Strokes are a leading cause of death and disability in the United States, predominantly caused by ischemic events. Ischemic strokes occur when a clot or other obstruction lodges in a blood vessel of the brain, restricting the movement of blood. Subsequent rapid cell death occurs and often leads to long term neurological deficits. Pial collaterals are a well-established determinant of patient outcome due to their unique ability to remodel into conductance arteries that can reroute blood back to the ischemic tissue. During development, pial collaterals arise within the pia mater and establish connections between distant arterioles of cerebral arteries. Under healthy conditions, these vessels are exposed to bidirectional blood flow, keeping them small and dormant. Following vascular obstruction, pial collaterals are exposed to unidirectional blood flow, triggering them to expand through an adaptive process termed, arteriogenesis, allowing for retrograde perfusion into the obstructed artery and its affected tissue. However, hyperacute arteriogenesis following ischemic stroke has been poorly investigated. The following dissertation aims to address this research gap and leverage the findings to develop therapeutics that enhance arteriogenesis. Previous research has revealed EphA4 restricts arteriogenesis through the Tie2 signaling axis, therefore this work sought to evaluate the endothelial cell (EC) specific role of the EphA4/Tie2 axis in acute arteriogenesis. EC-specific EphA4 KO mice displayed increased pial collateral size from 4.5 to 24-hours post-injury, which was associated with reduced tissue damage, improved cerebral blood flow, and enhanced motor function. Additionally, pharmaceutical stimulation of the Tie2 axis using Vasculotide, an angiopoietin-1 memetic peptide, replicates these findings. Administration of 3ug/kg Vasculotide to wildtype mice immediately after permanent middle cerebral artery occlusion leads to significantly larger pial collateral diameters, correlating with reduced tissue damage and improved functional recovery. Unlike Vasculotide, device stimulation using low intensity focused ultrasound failed to increase collateral diameter, despite resulting in profound neuroprotection. Taken together, this dissertation work demonstrates that the EphA4/Tie2 signaling pathway can be pharmacologically targeted to improve arteriogenesis following ischemic stroke. / Ph.D. / Worldwide, strokes are a leading cause of death and long-term disability with many cases being ischemic strokes, where a blood clot blocks blood flow to the brain. Without the critical oxygen and nutrients that the blood provides, cells in the affected region of the brain begin to rapidly die, leading to neurological deficits. While current treatments focus on removing the clot, it does not guarantee the restoration of blood flow to the damaged area. In contrast, our research focuses on pre-existing blood vessels in the brain, called pial collaterals, that can ease the loss of blood flow after stroke. These vessels, although relatively inactive under normal conditions, can enlarge after a stroke to reroute blood flow to the injured tissue. Thus, pial collateral growth is a critical process in the initial hours after stroke when this blood flow can prevent brain cells from dying. Previous work has shown EphA4, a receptor known for its role in nervous system development, restricts pial collateral size by inhibiting the Tie2 signaling pathway. Loss of EphA4 in endothelial cells allows for Tie2 receptor activation, increased pial collateral size, and decreased tissue damage. To explore therapeutic enhancement of pial collaterals, we administered Vasculotide, a drug that activates the Tie2 receptor, to wildtype mice expressing EphA4 after a stroke. The mice treated with Vasculotide displayed significantly larger pial collateral vessels one day after the stroke, compared to control mice. Moreover, Vasculotide-treated mice exhibited reduced tissue damage and performed better on behavioral assessments. In addition to pharmaceutical stimulation with Vasculotide, we also investigated the effects of low-intensity focused ultrasound (LIFU) on collateral size. LIFU treatment resulted in decreased tissue damage compared to untreated controls; however, it did not impact collateral size. These findings suggest that inhibiting EphA4 or stimulating Tie2 could serve as novel therapeutic targets to promote the expansion of pial collateral blood vessels, thereby restoring critical blood flow to injured areas of the brain.