MicroRNA-21-3p regulation of NADPH oxidase 4 and vascular endothelial growth factor A contributes to hemorrhage in cerebral cavernous malformations

Background Cerebral cavernous malformations (CCMs) are vascular anomalies prone to hemorrhage, leading to neurological deficits and reduced quality of life. Current therapies are limited, and molecular mechanisms underlying vascular instability remain incompletely understood. MicroRNAs have emerged as key regulators of vascular integrity. This study investigates the role of microRNA-21-3p (miR-21-3p) in modulating oxidative stress and angiogenesis in CCM. Methods Expression of miR-21-3p, NADPH oxidase 4 (NOX4), and vascular endothelial growth factor A (VEGFA) was assessed in endothelial cells and pericytes from CCM lesions of 20 sporadic patients using fluorescence in situ hybridization. Functional assays of proliferation, permeability, reactive oxygen species (ROS), migration, and tubule formation were performed in human brain microvascular endothelial cells (BMECs) and pericytes with CCM2 gene depletion, following miR-21-3p mimic or inhibitor intervention. In vivo effects on cerebral hemorrhage, vascular permeability, dilation, and angiogenesis were evaluated in ccm2 -knockdown zebrafish. Results CCM lesions exhibited decreased miR-21-3p and increased NOX4 and VEGFA compared to controls, correlating with hemorrhage. In vitro, miR-21-3p mimics suppressed proliferation, permeability, ROS generation, and angiogenic activity by directly targeting NOX4 and VEGFA. In vivo, miR-21-3p supplementation reduced cerebral hemorrhage, vascular leakage, dilation, and sprouting angiogenesis in zebrafish with ccm2 knockdown. Conclusion MiR-21-3p regulates vascular integrity in CCM by modulating NOX4-mediated oxidative stress and VEGFA-driven angiogenesis. Loss of miR-21-3p contributes to hemorrhage, while its restoration stabilizes vascular function. This study identifies miR-21-3p as a potential therapeutic target for CCM, bridging endothelial and pericyte biology with translational potential to reduce cerebral hemorrhage.