Abstract 14271: TLR3 as a Potential Target to Prevent Aortic Valve Stenosis

Abstract

Aortic stenosis (AS) is the most common valvular disease in the western world and is associated with severe morbidity and mortality. Our current concept of disease development suggests that local chronic inflammation drives fibrosis and calcification of the valve cups. It has recently been proposed that activation of pattern recognition receptors may be critical in this context. In a preliminary screening, we found Toll-like receptor 3 expression increased in human AS samples. Aim of this study was to investigate the role of TLR3 in the pathogenesis of AS. Isolated human valvular interstitial cells (VICs) were cultured and stimulated with the TLR3 agonist Poly I:C. To inhibit TLR-3 activation, a knockdown was performed using TLR3 small interfering RNA (siRNA) as well as the TLR3/ RNA Complex inhibitor C4a. Calcification was induced by changing the culturing media. RNA was isolated for gene expression analysis. Calcification was visualized by alizarin red staining. Wild type as well as TLR3 knockout mice underwent an ultrasound guided wire injury. After surgery, C4a was injected in WT mice to suppress TLR3 signaling. Valves were explanted and stained with hematoxylin/eosin or anti CD68. Upon stimulation of TLR3 with PolyI:C, TLR3 expression was consistently upregulated in VICs while incubation of VICs with the TLR3/ RNA complex inhibitor C4a resulted in a downregulation of TLR3. These results suggest that TLR3 may be subject to a potential detrimental positive feedback loop in these cells. Importantly, activation of TLR3 led to a marked increase in expression of the pro-osteogenic markers BMP-2 and RUNX2. TLR3 inhibition or knockdown blunted this response.TLR3 deficient mice revealed a significantly smaller aortic valve leaflet area as well as a reduced intravalvular infiltration with monocytes compared to WT. C4a treatment resulted in lower trans-aortic valve peak velocity levels. Further, C4a mice failed to develop AS upon injury. These findings not only support the endogenous role of TLR3 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 test this hypothesis and to decipher the exact mechanisms how TLR3 contributes to AS.