Therapeutic Inhibition of Toll-Like Receptor 3 Prevents Calcific Aortic Valve Disease

Background: There is still no effective pharmacological treatment for the prevention os Calcific aortic valve disease (CAVD). Mechanistically, it is caused by an osteoblastic phenotype switch of valvular interstitial cells (VICs), triggered by the activation of Toll-Like Receptor 3 (TLR3) by endogenous ligands released from the extracellular matrix by mechanical stress. We could show recently that the TLR3-interferon β axis is an evolutionarily conserved pathway governing calcification of the aortic valve. We aimed to translate our findings towards a therapeutic approach and explored TLR3 as pharmacological target.

Methods: A systematic literature research resulted in target candidates inhibiting TLR3. These were tested on TLR3 specific reporter cells for their inhibitory potential on IFNb and NFkB production. Gene- and protein expression was performed via qPCR and western blotting. An in vitro calcification assay was performed by cultivating cells in a pro calcific environment staining calcific deposits . To test toxicologic side effects BrdU and MTT assays were carried out. C57BL/6 mice were subjected to high-fat diet and drug treatment to induce aortic stenosis for four months. In vivo aortic valve function was analyzed via echocardiography. Valve morphology was analyzed histologically.

Results: In vitro, the activation of TLR3 caused calcification of valvular cells. The effects were decreased by inhibiting TLR3, highlighting the specificity of the mechanism. A literature screen resulted in a selection of 15 possible pharmaceutical targets inhibiting TLR3. These possible targets were screened for their TLR3 inhibiting efficiency resulting in four promising candidates: Levocetirizine (LCEZ), Amiodarone (AMD), Chloroquine (CHQ) and Sertraline (SER), showing decreased calcification in vitro without causing toxicity. Alongside these targets allele specific oligonucleotides (ASOs) against IRF3 were tested and found to be effective against calcification in vitro. To confirm results in vivo, C57BL/6 mice were subjected to high-fat diet and drug treatment to induce CAVD for four months. The treatment with TLR3 inhibitors prevented the development of CAVD in vivo resulting in decreased aortic valve gradients, leaflet thickness and calcification in echocardiographies and histological analysis.

Conclusion: The pharmacological inhibition of TLR3 prevents the osteoblastic phenotype switch and calcification in vitro and protects mice from the development of CAVD. The TLR3 axis is a promising target to counteract CAVD.

Publikationsverlauf

Artikel online veröffentlicht:
13. Februar 2024