Lung collagen cross-links in rats with experimentally induced pulmonary fibrosis

Abstract

Rats were intratracheally instilled with bleomycin or with silica (quartz) dust to induce lung fibrosis. Several weeks later, purified collagen chains (or collagen digests) were isolated from the lungs of these animals and from age-matched controls instilled intratracheally with saline solution, and the ratios of hydroxylysine to lysine and of the dysfunctional cross-links DHLNL to HLNL were quantified. Collagen from fibrotic lungs had significantly higher ratios of DHLNL:HLNL than did control lungs, 15.5 +/- 4.8 and 17.1 +/- 4.8 vs. 2.3 +/- 0.5 for the silica-instilled and the bleomycin-instilled animals, respectively. The hydroxylysine:lysine ratio was significantly increased for the alpha 1(I) chain, to a value 170% of that of lung collagen from control animals, and for several of its constituent CNBr peptides. Lung tissue was exhaustively digested with collagenase and specific cross-linked peptides were isolated and characterized. The cross-linked alpha 1(I) x alpha 1(I) peptide linked by the residues 87 x 16C, with a ratio of DHLNL:HLNL of 17:1, demonstrated that the increased hydroxylation of the dysfunctional cross-links in fibrotic lung collagen could be accounted for in part by increased hydroxylation of the lysine residue at position 16C of the C-terminal telopeptide of the collagen alpha 1(I) chain. It proved impossible to locate the corresponding N-terminal cross-linked fragment from alpha 1(I) x alpha 1(I) chains, 9N x 930, possibly due to further reactions of this material to form the material referred to as poly(CB6). Isolated poly (CB6) accounted for more than half of the total alpha 1(I)CB6 peptide expected in lung collagen, and had a hydroxylysine:lysine content 2.8 times greater in bleomycin-treated animals than in their age-matched controls. Evidence was also found for a cross-linked alpha 1(III) x alpha 1(I) peptide linking residue 87 from the alpha 1(III) chain with residue 16C from the alpha 1(I) chain; it also had an increased ratio of DHLNL:HLNL. We conclude that the increased hydroxylation of lysine observed in two different animal models of lung fibrosis occurs preferentially at the N- and C-terminal nonhelical extension peptides of the alpha 1(I) collagen chains, and that this apparent specificity of overhydroxylation of fibrotic collagen may have important structural and pathological consequences.

Collagen and elastin cross-linking is altered during aberrant late lung development associated with hyperoxia

“…Concerning the ECM cross-link status of mouse lungs following hyperoxia exposure, the abundance of the DHLNL collagen cross-link, as well as the DHLNL/HLNL, was increased in lungs from hyperoxia-exposed mice, suggestive of a profibrotic state (17,63). The application of BAPN concomitantly with hyperoxia blunted this increase in lung collagen accumulation and partially normalized the DHLNL/HLNL.…”

Always cleave up your mess: targeting collagen degradation to treat tissue fibrosis

“…Lysyl oxidase expression and the proportion of hydroxylated lysine residues are increased in fibrotic states (104). An increase in cross-linking and altered cross-linking patterns have been implicated in dermal fibrosis (17), in ARDS in the lung (86), in myocardial fibrosis (98), and in pulmonary fibrosis (56,142). A recent study demonstrated that inhibition of lysyl oxidase-like-2 prevented the development of bleomycin-induced lung fibrosis and reversed established fibrosis (6).…”

“…What is unclear from this technique is whether all collagen in the lung is degraded at similar rates or whether there are pools of collagen that are more or less accessible to degradation (55,58,90). It has been suggested that the increased ratio of more stable dihydroxylysinonorleucine cross-links to less stable hydroxylysinonorleucine crosslinks found in fibrotic lung tissue may protect "fibrotic collagen" from enzymatic degradation or mechanical damage (56). Further studies examining whether collagen that is incorporated into fibrillar scar is less prone to degradation than collagen that is part of normal tissue architecture are warranted.…”

Defective collagen crosslinking in bone, but not in ligament or cartilage, in Bruck syndrome: Indications for a bone-specific telopeptide lysyl hydroxylase on chromosome 17

“…Despite the large differences in lysyl hydroxylation of the telopeptides of collagen type I between skin and bone, for instance, less marked differences are seen in the hydroxylation of lysine residues in the triple helical part of the collagen molecule. To explain this difference, it has been proposed that two forms of LH exist: one that specifically hydroxylates Lys in the ␣-helix (we propose the term ''helical lysyl hydroxylase,'' HLH), and another capable of hydroxylating Lys residues in the telopeptides (telopeptide lysyl hydroxylase, TLH) (16)(17)(18)(19)(20)(21).…”