fructose-2</i>,<i>6-biphosphatase 3</i> (<i>Pfkfb3</i>)⁺, <i>Ras homolog family member J</i> (<i>Rhoj</i>)⁺, and <i>platelet-derived growth factor subunit B</i> (<i>Pdgfb</i>)⁺ ECs in both human and mouse lungs with fibrosis. Inhibiting this <i>Pfkfb3</i>⁺<i>Rhoj</i>⁺<i>Pdgfb</i>⁺ EC subset normalized IA, alleviated fibrosis, and restored regeneration in bleomycin (BLM)-injured mouse lungs. We found that m6A modification of <i>Foxo1</i> in the mouse vasculature promoted lung regeneration over fibrosis after PNX and BLM injury.