Abstract Body (Do not enter title and authors here): Background: We have described the role of angiogenic transdifferentiation as a response to ischemia. Specifically, in the setting of ischemia, a subset of tissue fibroblasts transdifferentiates into endothelial cells to contribute to angiogenesis. Here we demonstrate that angiogenic transdifferentiation, and the recovery from ischemia, requires an N6-methyladenosine (m6A) modification of messenger RNA (mRNA) in the ischemic tissue. METTL3 and METTL14 constitute the core m6A methyltransferase complex that catalyzes m6A deposition.
Methods: A murine hindlimb ischemia model was employed to evaluate vascular recovery post-ischemia, with blood flow assessed via laser Doppler imaging. m6A levels in RNA were quantified using RNA dot blot and ELISA-based assays. Capillary density was assessed by immunostaining for endothelial markers CD31 and CD144. Fibroblast lineage tracing utilized constitutive (FSP1Cre: R26R-EYFP) and inducible [Col1a2-CreER: Ai9 (RCL-tdT)] reporter mouse models. Additionally, a small molecule-based protocol induced fibroblast-to-endothelial cell transdifferentiation in human fibroblasts in vitro.
Results: At 7 days post-femoral artery ligation, m6A levels increased in ischemic muscle, predominantly within fibroblasts. Inhibition of m6A modification, via pharmacological METTL3 inhibition or fibroblast-specific METTL14 knockout, impaired vascular recovery, as demonstrated by decreased blood flow, enhanced tissue damage, and reduced capillary density. Lineage tracing studies revealed that impaired recovery in m6A-deficient mice was associated with reduced angiogenic transdifferentiation, evidenced by fewer fibroblast-derived endothelial cells. Consistently, in vitro experiments demonstrated that suppressing m6A hindered, whereas enhancing m6A promoted, fibroblast-to-endothelial cell transdifferentiation. Furthermore, elevating m6A levels using small molecules improved vascular recovery in the ischemia model.
Conclusions: mRNA m6A modification critically regulates microvascular recovery following ischemia, partially through facilitating angiogenic transdifferentiation. Targeting m6A modification may represent a novel therapeutic strategy for enhancing vascular repair in ischemic diseases.