VCOM Research Day Program Book 2023

Graduate Student Research Biomedical

13 Targeting EphA4/Tie2 Signaling Improves 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 Corresponding author:

1 Department of Biomedical Sciences and Pathobiology 2 Center for Regenerative Medicine, VMRCVM, Virginia Tech, Blacksburg 3 School of Neuroscience, 4Department of Chemistry, Virginia Tech, Blacksburg

Strokes are a leading cause of death and disability in the United States, with an increasing incidence in younger populations. Ischemic strokes account for the majority of stroke cases and are caused by a vascular obstruction, such as a blood clot, reducing cerebral blood flow. Current treatments focus on removing the blood clot, through either surgical intervention or thrombolysis. The formation and recruitment of a patient’s leptomeningeal anastomoses, or pial collateral vessels, is a major predictor of patient outcome following ischemic stroke, garnering them attention as a therapeutic target. Pial collaterals are distinctive arterioles that form during development in the pia mater of the brain and directly connect distal arterioles of cerebral arteries. Following a vascular obstruction, pial collaterals can expand and remodel through arteriogenesis, a unique adaptive process allowing for retrograde perfusion into the obstructed artery and its affected tissue. Despite their promise therapeutically, the mechanism of acute pial collateral

growth following an ischemic stroke remains poorly understood, therefore our research aims to fill this knowledge gap. Our novel pre-clinical findings show that crosstalk between the receptor tyrosine kinases, EphA4 and Tie2, play a major role in controlling collateral vessel growth following permanent middle cerebral artery occlusion. To investigate the role of EphA4 and its connection to Tie2 during arteriogenesis, we employed endothelial cell specific EphA4 KO ( EphA4 fl/fl/ Cdh5::Cre ERT2 ; KO) and wild type ( EphA4 fl/fl ; WT) mice. Compared to WT control mice, KO mice displayed a significant reduction in infarct volume (24.0±1.8mm3 vs 14.3±2.5mm3; n=9), which correlated with significantly larger pial collaterals as early as 4.5-hours (27.31±0.6um vs 32.41±0.8um; n=15) and up to 24-hours post-pMCAO (31.14±0.8um vs 36.7±0.9um; n=15). Analysis of mRNA expression in the pial surface 24-hours post pMCAO showed significantly higher fold change of Angiopoetin-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), inhibitors of Tie2 signaling, in WT compared to KO mice. To assess the role of Tie2 signaling in collateral growth, WT mice were treated with Vasculotide, an Angiopoeitin-1 mimetic peptide at a dose of either 3ug/kg or 150ug/ kg via tail vein injection immediately post-pMCAO. Compared to vehicle controls, mice receiving either dose of Vasculotide displayed decreased infarct volumes (Saline: 18.7±1.6mm3, 3ug/kg: 9.1±0.8mm3, and 150ug/kg: 11.4±1.4mm3) and significantly increased collateral vessels at 24-hours post-pMCAO (Saline: 29.6±0.8um, 3ug/kg: 37.2.0±0.6um, and 150ug/kg: 35.0±1.0um). These findings demonstrate that inhibition of EphA4 or stimulation of Tie2 may represent novel options for therapeutically enhancing pial collateral vessel growth following ischemic stroke.

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