Endothelial-Specific EphA4 Negatively Regulates Native Pial Collateral Formation and Re-Perfusion following Hindlimb Ischemia

Abstract

Leptomeningeal anastomoses play a critical role in regulating vascular re-perfusion following obstruction, however, the mechanisms regulating their development remains under investingation. Our current findings indicate that EphA4 receptor is a novel negative regulator of collaterogenesis. We demonstrate that EphA4 is highly expressed on pial arteriole collaterals at post-natal day (P) 1 and 7, then significantly reduced by P21. Endothelial cell (EC)-specific loss of EphA4, EphA4f/f/Tie2::Cre (KO), resulted in an increase in the density but not diameter of pial collaterals compared to WT mice. ECs isolated from KO mice displayed a 3-fold increase in proliferation, enhanced migration, tube formation and elevated levels of phospho(p)-Akt compared to WT ECs. Attenuating p-Akt, using LY294002, reduced the proliferative and migration effects in the KO ECs. RNAseq analysis also revealed altered expression patterns for genes that regulate cell proliferation, vascular development, extracellular matrix and immune-mediate responses, namely MCP-1, MMP2 and angiopoietin-1. Lastly, we show that induction of hindlimb ischemia resulted in accelerated re-perfusion, collateral remodeling and reduced tissue necrosis in the absence of ECspecific EphA4 compared to WT mice. These findings demonstrate a novel role for EphA4 in the early development of the pial collateral network and suggests a role in regulating vascular remodeling after obstruction.