Background: Aortic stenosis due to calcific aortic valve disease (CAVD) constitutes the most common valvular heart disease in the developed world. In symptomatic patients, without repair the prognosis 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. With the consequent goal of developing innovative treatment approaches for calcifying CAVD, we therefore aim to elucidate the unclear role of sphingolipids in the development of CAVD.

Methods: Human aortic valve samples of CAVD patients and controls (n=41) were analyzed by liquid chromatography–mass spectrometry (LC–MS) for comprehensive lipidomics.

Human aortic valve interstitial cells (hVICs) were stimulated with ceramide species. Calcification was detected by alizarin red staining and colorimetric calcium content assay.

NF-κB Pathway and NLRP3 activity were assessed by proteome profiler and ELISA. hVIC proliferation was analyzed by BrdU assay, while apoptosis was evaluated using flow cytometry. mRNA expression of bone-related proteins was determined by qPCR.

GW4869 was applied to inhibit ceramide biosynthesis by neutral sphingomyelinase (N-SMase). Pyrrolidinedithiocarbamate (PDTC) and MCC950 were used to inhibit NF-κB Pathway and NLRP3 activation respectively.

Results: Ceramide species Cer(d18:1/16:0) was increased in calcified aortic valve tissue of CAVD patients. Cer(d18:1/16:0) indeed enhanced hVIC calcification measured by alizarin staining and calcium content assay. Accordingly, ALP activity and mRNA expression of osteogenic genes RUNX2, ALPL and MSX2 were significantly increased. GW4869 reduced ox-LDL-induced VIC calcification which was reversed by Cer(d18:1/16:0). Futhermore, Cer(d18:1/16:0) enhanced NF-κB p65 phosphorylation and NLRP3 activation, while treatment with both PDTC and MCC950 protected against Cer(d18:1/16:0) induced calcification.

Conclusion and perspective:

Cer(d18:1/16:0) contributes to calcification in CAVD in a NF-κB/ NLRP3 dependent manner. To, first, validate our findings and, second, evaluate a therapeutic approach we will apply a mouse model using a conditional knockout of sphingomyelin phosphodiesterase 3 in aortic VICs and further assess the efficiency of GW4869 to dimmish aortic valve calcification in a PCSK9-AAV mice model of CAVD.