Genetic and Pharmacological Targeting of the EphA4/Tie2 Axis Results in Improved Pial Collateral Response following Ischemic Stroke
Background: Pial collaterals are a major determinant of patient outcome following ischemic stroke, however, targeted treatments for enhancing these blood vessels remain elusive. EphA4, a receptor tyrosine kinase, has been found to restrict the growth of these vessels, potentially through modulation of the Tie2 signaling pathway. The present study assessed the endothelial cell (EC)-specific role of EphA4 in arteriogenesis and the therapeutic potential of Vasculotide, a Tie2 agonist, after ischemic stroke. Methods: EC-specific EphA4 knockout mice and wildtype controls, as well as wildtype mice treated with vehicle or Vasculotide, were subjected to a permanent middle cerebral artery model of stroke. Infarct volume and functional recovery were assessed. Vessel painting coupled with immunohistochemistry was employed to assess collateral size and cellular changes. Bulk RNA sequencing of the pial surface was performed to identify unique transcriptional changes.  Results: Deletion of EphA4 in ECs resulted in increased collateral size and accelerated cellular remodeling, which correlated with reduced infarct volume, enhanced CBF recovery, and improved performance on behavioral assessments. These effects could be replicated in wildtype mice through the administration of 3?g/kg Vasculotide immediately following stroke. Treatment with 150?g/kg Vasculotide provided neuroprotection and increased collateral size but lacked the robust improvements in CBF recovery and motor recovery seen in the 3?g/kg treated group. Additionally, pial surfaces from 3?g/kg Vasculotide and EphA4 KO mice showed significant changes in mRNA transcripts associated with the Krt5/Krt14 pathway compared to WT controls.  Conclusion: The EphA4/Tie2 signaling axis mediates collateral growth and can be successfully therapeutically targeted by Vasculotide. Overall design: Stroke was induced in CD1 WT and EphA4 EC-Ko mice, then after 24hours, the contralateral and ipsilateral meningeal brain layer was dissected and RNA isolated for bulk sequencing.