Radiation Induces Valvular Interstitial Cell Calcific Response in an in vitro Model of Calcific Aortic Valve Disease

Manon Meerman, Rob Driessen, Nicole C.A. van Engeland, Irith Bergsma, Jacco L.G. Steenhuijsen, David Kozono, Elena Aikawa, Jesper Hjortnaes (Corresponding author), Carlijn V.C. Bouten (Corresponding author)

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Background: Mediastinal ionizing radiotherapy is associated with an increased risk of valvular disease, which demonstrates pathological hallmarks similar to calcific aortic valve disease (CAVD). Despite advances in radiotherapy techniques, the prevalence of comorbidities such as radiation-associated valvular disease is still increasing due to improved survival of patients receiving radiotherapy. However, the mechanisms of radiation-associated valvular disease are largely unknown. CAVD is considered to be an actively regulated disease process, mainly controlled by valvular interstitial cells (VICs). We hypothesize that radiation exposure catalyzes the calcific response of VICs and, therefore, contributes to the development of radiation-associated valvular disease. Methods and Results: To delineate the relationship between radiation and VIC behavior (morphology, calcification, and matrix turnover), two different in vitro models were established: (1) VICs were cultured two-dimensional (2D) on coverslips in control medium (CM) or osteogenic medium (OM) and irradiated with 0, 2, 4, 8, or 16 Gray (Gy); and (2) three-dimensional (3D) hydrogel system was designed, loaded with VICs and exposed to 0, 4, or 16 Gy of radiation. In both models, a dose-dependent decrease in cell viability and proliferation was observed in CM and OM. Radiation exposure caused myofibroblast-like morphological changes and differentiation of VICs, as characterized by decreased αSMA expression. Calcification, as defined by increased alkaline phosphatase activity, was mostly present in the 2D irradiated VICs exposed to 4 Gy, while after exposure to higher doses VICs acquired a unique giant fibroblast-like cell morphology. Finally, matrix turnover was significantly affected by radiation exposure in the 3D irradiated VICs, as shown by decreased collagen staining and increased MMP-2 and MMP-9 activity. Conclusions: The presented work demonstrates that radiation exposure enhances the calcific response in VICs, a hallmark of CAVD. In addition, high radiation exposure induces differentiation of VICs into a terminally differentiated giant-cell fibroblast. Further studies are essential to elucidate the underlying mechanisms of these radiation-induced valvular changes.

Original languageEnglish
Article number687885
Number of pages13
JournalFrontiers in Cardiovascular Medicine
Publication statusPublished - 30 Aug 2021


Funding. This work was supported by the Netherlands Heart Foundation (NHF-2011T024) and the Netherlands Scientific Council (NWO-92003572) (to JH); the Ministry of Education, Culture and Science for the Gravitation Program 024.003.013 Materials Driven Regeneration (to CB); and NIH grants R01 HL136431, R01 HL141917 and R01 HL147095 (to EA).

FundersFunder number
Nederlandse Organisatie voor Wetenschappelijk OnderzoekNWO-92003572
National Institutes of HealthR01 HL141917, R01 HL136431, R01 HL147095
Hartstichting, NederlandseNHF-2011T024
Ministerie van Onderwijs, Cultuur en Wetenschap


    • aortic valve disease
    • extracellular matrix
    • in vitro modeling
    • radiotherapy
    • valvular interstitial cells


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