RT Journal Article
SR Electronic
T1 An integrated multiomic and quantitative label-free microscopy-based approach to study pro-fibrotic signalling in <em>ex vivo</em> human precision-cut lung slices
JF European Respiratory Journal
JO Eur Respir J
FD European Respiratory Society
SP 2000221
DO 10.1183/13993003.00221-2020
VO 58
IS 1
A1 Muzamil Majid Khan
A1 Daniel Poeckel
A1 Aliaksandr Halavatyi
A1 Joanna Zukowska-Kasprzyk
A1 Frank Stein
A1 Johanna Vappiani
A1 Daniel C. Sevin
A1 Christian Tischer
A1 Nico Zinn
A1 Jessica D. Eley
A1 Natasja Stæhr Gudmann
A1 Thomas Muley
A1 Hauke Winter
A1 Andrew J. Fisher
A1 Carmel B. Nanthakumar
A1 Giovanna Bergamini
A1 Rainer Pepperkok
YR 2021
UL http://erj.ersjournals.com/content/58/1/2000221.abstract
AB 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 ex vivo 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 ex vivo 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 ex vivo 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 ex vivo 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.Multiomic and label-free imaging-based characterisation of ex vivo cultured human precision-cut lung slices (hPCLS) reveals that MMP signalling is a rate-limiting factor necessary for deposition of fibrillar collagen in ECM of hPCLS https://bit.ly/3rcUa0e