Abnormal angiogenesis and increased vascular permeability of subchondral bone are key mechanisms related to osteoarthritis (OA). However, the precise mechanisms responsible for heightened vascular permeability in OA remain unclear. The present study used proteomics to identify protein expression in damaged subchondral bone compared with normal subchondral bone. The results suggest that Ras homolog family member A (RhoA) may be associated with the vascular permeability of subchondral bone and ferroptosis in OA. The results of analysis of clinical samples indicated a significant increase in expression of RhoA in the subchondral bone of OA. This were consistent with the proteomics findings. We found through western blotting, RT‑PCR, and immunofluorescence that RhoA significantly increased the permeability of endothelial cells (ECs) by inhibiting inter‑EC adhesion proteins (zona occludens‑1, connexin 43 and Vascular endothelial‑Cadherin) and actin filaments. Furthermore, RhoA induced ferroptosis core proteins (glutathione peroxidase 4, solute carrier family 7 member 11 and acyl‑CoA synthase long‑chain family member 4, ACSL4) by influencing lipid peroxidation and mitochondrial function, leading to ferroptosis of ECs. This suggested an association between RhoA, ferroptosis and vascular permeability. Ferroptosis significantly increased permeability of ECs by inhibiting inter‑EC adhesion proteins. RhoA increased vascular permeability by inducing ferroptosis of ECs. In vivo, inhibition of RhoA and ferroptosis significantly mitigated progression of OA by alleviating cartilage degeneration and subchondral bone remodeling in mice with destabilization of the medial meniscus. In conclusion, the present findings indicated that RhoA enhanced vascular permeability in OA by inducing ferroptosis. This may serve as a novel strategy for the early prevention and treatment of OA.

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.