Figure 3. 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 multiple t-test of log transformed data using the Holm-Sidak method to adjust for multiple comparisons (two-tailed). SD was not assumed to be consistent between groups. (C–F) Hydroxyallysine-derived collagen cross-link analysis of IPF (n = 17 donors) and control (n = 15 donors) lung tissue, normalised to total collagen content, as determined by UHPLC-ESI-MS/MS. Each data point represents an individual donor. (C) Total immature divalent cross-links (DHLNL + HLNL); (D) Total mature trivalent cross-links (PYD + DPD); (E) Ratio of immature to mature cross links; (F) Ratio of DHLNL to HLNL immature divalent cross-links. Bars indicate median values. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 by Mann-Whitney t-test (two-tailed). (G, H) Scatterplots of paired data from Figure 1A and Figure 3C or Figure 3D showing dependency of mean tissue stiffness on total immature (r = 0.364, p=0.137) or total mature (r = 0.723, p=0.0007) collagen cross-link density, respectively (Multivariate correlation analysis including mean tissue stiffness, normalised collagen and immature and mature cross-links).