Nanoscale dysregulation of collagen structure-function disrupts mechano-homeostasis and mediates pulmonary fibrosis

  1. Mark G Jones  Is a corresponding author
  2. Orestis G Andriotis
  3. James JW Roberts
  4. Kerry Lunn
  5. Victoria J Tear
  6. Lucy Cao
  7. Kjetil Ask
  8. David E Smart
  9. Alessandra Bonfanti
  10. Peter Johnson
  11. Aiman Alzetani
  12. Franco Conforti
  13. Regan Doherty
  14. Chester Y Lai
  15. Benjamin Johnson
  16. Konstantinos N Bourdakos
  17. Sophie V Fletcher
  18. Ben G Marshall
  19. Sanjay Jogai
  20. Christopher J Brereton
  21. Serena J Chee
  22. Christian H Ottensmeier
  23. Patricia Sime
  24. Jack Gauldie
  25. Martin Kolb
  26. Sumeet Mahajan
  27. Aurelie Fabre
  28. Atul Bhaskar
  29. Wolfgang Jarolimek
  30. Luca Richeldi
  31. Katherine MA O'Reilly
  32. Phillip D Monk
  33. Philipp J Thurner
  34. Donna E Davies  Is a corresponding author
  1. Faculty of Medicine, University of Southampton, United Kingdom
  2. TU Wien, Austria
  3. Synairgen Research Ltd, United Kingdom
  4. Pharmaxis Ltd, Australia
  5. Firestone Institute for Respiratory Health, McMaster University and The Research Institute of St. Joe's Hamilton, Canada
  6. University of Southampton, United Kingdom
  7. University Hospital Southampton, United Kingdom
  8. University of Rochester School of Medicine and Dentistry, United States
  9. St. Vincent's University Hospital & UCD School of Medicine, University College Dublin, Ireland
  10. Fondazione Policlinico Universitario Agostino Gemelli IRCCS, Italy
  11. Mater Misericordiae University Hospital, Ireland
  12. University College Dublin, Ireland
8 figures, 1 video and 1 additional file

Figures

Figure 1 with 1 supplement
IPF lung tissue has increased stiffness which is not dependent on total collagen content.

(A) Tissue stiffness of control (n = 8 donors, ●) and IPF (n = 10 donors, ■) lung tissue determined by micro-indentation atomic force microscopy (AFM). Data presented as mean stiffness of each donor …

https://doi.org/10.7554/eLife.36354.003
Figure 1—figure supplement 1
IPF lung tissue total collagen content.

Total collagen content of control (n = 15) and IPF lung tissue (n = 17) normalised by total protein. Bars are mean ±s.e.m. p=0.70 by Student's t-test (two-tailed).

https://doi.org/10.7554/eLife.36354.004
Lysyl oxidase-like (LOXL) enzymes and amine oxidase activity are increased in IPF tissue.

(A–E) Expression of LOX, LOXL1, LOXL2, LOXL3 and LOXL4 was analysed in control (●) and IPF (■) lung tissue (n = 11 individual donors per group) using the ∆∆Ct method. Bars indicate geometric means. …

https://doi.org/10.7554/eLife.36354.005
Hydroxyallysine-derived collagen cross-link density is increased in IPF lung and is correlated with tissue stiffness.

(A, B) Lysyl hydroxylase 2 (LH2A and LH2B) expression was analysed in control (●) and IPF (■) lung tissue (n = 11 donors per group) using the ∆∆Ct method. Bars indicate geometric means. *p<0.05 by …

https://doi.org/10.7554/eLife.36354.006
Collagen fibrils from IPF tissue have altered fibril diameter and stiffness.

(A) Atomic force microscopy (AFM) height topography image of a collagen fibril and corresponding height topography long axis profile showing characteristic D-periodicity (~67 nm). (B) Force …

https://doi.org/10.7554/eLife.36354.007
In vitro modelling of fibrillar collagen production and cross-linking in IPF.

We utilised a long-term 3D in vitro model of lung fibrosis using primary human lung fibroblasts from patients with IPF treated with the pro-fibrotic cytokine TGF-β1 cultured for up to 6 weeks. (A–C) …

https://doi.org/10.7554/eLife.36354.008
Figure 6 with 1 supplement
Selective inhibition of LOXL2/LOXL3 reduces hydroxyallysine-derived collagen cross-links and tissue stiffness.

(A) PXS-S2A inhibition curves against LOXL2 and LOXL3 (upper panel), LOX, LOXL1, LOXL4 (middle panel), and all LOX/LOXL enzymes (lower panel). Downward arrows in (A–E, G) identify …

https://doi.org/10.7554/eLife.36354.009
Figure 6—figure supplement 1
Parallel plate compression testing and analysis of the in vitro fibrosis model: Example for a single experiment.

Output graphs of raw data from parallel plate compression testing: (A) force vs time, (B) displacement vs time, and (C) force vs displacement for the control, BAPN, or PXS-S2A-treated cultures.

https://doi.org/10.7554/eLife.36354.010
Selective LOXL2/LOXL3 inhibition modifies collagen fibril assembly.

IPF fibroblasts were grown for 6 weeks in the 3D in vitro model of fibrosis with vehicle control, concentrations of PXS-S2A or 1 mM BAPN, as indicated. (A) Representative images of histological …

https://doi.org/10.7554/eLife.36354.012
Figure 8 with 1 supplement
Selective LOXL2/LOXL3 inhibition reduces fibrosis and improves lung function in an in vivo model of lung fibrosis driven by TGF-β.

Rats received either empty vector control (AdDL) (n = 6) on Day 0 or adenoviral vector producing active TGF-β1 (AdTGF-β1) to induce progressive pulmonary fibrosis. AdTGF-β1 groups received either …

https://doi.org/10.7554/eLife.36354.013
Figure 8—figure supplement 1
Selective inhibition of LOXL2/LOXL3 with PXS-S3B reduces hydroxyallysine-derived collagen cross-links and tissue stiffness.

Peripheral lung fibroblasts from IPF patients (n = 3 donors) were used in the in vitro model of fibrosis. Either 1 mM BAPN, PXS-S3B (at the concentrations indicated) or vehicle control (0.1% DMSO) …

https://doi.org/10.7554/eLife.36354.014

Videos

Video 1
10x Time-lapse movie of an in vitro model fibrosis sample during testing by parallel plate compression.
https://doi.org/10.7554/eLife.36354.011

Additional files

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