Introduction: Aortic valve stenosis (AS) is the most common and most deadly valvular disease in humans. As the pathophysiological understanding of the disease development of AS is only incompletely understood, currently the only treatment option is the replacement of the valve. Recently, AS has been characterized as an inflammatory process, which involves immune cell infiltration and calcification of the valvular leaflets. In the context of cardiovascular disease, Ceramides (a sphingolipid subclass), mainly produced by neutral sphingomyelinase 2 (nSMase2), have been shown to be important regulators of inflammation as well as calcification. The role of nSMase2 in the pathophysiology of aortic valve disease, however, has never been investigated, yet.

Methods and Results: In order to induce AS in mice, a coronary angiography wire was introduced into the aortic valve over the right carotid artery. The wire was rotated inside the valve under ultrasound guidance to generate a defined lesion of the aortic valve for the induction of AS. The mice received the nSMase-inhibitor GW4869 at 2 mg/kg Body weight in DMSO or only DMSO intraperitoneally three times a week starting directly prior to the injury. In the intervention group one mouse died during the induction of the lesion resulting in n=6/8. The GW4869 injection reduced nSMase activity in the blood by half in 24 h and down to 20% after 4 weeks in the aortic tissue (A). After four weeks, the mice were sacrificed for histological analysis. We found that the treatment with GW4869 significantly reduced the aortic valve area (B). Immune histological staining for the macrophage antigen CD68 showed a significantly lower immune cell infiltration into the leaflets of valve after treatment with GW4869 (C). In vitro we used human valvular interstitial cells (VICs), which are known to drive calcification, and knocked down nSMase2 = SMPD3 and acid Sphingomyelinase 1 (SMDP1) as a control by use of siRNA transfection (D). Calcification was induced with pro-calcifying medium (2 mmol/l NaH₂PO₄ and 50 μg/ml L-Ascorbic acid) for 7 days and quantified by alizarine red staining which was dissolved by cetylpyridinium chloride. While knockdown of SMPD3 lead to as significant reduction of VIC calcification, no difference was observed after knockdown of SMPD1 (E).

Conclusion: In conclusion, our results indicate that nSMAse2 is involved in immune cell infiltration and valvular calcification, which are the two most important pathophysiological hallmarks of AS development. Pharmacological inhibition of nSMase2 with GW4869 is able to reduce valvular thickening and immune cell infiltration in vitro. Therefore, it is in our opinion a promising therapeutic concept to counteract the development of AS.