Abstract

Background

Our current concept of aortic valve stenosis (AS) development suggests that local chronic inflammation drives fibrosis and calcification of the valve cusps. This remodeling is driven by endothelial to mesenchymal transition (EndMT) of valvular endothelial cells (VECs) and by calcification of valvular interstitial cells (VICs). In a preliminary screening, we found Toll-like receptor 3 (TLR3) expression increased in human AS samples. In conclusion, this study aims to investigate the role of TLR3 in the pathogenesis of AS in human in vitro studies focusing on calcification as well as the process of EndMT.

Methods

The following experiments used commercial VICs and VECs from healthy donors. VICs were stimulated with the TLR3 agonist PolyIC. To inhibit TLR3 activation, a knockdown was performed using TLR3 small interfering RNA (siRNA) as well as the TLR3/ RNA Complex inhibitor C4a. Calcification was induced by incubating cells in a pro-calcifying medium containing 5% FBS, 1% P/S, 2 mmol/l disodium hydrogen phosphate, and 50 µg/ml ascorbic acid. RNA was isolated for gene expression analysis. Calcification was visualized by alizarin red staining.Different stimuli were tested to induce EndMT in VECs including TNFα and TGFβ/IL-1β.

Results

We confirmed TLR3 expression in cultured human VICs and VECs. Upon TLR3 stimulation with PolyIC, both VICs and VECs displayed a positive feedback with increased TLR3 expression. Concomitant treatment of PolyIC-stimulated VICs and VECs with the TLR3/RNA complex inhibitor (C4a) significantly blunted this response. The TLR3-mediated pro-inflammatory and calcifying response of VICs was significantly reduced by TLR3-inhibtion with C4a. VECs underwent EndMT in response to TGFβ/IL-1β and TNF-α, which was accompanied by increased and decreased expression of mesenchymal and endothelial markers. The EndMT effect was triggered immediately after TLR3 activation, while C4a treated VECs maintained the endothelial characteristics.

Conclusion

This study examined the effect of TLR3 induced calcification and EndMT in human VICs and VECs.Altogether, these findings not only support the role of endogenous TLR3 activation in the development of AS but suggest that specific TLR3 inhibition might be beneficial for the treatment or prevention of AS. Further studies are required to decipher the exact mechanisms how TLR3 contributes to AS.