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; Carlijn V C Bouten

doi:10.3389/fcvm.2021.687885

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

Front Cardiovasc Med. 2021 Aug 30:8:687885. doi: 10.3389/fcvm.2021.687885. eCollection 2021.

Authors

Manon Meerman¹, Rob Driessen^{2

3}, Nicole C A van Engeland^{2

3

4}, Irith Bergsma^{2

3}, Jacco L G Steenhuijsen⁵, David Kozono⁶, Elena Aikawa⁷, Jesper Hjortnaes¹, Carlijn V C Bouten^{2

3}

Affiliations

¹ Department of Cardiothoracic Surgery, Heart and Lung Division, Leiden University Medical Center, Leiden, Netherlands.
² Department of Biomedical Engineering, Soft Tissue Engineering and Mechanobiology (STEM), Eindhoven University of Technology, Eindhoven, Netherlands.
³ Institute for Complex Molecular Systems (ICMS), Eindhoven University of Technology, Eindhoven, Netherlands.
⁴ Åbo Akademi University, Faculty of Science and Engineering, Molecular Biosciences, Turku, Finland.
⁵ Department of Radiotherapy, Catherina Ziekenhuis Eindhoven, Eindhoven, Netherlands.
⁶ Department of Radiation Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, United States.
⁷ Center for Interdisciplinary Cardiovascular Sciences, Cardiovascular Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, United States.

Abstract

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.

Keywords: aortic valve disease; extracellular matrix; in vitro modeling; radiotherapy; valvular interstitial cells.