This study provides the following important information on the diagnosis and progression of important lung diseases:
Elastin is degraded by different proteases at different times. This degradation pattern adds to information already described by others
[34–36]. We selected a specific neoepitope as a candidate novel biomarker of lung disease, and developed an ELISA (ELM) for quantifying this unique target. To our knowledge, this assay is the first to quantify MMP degradation of elastin, in both in vitro generated material and in human fluids. This tool may provide value for other researchers and for the characterization of patients.
The ELN-441 neoepitope was generated by MMP-9 and -12 in a time dependent manner for soluble elastin, while in the case of insoluble elastin, only MMP-12 was able to generate the fragment.
The selected neoepitope of elastin was based on our MS analysis cleavage pattern by MMP-12, which is the protease known to be highly expressed in macrophages during lung inflammation.
By preliminary analysis in a limited number of patients, the ELM degradation marker exhibited highly specific diagnostic power, in particular for COPD with an AUC over 97%, and IPF with an AUC over 90%.
This study showed that elastin was degradable by MMPs, cathepsins as well as aggrecanases. The bulk of the identified peptides from in vitro cleaved soluble elastin was however generated by aggrecanases. This is an important observation as a recent publication highlighted the aggrecanases as important molecules in lung diseases
, and the fact that different proteases have different molecular characteristics
. It is well appreciated in cartilage pathologies that aggrecanase and MMP mediated cartilage destruction provide different molecular information
. Due to the sensitivity of the MS–technology, we identified elastin in the non-proteolytical fraction that was degraded in vitro. Whether this may indicate hot-spots for protein degradation, instability or artifacts during the purification procedure remains to be investigated. Interestingly, different numbers and fragments of identified peptides were obtained in the three different MS-laboratories. This may reflect different equipment and emphasizes the fragility of the current approach, and the necessity for cross-validation by multiple runs of the identified fragments in different cleavages by different equipments.
Although elastin fragments generated by aggrecanases and cathepsins were identified and may serve as biomarker targets for other indications, our study focused on the activity of MMP-9 and -12 because these proteases are expressed in respiratory diseases
[15, 16]. To some extent the two MMPs have a similar degradation profile, cleaving at many of the same sites, although some unique sites were also identified. Measuring the release of ELN-441 from elastin-rich tissues emphasized that only MMP-12, and not MMP-9, is able to degrade elastin from insoluble lung and that MMP-12 is faster to degrade elastin from aorta than MMP-9.
Of the elastin cleavages, 73% involved alanine, valine and glycine, of which glycine was predominant. This was as expected, alanine, valine and glycine compose 3 out of the four main amino acids in elastin. The fourth amino acid is proline. Alanine, valine and glycine are the amino acids with the shortest molecular chain leading to the smallest steric hindrance and probably easy accessible. This might be the reason for the reduced cleavage at proline, since it contains a pyrrolidin ring. Half of the amino acids making up elastin are hydrophobic which matches the outcome that half of the amino acids involved in the cleavage of elastin are hydrophobic. Interestingly the majority of the cleavages of the hydrophobic amino acids took place at the NH2-group of the amino acid. The opposite was observed for the hydrophilic amino acids in which the COOH-group was the preferred cleavage site. Cleavage sites involving glycine specially glycine-valine and glycine-glycine are not protease specific since aggrecanases, cathepsins and MMPs recognize these sites. Aggrecanases differ from the other proteases by degrading elastin at different cleavage sites such as between leucine-alanine and arginine-phenylalanine. Aggrecanase generated neoepitopes may therefore have a different diagnostic profile than for example MMPs. The cleavage products are dependent on incubation time, amount of protease and the stability of the peptide, as observed by others
Elastin degradation has been investigated by several groups
[14, 41–45] conducting analyses of the cleavage pattern and of proteases involved as a consequence of inflammation and macrophage activity. When analyzing the MMP-12 degradation of tropoelastin Taddese et al. and Heinz et al. both identified the ELN-441 fragment
[34, 36]. Barroso et al. did not identify ELN-441, but observed that the amount of degradation peptides is highly related to the amount of protease
. Furthermore, it has been shown that elastin degradation fragments, in particular a MMP-12 generated repeated sequence fragment, acts as a chemo-attractant for monocytes and fibroblasts in vitro
[41, 42] and that autoimmune response to elastin fragments has been identified
A battery of proteases, in particular MMPs, has been shown to be important mediators in lung disease. MMP and neutrophil elastase expression was investigated in patients with COPD and healthy controls using bronchoalveolar lavage fluid to analyse macrophage expression of the different MMPs
. It was found that MMP-9, MMP-8, and MMP-1 along with neutrophil elastase were significantly increased in COPD patients compared with healthy controls. Another group also showed that MMP-12 is necessary for macrophage recruitment in the lungs of smoke-exposed mice since MMP-12 knockout mice failed to develop inflammation in response to cigarette smoke
. Interestingly, a study investigated the ability of human and mouse monocyte-derived macrophages to degrade elastin ex vivo, concluded that MMP-12 may not be an elastolytic enzyme but is rather an inducer of an unknown pathway that activates elastin-degrading enzymes
. These data are in contrast to our in vitro data clearly showing that insoluble elastin may be degraded by MMP-12 but not MMP-9. The role of MMP-9 in cigarette smoke-induced COPD was investigated in study including MMP-9 over-expressing mice, MMP-9 knockout mice, and in patients that had undergone lung transplantation
. Data showed that MMP-9 expression was not correlated to severity of disease, albeit in the mouse models an integrated part of the disease. Our findings show that MMP-9 was not able to generate the ELN-441 fragment from insoluble elastin, when assessed using the ELM assay.
In the present study we identified the ELN-441 biomarker as diagnostically sensitive for COPD and IPF, as compared to controls. This suggests that the hypothesis stating that lung destruction is driven by MMP-9 and MMP-12 is valid and can be quantified. The diagnostic power of ELN-441 was higher for COPD than IPF, which is in accordance with the recent research in the field of MMPs and elastin in COPD, and that the main pathologic problem in IPF might be pulmonary fibrosis and not elastin degradation. However, one complicating factor in the use of ELN-441 is that elastin expression is not restricted to the lung tissues, as arteries, skin and tendons have been shown to express this protein
. Thus, several co-morbidities may influence the systemic levels of ELN-441. Further investigations are needed to determine each tissue’s contribution to the total pool of the ELN-441 neoepitope, and possibly other ELN epitopes.
One major limitation of the current clinical study of ELN-441 was the relatively small sample size and the limited clinical information obtained. Thus these preliminary findings need to be validated in larger clinical settings for the diagnostic utility, and also for prognostic potential.
Other researchers have investigated an array of biomarkers in induced sputum, exhaled air condensate, bronchial biopsy, bronchoalveolar lavage fluid, urine and peripheral blood that could be used as diagnostic and prognostic tools for lung diseases
. There is, however, a relative lack of information about how these biomarkers relate to disease severity and to other disease measurements such as FEV1, how reproducible they are, and how they may be affected by therapies. Desmosine and isodesmosine have been extensively discussed as biomarkers of elastin turnover since they are unique to human elastin. ELISA measurements of desmosine and isodesmosine in serum have, however, been shown to be incapable of discriminating between normal and COPD subjects
. Others have investigated serological biomarkers of the elastin-derived peptides (EDPs), which have been found elevated in plasma of patients with COPD, but it is unclear whether these peptides reflect elastin turnover in the lung or in other compartments of the body such as the arteries
[5, 10]. Nevertheless, a correlation between EDPs and lung damage on computed tomographic scans has been shown
. EDPs are detected with use of polyclonal antibodies making the method less sensitive than assays using monoclonal antibodies. Several other serological biomarkers have been investigated such as; fibrinogen, C-reactive protein, Trolox equivalent antioxidant capacity, CXCR2, TGF-beta, TNF-α
 and Clara cell secretory protein-16
. Of these, only C-reactive protein and TNF-α showed a relationship with FEV1-based disease staging criteria of COPD in a meta-analysis by Franciosi et al.
. However the separation was small, demonstrating poor sensitivity and diagnostic potential. Ultimately, a panel of biomarkers may be needed to characterize different aspects of lung disease in patients, and for prognosis, diagnosis and assessment of efficacy of intervention.
The neoepitope technology, measuring of specific protein degradation fragments, allows for assessment of specific proteolytic activity in given tissues, provided that the sequence is unique for one or fewer proteases. The present assay quantifies the peptide in elastin which is cleaved at the 441 position, and not the elongated peptide containing an extra amino acid at the cleavage site, nor intact elastin. Thus, this assay allows for quantification of one specific sub-pool of the elastin molecule—namely the soluble, degraded one. The sequence was MMP specific, whereas other fragments identified seems to be specific for aggrecanases and cathepsins. Other assays will have to be developed to allow the quantification of these epitopes, providing different biological or pathological information.
In conclusion, a robust assay has been developed using a specific monoclonal antibody for detection of ELN-441, a MMP-9 and -12 generated fragment of elastin. It was demonstrated that this fragment was significantly elevated in COPD and IPF patients and has high diagnostic potential. Further, larger, clinical studies are needed to confirm the diagnostic value and also to evaluate the prognostic potential in lung disease, and the potential utility of this neoepitope in other diseases in which elastin degradation may be a pivotal pathological feature.