Date Published: January 11, 2017
Publisher: Public Library of Science
Author(s): Daniel Guldager Kring Rasmussen, Jannie Marie Bülow Sand, Morten Asser Karsdal, Federica Genovese, Matthew Bogyo.
A high level of extracellular matrix (ECM) turnover characterizes several lung diseases with fibrotic features. Type III collagen is one of the most abundant collagens in lung parenchyma, and cathepsins play a role in lung pathology, being responsible for tissue remodeling. In this study, we explore the diagnostic features of neo-epitope fragments of type III collagen generated by cathepsins that could reflect the pathological tissue turnover in patients with different diseases. A novel enzyme-linked immunosorbent assay (ELISA) measuring cathepsins B, L, S and K -generated type III collagen fragments (C3C) was developed for assessment in serum and plasma. The assay was biologically validated in serum from patients with chronic obstructive pulmonary disease (COPD). Serological levels of C3C were significantly elevated in patients with COPD compared to healthy controls (p = 0.0006). Levels of C3C in serum and heparin plasma of COPD patients had a highly significant correlation (R2 = 0.86, p<0.0001). The data suggests that the C3C fragment is elevated in patients with COPD compared to healthy controls.
Fibrillar collagens, such as type I and III collagen, are some of the most prominent collagens in the extracellular matrix (ECM) of the lung [1–3]. Fibrillar collagens provide tensile strength which enables enlargement of the lung components such as the alveoli, vessels, and connective tissue sheaths. An important requirement for balanced remodeling of the ECM is a tight control of enzymes involved in its turnover. Fibrotic diseases have an imbalance between formation and degradation which leads to an altered composition of the ECM thereby causing an abnormal tissue function [4,5]. Plenty of studies have presented findings suggesting the involvement of cathepsins in tissue turnover in various organs, such as the lungs. As an example for pathologies of the lung, cathepsins are upregulated in patients with chronic obstructive pulmonary disease (COPD)[6,7], idiopathic pulmonary fibrosis (IPF)  and lung cancer [9–11]. Some studies suggested that cathepsins B and K are implicated in the mechanisms leading to invasion of lung adenocarcinomas [10,12]. In patients with COPD, cathepsin S and L were shown to be involved in degradation of the extracellular matrix mediated by macrophages [13,14]. Structural changes resembling COPD were seen in two transgenic mouse strains overexpressing either IL-13 or IFN- γ [15,16]. In these models cathepsins B, L, S, and K were upregulated and caused lung epithelial apoptosis, lung inflammation, and airspace enlargement [15,16]. In an attempt to assess dynamic turnover of the extracellular matrix by cathepsins, we developed a novel neo-epitope assay, which utilizes a monoclonal antibody targeting a specific neo-epitope of type III collagen generated by cathepsins B, L, S and K and measured the levels of this fragment in samples from patients with different lung pathologies.
We developed and characterized the novel competitive ELISA C3C using a monoclonal antibody detecting neo-epitope fragments of type III collagen generated by the proteases cathepsins B, L, S and K. The C3C assay specifically recognized the human sequence GLPGTGGPPG, from residue 642 towards 651, and was able to measure fragments containing this sequence in human serum and plasma. The assay was robust and all technical parameters were accepted except when abnormally high levels of biotin were present. The biological relevance of this particular neo-epitope was confirmed in samples from a previously described cohort of COPD patients. Our data suggest that there is an elevated level of cathepsin B, L, S or K activity and/or an upregulated type III collagen production in patients with COPD giving rise to a higher level of type III collagen fragments containing the C3C neo-epitope.