Targeting transglutaminase 2 partially restores extracellular matrix structure but not alveolar architecture in experimental bronchopulmonary dysplasia

Abstract

The generation, maturation and remodelling of the extracellular matrix (ECM) are essential for the formation of alveoli during lung development. Alveoli formation is disturbed in preterm infants that develop bronchopulmonary dysplasia (BPD), where collagen fibres are malformed, and perturbations to lung ECM structures may underlie BPD pathogenesis. Malformed ECM structures might result from abnormal protein cross-linking, in part attributable to the increased expression and activity of transglutaminase 2 (TGM2) that have been noted in affected patient lungs, as well as in hyperoxia-based BPD animal models. The objective of the present study was to assess whether TGM2 plays a causal role in normal and aberrant lung alveolarization. Targeted deletion of Tgm2 in C57BL/6J mice increased septal thickness and reduced gas-exchange surface area in otherwise normally developing lungs. During aberrant lung alveolarization that occurred under hyperoxic conditions, collagen structures in Tgm2 mice were partially protected from the impact of hyperoxia, where normal dihydroxylysinonorleucine and hydroxylysylpiridinoline collagen cross-link abundance was restored; however, the lung alveolar architecture remained abnormal. Inhibition of transglutaminases (including TGM2) with cysteamine appreciably reduced transglutaminase activity in vivo, as assessed by N -(γ-l-glutamyl)-l-lysine abundance and TGM catalytic activity, and restored normal dihydroxylysinonorleucine and hydroxylysylpiridinoline collagen cross-link abundance under pathological conditions. Furthermore, a moderate improvement in alveoli size and gas-exchange surface density was noted in cysteamine-treated mouse lungs in which BPD was modelled. These data indicate that TGM2 plays a role in normal lung alveolarization, and contributes to the formation of aberrant ECM structures during disordered lung alveolarization.

Mouse genetic background impacts susceptibility to hyperoxia‐driven perturbations to lung maturation

“…Newborn mouse pups of all strains were randomized to litter sizes of either four or five, which are smaller than the litter sizes generally used (a litter size of six is generally used by the investigators for studies on C57BL/6J mice), however, were used to match the smaller litter sizes of the DBA/2J mouse strain. Mouse pups were exposed to normoxic (21% O 2 ) or hyperoxic (85% O 2 ) conditions from the day of birth (designated P1) to the 14th day of postnatal life (P14), in a well‐established experimental animal model of BPD using the Pro‐Ox oxygen control and delivery system of BioSpherix (Parish, NY) .…”

Minoxidil Cannot Be Used To Target Lysyl Hydroxylases during Postnatal Mouse Lung Development: A Cautionary Note

“…The pixel density of immunoblot bands were determined by densitometry, and the pixel density of the PLOD1 band was related to the pixel density of the corresponding β-actin band in the same lane, using Fiji software (Schindelin et al, 2012). hydroxylysinonorleucine (HLNL) was determined using the protocol described previously described by the authors (Brinckmann et al, 2005, Nave et al, 2014, Mižíková et al, 2018. The collagen cross-link nomenclature employed in the present study refers to the reduced variants of the intermediate collagen cross-links DHLNL and HLNL.…”

“…Design-based stereology serves at the gold standard for the quantitative assessment of the lung architecture, following the American Thoracic Society/European Respiratory Society recommendations for quantitative assessment of lung structure (Hsia et al, 2010). The stereological approach employed in the present study was described by the authors in detail previously , Mižíková et al, 2018. Mouse lungs were fixed by intratracheal instillation of 1.5%…”

“…For this reason, families of ECM cross-linking enzymes have received much attention in the context of lung development, including lysyl oxidases (Bland et al, 2007, Kumarasamy et al, 2009, lysyl hydroxylases , and transglutaminases . To this end, a combination of gene ablation and pharmacological intervention studies have highlighted roles for lysyl oxidases and transglutaminases (Mižíková et al, 2018) in normal lung development, and stunted lung development associated with BPD. However, to date, no function for lysyl hydroxylases in normal or aberrant lung development has been validated.…”

Studies on differentiation‐dependent expression and activity of distinct transglutaminases by specific substrate peptides using three‐dimensional reconstituted epidermis

“…For our investigations on biochemical studies on TGs, we developed and used 12‐mer peptides as ‘minimum substrate sequences' identified from a random peptide library . These peptides have been widely used as tools to efficiently visualize enzyme activity in several tissues including skin epidermis . Moreover, possible substrates have been identified in feasible and efficient procedures .…”