Department of Pharmaceutical Technology and Biopharmacy, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV, Groningen, The Netherlands.
MATRIX Research Group, Department of Pathology and Medical Biology, University Medical Center Groningen, University of Groningen, Hanzeplein 1, 9713 GZ, Groningen, The Netherlands.
Sci Rep. 2022 Aug 17;12(1):13940. doi: 10.1038/s41598-022-18116-9.
Dupuytren's disease is a chronic, progressive fibroproliferative condition of the hand fascia which results in digital contraction. So far, treatments do not directly interfere with the (myo)fibroblasts that are responsible for the formation of the collagen-rich cords and its contraction. Here we investigated whether verteporfin (VP) is able to inhibit the activation and subsequent differentiation of DD nodular fibroblasts into myofibroblasts. Fibroblasts were isolated from nodules of 7 Dupuytren patients. Cells are treated (1) for 48 h with 5 ng/ml transforming growth factor β1 (TGF-β1) followed by 48 h with/without 250 nM VP in the absence of TGF-β1, or treated (2) for 48 h with TGF-β1 followed by 48 h with/without VP in the presence of TGF-β1. mRNA levels were measured by means of Real-Time PCR, and proteins were visualized by means of Western blotting and/or immunofluorescence. Quantitative data were statistically analyzed with GraphPad Prism using the paired t-test. We found that fibroblasts activated for 48 h with TGF-β1 show a decrease in mRNA levels of COL1A1, COL3A1, COL4A1, PLOD2, FN1EDA, CCN2 and SERPINE1 when exposed for another 48 h with VP, whereas no decrease is seen for ACTA2, YAP1, SMAD2 and SMAD3 mRNA levels. Cells exposed for an additional 48 h with TGF-β1, but now in the presence of VP, are not further activated anymore, whereas in the absence of VP the cells continue to differentiate into myofibroblasts. Collagen type I, fibronectin-extra domain A, α-smooth muscle actin, YAP1, Smad2 and Smad3 protein levels were attenuated by both VP treatments. We conclude that VP has strong anti-fibrotic properties: it is able to halt the differentiation of fibroblasts into myofibroblasts, and is also able to reverse the activation status of fibroblasts. The decreased protein levels of YAP1, Smad2 and Smad3 in the presence of VP explain in part the strong anti-fibrotic properties of VP. Verteporfin is clinically used as a photosensitizer for photodynamic therapy to eliminate abnormal blood vessels in the eye to attenuate macular degeneration. The antifibrotic properties of VP do not rely on photo-activation, as we used the molecule in its non-photoinduced state.
掌腱膜挛缩症是一种手部筋膜的慢性、进行性纤维增生性疾病，会导致手指挛缩。到目前为止，治疗方法并不能直接干预导致富含胶原的索状结构形成及其收缩的（肌）成纤维细胞。在这里，我们研究了维替泊芬（VP）是否能够抑制 DD 结节成纤维细胞的激活和随后向肌成纤维细胞的分化。从 7 名掌腱膜挛缩症患者的结节中分离出成纤维细胞。细胞（1）用 5ng/ml 转化生长因子 β1（TGF-β1）处理 48 小时，然后在无 TGF-β1 的情况下再用 250nM VP 处理 48 小时，或（2）用 TGF-β1 处理 48 小时，然后在有 TGF-β1 的情况下再用 VP 处理 48 小时。通过实时 PCR 测量 mRNA 水平，并通过 Western blot 和/或免疫荧光观察蛋白质。使用 GraphPad Prism 通过配对 t 检验对定量数据进行统计学分析。我们发现，用 TGF-β1 激活 48 小时的成纤维细胞在暴露于 VP 48 小时后，COL1A1、COL3A1、COL4A1、PLOD2、FN1EDA、CCN2 和 SERPINE1 的 mRNA 水平下降，而 ACTA2、YAP1、SMAD2 和 SMAD3 的 mRNA 水平没有下降。在用 TGF-β1 进一步处理 48 小时后，但现在有 VP 存在的情况下，细胞不再进一步激活，而在没有 VP 的情况下，细胞继续分化为肌成纤维细胞。I 型胶原蛋白、纤维连接蛋白外显子 A、α-平滑肌肌动蛋白、YAP1、Smad2 和 Smad3 蛋白水平均被两种 VP 处理所减弱。我们得出的结论是，VP 具有很强的抗纤维化特性：它能够阻止成纤维细胞向肌成纤维细胞的分化，并且还能够逆转成纤维细胞的激活状态。VP 存在时 YAP1、Smad2 和 Smad3 的蛋白水平降低部分解释了 VP 具有很强的抗纤维化特性。维替泊芬在临床上用作光动力疗法的光敏剂，以消除眼睛中异常的血管，从而减轻黄斑变性。VP 的抗纤维化特性不依赖于光激活，因为我们在非光诱导状态下使用该分子。
