MicroRNA-21-3p regulation of NOX4 and VEGFA contributes to hemorrhage in cerebral cavernous malformations

Objective: MicroRNAs regulate the brain vascular integrity and are involved in the lesion development of cerebral cavernous malformations (CCM). This study examines the role of microRNA-21-3p in CCM-related cerebral hemorrhage and its underlying mechanisms. Methods: The expression of miRNA-21-3p and its target genes of NADPH oxidase 4 (NOX4) and vascular endothelial growth factor A (VEGFA) in brain microvascular endothelial cells (BMECs) and pericytes were assessed in cavernous malformation lesions of 20 sporadic CCM patients by fluorescence in situ hybridization. The association of their expression with hemorrhage manifestation was evaluated. Cell proliferation, permeability, reactive oxygen species (ROS), migration, tubule formation, and the expression of NOX4 and VEGFA were assessed in CCM2 gene-depleted human BMECs and pericytes after miRNA-21-3p intervention. Cerebral hemorrhage, vascular permeability, vascular dilation, and angiogenesis after miRNA-21-3p intervention were evaluated in the ccm2 gene-knockdown zebrafish. Results: Decreased miRNA-21-3p and increased NOX4 and VEGFA were shown in BMECs and pericytes of the CCM lesions compared to peri-lesion normal vessels from epilepsy patients, which were also correlated with the presence of cerebral hemorrhage in CCM patients. Increasing miRNA-21-3p attenuated cell proliferation, permeability, ROS expression, cell migration, and tubule formation by targeting NOX4 and VEGFA in CCM2 gene-depleted BMECs and pericytes. In vivo studies revealed that increasing miRNA-21-3p reduced cerebral hemorrhage, vascular permeability, vascular dilation, angiogenesis, and the overexpression of nox4 and vegfa in ccm2 gene-knockdown zebrafish. Conclusion: MiRNA-21-3p can be a novel therapeutic target by regulating NOX4 and VEGFA, thereby stabilizing vascular integrity and reducing cerebral hemorrhage in CCM lesions.