Lysyl oxidase activity is dysregulated during impaired alveolarization of mouse and human lungs

Abstract

Rationale: Disordered extracellular matrix production is a feature of bronchopulmonary dysplasia (BPD). The basis of this phenomenon is not understood.

Objectives: To assess lysyl oxidase expression and activity in the injured developing lungs of newborn mice and of prematurely born infants with BPD or at risk for BPD.

Methods: Pulmonary lysyl oxidase and elastin gene and protein expression were assessed in newborn mice breathing 21 or 85% oxygen, in patients who died with BPD or were at risk for BPD, and in control patients. Signaling by transforming growth factor (TGF-beta) was preemptively blocked in mice exposed to hyperoxia using TGF-beta-neutralizing antibodies. Lysyl oxidase promoter activity was assessed using plasmids containing the lox or loxl1 promoters fused upstream of the firefly luciferase gene.

Measurements and main results: mRNA and protein levels and activity of lysyl oxidases (Lox, LoxL1, LoxL2) were elevated in the oxygen-injured lungs of newborn mice and infants with BPD or at risk for BPD. In oxygen-injured mouse lungs, increased TGF-beta signaling drove aberrant lox, but not loxl1 or loxl2, expression. Lox expression was also increased in oxygen-injured fibroblasts and pulmonary artery smooth muscle cells.

Conclusions: Lysyl oxidase expression and activity are dysregulated in BPD in injured developing mouse lungs and in prematurely born infants. In developing mouse lungs, aberrant TGF-beta signaling dysregulated lysyl oxidase expression. These data support the postulate that excessive stabilization of the extracellular matrix by excessive lysyl oxidase activity might impede the normal matrix remodeling that is required for pulmonary alveolarization and thereby contribute to the pathological pulmonary features of BPD.

Figure 1.

Elastin production and cross-linking are dysregulated in the injured developing mouse lung. (A) Hart's stain for elastin in air-exposed mouse pup lungs, indicating punctate elastin foci (arrows) in the developing septa at Postnatal Day (P)7 and P28 in the lungs of pups exposed to 21% O₂ that are absent in the lungs of pups exposed to 85% O₂. (B) Assessment of collagen morphology by Masson's trichrome stain (a and b) in the developing septa of P7 mouse pups exposed to 21% O₂ or 85% O₂. Collagen was also assessed by picrosirius red staining observed under polarized light in mouse pups exposed to (c) 21% O₂ or (d) 85% O₂. Both (C) soluble elastin and (D) desmosine were elevated in 85% O₂-exposed pups (open bars) compared with 21% O₂-exposed pups (solid bars) at P28 (n = 5). (E) Expression of elastin (the eln gene), fibulin-5 (the fbln5 gene), and emilin-1 (the emilin1 gene) mRNA monitored by semiquantitative reverse transcriptase–polymerase chain reaction in the first month of postnatal life of pups exposed to 21% O₂ or 85% O₂. The constitutively expressed hspa8 and gapdh genes served as controls for loading equivalence. *P < 0.01.

Figure 2.

Lysyl oxidase mRNA and protein expression is dysregulated in the injured developing mouse lung. (A) Expression of lysyl oxidase mRNA was monitored by semiquantitative reverse transcriptase–polymerase chain reaction where the constitutively expressed hspa8 gene served as a loading control. These data were quantified by densitometric analysis in B. (C) Expression of lysyl oxidase protein was monitored by immunoblot, using constitutively expressed α-tubulin as a loading control. These data were quantified by densitometric analysis in D. (E) Lysyl oxidase activity, assessed by an Amplex Red–based assay, was elevated in whole lung extracts from 85% O₂-exposed pups (open bars) compared with 21% O₂-exposed pups (solid bars) at P14 and P28. * P < 0.01 (n = 4 for A and C, and n = 5 for E). AFU = arbitrary fluorescence units.

Figure 3.

Lysyl oxidase protein expression is dysregulated in the septa of oxygen-injured mouse pup lungs. Increased staining intensity is observed for Lox, LoxL1, and LoxL2 in the septa of mice exposed to 85% O₂ compared with 21% O₂-exposed mouse pups at P14. Antibody specificity was confirmed by preadsorption of antibodies with a competing peptide, which had been used as the immunogen for antibody generation. Results for LoxL4 are omitted as no immunoreactivity was observed with the anti-LoxL4 antibody on mouse lung sections.

Figure 4.

Lysyl oxidase mRNA expression can be modulated in vitro by oxygen and transforming growth factor (TGF)-β. (A) Murine NIH/3T3 fibroblast-like cells and (B) human pulmonary artery smooth muscle cells were maintained under hyperoxic (85% O₂) or normoxic (21% O₂) conditions for 24 hours, before addition of TGF-β (2 ng/ml) for an additional 24 hours, after which mRNA was isolated and assessed for lysyl oxidase gene expression. Whole-lung mRNA from mouse or human lungs, respectively, served as a positive control for the polymerase chain reactions (PCR) (Pos.), whereas expression of the hspa8 gene was used as a loading control. The PCR amplicons derived from two separate cell cultures are represented for each condition. For quantification, densitometric data for amplicons derived from six different cell cultures per condition were averaged; ^¶ P < 0.01 comparing 85% O₂ versus 21% O₂ exposures in the presence of vehicle; ^§ P < 0.01 comparing TGF-β–stimulated versus unstimulated cells with 21% oxygen or 85% oxygen exposure; * P < 0.01 comparing 85% O₂ versus 21% O₂ exposures 24 hours after TGF-β stimulation.

Figure 5.

Hyperoxia induced autocrine activation of lysyl oxidase promoters in NIH/3T3 through transforming growth factor (TGF)-β. (A) Murine NIH/3T3 fibroblast-like cells and human HFL1 fibroblast-like cells were transfected with plasmid constructs in which the mouse lox or human loxl1 promoters had been inserted, upstream of a firefly luciferase gene. The cells were maintained under hyperoxic (85% O₂) or normoxic (21% O₂) conditions for 24 hours, before addition of TGF-β (2 ng/ml) for an additional 12 hours, after which cell extracts were assessed for firefly luciferase activity. To control for the effects of ligand stimulation and hyperoxia on the baseline transcriptional activity of cells, values were normalized for the transcriptional activity of the pGL3-control vector (20). (B) Murine NIH/3T3 fibroblast-like cells and human HFL1 fibroblast-like cells were treated as described in A, except that medium was supplemented with nonimmune IgG or anti–TGF-β1,2,3–neutralizing antibodies over the entire time course of the experiment (10 μg/ml). *P < 0.01 (n = 5). ALU = arbitrary luminescence units; n.i. = nonimmune.

Figure 6.

Treatment of neonatal mice with transforming growth factor (TGF)-β1,2,3–neutralizing antibodies suppressed the induction of lox gene expression by hyperoxia. Neonatal mice were treated either with control IgG or anti–TGF-β1,2,3–neutralizing antibodies before exposure to hyperoxic (85% O₂) or normoxic (21% O₂) conditions for 10 days, and then killed. The expression of lysyl oxidases was assessed in mRNA from mouse lungs by (A) semiquantitative reverse transcriptase–polymerase chain reaction (RT-PCR), where expression of the hspa8 gene was used as a loading control; or (B) real-time quantitative RT-PCR (n = 5); or (C) by immunohistochemistry. Antibody specificity was confirmed by preadsorption of antibodies with a competing peptide, which had been used as the immunogen for antibody generation (insets). Additionally, (D) lung sections from mice were screened for assessment of elastin deposition in the developing septa by Hart's elastin stain. *P < 0.01.

Figure 7.

The expression of Lox and LoxL1 was elevated in the lungs of neonatal patients who died with BPD or were at risk for BPD. The expression of lysyl oxidases was assessed in the lungs of patients with BPD or at risk for BPD, as well as in control lungs. A low-power (200-μm scale) view of representative sections from (A) patients in control group 2 (the histopathology of six patients [Table 1] was examined; in this case, sections from patient 11 are illustrated) and from (C) patients in the BPD group (the histopathology of seven patients [Table 2] was examined; in this case, sections from patient 24 are illustrated), stained for LoxL1. (B, D) High-power views (50-μm scale) are also illustrated for LoxL1 staining in the same sections (the magnified area is demarcated in the low-power view by a black box). Representative medium-power views (100-μm scale) are illustrated for the same two patients, stained for Lox (E, F), with the corresponding high-power views (50-μm scale) illustrated in F and H (the magnified area is demarcated in the low-power view by a black box). (I, J) Antibody specificity was confirmed by preadsorption of antibodies with a competing peptide, which had been used as the immunogen for antibody generation, before staining a section of lung tissue from the same patient with BPD. Staining was consistently more intense in patients with BPD, and the sections illustrated are representative of the trends observed in a total of five patients assessed per group (as indicated in Tables 1 and 2). The expression of lysyl oxidase mRNA was also assessed in mRNA isolated from the lungs of seven patients in control group 1 (red), seven patients with BPD or at risk for BPD (green) and five patients in control group 2 (yellow) by quantitative real-time reverse transcriptase–polymerase chain reaction (K). The bars represent the data range and the boxes represent lower and upper quartiles. The line within the quartile box indicates the median; *P < 0.01.