Actin polymerization drives endogenous MMP-9 upregulation and blood-brain barrier disruption in ischemic brain endothelial cells.

Blood-brain barrier (BBB) disruption is a critical early event in ischemic stroke. Matrix metalloproteinase-9 (MMP-9) drives BBB breakdown through proteolytic degradation of junctional proteins; however, the upstream mechanisms triggering endogenous MMP-9 upregulation within brain endothelial cells before inflammatory cell infiltration remain poorly understood. Here, we identify actin polymerization as a functional upstream driver of endothelial MMP-9 activation and BBB disruption under ischemic conditions. Using an oxygen-glucose deprivation (OGD) model in bEnd.3 cells, we found that 6 h of OGD induced significant actin remodeling concurrent with upregulation of both MMP-9 expression and enzymatic activity, reduced the total protein levels of occludin, ZO-1, and VE-cadherin, disrupted their membrane localization, and increased transendothelial permeability. Bidirectional pharmacological modulation of actin dynamics using jasplakinolide (an F-actin stabilizer) and latrunculin B (a polymerization inhibitor) correspondingly amplified or attenuated all these changes. Crucially, lentivirus-mediated shRNA knockdown of MMP-9 significantly preserved junctional protein expression, maintained transendothelial electrical resistance, and reduced barrier permeability even under jasplakinolide-enhanced polymerization, establishing MMP-9 as a necessary mediator of actin-driven barrier disruption. MMP-9 knockdown also attenuated F-actin accumulation, suggesting a self-amplifying feedback loop. These findings delineate an endothelial-autonomous actin-MMP-9-BBB disruption axis that operates independently of inflammatory cell infiltration, identifying endothelial actin homeostasis as a potential therapeutic target for early barrier protection in ischemic stroke.