Abstract Body (Do not enter title and authors here): Background: Calcific aortic valve disease (CAVD) is the leading cause of valvular heart intervention yet lacks effective pharmacological therapies. Ceramides are mediators of inflammation and lipotoxicity in cardiometabolic disorders; however, their role in valvular calcification remains poorly understood. This study aimed to clarify the pathogenic relevance of ceramide accumulation in human CAVD, dissect underlying molecular mechanisms, and evaluate the therapeutic efficacy of targeting ceramide de novo synthesis using a reverse translational approach.

Methods: We integrated lipidomics and proteomics to characterize ceramide accumulation and related inflammatory signaling in calcified versus non-calcified human aortic valves. In vitro, human valvular interstitial cells (hVICs) were used to assess pro-calcific effects of ceramides and synthesis inhibition via serine palmitoyltransferase (SPT) blockade using Myriocin. In vivo, we employed a murine CAVD model (ApoE-KO with PCSK9-AAVDY), to replicate chronic ceramide overload and degenerative pathophysiology of human disease. Effects of SPT inhibition were assessed by echocardiography, lipidomics, histology and single-cell RNA sequencing (scRNA-seq).

Results: Human calcified valves showed pronounced accumulation of long-chain ceramides, correlating with NF-κB activation and endothelial-to-mesenchymal transition (EndMT). In vitro, ceramide induced hVIC calcification in an NF-κB-dependent manner, while inhibition of SPT attenuated hVIC calcification. Translating these findings in vivo, SPT inhibition reduced systemic and valvular ceramide levels without affecting cholesterol, leading to reduced aortic valve calcification and improved valve hemodynamics. scRNA-seq confirmed reduced NF-κB activation and EndMT under Myriocin, along with a shift in VIC phenotypes toward less osteogenic states. Hallmark long-chain C18:0 ceramide species associated with pathogenicity in the murine model correlated with pro-inflammatory and pro-calcific markers in human aortic valves, highlighting cross-species relevance and translational significance.

Conclusions: Ceramide-driven lipotoxicity, mediated through NF-κB-EndMT axis, is a key driver of CAVD pathogenesis. Targeting ceramide de novo synthesis attenuates valvular calcification independent of cholesterol metabolism and represents a novel therapeutic strategy for this unmet need.