Virginia Research Day 2022

Graduate Student Research Biomedical

07 Endothelial Cell Specific EPHA4 Inhibits Acute PIAL Collateral Response Following Ischemic Stroke

Alexandra M. Kaloss 1 ; Nathalie A. Groot 1 ; Kennedie Lyles 3 ; Jackie Zhu 4 ; John B. Matson 4 ; Michelle H. Theus 1,2,3 Corresponding author: akaloss@vt.edu

Strokes are a leading cause of death and long- term disability in the United States, afflicting nearly 800,00 Americans annually. The majority of these stroke cases are ischemic in nature, where a vascular obstruction, such as a blood clot, causes reduced cerebral blood flow. Current ischemic stroke treatments are limited to surgical removal of the clot or thrombolysis. Alternatively, leptomeningeal anastomoses, or pial collateral vessels, are a major determinant of patient outcome following ischemic stroke and have garnered attention as a therapeutic target. These vessels form during development in the pia mater of the brain and connect distal arterioles of cerebral arteries. Following occlusion, pial collaterals remodel and expand through arteriogenesis, an adaptive process that allows for retrograde perfusion into the obstructed artery and its affected tissue. However, the mechanisms underlying acute injury-induced arteriogenesis following ischemic stroke has been poorly investigated, therefore our research aims to fill this knowledge gap. Our novel pre-clinical murine findings suggest 1 Department of Biomedical Sciences and Pathobiology 2 Center for Regenerative Medicine, VMRCVM, Virginia Tech 3 School of Neuroscience 4 Department of Chemistry, Virginia Tech

EphA4, a receptor tyrosine kinase, plays a role in restricting arteriogenesis following permanent middle cerebral artery occlusion (pMCAO). To investigate the role of endothelial cell specific EphA4 in acute arteriogenesis, we utilized 3-month old conditional endothelial cell (EC)-specific EphA4 knockout ( EphA4 fl/ fl /Cdh5::Cre ERT2 ; KO) and wild type ( EphA4 fl / fl; WT) mice. We find that KO mice lacking EphA4 displayed reduced infarct volume one day following pMCAO, compared to WT controls (24.0±1.8mm3 vs 14.3±2.5mm3; n=9). This reduction in tissue damage correlated with larger ipsilateral pial collateral vessels in the KO mice as early as 4.5hrs (27.31±0.6um vs 32.41±0.8um; n=15) and up to 24hrs post- pMCAO (31.14±0.8um vs 36.7±0.9um; n=15). Additionally, our previous work has indicated that EphA4 may exert its inhibitory function on arteriogenesis by hindering the Tie2 signaling pathway. To identify gene expression changes of the collateral vessels and connecting cerebral arteries, the pial surface was carefully isolated from four mice per sample and pooled.

Analysis of mRNA expression in the pial surface 24-hours post-pMCAO revealed that WT mice had significantly higher fold change of angiopoietin-2 (1.4±0.2 vs 0.8± 0.09; n=5) and Tie1 (5.3±1.8 vs 0.9±0.2; n=5) compared to KO mice, indicating potential inhibition of Tie2 signaling. Promotion of Tie2 signaling via administration of Vasculotide, an angiopoietin-1 mimetic peptide, resulted in greater migration of KO cells in vitro compared to vehicle control treated cells. These findings demonstrate that EC-specific EphA4 negatively regulates collateral growth and inhibition of this receptor or activation of Tie2 could serve as novel therapeutic strategies for improving collateral response following ischemic stroke.

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