Sphingolipid-Driven Lipotoxicity Promotes Calcific Aortic Valve Disease Through ER-Stress–NF-κB–EndMT Signaling and Is Therapeutically Targetable Through Serine Palmitoyltransferase Inhibition

Background Calcific aortic valve disease (CAVD) lacks disease-modifying therapy. LDL-cholesterol lowering does not attenuate valvular calcification, suggesting cholesterol-independent drivers. Thus, we tested whether ceramide accumulation drives CAVD and whether inhibiting sphingolipid de novo synthesis is therapeutic. Methods We integrated lipidomics and proteomics of calcified and non-calcified human aortic valves with matched plasma. Mechanistic effects of ceramides were tested in primary human valvular interstitial cells (VICs). Therapeutic efficacy of serine palmitoyltransferase (SPT) inhibition with Myriocin was evaluated in ApoE −/− mice with PCSK9 DY -AAV–driven hyperlipidemia on Western diet using preventive treatment and therapeutic initiation after echocardiographically verified gradient elevation. Myriocin was additionally tested in a normolipidemic wild-type aortic valve wire-injury model. Hemodynamics, valve structure, lipid composition, and cell-state transitions were assessed by echocardiography, histology, lipidomics, and single-cell RNA sequencing. Results\ Calcified human valves showed enrichment of long-chain ceramides linked to NF-κB, endothelial-to-mesenchymal transition (EndMT), and ER-stress/unfolded protein response (UPR) programs, whereas matched plasma sphingolipids were not elevated. In hVICs, long-chain C18 ceramide induced GRP78/BiP, CHOP, and ATF4, activated NF-κB, and promoted dose-dependent calcification. In vivo , SPT inhibition with Myriocin lowered valvular ceramides, reduced leaflet inflammation and mineralization, and improved cusp separation and transvalvular hemodynamics without lowering plasma LDL-cholesterol. In a therapeutic protocol initiated after gradient elevation, Myriocin halted further hemodynamic progression, providing the first evidence that de novo sphingolipid synthesis can be targeted after established CAVD onset in vivo . Myriocin also attenuated injury-induced stenosis in normolipidemic mice. Single-cell RNA sequencing showed reduced NF-κB/EndMT and ER-stress signatures and shifted VIC states away from osteogenic phenotypes. Conclusions Human valve lipidomics and human VIC experiments identify ceramide-driven lipotoxicity as a driver of an ER-stress–NF-κB–EndMT axis in CAVD. In vivo , de novo sphingolipid synthesis is an actionable, LDL-independent target capable of modifying established hemodynamic disease progression, supporting SPT inhibition as a tractable strategy for medical CAVD therapy. What is new? Calcified human aortic valves show local long-chain ceramide enrichment linked to NF-κB, EndMT, and ER-stress/UPR programs, whereas matched plasma lacks a corresponding sphingolipid increase. C18 ceramide directly promotes ER-stress– and NF-κB-dependent calcification in primary human valvular interstitial cells. To our knowledge, this is the first demonstration that pharmacologic inhibition of de novo sphingolipid synthesis, initiated after echocardiographically verified gradient elevation, attenuates further hemodynamic progression of CAVD in vivo . What are the clinical implications? CAVD progression may involve valve-local ceramide lipotoxicity rather than LDL cholesterol alone. SPT inhibition provides preclinical proof-of-concept for LDL-independent disease modification, including established hemodynamic disease. Clinically translatable SPT inhibitors or targeted delivery strategies warrant evaluation as medical approaches to slow calcific aortic stenosis progression.