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Local photocrosslinking of native tissue matrix regulates lung epithelial cell mechanosensing and function

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Abstract

Within most tissues, the extracellular microenvironment provides mechanical cues that guide cell fate and function. Changes in the extracellular matrix such as aberrant deposition, densification and increased crosslinking are hallmarks of late-stage fibrotic diseases that often lead to organ dysfunction. Biomaterials have been widely used to mimic the mechanical properties of the fibrotic matrix and study pathophysiologic cell function. However, the initiation of fibrosis has largely been overlooked, due to challenges in recapitulating early stages of disease progression within the native extracellular microenvironment. Here, using visible-light-mediated photochemistry, we induced local crosslinking and stiffening of extracellular matrix proteins within ex vivo mouse and human lung tissue. In ex vivo lung tissue of epithelial cell lineage-traced mice, local matrix crosslinking mimicked early fibrotic lesions that increased alveolar epithelial cell mechanosensing, differentiation, and nascent protein deposition and remodelling. However, the inhibition of cytoskeletal tension, mechanosensitive signalling pathways or integrin engagement reduced epithelial cell spreading and differentiation. Our findings emphasize the role of local extracellular matrix crosslinking and nascent protein deposition in early stage tissue fibrosis and have implications for ex vivo disease modelling and applications to other tissues.

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Fig. 1: Dityrosine photochemistry enables the local crosslinking of ECM proteins in vitro.
Fig. 2: Dityrosine photochemistry enables the local crosslinking of ECM proteins and stiffening of ex vivo tissues.
Fig. 3: ECM stiffening of ex vivo tissue directs AT2–AT1 cell differentiation through enhanced mechanosensing.
Fig. 4: Tissue stiffening induces alveolar transitional cell states in mouse and human tissue.
Fig. 5: Integrin β1 is required for AT2–AT1 cell differentiation on nascent protein deposition.
Fig. 6: Local stiffening of native ECM regulates lung epithelial cell function via mechanosensing and nascent protein deposition.

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The data that support the findings of this study are available in the article, Extended Data Figs. 1–7 and Supplementary Information. Source data are provided with this paper.

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Acknowledgements

This work was partially supported by funding from the NIH (R00-HL151670 and R35GM157063 to C.L., NIH T32 GM1404 to D.W.A., NHLBI T32 HL007749 to M.L.T., HL124322 to B.M.B, T32DE00705745 to M.M.H., and R35HL160770 and R56ES035710 to R.L.Z.), the American Lung Association (IA-939940 to C.L.), the David and Lucile Packard Foundation (to C.L.), and the National Science Foundation (NSF CMMI-1751898 to L.H.). We also thank S. Huang for his assistance with human tissue sample collection.

Author information

Author notes

  1. These authors contributed equally: Donia W. Ahmed, Matthew L. Tan.

Authors and Affiliations

  1. Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, USA

    Donia W. Ahmed, Jackson Gabbard, Esther Gao, Michael M. Hu, Firaol S. Midekssa, Miriam Stevens, Fulei Wuchu, Minal Nenwani, Jingyi Xia, Deepak Nagrath & Brendon M. Baker

  2. Department of Materials Science and Engineering, University of Michigan, Ann Arbor, MI, USA

    Matthew L. Tan, Avinava Roy & Claudia Loebel

  3. School of Biomedical Engineering, Science and Health Systems, Drexel University, Philadelphia, PA, USA

    Yuchen Liu & Lin Han

  4. Department of Orthopedics, University of Michigan, Ann Arbor, MI, USA

    Adam Abraham

  5. Cellular and Molecular Biology Program, University of Michigan, Ann Arbor, MI, USA

    Rachel L. Zemans

  6. Department of Internal Medicine, University of Michigan, Ann Arbor, MI, USA

    Rachel L. Zemans

  7. Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USA

    Claudia Loebel

  8. Center for Precision Engineering for Health, Philadelphia, PA, USA

    Claudia Loebel

Authors
  1. Donia W. Ahmed
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  2. Matthew L. Tan
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  3. Yuchen Liu
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  4. Jackson Gabbard
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  5. Esther Gao
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  6. Avinava Roy
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  7. Michael M. Hu
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  8. Firaol S. Midekssa
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  9. Miriam Stevens
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  10. Fulei Wuchu
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  11. Minal Nenwani
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  12. Jingyi Xia
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  13. Adam Abraham
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  14. Deepak Nagrath
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  15. Lin Han
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  16. Rachel L. Zemans
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  17. Brendon M. Baker
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  18. Claudia Loebel
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Contributions

C.L. supervised the project and interpreted the data findings, and wrote the paper with D.W.A. and M.L.T. D.W.A. developed the photocrosslinking strategy for the ECM stiffening of PCLS and performed all time-lapse imaging for cell motility analysis. M.L.T. conducted all human PCLS experiments, birefringence imaging, mechanotransduction perturbations (YAP and pFAK), and metabolism experiments (ELISA and glucose/lactate assays). D.W.A. conducted the integrin-specific perturbation experiments and ECM deposition experiments and analysis. M.L.T. and D.W.A. equally contributed to the photostiffening characterization of hydrogels (rheology, PIV and dityrosine fluorescence). Y.L. and L.H. conducted and interpreted the PCLS AFM characterization experiments. M.M.H. conducted the gelatin AFM characterization. J.G. performed the cell motility analyses for time-lapse imaging data. E.G. assisted in the imaging analysis and characterization of photocrosslinking strategy on PCLS. F.S.M. conducted the fibrin photocrosslinking mechanical characterization. M.S. assisted in PCLS preparation for the experiments. F.W., M.N. and D.N. conducted and interpreted the glucose/lactate experiments and analysis. J.X. contributed to the development of the SPC-MetRS mice model. A.A. provided equipment and assistance with the birefringence imaging of PCLS. R.L.Z. provided mice to breed SPC-mTmG mice for PCLS experiments. B.M.B. and R.L.Z. helped interpret the data findings. A.R. performed live cell imaging of fibroblasts.

Corresponding author

Correspondence to Claudia Loebel.

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Competing interests

The authors declare no competing interests.

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Nature Materials thanks Janette Burgess, Sanjay Kumar and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Characterization of dityrosine cross-linking in hydrogels.

a. Representative PIV plots bead displacement of gelatin hydrogels (50, 100, and 150 mg/mL), collagen type I hydrogels (1, 3 and 6 mg/mL), and fibrin hydrogels (5. 10 and 20 mg/mL) on blue light exposure (3.6 J/cm2, scale bar 100 µm). b. Representative heat maps of gelatin hydrogels before (OFF) and after (ON) local exposure to blue light, far red light (670 nm) and blue light without photo-initiator (PI) (scale bar 100 µm). c. Quantification of local indentation moduli of gelatin hydrogels (150 mg/mL) before (OFF) and after (ON) on exposure to blue light (0.13 mM Ru, 20 mW/cm2 for 3 minutes, 3.6 J/cm2) by atomic force microscopy nanoindentation (spherical tip radius of 6.79 µm, n = 27 measurements from 1 representative hydrogel per condition, ****p<0.0001, two-tailed unpaired Student’s t-test with Welch’s correction). d. Representative heat maps of tyramine-functionalized hyaluronic acid (HA-Tyramine) hydrogels and methacrylated hyaluronic acid before and after local exposure to blue light (scale bar 100 µm). e. Representative time sweeps for storage (G’) and loss (G’’) modulus of HA-Tyramine hydrogels and methacrylated hyaluronic acid (meHA) hydrogels after blue light exposure. f. Quantification of storage moduli (G’) of bovine gelatin hydrogels (150 mg/mL) before (OFF) and after (ON) exposure to blue light (20 mW/cm2 blue light for 3 minutes, 3.6 J/cm2) with 0.13 mM and 0.26 mM ruthenium (Ru) (n = 9 hydrogels per group (0.13mM) and n=5 hydrogels per group (0.26mM), **P = 0.0082, ns= not significant, *P = 0.0386 between 0.13mM OFF/ON, *P = 0.0213 between 0.26mM OFF/ON by one-way ANOVA with Tukey’s multiple comparisons test). g. Quantification of storage moduli (G’) of gelatin hydrogels (porcine, 150 mg/mL): after incubation in PBS containing 0.13 mM Ru for 5, 10, 20 and 30 min prior exposure to blue light (n = 3 hydrogels per group, mean ± s.d, n.s = not significant, one-way ANOVA with Tukey’s multiple comparisons test); after incubation in PBS containing 0.13 mM Ru and 10, 20, or 40 mM SPS for 10 minutes prior to exposure to blue light (n = 3 hydrogels (10 mM) and n = 4 hydrogels (20, 40 mM), mean ± s.d, n.s = not significant, one-way ANOVA with Tukey’s multiple comparisons test); at increasing blue light dosage (0.9 - 3.6 J/cm2) (n = 3 hydrogels per group, mean ± s.d, n.s = not significant, one-way ANOVA with Tukey’s multiple comparisons test). For box plots (1C and 1F), the centre line represents the median, the box limits are upper and lower quartiles, and the whisker limits are the minimum and maximum points on the plots.

Source data

Extended Data Fig. 2 Characterization of dityrosine cross-linking in tissue.

a. Representative heat maps and quantification of dityrosine fluorescence of murine liver and skin tissues before (CTRL) and after (STIFF) blue light exposure (n = 3 slices from one mouse, mean ± s.e.m, 0.13 mM Ru, 20 mW/cm2 for 3 minutes, 3.6 J/cm2, scale bar 100 µm) b. Representative heat maps of dityrosine fluorescence and PIV plots of bead displacement of murine liver and lung tissues before and after blue light exposure (scale bar 100 µm). c. Representative heat maps of dityrosine fluorescence in lung tissue before (CTRL) and upon exposure with far red light (scale bar 100 µm). d. Quantification of light intensity over increasing PCLS thickness (100, 300, 600 μm) (n = 3 PCLS per thickness, mean ± s.d.).

Source data

Extended Data Fig. 3 Tunability of dityrosine crosslinking in lung tissue.

a. Representative heat maps and quantification of dityrosine fluorescence of murine PCLS before and after blue light exposure at increasing light intensities (2.3 - 4.8 J/cm2) (n = 3 images from 3 PCLS, mean ± s.e.m, scale bar 100 µm). b. Representative heat maps and quantification of dityrosine fluorescence of murine PCLS before and after blue light exposure with 0 mM, 0.13 mM, and 0.26 mM Ru) (n = 3 PCLS, mean ± s.e.m, scale bar 100 µm).

Source data

Extended Data Fig. 4 Characterization of AT2 and AT1 markers in control PCLS.

a. Representative fluorescent images of LAMP3 and PDPN staining in CTRL murine PCLS over a 5-day culture period (scale bar 100 μm). b. Quantification of AT2-specific marker lysosomal associated membrane protein 3 (LAMP3) positive cells in murine PCLS up to day 5 day after PCLS preparation (n = 10 images per timepoint from one representative mouse, *p=0.0195, ns = not significant by Kruskal-Wallis test with Dunn’s multiple comparisons). For all box plots, the centre line represents the median, the box limits are upper and lower quartiles, and the whisker limits are the minimum and maximum points on the plots.

Source data

Extended Data Fig. 5 Cell motility in response to tissue stiffening.

a. Representative live SftpcGFP cell fluorescent images of lineage-traced cells in CTRL and STIFF regions of murine PCLS for up to 48 hours (white arrows for representative cells, scale bar 100 µm) with respective migration and rosette plots over 48 hours and analyses of displacement and directionality ratio (n = 60 cells per group, **p=0.0033, ****p<0.001 by two-tailed unpaired Mann Whitney test). b. Quantification of average 3T3 cell speed with (+) versus without (-) blue light exposure over 1 hour (n = 14 images of 3 independent experiments, ns = not significant by unpaired Student’s t-test with Welch’s correction). c. Representative fluorescent images and quantification of normalized mean dityrosine immunofluorescence in CTRL and STIFF regions of embedded mouse lung fibroblasts in hydrogels (n = 8 images per group (CTRL, STIFF) from one representative hydrogel, ns = not significant by two-tailed unpaired Mann Whitney test, scale bar 50 µm). For all box plots, the centre line represents the median, the box limits are upper and lower quartiles, and the whisker limits are the minimum and maximum points on the plots.

Source data

Extended Data Fig. 6 Metabolic and secretory analyses in response to tissue stiffening.

a. Quantification of extracellular lactate and glucose concentrations in 24 hours culture of murine CTRL and STIFF PCLS (day 3-4, n = 3 mice, ****p<0.0001 by two-tailed unpaired Mann Whitney test). b. Quantification of extracellular cytokine concentrations (Interleukin-1 beta (IL-1β), Interleukin-6 (IL-6), Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), Interferon-Gamma Inducible Protein 10 (IP-10), Granulocyte Colony-Stimulating Factor (G-CSF), Tumor Necrosis Factor-alpha (TNF-α), S100 calcium binding protein A8 (S100A8), Macrophage inflammatory protein 2 (MIP-2), Macrophage Inflammatory Protein 3 (MIP-3), and Monokine Induced Gamma Inteferon (MIG) in 24 hours culture of murine CTRL and STIFF PCLS (day 4-5, n = 3 mice, p as indicated, ns= not significant by two-tailed unpaired Mann Whitney test). For all box plots, the centre line represents the median, the box limits are upper and lower quartiles, and the whisker limits are the minimum and maximum points on the plots.

Source data

Extended Data Fig. 7 Integrin signaling in response to tissue stiffening.

a. Representative fluorescent images and quantification of integrin β4 (ITGβ4) immunostaining and of integrin β4/GFP+ area in CTRL and STIFF regions of murine PCLS at day 5 (n = 24 images (CTRL) and 27 images (STIFF) from 5 mice, p as indicated by two-tailed unpaired Mann Whitney test, scale bar 30 µm). b. Representative fluorescent images and quantification of GFP+ cell area in STIFF regions of murine PCLS treated without (0 µg/mL) or with 5, 10 or 15 µg/mL integrin β4 function perturbing antibodies for 2 days (scale bar 100 µm, n = 149 images from 7 mice (0 µg/mL), 5 images (5, 10, 15 µg/mL) from one representative mouse, ns = not significant by unpaired Kruskal-Wallis test with Dunn’s multiple comparisons). c. Representative fluorescent images and quantification of GFP+ cell area in STIFF regions of murine PCLS treated without (0 µg/mL) or with 5, 10 or 15 µg/mL integrin β1 function perturbing antibodies for 2 days (scale bar 100 µm, n = 149 images per group from 7 mice (0 µg/mL) and n = 4 images from one representative mouse (5, 10 and 15 µg/mL), *p=0.0249 between 0 and 5 µg/mL, *p=0.022 between 0 and 15 µg/mL, **p=0.0089 between 0 and 10 µg/mL, ns = not significant by unpaired Kruskal-Wallis test with Dunn’s multiple comparisons). d. Quantification of LAMP3+GFP+ and PDPNhi of GFP+ area cells in CTRL and STIFF regions of murine PCLS without or with 10 µg/mL integrin β1 function perturbing antibody (β1i) for 2 days (LAMP3: n = 40 images from 5 mice (CTRL-β1i), 24 images from 3 mice (CTRL+β1i), ns = not significant by two-tailed unpaired Mann Whitney test; PDPN: n = 24 images from 3 mice (CTRL - β1i), 32 images from 4 mice (CTRL+ β1i), ns = not significant by by two-tailed unpaired Mann Whitney test). e. Quantification of LAMP3+GFP+ cells per ROI in CTRL and STIFF regions of murine PCLS without or with 10 µg/mL integrin β4 function perturbing antibody (+β4i) for 2 days (days (n = 40 images per group (CTRL, STIFF) from 5 mice, and n = 24 images per group (CTRL + β4i, STIFF + β4i) from 3 mice, ****p<0.0001, **p=0.0077 between CTRL/STIFF -β4i and p=0.0087 between CTRL/STIFF +β4i, and ns = not significant by unpaired Kruskal-Wallis test with Dunn’s multiple comparisons and quantification of PDPNhi of GFP+ area in CTRL and STIFF regions of murine PCLS without or with 10 µg/mL integrin β4 function perturbing antibody (+β4i) for 2 days (n = 23 images per group (CTRL, STIFF) from 3 mice, and n = 23 images per group (CTRL + β4i, STIFF + β4i) from 3 mice, *p=0.025 and ns = not significant by unpaired Kruskal-Wallis test with Dunn’s multiple comparisons). For all box plots, the centre line represents the median, the box limits are upper and lower quartiles, and the whisker limits are the minimum and maximum points on the plots.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–12.

Reporting Summary

Supplementary Video 1

Ex vivo time-lapse video of SftpcGFP cells in the CTRL and STIFF regions of PCLS. The circles indicate cells over a 48-h time lapse. Scale bar, 100 µm.

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Ahmed, D.W., Tan, M.L., Liu, Y. et al. Local photocrosslinking of native tissue matrix regulates lung epithelial cell mechanosensing and function. Nat. Mater. (2025). https://doi.org/10.1038/s41563-025-02329-0

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