European Molecular Biology Laboratory, Heidelberg, Germany.
Discovery Biology, Cellzome GmbH, GSK, Heidelberg, Germany.
Eur Respir J. 2021 Jul 1;58(1). doi: 10.1183/13993003.00221-2020. Print 2021 Jul.
Fibrosis can affect any organ, resulting in the loss of tissue architecture and function with often life-threatening consequences. Pathologically, fibrosis is characterised by the expansion of connective tissue due to excessive deposition of extracellular matrix (ECM) proteins, including the fibrillar forms of collagen. A significant limitation for discovering cures for fibrosis is the availability of suitable human models and techniques to quantify mature fibrillar collagen deposition as close as possible to human physiological conditions.Here we have extensively characterised an cultured human lung tissue-derived, precision-cut lung slices (hPCLS) model using label-free second harmonic generation (SHG) light microscopy to quantify fibrillar collagen deposition and mass spectrometry-based techniques to obtain a proteomic and metabolomic fingerprint of hPCLS in culture.We demonstrate that hPCLS are viable and metabolically active, with mesenchymal, epithelial, endothelial and immune cell types surviving for at least 2 weeks in culture. Analysis of hPCLS-conditioned supernatants showed a strong induction of pulmonary fibrosis-related ECM proteins upon transforming growth factor-β1 (TGF-β1) stimulation. This upregulation of ECM proteins was not translated into an increased deposition of fibrillar collagen. In support of this observation, we revealed the presence of a pro-ECM degradation activity in our cultures of hPCLS, inhibition of which by a metalloproteinase inhibitor resulted in increased collagen deposition in response to TGF-β1 stimulation.Together the data show that an integrated approach of measuring soluble pro-fibrotic markers alongside quantitative SHG-based analysis of fibrillar collagen is a valuable tool for studying pro-fibrotic signalling and testing anti-fibrotic agents.
纤维化可影响任何器官，导致组织结构和功能丧失，常伴有危及生命的后果。从病理学角度来看，纤维化的特征是由于细胞外基质（ECM）蛋白的过度沉积，导致结缔组织扩张，其中包括胶原的纤维形式。发现纤维化治疗方法的一个显著限制是缺乏合适的人类模型和技术，无法尽可能接近人体生理条件来定量成熟的纤维状胶原蛋白沉积。在这里，我们使用无标记的二次谐波产生（SHG）共聚焦显微镜广泛表征了源自培养的人肺组织的精密切割肺切片（hPCLS）模型，以定量纤维状胶原蛋白沉积，并使用基于质谱的技术获得 hPCLS 在培养中的蛋白质组学和代谢组学特征。我们证明 hPCLS 具有活力和代谢活性，至少在培养 2 周内，间充质细胞、上皮细胞、内皮细胞和免疫细胞存活。分析 hPCLS 条件培养基显示，转化生长因子-β1（TGF-β1）刺激后，肺纤维化相关 ECM 蛋白强烈诱导。这种 ECM 蛋白的上调并没有转化为纤维状胶原蛋白沉积的增加。支持这一观察结果，我们在 hPCLS 的培养物中发现了存在一种促进 ECM 降解的活性，通过金属蛋白酶抑制剂抑制这种活性可导致 TGF-β1 刺激时胶原蛋白沉积增加。综上所述，这些数据表明，测量可溶性促纤维化标志物与定量基于 SHG 的纤维状胶原蛋白分析相结合的综合方法是研究促纤维化信号和测试抗纤维化剂的有价值工具。
