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.

中文翻译：

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辐射在钙化性主动脉瓣疾病的体外模型中诱导瓣膜间质细胞钙化反应。

背景：纵隔电离放疗与瓣膜疾病风险增加有关，其病理特征类似于钙化性主动脉瓣疾病 (CAVD)。尽管放疗技术取得了进步，但由于接受放疗的患者生存率提高，诸如放疗相关瓣膜病等并发症的患病率仍在增加。然而，辐射相关瓣膜疾病的机制在很大程度上是未知的。CAVD 被认为是一种主动调节的疾病过程，主要由瓣膜间质细胞 (VIC) 控制。我们假设辐射暴露会催化 VIC 的钙化反应，因此有助于辐射相关瓣膜疾病的发展。方法和结果：为了描绘辐射与 VIC 行为（形态、钙化和基质转换）之间的关系，建立了两种不同的体外模型：（1）在对照培养基（CM）或成骨培养基中的盖玻片上二维（2D）培养 VIC (OM) 并用 0、2、4、8 或 16 格雷 (Gy) 辐照；(2) 设计了三维 (3D) 水凝胶系统，加载 VIC 并暴露于 0、4 或 16 Gy 的辐射。在这两种模型中，在 CM 和 OM 中观察到细胞活力和增殖的剂量依赖性降低。辐射暴露导致肌成纤维细胞样形态学变化和 VIC 分化，其特征是αSMA 表达降低。钙化，由碱性磷酸酶活性增加定义，主要存在于暴露于 4 Gy 的 2D 辐照 VIC 中，而在暴露于更高剂量后，VIC 获得了独特的巨大成纤维细胞样细胞形态。最后，如胶原染色减少和 MMP-2 和 MMP-9 活性增加所示，基质转换受 3D 辐照 VIC 中辐射暴露的显着影响。结论：目前的工作表明，辐射暴露增强了 VIC 的钙化反应，这是 CAVD 的标志。此外，高辐射暴露诱导 VIC 分化为终末分化的巨细胞成纤维细胞。进一步的研究对于阐明这些辐射引起的瓣膜变化的潜在机制至关重要。如胶原染色减少和 MMP-2 和 MMP-9 活性增加所示。结论：目前的工作表明，辐射暴露增强了 VIC 的钙化反应，这是 CAVD 的标志。此外，高辐射暴露诱导 VIC 分化为终末分化的巨细胞成纤维细胞。进一步的研究对于阐明这些辐射引起的瓣膜变化的潜在机制至关重要。如胶原染色减少和 MMP-2 和 MMP-9 活性增加所示。结论：目前的工作表明，辐射暴露增强了 VIC 的钙化反应，这是 CAVD 的标志。此外，高辐射暴露诱导 VIC 分化为终末分化的巨细胞成纤维细胞。进一步的研究对于阐明这些辐射引起的瓣膜变化的潜在机制至关重要。