https://doi.org/10.1007/s00392-025-02625-4
1Universitätsklinikum Schleswig-Holstein Medizinische Klinik II / Kardiologie, Angiologie, Intensivmedizin Lübeck, Deutschland; 2Universität zu Lübeck Center of Brain, Behavior and Metabolism - Bioanalytic Core Facility Lübeck, Deutschland; 3Universitätsklinikum Schleswig-Holstein Campus Lübeck Institut für Humangenetik Lübeck, Deutschland; 4Universitätsklinikum Schleswig-Holstein Campus Lübeck Institut für Kardiogenetik Lübeck, Deutschland; 5Universitätsklinikum Schleswig-Holstein Campus Lübeck Sektion für Translationale Chirurgische Onkologie und Biomaterialbank Lübeck, Deutschland; 6Universität zu Lübeck Medical Systems Biology - Bioinformatic Service Unit Lübeck, Deutschland; 7Asklepios Westklinikum Rissen Abteilung für Kardiologie Hamburg, Deutschland; 8Universitäres Herz- und Gefäßzentrum Hamburg Klinik für Kardiologie Hamburg, Deutschland; 9Deutsches Herzzentrum der Charite (DHZC) Klinik für Herz-, Thorax- und Gefäßchirurgie Berlin, Deutschland
Background/Introduction
Calcific aortic valve disease (CAVD) is the most prevalent valvular heart disease requiring therapeutic intervention. Currently, treatment options are restricted to surgical or catheter-based procedures due to the lack of pharmacotherapeutic options to prevent or stop the development or progression of CAVD. Cellular accumulation of sphingolipids is commonly linked to a pro-inflammatory response associated with lipotoxicity and inflammatory processes might be crucial in CAVD.
Purpose
With the subsequent goal of developing innovative treatment approaches for CAVD, we aim to elucidate the unclear role of sphingolipids in the development of CAVD.
Methods
Human aortic valve and blood plasma samples of CAVD patients and controls (n=29) were analysed by liquid chromatography–mass spectrometry (LC–MS) for lipidomics and proteomics.
Human aortic valve interstitial cells (hVICs) were stimulated with ceramides and oxidized LDL. Calcification was detected by Alizarin red staining. NF-κB pathway was assessed by proteome profiler. Myriocin and GW4869 were applied to inhibit sphingolipid biosynthesis. PDTC and MCC950 were used to inhibit NF-κB pathway and NLRP3 activation.
ApoE-/- mice received a gain-of-function PCSK9 adeno-associated virus vector and fed high cholesterol diet for 21 weeks. Myriocin and GW4869 were applied orally over the treatment period to inhibit sphingolipid metabolism in vivo. Murine echocardiography was used to measure transvalvular velocity and left ventricular function.
Tissue changes like calcification or lipid accumulation in valve tissue were determined by histological staining. Murine aortic valve tissue was analysed using LC-MS for lipidomics or used for single cell RNA sequencing to identify cellular heterogeneity.
Results
A significantly altered sphingolipid profile was observed in human aortic valve tissue of CAVD patients. Ceramide species e.g. Cer (17:1;2O/16:0) were increased (Fig. 1A). Proinflammatory target proteins identified by aortic valve tissue proteomics correlate with sphingolipid accumulation with reference to lipotoxicity (Fig. 1B).
Verifying the biological relevance of these findings in vitro, C2-Cer (d18:1/2:0) enhanced hVIC calcification (Fig. 2A-B). Furthermore, C2-Cer (d18:1/2:0) enhanced NF-κB p65 phosphorylation and NLRP3 activation, while treatment with both PDTC and MCC950 were capable to protect against C2-Cer (d18:1/2:0) induced calcification (Fig.2C). Supporting the role of sphingolipid biosynthesis in hVIC calcification, Myriocin and GW4869 both reduced ox-LDL-induced hVIC calcification which was reinduced by C2-Cer (d18:1/2:0) (Fig. 2D).
Finally, Myriocin reduced static and haemodynamic parameters of calcific aortic valve stenosis and prevents aortic valve sphingolipidome changes in ApoE-/- + PCSK9-AAV-mice model (Fig.3A-D). Single cell RNA sequencing of murine aortic valve tissue reveals disease specific expression patterns of murine valvular endothelial cells (mVEC) and murine valvular interstitial cells (mVIC), the presence of epithelial-to-mesenchymal transition and the interaction with immune cells.
Conclusion
CAVD is accompanied by an accumulation of ceramides causing proinflammatory lipotoxicity in human aortic valve tissue. Pharmacological modulation of sphingolipid metabolism is an effective approach to prevent the development and progression of aortic valve calcification in vivo and should be considered as a future therapeutic strategy in CAVD patients.