Determinants and effects of collagen cross-linking enzyme dysregulation in idiopathic pulmonary fibrosis

Abstract

Progressive accumulation of abnormal extracellular matrix (ECM) is a key pathologic event in idiopathic pulmonary fibrosis (IPF). Repetitive micro-injuries to a genetically susceptible alveolar epithelium are thought to incite an abnormal wound healing response, propagating fibrogenesis through the induction of pro-fibrotic signals of which TGFβ is considered the prototype. Beyond ECM deposition, alterations to collagen nanostructure such as increased pyridinoline cross-linking are critical to the loss of ECM compliance in IPF, resulting from upregulation of collagen cross-linking enzymes such as lysyl hydroxylase 2 (PLOD2) and the lysyl oxidase-like (LOXL) family.
It was hypothesised that pathological remodelling and increased tissue stiffness in IPF involves aberrant expression of these collagen cross-linking enzymes, caused by dysregulation of core upstream signalling pathways. The aims of this study were therefore to identify the cellular provenance of key ‘bone-type’ collagen cross-linking enzymes, the upstream regulatory pathways which promote their expression, and the biomechanical consequences of their dysregulation.
Through human tissue analyses integrating regionally selective RNA sequencing LOXL2 was established as the LOX(L) enzyme with greatest expression in IPF fibroblastic foci, and a strong correlation between LOXL2 and PLOD2 expression was observed, with co-localisation identified through in-situ mRNA hybridisation. Hypoxia Inducible Factor (HIF) pathway stabilisation was found to be a potent inducer of these ‘bone-type’ collagen cross-linking enzymes using 2D in vitro experiments, and exhibited synergistic effects with TGFβ signalling. Yet HIF pathways, rather than TGFβ signalling, disproportionately induced collagen cross-linking expression compared to collagen fibrillogenesis. The presence of HIF pathway activation within IPF tissue was confirmed through immunohistochemical analysis, correlating to areas of PLOD2 and LOXL2 mRNA expression. Using an in vitro 3D fibroblastic focus model, HIF pathway stabilisation was observed to profoundly alter collagen structure-function, with increases in pyridinoline collagen cross-linking and matrix stiffness, and a reduction in collagen fibril diameter, that were comparable to changes seen within IPF tissue. Gene silencing of HIF1/2α or LOXL2, and catalytic inhibition of LOX(L) activity, was capable of modulating the pathogenetic HIF-mediated changes to ECM stiffness, suggesting these pathways may offer novel therapeutic targets.
In summary HIF pathway stabilisation is a key regulatory promoter of ‘bone-type’ collagen cross-linking and, in conjunction with TGFβ signalling, may act as a ‘second-hit’ insult to drive pathological remodelling of ECM within the IPF lung. Therapeutic applications which target HIF pathways could normalise these matrix changes and prevent progressive fibrosis.

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