https://doi.org/10.1007/s00392-024-02526-y
1Universitätsklinikum Schleswig-Holstein Medizinische Klinik II / Kardiologie, Angiologie, Intensivmedizin Lübeck, Deutschland; 2Universität zu Lübeck Center for Brain, Behavior and Metabolism - Bioanalytic Core Facility Lübeck, Deutschland; 3Universitätsklinikum Schleswig-Holstein, Klinik für Chirurgie Sektion für Translationale Chirurgische Onkologie und Biomaterialbanken Lübeck, Deutschland; 4Universität zu Lübeck Group for Medical Systems Biology Lübeck, Deutschland; 5Deutsches Herzzentrum der Charite (DHZC) Klinik für Herz-, Thorax- und Gefäßchirurgie Berlin, Deutschland; 6Universitätsklinikum Schleswig-Holstein Institut für Kardiogenetik Lübeck, Deutschland
Background/ Introduction:
Aortic stenosis resulting from calcific aortic valve disease (CAVD) is the most common valvular heart disease. In symptomatic patients, the prognosis without any intervention is poor. Treatment to date is based on surgery or catheter-based interventions. Pharmacological therapy options to prevent the progression of valve calcification are not available. In vascular smooth muscle cells, cellular accumulation of sphingolipids is associated with a proinflammatory response in the sense of lipotoxicity. Chronic inflammatory processes are indeed also essential for aortic valve calcification.
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=26) were analysed by liquid chromatography–mass spectrometry (LC–MS) for lipidomics and proteomics. Human aortic valve interstitial cells (hVICs) were stimulated with ceramides. Calcification was detected by alizarin red staining. mRNA expression of bone-related proteins was determined by qPCR. 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. Calcification degree of murine aortic valve was determined by histological staining.
Results:
A significantly altered sphingolipid profile was observed in aortic valve tissue of CAVD patients. Ceramide species e.g. Cer (17:1;2O/16:0) were increased (Fig. 1A). Furthermore, proteomics analyses reveal an increased activity of inflammatory signaling pathways in the calcified valve tissue (Fig. 2A).
Verifying the biological relevance of these findings in vitro, C2-Cer (d18:1/2:0) enhanced hVIC calcification (Fig. 3A-C). Accordingly, ALP activity and mRNA expression of osteogenic genes RUNX2, ALPL and MSX2 were increased. 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. Supporting the role of sphingolipid biosynthesis in hVIC calcification, Myriocin and GW4869 both reduced ox-LDL-induced hVIC calcification which was reversed by C2-Cer (d18:1/2:0) (Fig. 4A).
Finally, Myriocin reduced the degree of calcification and transvalvular jet velocity of aortic valve in the PCSK9-AAV-ApoE-/- mice model.
Conclusions:
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.