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Introduction: Piezo1 is a mechanosensitive ion channel that gates calcium and sodium influx in response to membrane stretch or increased shear stress. We previously found that selective deletion of endothelial Piezo1 in mice leads to reduced brain perfusion. The present study tested the hypothesis that activation of endothelial Piezo1 promotes acute vasodilation of leptomeningeal arterioles in vivo. Methods: Endothelial Piezo1 deletion (EC Piezo1 KO) was induced by tamoxifen treatment of Cdh5(Pac)CreERT2; Piezo1 fl/fl mice. Tamoxifen treatment of Piezo1 fl/fl littermates was used as control. After one month, mice underwent intravital fluorescence imaging of leptomeningeal vasculature viewed through a thinned skull (Leica DMI8 microscope, isoflurane and heat support provided). Before imaging, 50–80 μL of an aggregation-induced emission (AIE) dye was injected via the jugular vein for in vivo vessel-tracking (imaged at 4Hz, 2 min duration). Distalmost arterioles (DMA) and leptomeningeal collaterals (LMC) on the parietal cortex were imaged before and after delivery of a Piezo1 agonist (Yoda 1, 1.25 mg/kg, i.p.). Image stacks were measured with the VasoMetrics plugin (ImageJ). RNAscope was used to determine Piezo1 mRNA expression in brain vasculature from mouse and human tissues. Results: Resting diameter of LMCs and DMAs were not different between groups (LMC: 20.9 ± 4.5 mm (WT) vs. 23.4 ± 5.8 mm (EC Piezo1 KO) (P=0.65, n=10-15 vessels from 2-3 mice); DMA: 28.9 ± 6.6 mm (WT) vs. 30.7 ± 7.2 mm (EC Piezo1 KO) (P=0.93, n=10-15 vessels from 2-3 mice). Following Yoda 1 administration, LMC from WT mice dilated (7.4 ± 6.23%) whereas LMC from EC Piezo1 KO mice constricted (-6.7 ± 9.4%; P=0.001). DMAs showed a similar but less robust trend. DMAs from WT mice dilated (3.1 ± 4.5%), whereas DMAs from EC Piezo1 KO mice slightly constricted (-0.2 ± 4.7%; P=0.088). RNAscope showed Piezo1 expression in endothelium and smooth muscle of brain arterioles of both mouse and human autopsy samples. Conclusions: These studies support our proposed model that activation of EC Piezo1 can promote cerebral vasodilation. Additionally, our finding that Yoda 1 promotes vasoconstriction of cerebral arterioles in EC Piezo1 KO mice (particularly LMC arterioles) is consistent with an opposing constriction role of Piezo1 in the smooth muscle. We conclude that net effect of Piezo1 activation is determined by the balance of opposing effects from EC and SMC Piezo1 channels.