FOXO1 inhibitor(compound 10)

Calcific aortic valve disease (CAVD) is a progressive and life-threatening condition characterized by fibrocalcific remodeling of the valve leaflets. Valvular interstitial cells (VICs) are central mediators of calcific aortic valve disease (CAVD), as their osteogenic trans-differentiation drives pathological matrix remodeling and calcium deposition within the valve leaflets. Human-induced pluripotent stem cell-derived valvular interstitial cells (hiVICs) represent a promising patient-specific platform for disease modeling. While primary VICs (pVICs) readily undergo mineralization under osteogenic stimulation, hiVICs fail to calcify in conventional two-dimensional (2D) cultures. Our data suggests that the inability of hiVICs to calcify in 2D culture is related to FOXO1 (Forkhead box protein O1) activity, which suppresses the osteogenic transcriptional program by inhibiting RUNX2 (Runt-related transcription factor 2). To address this limitation, we then developed a three-dimensional tissue ring construct using hiVICs. When cultured in osteogenic medium, these constructs exhibited robust calcification, as confirmed by Alizarin Red and Von Kossa staining. FOXO1 was also identified as a mediator of calcification in the tissue ring constructs. Metformin treatment restored FOXO1 expression and inhibited calcification, while AS1842856, a selective FOXO1 inhibitor, exacerbated tissue construct mineralization and led to a near-complete tissue collapse. In summary, we establish a functional 3D hiVIC-based model of CAVD enabling mechanistic investigation and pharmacological screening and identify FOXO1 as a critical regulator of osteogenic transition. Graphical Abstract

The induced pluripotent stem cells derived mesenchymal stem cells (iMSCs) have shown great promise for bone tissue regeneration in critical‐sized calvarial defects. Still, the roles of FKBP5 in its osteogenesis are rarely known. It was observed that FKBP5 increased rapidly in iMSCs following osteogenic differentiation. To elucidate its role, FKBP5 was knocked down or overexpressed by lentivirus infection. Interestingly, the down‐regulation of FKBP5 impaired the osteogenesis of iMSCs, whereas the up‐regulation of FKBP5 promoted it. Proteomics analysis of iMSCs/oeFKBP5 and iMSCs/oeNC revealed that the protein variances are enriched in several signalling pathways associated with osteogenesis. Notably, the PI3K‐AKT signalling pathway was enriched highly at both D4 and D14. Co‐immunoprecipitation results demonstrated that the binding proteins of FKBP5 are AKT and pS473‐AKT, but not PI3K/p‐PI3K or FOXO1/pS256‐FOXO1; however, the ratios of pS473‐AKT/AKT and pS256‐FOXO1/FOXO1 were down‐regulated by FKBP5. FOXO1 inhibitor AS1842367 lessened the enhanced osteogenesis by FKBP5. Moreover, in a rat critical‐sized calvarial defect model, the iMSCs/oeFKBP5 delivery exhibited improved bone regeneration capability than iMSCs/oeNC in vivo. In conclusion, our findings first revealed that FKBP5 promotes the osteogenic differentiation of iMSCs partially through the FKBP5‐AKT‐FOXO1 pathway and presents a promising approach to iMSCs transplantation for clinical bone defects.