Regulation of H-type angiogenesis and permeability in the subchondral bone of osteoarthritis: the role of Slit3 and the Robo4/Rac1-GTP/ROS axis

Subchondral bone H-type angiogenesis and increased permeability are critical in osteoarthritis (OA) progression, yet the underlying regulatory mechanisms remain unclear. This study utilized proteomic analysis to identify significantly elevated expression of Slit guidance ligand 3 (Slit3) and Ras-related C3 botulinum toxin substrate 1 (Rac1) in the subchondral bone of patients with OA, both associated with angiogenesis and permeability. Experimental validation revealed that Slit3 and Rac1-GTP, rather than total Rac1, drive increased H-type angiogenesis and permeability. To further investigate, we exogenously added Slit3 recombinant protein to mimic the effect of non-ECs secreting Slit3 in the subchondral bone microenvironment on endothelial cells (ECs). Exogenous Slit3 significantly promoted migration, tube formation, and permeability in H-type ECs. An anti-Roundabout guidance receptor 4 (Robo4) antibody inhibited these effects and suppressed Rac1-GTP expression and reactive oxygen species (ROS) levels in ECs. Further investigation revealed that Rac1-GTP and ROS inhibitors could block the effects of exogenous Slit3 on H-type endothelial cell migration, tube formation, and permeability. In in vivo experiments, knockout of Slit3 aggravated early OA in destabilization of the medial meniscus (DMM) mice by altering cartilage and subchondral bone structure but alleviated late-stage OA. Moreover, Slit3 promoted H-type angiogenesis and permeability in the subchondral bone of DMM mice through the Robo4/Rac1-GTP signaling pathway, consistent with in vitro findings. Collectively, this study demonstrates that Slit3 mediates increased H-type angiogenesis and permeability in OA subchondral bone via the Robo4/Rac1-GTP/ROS signaling axis. Modulating Slit3 may offer stage-specific therapeutic strategies for OA.