To our knowledge, this is the first study investigating the serological profile of collagen turnover in fibrotic lung diseases. We tested a range of novel ECM degradation serum markers in patients with COPD or IPF and in healthy controls. Interestingly, significant differences in marker levels were seen between healthy and disease-affected individuals, and also between the two lung diseases. The markers of ECM degradation provided diagnostic information and suggest that tissue degradation is highly elevated in patients with mild COPD and mild to severe IPF. C1M, C3M, C5M and C6M showed highly statistically significant power to discriminate between IPF and mild COPD patients versus healthy individuals, while C4M and C3A were only able to diagnose COPD patients. The fact that four out of six collagen degradation markers were able to detect even a mild form of IPF is interesting, since it is this patient group that will benefit the most from a diagnosis. These preliminary findings need to be validated in larger clinical settings. This study, although the first and small, has identified collagen turnover markers with the potential to separate healthy individuals from COPD and IPF patients. In future these markers may assist in the identification of patients that are fast progressers and may respond to a given intervention.
Endopeptidases play a major role in the degradation of ECM proteins such as collagens and proteoglycans.26,37,38
MMP-2 and -9, in particular, have been shown to be highly up-regulated in connective tissue diseases leading to fibrosis.39–41
A wide range of ADAMs/ADAMTSs are expressed in the lung42
and many have been associated with different respiratory diseases such as COPD, IPF and asthma.20
COPD has been coupled with changes in ADAM-33, ADAM-17, and ADAMTS-4 expression,43–45
while only a weak association has been observed for a few ADAMs/ADAMTSs in IPF.46
The C3A marker showed elevated levels of ADAMTS-4-mediated degradation of type III collagen in COPD but not IPF patients. This finding is in agreement with previous experiments showing that ADAMTS-4 is up-regulated in COPD while there is no evidence of a relationship in IPF.45,46
This highlights that the differences in the pathological processes and the proteases involved in these two diseases result in distinct tissue turnover profiles.
Fibrotic lungs have an overall increased ECM turnover rate, but the normal balance between formation and degradation is changed, leading to increased deposition and increased degradation. The change in remodeling balance is not the same for different types of collagen; a key feature is the increased deposition and degradation of type I collagen in pulmonary fibrosis.4,5,47
This is in line with our C1M results showing significantly elevated levels in both patients with mild to severe IPF and mild COPD and thus indicating an increased degradation of type I collagen. Contradictory results have been published in relation to type III collagen deposition in pulmonary fibrosis, demonstrating that both up—and down-regulation of type III collagen occurs.4–6
The serum C3M data presented here suggest an increased type III collagen degradation level in both IPF and mild COPD, indicating a high level of formation of type III collagen as well. Indications of a decreased content of type V collagen in fibrotic rat lungs have been presented.48
However, Parra et al demonstrated that type V collagen levels in biopsies from IPF patients increased with disease severity,49
supporting an increased degradation of deposited type V collagen indicated by our C5M data. Type IV and type VI collagen expression, as well as protein levels, have been reported to be elevated in lung tissue of patients with COPD50
further supporting the findings of our biochemical marker study.
Desmosine, isodesmosine, KL-6, CCL18, SP-A and SP-D have all been extensively discussed as serological markers of pulmonary diseases.17,21–23
However, none of these markers have demonstrated optimal sensitivity and diagnostic value. Thus, more sensitive and accurate biochemical markers are needed for fibrotic lung diseases. Stratification of patients with pulmonary fibrosis is most likely not feasible using a single marker since would require a marker specific for a protein only expressed during lung fibrosis. A panel of markers reflecting different pathophysiological processes involved in pulmonary fibrosis during the two different diseases will almost certainly be required for diagnosis, prognosis and assessment of the efficacy of interventions.
The systemic level of a biochemical marker is the sum of all tissue sites generating this one fragment, and also depends on the extent of disease- affected tissue, the aggressiveness of the disease and the protein specificity of the fragment. This was elegantly investigated by Meulenbelt et al52
who demonstrated that the level of a MMP-generated fragment of the signature protein of cartilage, type II collagen, was correlated to the number of affected joints in osteoarthritis. Collagen expression is not restricted to the lung tissue, but is found ubiquitously throughout the body. Thus, several co-morbidities may influence the systemic level of fragments produced by MMP degradation of collagen molecules. Further investigations are needed to determine the individual contribution of different tissues to the total pool of collagen neoepitopes.
There are several limitations with the current study. The sample size was very small, and thus the findings are preliminary. The lack of information available for patients does not allow for further analysis and correlation with clinical parameters. Furthermore, this is a cross-sectional study and the prognostic value of the biomarkers could not be validated.
In conclusion, by using protein fingerprint technology, we have developed assays measuring novel biochemical markers which enable us to assess peptides generated during degradation of collagens in pulmonary fibrosis. These markers were able to distinguish between healthy controls and patients with mild COPD and/or IPF in a small clinical population. The collagen degradation markers demonstrated promising discriminative diagnostic power and may provide an improved tool for identification of those patients most in need of treatment, as well as for monitoring potential efficacy of interventions. These data need to be validated in larger clinical settings.