Knockdown of RGMa reduces vascular calcification by inhibiting the AKT signaling pathway

Vascular calcification (VC), pathologically characterized by ectopic calcium-phosphate deposition, demonstrates a significant correlation with cardio-cerebrovascular morbidity and adverse clinical outcomes. Despite its clinical relevance, current interventions to prevent and treat VC progression remain limited in efficacy. Numerous studies have demonstrated that Repulsive Guidance Molecule A (RGMa) plays a pivotal role in cardio-cerebrovascular diseases. This investigation aimed to elucidate the role of RGMa in VC and to explore the underlying molecular mechanisms. This study systematically investigated the dynamic alterations in RGMa expression during VC progression through integrated in vitro and in vivo experimental models. The functional regulatory role of RGMa in VC pathogenesis and its molecular mechanistic basis were further elucidated via lentiviral short hairpin RNA (shRNA)-mediated RGMa knockdown and adeno-associated virus serotype 9 (AAV9)-delivered gene silencing, combined with pharmacological inhibition of AKT signaling using the selective inhibitor MK-2206. Alizarin Red S staining and calcium content assays demonstrated that VC and vascular smooth muscle cell (VSMC) calcification were successfully induced in vivo (via subcutaneous vitamin D3 overdose) and in vitro (through high-phosphate medium culture), respectively. Western blot (WB) and immunofluorescence (IF) analyses revealed a significant upregulation of RGMa expression in calcified VSMCs. We successfully constructed a VSMC-specific RGMa-targeting AAV, and RGMa knockdown effectively attenuated VC. These findings indicate that RGMa plays a pivotal role in VSMC calcification. Finally, we investigated whether RGMa exerts its effects via the AKT pathway. In the in vitro model, treatment with the AKT inhibitor MK-2206 suppressed osteogenic marker expression in high phosphate-treated VSMCs without reversing RGMa upregulation. This study indicates that RGMa may promote VC by modulating AKT activation, highlighting RGMa as a promising new molecular target for the treatment of VC.