Structured wound angiogenesis instructs mesenchymal barrier compartments in the regenerating nerve [bulkRNASeq]

Organ injury stimulates the formation of new capillaries to restore blood supply raising questions about the potential contribution of neoangiogenic vessel architecture to the healing process. With single-cell mapping we resolved the properties of endothelial cells that organize a polarized scaffold at the repair site of lesioned peripheral nerves. Transient reactivation of an embryonic guidance program is required to orient neovessels across the wound. Manipulation of this structured angiogenic response through genetic and pharmacological targeting of Plexin-D1/VEGF pathways within an early window of repair has long-term impact on configuration of the nerve stroma. Neovessels direct nerve-resident mesenchymal cells to mold a provisionary fibrotic scar by assembling an orderly system of stable barrier compartments that channel regenerating nerve fibers and shield them from the persistently leaky vasculature. Thus, guided and balanced repair angiogenesis enables the construction of a “bridge” microenvironment conducive for axon regrowth and homeostasis of the regenerated tissue. Endothelial cells genetically labeled with R26-TomatoLSL (Jax stock# 007914) reporter under the control of Cdh5(PAC)-CreERT2 (PMID: 20445537) were FACS-isolated from pooled uninjured sciatic nerves of adult mice or from the lesion site ("bridge" region) of sciatic neves at 7 or 14 days post-injury (dpi), and profiled by bulk RNA-seq. In a separate experiment, endothelial cells expressing Kdr-Cherry reporter (Jax stock# 018542) were FACS-isolated from the bridge region of sciatic nerves from control or Chd5::CreER; Plxnd1fl/fl mice (endothelial-specific deletion of Plexin-D1) at 7dpi.