desmosine and Pulmonary-Fibrosis

Desmosine and isodesmosine (DID) are unique elastin crosslinks that may serve as biomarkers for elastic fiber degradation in chronic obstructive pulmonary disease. Previously, our laboratory found that the ratio of free to peptide-bound DID in bronchoalveolar lavage fluid (BALF) showed a significant positive correlation with the extent of airspace enlargement in an elastase model of pulmonary emphysema. To further evaluate this hypothesis, our laboratory measured this ratio in a bleomycin (BLM) model of pulmonary fibrosis, which involved different microarchitectural changes than those associated with pulmonary emphysema.. Syrian hamsters were instilled intratracheally with 1.0 unit BLM in 0.2 ml of normal saline (controls received the vehicle alone), and BALF was analyzed for both free and total DID, using a combination of liquid chromatography and tandem mass spectrometry.. Total BALF DID was significantly increased in hamsters receiving BLM at 1 week post-treatment (92 vs 13 pg/ml; p < 0.001), consistent with elastic fiber degradation. However, in contrast to elastase-induced emphysema, free/bound DID was lower in BLM-treated animals compared to controls at both 1 week (0.76 vs 0.84) and 2 weeks post-treatment (0.69 vs 0.86), though the differences were not statistically significant.. These results indicate that it may be possible to identify specific pulmonary microarchitecture changes, based on the ratio of free to peptide-bound DID. It is speculated that the proportionate decrease in free DID in BLM-induced fibrosis may be due to preservation of intact elastic fibers as the lung injury progresses.

Topics: Animals; Bleomycin; Bronchoalveolar Lavage Fluid; Cricetinae; Desmosine; Elastic Tissue; Emphysema; Female; Isodesmosine; Lung; Lymphocyte Count; Neutrophils; Pancreatic Elastase; Proteins; Pulmonary Fibrosis

Increased expression of matrix metalloproteinases, particularly gelatinase B (MMP-9), has been described in the lungs in pulmonary fibrosis. Intratracheal bleomycin is often used experimentally to produce lesions resembling human fibrosing alveolitis. To assess the role of gelatinase B in bleomycin-induced fibrosing alveolitis, we instilled bleomycin intratracheally into gelatinase B-deficient mice and gelatinase B+/+ littermates. Twenty-one days after bleomycin the two groups of mice were indistinguishable in terms of pulmonary histology and total lung collagen and elastin. However, the lungs of gelatinase B-deficient mice showed minimal alveolar bronchiolization, whereas bronchiolization was prominent in the lungs of gelatinase B+/+ mice. Gelatinase B was identified immunohistochemically in terminal bronchiolar cells and bronchiolized cells 7 and 14 days after bleomycin in gelatinase B+/+ mice, and whole lung gelatinase B mRNA was increased at the same times. Many bronchiolized cells displayed Clara cell features by electron microscopy. Some bronchiolized cells stained with antibody to helix transcription factor 4, a factor associated with the ciliated cell phenotype. Thus, fibrosing alveolitis develops after intratracheal bleomycin irrespective of gelatinase B. However, gelatinase B is required for alveolar bronchiolization, perhaps by facilitating migration of Clara cells and other bronchiolar cells into the regions of alveolar injury.

Topics: Animals; Bleomycin; Bronchi; Bronchoalveolar Lavage Fluid; Cell Count; Desmosine; Genotype; Hydroxyproline; Immunohistochemistry; Instillation, Drug; Intubation, Intratracheal; Lung; Lymphocytes; Macrophages; Matrix Metalloproteinase 7; Matrix Metalloproteinase 9; Mice; Mice, Inbred Strains; Mice, Knockout; Microscopy, Electron; Neutrophils; Pulmonary Alveoli; Pulmonary Fibrosis

Sprague-Dawley rats were exposed for 6 h daily to 0.8 ppm of ozone and 14.4 ppm of nitrogen dioxide. Approximately 7 to 10 wk after the initiation of exposure, animals began to demonstrate respiratory insufficiency and severe weight loss. About half of the rats died between Days 55 and 78 of exposure; no overt ill effects were observed in animals exposed to filtered air, to ozone alone, or to nitrogen dioxide. Biochemical findings in animals exposed to ozone and nitrogen dioxide included increased lung content of DNA, protein, collagen, and elastin, which was about 300% higher than the control values. The collagen-specific crosslink hydroxy-pyridinium, a biomarker for mature collagen in the lung, was decreased by about 40%. These results are consistent with extensive breakdown and remodeling of the lung parenchyma and its associated vasculature. Histopathologic evaluation showed severe fibrosis, alveolar collapse, honeycombing, macrophage and mast cell accumulation, vascular smooth muscle hypertrophy, and other indications of severe progressive interstitial pulmonary fibrosis and end-stage lung disease. This unique animal model of progressive pulmonary fibrosis resembles the final stages of human idiopathic pulmonary fibrosis and should facilitate studying underlying mechanisms and potential therapy of progressive pulmonary fibrosis.

Topics: Administration, Inhalation; Animals; Collagen; Desmosine; Disease Models, Animal; DNA; Elastin; Environmental Exposure; Hydroxyproline; Lung; Male; Nitrogen Dioxide; Ozone; Proteins; Pulmonary Fibrosis; Pyridines; Rats; Rats, Sprague-Dawley; Survival Rate

To evaluate the concept that lung elastin degradation is accelerated in homozygous alpha-1-antitrypsin (AAT) deficient persons, we prepared acid hydrolysates of urine and used a radioimmunoassay for desmosine to measure urine concentrations of this elastin-specific cross-link in such persons and in control subjects. Excretion of desmosine in 17 homozygous AAT-deficient (PiZZ) patients with emphysema was compared with that in 27 patients with interstitial lung diseases (16 sarcoid, 5 idiopathic pulmonary fibrosis, 6 other interstitial lung diseases) and 26 healthy subjects. Both smokers and nonsmokers were present in all groups. Urinary desmosine concentration (microgram/100 mg creatinine) was 2.35 +/- 0.93 in the PiZZ patients, 2.49 +/- 1.01 in those with interstitial lung disease, and 2.05 +/- 0.54 in the healthy control subjects (p greater than 0.1, all comparisons). Because abnormal pulmonary elastolysis may be largely completed before symptoms of emphysema develop in AAT-deficient persons, we also tested 6 asymptomatic adults with homozygous AAT deficiency (PiZZ) and 5 PiZZ children. Urine desmosine (microgram/100 mg creatinine) was not significantly elevated in either group compared with that in the age-matched control subjects, although children (PiZZ and age-matched controls) showed higher excretions than did adults (6 asymptomatic PiZZ adults, 2.60 +/- 0.91; 5 PiZZ children, 3.27 +/- 0.62; 10 control children, 3.61 +/- 0.62). These data suggest that pathologic lung elastolysis in the PiZZ subject may constitute too small a fraction of total-body elastin turnover to be detected by this method.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adult; alpha 1-Antitrypsin Deficiency; Amino Acids; Child; Desmosine; Disease Susceptibility; Female; Homozygote; Humans; Male; Middle Aged; Phenotype; Pulmonary Emphysema; Pulmonary Fibrosis

Cross-linked elastin synthesis was measured in the intratracheal bleomycin model of interstitial pulmonary fibrosis by incorporation of 14C-lysine into the elastin-specific crosslinks, desmosine and isodesmosine. Detection of the labeled crosslinks was facilitated by development of a highly sensitive assay utilizing thin-layer electrophoresis. The results indicate that crosslinked elastin synthesis is significantly elevated from controls (p less than 0.05) at 1 to 3 weeks after exposure to bleomycin and returns to normal by 5 weeks. The increases in labeled elastin synthesis are not directly related to changes in either total lung protein synthesis or the pool size of the 14C-lysine. In comparison with collagen and glycosaminoglycan synthesis in this model of lung injury, maximal increases in cross-linked elastin formation occur later, but overlap with the elevated synthesis of these other connective tissue components. The marked increase from normal in cross-linked elastin synthesis in this model suggests that this tissue component is an important part of the fibrotic response of the pulmonary parenchyma and may play a role in the observed alterations in lung structure and function.

Topics: Amino Acids; Animals; Bleomycin; Carbon Radioisotopes; Cricetinae; Desmosine; Elastin; Female; Isodesmosine; Lysine; Mesocricetus; Pulmonary Fibrosis

Proline analogs inhibit collagen biosynthesis and prevent accumulation of collagen in tissues. The antifibrotic effects of three proline analogs, cis-hydroxyproline, L-azetidine-2-carboxylic acid, and L-3,4-dehydroproline, were compared in a rat oxygen toxicity model. The specificity of these agents for collagen was examined by measuring their effects on noncollagen protein and elastin accumulation in the lung. Increased lung collagen was produced by exposing rats to 95% O2 for 60 hr followed by a 2-week recovery period. Animals were treated with the proline analogs for the 2-week period. Oxygen exposure in untreated animals increased lung collagen 26% above air-breathing controls, and this increase was prevented by all three analogs. Increased noncollagen protein was also prevented by these agents, suggesting they were not entirely specific for collagen. Elastin accumulation, however, was not inhibited by cis-hydroxyproline. It was concluded that proline analogs were antifibrotic, but affected the metabolism of noncollagen protein.

Topics: Animals; Azetidinecarboxylic Acid; Desmosine; Elastin; Hydroxyproline; Male; Organ Size; Oxygen; Proline; Proteins; Pulmonary Fibrosis; Rats; Rats, Inbred Strains

Intratracheal administration of bleomycin caused pulmonary fibrosis in rats. Bleomycin sulfate (640 micro grams/165 g b.wt. in 0.5 ml of sterile saline) was instilled Intratracheally into male Fisher 344 rats (169 +/- 5 g), whereas control animals received 0.5 ml of sterile saline by the same route. One, 3, 7, 14, 28 and 322 days after instillation, the animals were killed, the lungs were homogenized in 6 M urea, 0.01 M NaCl, 0.001 M potassium phosphate (pH 8.3) and the homogenates were subjected to ultracentrifugation. The 106,000 x g supernate was assayed for lysyl oxidase activity. Total lung hydroxyproline and desmosine content was determined at each time point. Lysyl oxidase specific activity in the lung was elevated significantly 3 days after bleomycin treatment, peaked 14 days after bleomycin treatment at 230% above the control value and was returned toward normal 28 days after treatment. The increase of lysyl oxidase activity preceded the maximal increase of total lung hydroxyproline and desmosine which occurred 28 days after bleomycin instillation.

Topics: Amino Acid Oxidoreductases; Animals; Bleomycin; Desmosine; Hydroxyproline; Lung; Male; Protein-Lysine 6-Oxidase; Pulmonary Fibrosis; Rats; Rats, Inbred F344