5-HT2B Antagonism Inhibits Strain- and Cytokine-Dependent Formation of Calcific Nodules by Aortic Valve Interstitial Cells

Abstract

The progression of aortic valve (AV) disease is often characterized by the formation of calcific nodules on thickened AV leaflets, limiting the biomechanical function of the valve. In these cases, the association of extracellular Ca2+ with phosphates remaining in cellular debris within the decellularized scaffolds has been proposed to lead to the nucleation and growth of calcific nodules. In native tissue, calcification is thought to be a more active process involving AV interstitial cells (AVICs). AVICs have been shown to form nodule-like structures in vitro through differentiation to a phenotype with osteogenic character. Additionally, in vitro nodules are characterized by activated smooth muscle α-actin (αSMA) positive AVICs and high levels of apoptosis [1–2]. Mechanical strain has also been shown to influence nodule formation in excised AV leaflets [3]. Our lab has recently developed a model system that recapitulates the formation of calcific nodules in vitro [4]. AVICs treated with TGF-β1 for 24 h prior to the addition of 15% cyclic strain in a Flexcell strain system form nodules that appear to be dependent upon the initiation of AVIC activation. These observations are consistent with previous studies that have shown that αSMA expression is required for nodule formation by AVICs in static culture, with statins shown to inhibit in vitro nodule formation [1]. However, retrospective epidemiological studies have shown that these drugs may not be as effective in preventing calcific valve disease in patients [5]. Additionally, the molecular target and relevant pathways for statins in AVICs remain largely unknown. Therefore, a therapeutically relevant target to prevent AVIC activation and subsequent nodule formation is greatly needed. In this study we investigated the ability of antagonists to 5-HT2B, a receptor known to be upstream of TGF-β1, to oppose strain- and TGF-β1-induced AVIC activation and nodule formation. We also assessed the efficacy of an antagonist to a receptor, the angiotensin II type I receptor (AT1R), known to crosstalk with both 5-HT2B and TGF-β1 signaling in other cell types in inhibiting AVIC nodule formation. Our results indicate that 5-HT2B antagonism inhibits AVIC activation and nodule formation by blocking non-canonical TGF-β1 signaling, whereas AT1R antagonism does not inhibit these outcomes. We believe that the results of this study may indicate novel therapeutic targets to prevent the progression of AV calcification.