Abstract
Fibrosis can affect any organ resulting in the loss of tissue architecture and function with often life-threatening consequences. Pathologically, fibrosis is characterised by expansion of connective tissue due to excessive deposition of extracellular matrix proteins (ECM), 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 anex vivocultured human lung tissue-derived, precision-cut lung slices model (hPCLS) using label-free second harmonic (SHG) light microscopy to quantify fibrillar collagen deposition and mass spectrometry-based techniques to obtain a proteomic and metabolomic fingerprint of hPCLS inex vivoculture.
We demonstrate that hPCLS are viable and metabolically active with mesenchymal, epithelial, endothelial, and immune cell types surviving for at least 2 weeks inex vivoculture. Analysis of hPCLS-conditioned supernatants showed a strong induction of pulmonary fibrosis-related ECM proteins upon TGFß1 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 ourex vivocultures of hPCLS, inhibition of which by metalloproteinase inhibitor resulted in increased collagen deposition in response to TGFß1 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 antifibrotic agents.
Footnotes
This manuscript has recently been accepted for publication in theEuropean Respiratory Journal. It is published here in its accepted form prior to copyediting and typesetting by our production team. After these production processes are complete and the authors have approved the resulting proofs, the article will move to the latest issue of theERJonline. Please open or download the PDF to view this article.
Conflict of interest: Dr. Khan has nothing to disclose.
Conflict of interest: Dr. Poeckel has nothing to disclose.
Conflict of interest: Dr. Halavatyi has nothing to disclose.
Conflict of interest: Mrs. Kasprzyk has nothing to disclose.
Conflict of interest: Dr. Stein has nothing to disclose.
Conflict of interest: Dr. Vappiani has nothing to disclose.
Conflict of interest: Dr. Sevin has nothing to disclose.
Conflict of interest: Dr. Tischer has nothing to disclose.
Conflict of interest: Dr. Zinn has nothing to disclose.
Conflict of interest: Dr. Eley has nothing to disclose.
Conflict of interest: Dr. Gudmann has nothing to disclose.
Conflict of interest: Dr. Muley has nothing to disclose.
Conflict of interest: Dr. Winter has nothing to disclose.
Conflict of interest: Dr. Fisher reports grants from GlaxoSmithKline, during the conduct of the study; grants from Pfizer, grants from Nuformix, grants from Genentech, outside the submitted work;.
Conflict of interest: Dr. Nanthakumar has nothing to disclose.
Conflict of interest: Dr. Bergamini has nothing to disclose.
Conflict of interest: Dr. Pepperkok has nothing to disclose.
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- ReceivedFebruary 3, 2020.
- AcceptedDecember 9, 2020.
- Copyright ©ERS 2020