The Role of Agrin as a Downstream Mediator of Slit3 in Osteogenic-Angiogenic Interaction Under Osteoporosis

Abstract Osteoporosis, characterized by an imbalance between bone formation and bone resorption, is currently treated mainly by inhibiting resorption or promoting formation. However, these therapeutic strategies are often limited in efficacy because they fail to simultaneously target the coupled osteogenic and angiogenic processes in bone metabolism. In recent years, Slit3 has emerged as a key regulator of osteo-angiogenic interaction and is now considered a promising therapeutic target for metabolic bone disease. In the present study, we performed in-vitro and in-vivo experiments, including employing a human cell-derived microfluidic organ-on-a-chip (OoC) platform to investigate the upstream and downstream regulatory relationships within the Slit3-Agrin axis in osteo-angiogenic interaction, performing functional assays to elucidate the signaling pathway between Slit3 and Agrin, and establishing a pathological animal model to validate the osteogenic effect of this axis. The results showed that Slit3 mediates the nuclear translocation of β-catenin via the Robo1 receptor. Subsequently, β-catenin binds to TCF3 to activate the transcription of Agrin. Agrin then binds to LRP4 to negatively regulate the canonical Wnt/β-catenin signaling pathway, thereby orchestrating osteogenesis. This study reveals that the Slit3-Agrin axis serves as a core regulator of the bone-vascular unit and demonstrates the promising potential of OoC-based systems for developing targeted therapies.