Abstract
The extracellular matrix of cirrhotic liver tissue is highly crosslinked. Here we show that advanced glycation end-products (AGEs) mediate crosslinking in liver extracellular matrix and that high levels of crosslinking are a hallmark of cirrhosis. We used liquid chromatography–tandem mass spectrometry to quantify the degree of crosslinking of the matrix of decellularized cirrhotic liver samples from patients and from two mouse models of liver fibrosis and show that the structure, biomechanics and degree of AGE-mediated crosslinking of the matrices can be recapitulated in collagen matrix crosslinked by AGEs in vitro. Analyses via cryo-electron microscopy and optical tweezers revealed that crosslinked collagen fibrils form thick bundles with reduced stress relaxation rates; moreover, they resist remodelling by macrophages, leading to reductions in their levels of adhesion-associated proteins, altering HDAC3 expression and the organization of their cytoskeleton, and promoting a type II immune response of macrophages. We also show that rosmarinic acid inhibited AGE-mediated crosslinking and alleviated the progression of fibrosis in mice. Our findings support the development of therapeutics targeting crosslinked extracellular matrix in scarred liver tissue.
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Data availability
The main data supporting the results in this study are available within the paper and its Supplementary Information. The raw RNA-seq data are available at the Sequence Read Archive database via the accession numbers PRJNA852213 and PRJNA856261. Source data are provided with this paper.
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Acknowledgements
We thank W. Wang, F. Wei and T. Yu at the Center of Pharmaceutical Technology, Tsinghua University for assistance in HPLC/MS assay; the sequencing core facility, Tsinghua University Cryo-EM Facility of the China National Center for Protein Sciences (Tsinghua University, Beijing, China) for help in Cryo-EM experiments; L. Bingyu at the Imaging Core Facility, Technology Center for Protein Sciences (Tsinghua University, Beijing, China) for assistance in operating Imaris 9.7 and Amira 20.2; J. Wang and Y. Sun at the Cell Biology Facility, Center of Biomedical Analysis (Tsinghua University, Beijing, China) for assistance with confocal microscopy; and the Laboratory Animal Resources Center (Tsinghua University, Beijing, China) for technical support. Some of the illustrations were created with reference to pictures in BioRender.com and ref. 19. This work was financially supported by the National Natural Science Foundation of China (82125018).
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C.L. and Y.D. conceived and designed the research. C.L. and W.K. performed the AQMC assays, collagen matrix construction, BMDM studies and animal experiments. Z.L. performed the optical tweezer experiments. W.K. and S.W. performed the cryo-EM experiments and established 3D collagen fibril models with the guidance of X.L. W.Y. and C.X. provided the clinical tissue samples and helped with the clinical consultation. P.Z. helped with the animal studies. K.L. prepared the illustrations. Y.N. helped with the preparation of stiffened collagen matrix. X.H. provided the guidance for the BMDM studies. C.L. and Y.D. wrote the paper. Y.D. is the principal investigator of the supporting grants.
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Extended data
Extended Data Fig. 1 Schematic of developing Absolute Quantification of Matrix-specific Crosslinking (AQMC) method.
a, Flow chart of quantifying crosslinking degree of liver decellularized ECM using AQMC method (detailed in Methods section). (1) Harvesting the liver decellularized ECM. Quality control is performed to verify the complete de-cell process without cellular component left. (2) Degradation of the liver ECM into fragments. (3) Complete degradation of the fragments into dipeptides and amino acids. (4) Enrichment of the crosslinking products. (5) Absolute quantification of the mole quantity of crosslinking products and hydroxyproline using LC-MS/MS. (6) Calculation of the crosslinking degree. The crosslinking degree of a certain type of crosslinking is defined as the molar ratio of crosslinking products and tropocollagen molecules. The mole quantity of triple-helix tropocollagen molecules is calculated according to the content of hydroxyproline as detailed in Methods section. b, Schematic of preparing mouse liver decellularized ECM using the whole-liver-perfusion method. The decellularization buffer is perfused into the liver from the hepatic portal vein and effused out from the inferior vena cava, as detailed in Methods section. c, Quality control of the mouse liver decellularization ECM performed by HE, Sirius Red, and cell nuclei staining. Nuclear components in liver tissue were removed by decellularization, whereas main ECM components (that is, collagen fibrils) were preserved as indicated by Sirius Red staining. Scale bars, 100 μm. d, Dry weight of liver decellularized ECM samples used for AQMC test. The mean ± s.e.m. values are marked on the plot.
Extended Data Fig. 2 MS/MS fragmentation spectra of standard crosslinking products.
a, LOX crosslinking product, pyridinoline (PYD), is marked by green colour. b, TGM crosslinking product, γ-Glutamyl-ε-Lysine (γ-GLY-ε-LYS), is marked by red colour. c-f, AGE crosslinking products are marked by blue colour. Total crosslinking degree of AGE crosslinking is quantified by the molar sum of CML (c), CEL (d), glucosepane (e), and pentosidine (f). The MH + labels indicate the protonated forms of precursor ions. Square labels above ions peaks are matched to their suggested fragment ion structures used to identify the crosslinking products.
Extended Data Fig. 3 Quantification of ECM crosslinking in livers from HFCDAA diet-induced liver fibrosis (HF fibrosis) model.
a, Schematic of preparing liver decellularized ECM from mice with HFCDAA diet-induced liver fibrosis. b, Representative images of Sirius red staining in livers from healthy mice and HF-fibrosis mice. Scale bars, 200 μm. c, Representative bright-field image of liver decellularized matrix harvested from HF-fibrosis mice. Scale bar, 1 cm. d, Representative SEM images of liver decellularized ECM from HF-fibrosis mice. Scale bars, 2 μm. e-j, Quantification of the crosslinking degree in liver ECM from HF-fibrosis mice using AQMC method. Data are shown in mole quantity of specific crosslinks normalized to mole quantity of tropocollagen molecules. e, LOX crosslinking degree quantified by pyridinoline (n ≥ 8); f, TGM crosslinking degree quantified by γ-Glutamyl-ε-Lysine (n ≥ 8); g, AGE crosslinking degree quantified by CML (h), CEL (i) and glucosepane (j) (n ≥ 8); Pentosidine is not detectable in this HF-fibrosis model. Results are presented as means ± s.e.m. The statistical analysis was performed using two-tailed unpaired t-test. All n values represent pieces of liver decellularized matrix obtained from at least 4 biologically independent mice.
Extended Data Fig. 4 Reagent residuals in the reconstructed AGE-crosslinked collagen matrix have negligible effects on macrophages’ response.
a, Characterization of reagent residuals in collagen matrix. b, Macrophages’ response to endotoxin of different concentrations. Verified by relative mRNA expression of Nos2 (b), Il6 (c), and Il1b (d). The detailed results in upper panels within the endotoxin concentration range of 0-0.05 EU·ml−1 are shown in corresponding panels below (n = 3, biologically independent samples per group). The blue dashed lines indicate the endotoxin residue levels in the reconstructed collagen matrix (~0.03 EU·ml−1). These results indicate that endotoxin residues in the in vitro collagen matrix keep at very low levels and cannot induce the unexpected response of macrophages. The statistical analysis was performed using a one-way ANOVA with Turkey test. Results are presented as mean ± s.d.
Extended Data Fig. 5 Decreased expression of HDAC3 in macrophages is observed when inhibiting F-actin organization by applying increased osmotic pressure.
a-e, Characterization of F-actin organization and HDAC3 expression in macrophages grown on 2D substrate. Cells were untreated or treated by PEG-induced osmotic pressure for 24 h (as detailed in Methods section). a, Representative images of F-actin (red) and G-actin (green) in macrophages with or without the treatment by the increased osmotic pressure. Scar bars, 10 μm. b, Statistical analysis of F/G-actin ratio in macrophages as shown in (a) (n ≥ 14, number of cells analysed per condition). c, Representative images of HDAC3 staining in macrophages. Top panel, HDAC3 (yellow), F-actin (green), cell nuclear (blue). Bottom panels, Colour-coded images of HDAC3. Colour bar indicates pixel intensity values. Scale bars, 10 μm. d, Statistical analysis of total HDAC3 intensity per cell as shown in (c) (n = 10, number of cells analysed per condition). e, Western blot analysis of HDAC3 expression in macrophages. Data are representative of three independent experiments. f-i, Characterization of F-actin organization and HDAC3 expression in macrophages grown on collagen matrix. NC (grey): macrophages grown on non-crosslinked collagen matrix; NC + Pressure (cyan): macrophages grown on non-crosslinked collagen matrix with treatment by PEG-induced osmotic pressure for 24 h; AGE (blue): macrophages grown on AGE-crosslinked collagen matrix. f, Representative images of F-actin (red) and G-actin (green) in macrophages grown on collagen matrix with or without the treatment by increased osmotic pressure. Scar bars, 4 μm. g, Representative images of HDAC3 staining in macrophages. Top panel, HDAC3 (yellow), F-actin (green), cell nuclear (blue). Bottom panels, Colour-coded images of HDAC3. Colour bar indicates pixel intensity values. Scale bars, 4 μm. h, Statistical analysis of F/G-actin ratio in macrophages as shown in (f) (n = 11, number of cells analysed per condition). i, Statistical analysis of total HDAC3 intensity per cell as shown in (c) (n = 11, number of cells analysed per condition). The statistical analysis was performed using two-tailed unpaired t-test in (b, d) and using a one-way ANOVA with Turkey test in (h, i). Results are presented as mean ± s.e.m.
Extended Data Fig. 6 Quantification of AGE crosslinking degree in liver ECM from mice with HFCDAA diet-induced liver fibrosis (HF fibrosis) after RA treatment.
a, Schematic of quantifying AGE crosslinking in liver decellularized ECM from HF-fibrotic mice after RA treatment. b, Representative bright-field images of liver decellularized ECM. Scale bars, 1 cm. c, Statistical analysis of the dry weight of entire liver decellularized ECM (n ≥ 4, biologically independent mice per group). d-g, Quantification of AGE crosslinking degree of liver decellularized ECM using AQMC method. d, Total AGE crosslinking degree; e, CML; f, CEL; g, Glucosepane (n = 6, pieces of liver decellularized ECM derived from at least 3 biologically independent mice). Results are presented as mean ± s.e.m. The statistical analysis was performed using two-tailed unpaired t-test.
Extended Data Fig. 7 Comparative analysis of ECM crosslinking degree from cirrhotic patients’ liver samples with different disease history.
a, Results from cirrhotic patients with (n = 9, pieces of liver ECM obtained from 5 independent patients) or without (n = 9, pieces of liver ECM obtained from 4 independent patients) viral hepatitis. b, Results from cirrhotic patients with (n = 12, pieces of liver ECM obtained from 6 independent patients) or without (n = 6, pieces of liver ECM obtained from 3 independent patients) carcinoma. The statistical analysis was performed using two-tailed unpaired t-test between the groups with or without viral hepatitis (a), and with or without carcinoma (b). Exact P values are marked on the plots. Results are presented as mean ± s.e.m.
Extended Data Fig. 8 TGM crosslinking affects structural and mechanical properties of collagen matrix in a way different from AGE crosslinking.
a, Schematic of reconstructing TGM-crosslinked collagen matrix in vitro. NC, Non-crosslinked collagen matrix; TGMlow, TGM-crosslinked collagen matrix with a low crosslinking degree; TGMhi, TGM-crosslinked collagen matrix with a high crosslinking degree; TGMhi + cys, collagen matrix with additional cystamine to inhibit TGMhi treatment. b, Quantification of TGM crosslinking degree of reconstructed collagen matrix using AQMC method. Data are shown in mole quantity of γ-Glutamyl-ε-Lysine normalized to mole quantity of tropocollagen molecules (n ≥ 6, independent collagen matrix samples). TGMhi collagen matrix is selected for the following assays in c-g (shown as TGM-crosslinked). c, Representative SEM images of non-crosslinked and TGM-crosslinked collagen matrix. Scale bars, 2 μm. d, Representative SEM images of macrophages grown on non-crosslinked and TGM-crosslinked collagen matrix. The bottom panels show protrusions of macrophages binding adjacent collagen fibrils. Scale bars, 5 μm. e, Statistical analysis of fibril diameter (n ≥ 123, number of fibrils randomly selected from at least 5 fields of SEM images). f, Statistical analysis of the Young’s modulus of non-crosslinked and TGM-crosslinked collagen matrix measured by AFM (n ≥ 171, points of measurement randomly selected from at least 10 fields of samples). g, Statistical analysis of the roundness of macrophages grown on non-crosslinked and TGM-crosslinked collagen matrix (n = 6, number of cells analysed per condition). The statistical analysis was performed using a one-way ANOVA with Turkey test in (b) and using two-tailed unpaired t-test in (e-g). Results are presented as mean ± s.e.m.
Supplementary information
Supplementary Information
Supplementary discussion, methods, figures, tables, references and video captions.
Supplementary Video 1
3D reconstruction model of a non-crosslinked collagen fibril.
Supplementary Video 2
3D reconstruction model of an AGE-crosslinked collagen fibril.
Supplementary Video 3
3D reconstruction model of collagen fibril where AGE crosslinking was inhibited by RA (AGE + RA).
Supplementary Video 4
3D view of F-actin in a macrophage that co-localized with collagen fibrils in the non-crosslinked matrix.
Supplementary Video 5
3D view of F-actin in a macrophage that co-localized with collagen fibrils in the AGE-crosslinked matrix.
Supplementary Video 6
Time-lapse imaging showing the displacement of non-crosslinked collagen fibrils caused by force applied by an optical trap.
Supplementary Video 7
Time-lapse imaging showing the displacement of AGE-crosslinked collagen fibrils caused by force applied by an optical trap.
Supplementary Video 8
Time-lapse imaging showing the displacement of AGE + RA collagen fibrils caused by force applied by an optical trap.
Source data
Source Data for Figs. 1–8 and Extended Data Figs. 1–8
Source data for all main figures and Extended Data figures, and unprocessed gels for Supplementary Fig. 14.
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Lyu, C., Kong, W., Liu, Z. et al. Advanced glycation end-products as mediators of the aberrant crosslinking of extracellular matrix in scarred liver tissue. Nat. Biomed. Eng 7, 1437–1454 (2023). https://doi.org/10.1038/s41551-023-01019-z
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DOI: https://doi.org/10.1038/s41551-023-01019-z
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