Disrupting Sphingosine-1-Phosphat signaling to reduce calcification in aortic valve disease

M. Hering (Düsseldorf)¹, M. Benkhoff (Düsseldorf)¹, M. Barcik (Düsseldorf)¹, P. Mourikis (Düsseldorf)¹, J. Dahlmanns (Düsseldorf)², P. Kahmann (Düsseldorf)³, P. Wollnitzke (Düsseldorf)⁴, T. Huckenbeck (Düsseldorf)⁵, M. Kelm (Düsseldorf)¹, R. Ahrends (Wien)⁶, B. Levkau (Düsseldorf)⁴, A. Polzin (Düsseldorf)^1
1Universitätsklinikum Düsseldorf Klinik für Kardiologie, Pneumologie und Angiologie Düsseldorf, Deutschland; ²Universitätsklinikum Düsseldorf Department of Cardiology, Pulmonology, and Vascular Medicine Düsseldorf, Deutschland; ³Universitätsklinikum Düsseldorf Klinik f. Kardiologie, Pneumologie und Angiologie Düsseldorf, Deutschland; ⁴Universitätsklinikum Düsseldorf Institut für Molekulare Medizin III Düsseldorf, Deutschland; ⁵Universitätsklinikum Düsseldorf Division of Cardiology, Pulmonology and Vascular Medicine Düsseldorf, Deutschland; ⁶Institut für Analytische Chemie Wien, Österreich

Background: Aortic valve stenosis (AVD) is predominantly characterized by progressive valve calcification and associated with high mortality and morbidity. Currently, there are no pharmacological interventions available to reduce the development and progression of AVD. Due to its important role in osteogenic signaling, we investigated the role of sphingosine-1-phosphate (S1P) in the valve calcification and deterioration of AVD.

Methods: Levels of S1P in explanted human aortic valves (AVs) and human Valvular Interstitial Cells (hVICs) were analyzed via LC-MS. Cardiovascular magnetic resonance (CMR) imaging was used to measure maximum velocity and maximum wall shear stress at the side of the AV in healthy and AVD patients. Progressive calcification in the context of S1P signaling under biomechanical stress was investigated in hVICs. In this AVD in vitro model S1P signaling was stimulated or inhibited to determine the underlying mechanism. In order to characterize stenosis progression, calcium accumulation and the expression or phosphorylation of osteogenic marker genes were examined.

Results: LC-MS measurements of human AV tissue revealed higher S1P concentrations in stenotic AVs compared to healthy control samples. Additionally, increased expression of S1P forming SphK1 was observed in calcified AVs. At the side of stenotic AVs significantly increased maximum velocity and maximum wall shear stress (axial and circumferential) were measured using CMR imaging. In the in vitro AVD model biomechanical stretching stimulated S1P production by activating SphK1 in hVICs, as measured by C17-S1P formation using LC-MS. Further in vitro investigations showed strongly enhanced hVIC calcification after S1P stimulation by activating S1P Receptor 2 (S1PR2). PCR and Western blot analyses identified osteoblastic differentiation and calcification of hVICs via the RUNX2/OPG and GSK3β-Wnt-β-catenin signaling pathways.

Conclusion: Biomechanical stress in AV tissue leads to elevated S1P concentrations by activating its production via SphK1 which contributes to enhanced calcification due to stimulation of S1PR2 signaling. S1PR2 antagonists and SphK1 inhibitors may offer viable pharmacological approaches for the prophylactic treatment or disease modification of AVD.