1Universitätsklinikum Schleswig-Holstein Medizinische Klinik II / Kardiologie, Angiologie, Intensivmedizin Lübeck, Deutschland; 2Bioanalytic Core Facility. Center of Brain, Behavior and Metabolism (CBBM) Universität zu Lübeck Lübeck, Deutschland; 3Deutsches Herzzentrum der Charite (DHZC) Klinik für Herz-, Thorax- und Gefäßchirurgie Berlin, Deutschland; 4Institut für Kardiogenetik Universität zu Lübeck Lübeck, Deutschland
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 in a reverse translational approach by analyzing human aortic valve samples and applying a novel PCSK9-AAV mice model 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. Ceramide synthesis was inhibited in vivo in a novel PCSK9-AAV mice model of CAVD by GW4869, an inhibitor of neutral sphingomyelinase. Valve calcification was detected by echocardiography and post-mortem alizarin red staining and colorimetric calcium content assay. Inflammatory NF-κB Pathway and NLRP3 inflammasome activation 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.
Results: Ceramide species Cer(d18:1/16:0) was increased in calcified aortic valve tissue of CAVD patients and CAVD PCSK9-AAV mice. 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). Strikingly, GW4869 treatment ameliorated CAVD in PCSK9-AAV mice reducing maximum transvalvular gradient in echocardiography and calcium content as detected by histochemistry. This was accompanied by a reduction of lipotoxic sphingolipid species accumulation in aortic valve tissue of GW4869 treated mice and subsequent reduction of NF-κB and NLRP3 inflammasome activation.
Conclusion:
Ceramides accumulate in aortic valve tissue of CAVD patients. Inhibition of ceramide synthesis by GW4869 ameliorates hVIC calcification and CAVD proprietarily by reducing lipotoxic burden in vivo. Thus, targeting sphingolipid metabolism constitutes a promising therapeutic approach in CAVD.