Aortic valve stenosis (AS) is the most common valve diseases in the western world requiring therapeutic intervention. It is believed that AS is a highly regulated disease influenced by resident and non-resident effectors. Recent studies suggest that inflammatory processes play a major role in the disease initiation processes. It has been shown that activation of Toll like receptor 3 (TLR3) can induce an osteogenic response in isolated human valvular interstitial cells.  TLR3 is a pattern recognition receptor, which is mainly expressed in myeloid cells, but also present in endothelial cells. Upon stimulation with double stranded RNA TLR3 signaling leads to expression of pro-inflammatory cytokines via the activation of NFκB. The aim of this study is to investigate the role of TLR3 in the pathogenesis of aortic valve stenosis.

Methods:

Ultrasound guided wire injury was used to induce AS in Wildtype-, ApoE- and TLR3/ApoE ^-/^- and TLR3^-/^- mice. Stenosis development was confirmed via ultrasound examinations at regular intervals. TLR3/RNA- Complex inhibitor C4a was injected every 48h after surgery in WT mice to supress TLR-3 signaling after wire injury. Valves were explanted after 6 weeks and stained with hematoxylin/eosin (aortic valve area) or anti-68 (macrophage infiltration). Vascular interstitial cells (VICs) were cultured and stimulated with TLR3 agonist polyIC. RNA was isolated for gene expression analysis.

Results:

To elucidate whether endogenous TLR3 activation is required for AS development we subjected TLR3/ApoE double- and ApoE knockout mice to our model of wire-induced AS. Interestingly, TLR3 deficient mice did not develop AS after wire injury. Trans aortic valve peak velocity levels were significantly lower than in ApoE mice and did not increase after wire injury. TLR3 single knockout mice showed similar results. Histological analysis revealed a significantly smaller aortic valve leaflet area and a reduced intravalvular infiltration with monocytes in TLR3 deficient mice. In order to pharmacological inhibit TLR3, WT mice were treated with C4a after wire injury. Mice treated with C4a failed to develop AS and trans-aortic valve peak velocity levels were significantly lower in C4a mice. Histological analysis confirmed these results and showed thinner aortic valves and decreased macrophage infiltration in C4a mice comparted to control animals. For in vitro experiments valvular interstitial cells were stimulated with polyIC to clarify whether TLR3 signalling could induce intravalvular calcification. Upon polyIC treatment TLR3 was strongly upregulated in VICs which went along with an induction of pro-osteogenic markers BNP-2 and RUNX.

Conclusion:

These first results show evidence that TLR3 signaling is involved in the development of AS in mice via an induction of pro-osteogenic genes in valvular interstitial cells. Since C4a prevents AS development in mice, TLR3 is a promising target for future therapeutic interventions.