Beyond typical lipid suspects - identification of sphingolipid metabolism as a pharmacotherapeutic target in calcific aortic valve disease

Abstract

With increasing prevalence of calcific aortic valve disease (CAVD) in aging populations, an urgent need exists for disease-modifying therapy beyond interventional or surgical approaches. Although atherosclerosis and CAVD share lipid accumulation and inflammation, LDL-lowering fails to modify valvular calcification, suggesting cholesterol-independent drivers. Sphingolipids are potent mediators of metabolic inflammation and cell stress that may contribute to CAVD pathogenesis.

This study investigated the pathogenic relevance of sphingolipid accumulation in CAVD, underlying mechanisms and therapeutic efficacy of targeting sphingolipid synthesis in a reverse-translational approach.

We integrated lipidomics and proteomics of calcified human aortic valves with matched plasma to identify tissue- and disease-associated sphingolipids and inflammatory markers. Functional relevance was validated in a human valvular interstitial cell (hVIC) calcification model by stimulation with ceramides and precursors and inhibition of key metabolic pathways. Calcification, osteogenic reprogramming, NF-κB-mediated inflammation and ER stress were quantified by staining, enzyme assays, qPCR and protein analyses. Therapeutic efficacy was examined in ApoE-/-+PCSK9 AAVDY mice with metabolically induced CAVD using Myriocin, an inhibitor of sphingolipid de novo synthesis, in preventive and early disease stages. Valve structure and function, lipid composition and cell-state transitions were analyzed via echocardiography, histology, lipidomics and single-nucleus RNA sequencing (snRNA-seq).

Human calcified valves showed accumulation of long-chain ceramides correlating with NF-κB activation, endothelial-to-mesenchymal transition (EndMT) and osteogenic marker expression. In vitro, ceramide and precursor stimulation induced hVIC calcification dose-dependently. This effect was abolished by NF-κB inhibition, ER-stress suppression or blockade of sphingolipid synthesis, underlining functional relevance. In vivo, echocardiography revealed CAVD changes without treatment (e.g., increased valve thickness), while Myriocin improved cusp separation, Vmax and dPmean in preventive and early-disease settings, indicating valve rescue. Histologically, Myriocin reduced ceramide accumulation and mineralization, preserving valve morphology. Long-chain ceramides were conserved across species and reduced to near wild-type by Myriocin. snRNA-seq showed reduced EndMT, decreased NF-κB signaling, attenuated ER stress and a VIC shift toward quiescent phenotypes. Importantly, benefits occurred without lowering plasma LDL, indicating a cholesterol-independent mechanism.

Our research links sphingolipid-mediated lipotoxicity in calcified human valves via cross-species-validated signaling to proof-of-concept that modulation of sphingolipid metabolism mitigates CAVD progression in a cholesterol-independent manner, establishing the ceramide-inflammation axis as a promising CAVD therapy target.