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 pathological event in ischemic stroke. Matrix metalloproteinase-9 (MMP-9) is a well-established effector of junctional protein degradation and barrier breakdown. While circulating MMP-9 derived from neutrophils has been extensively studied, the mechanisms underlying endogenous MMP-9 upregulation within brain endothelial cells during early ischemia remain poorly defined. Here, we provide evidence that actin polymerization functionally contributes to endothelial MMP-9 upregulation under ischemic conditions. Using an oxygen–glucose deprivation (OGD) model in mouse brain microvascular endothelial cells (bEnd.3), time-course analysis identified 6 h as a critical window at which cells remained viable but exhibited significant actin remodeling. At this time point, both intracellular and secreted MMP-9 levels were significantly increased, concurrent with reduced expression and disrupted membrane localization of occludin, ZO-1, and VE-cadherin, as well as increased transendothelial permeability to both 4.4-kDa and 70-kDa tracers. Pharmacological modulation of actin dynamics bidirectionally regulated these changes: jasplakinolide further amplified MMP-9 expression, exacerbated junctional protein loss, and increased barrier permeability, whereas latrunculin B significantly suppressed MMP-9 upregulation, preserved junctional protein integrity, and reduced permeability. These findings indicate that excessive actin polymerization in brain endothelial cells is functionally linked to MMP-9 activation and early BBB destabilization, independent of inflammatory cell infiltration. These in vitro findings suggest an endothelial-autonomous mechanism that may contribute to early BBB dysfunction