Thoracic Radiation Induces Toll-Like Receptor–Mediated Calcific Aortic Valve Disease

Background: Cardiovascular disease after adjuvant thoracic radiation has become the leading nonmalignant cause of death in cancer survivors. The incidence of calcific aortic valve disease (CAVD) is considerably increased after thoracic radiotherapy. It remains unknown how radiation promotes the onset of calcific aortic valve disease.

Purpose: We hypothesized that radiation causes release of danger associated molecular patterns (DAMPs). Activation of the pattern recognition receptor Toll-like receptor 3 (TLR3) promotes osteoblastic phenotype switch of valvular interstitial cells (VICs).

Methods: Valvular interstitial cells isolated from human aortic valves underwent radiation therapy in presence of TLR3 agonist poly (I:C) or a TLR3/dsRNA complex inhibitor. Transcriptome was analyzed via next generation sequencing. Radiated cells were analyzed for alkaline phosphatase activity and calcific nodule formation. Cell cycle analysis via flow cytometry was performed after radiation. ApoE^−/− and ApoE^−/−/TLR3^−/− mice underwent thoracic radiation. Aortic valve morphology, valvular plaque burden, calcification and leaflet thickness was evaluated via histological analyses and micro-CT. Aortic valve function was analyzed using transthoracic echocardiography.

Results: Radiated VICs showed enhanced osteoblastic activity with increased Runx2 and BMP2 expression and calcific nodule formation. In parallel, we found activation of the TLR3 pathway and downstream signaling. Transcriptome analysis revealed osteoblastic gene expression resembling human osteoblasts after TLR3 stimulation. TLR3 inhibition resulted in prevention of osteogenic phenotype switch upon radiation.

In vivo, thoracic radiation of ApoE^−/− mice resulted in increased leaflet thickness, valvular plaque burden and valvular calcification. Transthoracic echocardiography revealed decreased aortic valve opening diameters (mm: 0.81 ± 0.05 vs. 0.95 ± 0.03, p = 0.04) and significant leaflet thickening (mm: 0.09 ± 0.01 vs. 0.07 ± 0.004, p = 0.03). However, these changes were missing completely in radiated ApoE^−/−/TLR3^−/− mice.

Conclusions: Radiation activates TLR3 and leads to an osteoblastic phenotype switch of VICs, whereas inhibition of TLR3 prevents from calcification. ApoE^−/−/TLR3^−/− mice show no phenotype of CAVD after radiation. We describe a novel pathomechanism for the onset of CAVD after radiation and reveal a potential pharmacological target for the prevention of radiation induced CAVD.