The role of ROCK1/MLC/NMMHC IIA-actin signaling in ischemic stroke-induced blood-brain barrier disruption: implications for therapeutic intervention.

BACKGROUND

Disruption of the blood-brain barrier (BBB) is a key event in the onset of ischemic stroke (IS), primarily driven by endothelial cytoskeletal rearrangement. The interaction between non-muscle myosin heavy chain IIA (NMMHC IIA) and actin, along with the ROCK/MLC pathway, is central to this cytoskeletal reorganization. While our previous studies have shown that the Caspase-3/ROCK1/MLC/NMMHC IIA-actin positive feedback loop mediates H2O2-induced neuronal apoptosis, its role in cerebral ischemia-reperfusion (I/R) injury and BBB disruption remains unclear.

METHODS

In vivo, we used endothelial-specific NMMHC IIA conditional knockdown mice, NMMHC IIA-inducible endothelial conditional knock-in mice and C57BL/6J to establish a middle cerebral artery occlusion/reperfusion model. In vitro, we employed brain microvascular endothelial cells in an oxygen-glucose deprivation/reoxygenation model. The effects of the NMMHC IIA inhibitor blebbistatin, the ROCK1 inhibitor Y-27632, and the actin depolymerizer cytochalasin D were assessed for their impact on I/R-induced activation of the ROCK/MLC/NMMHC IIA-actin pathway, tight junction proteins (TJs) degradation, and brain damage.

RESULTS

Inhibition of NMMHC IIA expression and stress fiber depolymerization significantly reduced NMMHC IIA-actin interactions, suppressed the ROCK/MLC pathway, decreased TJs degradation, and alleviated cerebral I/R injury. Conversely, overexpression of NMMHC IIA further exacerbated cerebral I/R injury and BBB disruption and amplified activation of the ROCK1/MLC pathway. Y-27632 inhibited the ROCK/MLC/NMMHC IIA-actin pathway, mitigating I/R-induced BBB disruption.

CONCLUSIONS

This study reveals that the ROCK1/MLC/NMMHC IIA-actin pathway is implicated in I/R-induced BBB disruption and operates as a positive feedback loop. These findings offer a promising therapeutic strategy for the treatment of IS and BBB damage.