Piezo1–Src signaling rapidly disrupts brain endothelial barrier integrity

BACKGROUND: Blood-brain barrier integrity is essential for central nervous system homeostasis, yet the mechanisms underlying ultra-acute barrier dysfunction after abrupt hemodynamic changes remain incompletely understood. Piezo1 is a mechanosensitive Ca²⁺ channel expressed in endothelial cells and activated by mechanical stimuli such as shear stress, but its contribution to rapid brain endothelial barrier disruption remains unclear. We investigated whether Piezo1 activation induces an ultra-acute disruption of the endothelial barrier and explored the underlying signaling mechanisms. METHODS: Human brain microvascular endothelial cells (HBEC-5i) were stimulated with the Piezo1 agonist Yoda1 in the presence or absence of the Src inhibitor saracatinib, followed by barrier-function assessment and phosphoproteomic profiling. To assess in vivo relevance, vascular permeability was evaluated within minutes after reperfusion in a mouse cerebral ischemia-reperfusion model treated with the mechanosensitive channel inhibitor GsMTx4. RESULTS: Comprehensive phosphoproteomic profiling after Piezo1 activation revealed enrichment of phosphorylation events associated with cell junctions, adhesion, and cytoskeletal organization. Yoda1 induced rapid phosphorylation of junction-associated proteins, including occludin at Tyr287, and caused acute barrier disruption in HBEC-5i cells. Saracatinib attenuated occludin Tyr287 phosphorylation and preserved barrier integrity, supporting the involvement of Src-family kinase-sensitive signaling in this response. In mice, GsMTx4 significantly attenuated vascular permeability within minutes after reperfusion, consistent with the in vitro observations and supporting a role for mechanosensitive channel-associated signaling in ultra-acute blood-brain barrier leakage immediately after flow restoration. CONCLUSIONS: This study provides the first phosphoproteomic characterization of rapid phosphorylation changes associated with Piezo1 activation in human brain endothelial cells. Guided by these phosphoproteomic signatures, our data support the involvement of saracatinib-sensitive signaling, accompanied by occludin Tyr287 phosphorylation, in rapid endothelial barrier disruption. These findings suggest that Piezo1-associated signaling may contribute to ultra-acute BBB dysfunction and provide a rational for further investigation in the context of abrupt hemodynamic stress and cerebral revascularization.