Reelin promotes cerebral angiogenesis via mTOR/HIF-1α-mediated transcriptional upregulation of Netrin-4

Cerebral angiogenesis is essential for brain development and the maintenance of homeostasis. The secreted glycoprotein Reelin has been implicated in this process; however, the underlying mechanisms, particularly its functional relationship with the axon guidance molecule Netrin-4, remain incompletely understood. In this study, we used Reln^⁻/⁻ mouse models and bEnd.3 brain microvascular endothelial cells. Experimental approaches included western blotting, immunofluorescence, quantitative PCR (qPCR), in vitro tube formation assays, chromatin immunoprecipitation (ChIP), and dual-luciferase reporter assays. Functional validation was performed by intracerebroventricular injection of recombinant Reelin protein, the mTOR inhibitor rapamycin, and recombinant Netrin-4. Compared with wild-type mice, Reln^⁻/⁻ mice exhibited significantly reduced Netrin-4 expression and markedly impaired angiogenic capacity. These defects were rescued by exogenous Reelin through the Dab1/mTOR/HIF-1α pathway. Direct supplementation with Netrin-4 similarly restored angiogenesis. In vitro experiments confirmed the conservation of this pathway, and ChIP analysis together with luciferase reporter assays demonstrated direct binding of HIF-1α to the Netrin-4 promoter. In summary, this study identifies the Reelin-Dab1-mTOR-HIF-1α-Netrin-4 axis as a signaling pathway that is associated with cerebral angiogenesis during development.