Hypoxic Preconditioning Upregulates Connexin 43 in Neural Stem Cells to Enhance Host Angiogenesis After Transplantation -Evidence from a Rat Model of Cerebral Palsy.

Hypoxic preconditioning is commonly used to improve the therapeutic efficacy of neural stem cells (NSCs) transplantation; however, the mechanisms by which hypoxia regulates intercellular communication in NSCs remain incompletely understood. As connexin 43 (Cx43) is a key component of gap junctional intercellular communication (GJIC), we investigated whether hypoxic preconditioning modulates Cx43 expression and function in NSCs and whether this pathway contributes to NSCs-mediated angiogenesis in a rat model of cerebral palsy (CP). In vitro, NSCs exposed to 1% oxygen for different durations showed maximal upregulation of Cx43 mRNA and protein after six hours of hypoxic preconditioning without inducing cell necrosis. Functional analyses demonstrated that hypoxia significantly enhanced Cx43-mediated GJIC and hemichannel activity. Following transplantation, hypoxia-preconditioned NSCs increased Cx43 expression in the perilesional region of CP rats, with peak levels observed at 1 week post-transplantation and prominent localization at the graft-host interface. Importantly, transplantation of hypoxia-preconditioned NSCs increased perilesional vessel density and proliferating endothelial cells, whereas shRNA-mediated Cx43 knockdown abolished the effects. These findings demonstrate that hypoxic preconditioning enhances Cx43 expression and function in NSCs, thereby promoting intercellular communication and contributing to NSCs-induced angiogenesis in CP rats. Targeting Cx43 signaling may represent a promising strategy to improve the therapeutic efficacy of NSCs-based therapies.