Single-nucleus and machine-learning integration identifies HLA-DRA and FTL as immune-metabolic axes and traditional Chinese medicine-targetable hubs in calcific aortic valve disease.

Calcific aortic valve disease (CAVD) is driven by immune–metabolic dysregulation and osteogenic remodeling, yet actionable molecular targets remain limited. This study integrated multimodal computational analyses with experimental validation to identify functional biomarkers and therapeutic candidates. Single-nucleus RNA sequencing of human aortic valves was combined with CellChat, Monocle 3 and consensus NMF to define immune–stromal interactions and gene programs. A 10 gene classifier was developed using multi model machine learning and interpreted with SHAP. Cross cohort validation, immune deconvolution and network enrichment were applied. Network pharmacology, docking and 50 ns MD simulations were used to identify ligand candidates. Experimental validation was performed in human VICs using osteogenic stimulation, siRNA knockdown and small molecule treatments, followed by RT qPCR, Western blot, Alizarin Red S staining and ALP and proliferation assays. Computational analyses consistently prioritized HLA DRA and FTL as stable, high AUC biomarkers linked to macrophage rich and MHC II enriched microenvironments. Network analyses anchored HLA DRA to adaptive immunity and FTL to iron metabolic pathways. Docking and MD identified Chrysin, α Tocopherol, Colchicine and Diallyl trisulfide as structurally compatible ligands. In vitro, osteogenic stimulation markedly upregulated HLA DRA, FTL, RUNX2 and ALPL. siRNA knockdown of HLA DRA or FTL reduced mineral deposition, ALP activity and VIC proliferation. Predicted small molecule ligands decreased HLA DRA and FTL expression and attenuated calcification. HLA DRA and FTL function as immune–metabolic drivers of VIC osteogenic remodeling and represent viable therapeutic targets. This integrated systems to experimental framework highlights natural compound candidates with potential relevance for CAVD therapy.