MMP mediated type V collagen degradation (C5M) is elevated in ankylosing spondylitis

Abstract

Objectives

Type V collagen has been demonstrated to control fibril formation. The aim of this study was to develop an ELISA capable of detecting a fragment of type V collagen generated by MMP-2/9 and to evaluate the assay as biomarker for ankylosing spondylitis (AS).

Design and methods

A fragment unique to type V collagen and generated by both MMP-2/9 cleaved at the amino acid position 1317 (C5M) was selected for ELISA development. 40 AS patients and 40 age-matched controls were evaluated.

Results

An ELISA detecting C5M with inter- and intra-assay variations of 9.1% and 4.4% was developed. C5M levels were significantly higher in AS patients compared to controls, 229% (p < 0.0001). The diagnostic AUC was 83%.

Conclusions

This ELISA is the first for detecting type V collagen degradation. AS patients had highly elevated levels of MMP mediated type V collagen degradation. The prognostic and diagnostic values need to be further investigated in additional clinical settings.

Introduction

Ankylosing spondylitis (AS) is a form of chronic inflammation of the spine and the sacroiliac joints. Over time, chronic inflammation of the spine can lead to a complete cementation of the vertebrae [1], a process with a molecular pathology that still remains to be investigated and fully understood. AS is also a systemic disease, affecting other tissues throughout the body including inflammation in or injury to other joints, as well as to organs, such as eyes, heart, lungs, and kidneys [2], [3]. The inflammation related processes in AS involving multiple tissues seems to involve molecular processes including increased MMP activity and collagen deposition seen in most types of connective tissue turnover.

Extracellular matrix (ECM) remodeling is a key process of tissue homeostasis [4], [5], [6], [7]. Specific proteolytic activities are a prerequisite for a range of cellular functions and interactions within the ECM [8]. These specific activities are precisely coordinated under normal physiological situations, with a specified sequence of events resulting in controlled tissue turnover. In pathological situations, with special emphasis on connective tissue diseases, the normal repair–response relationship is disturbed [9], leading to excessive remodeling and tissue turnover. The consequence of this ECM remodeling is the release of a range of degradation products of proteins, neoepitopes, generated by the proteases expressed locally in the pathologically affected area. These protein degradation fragments may serve as molecular biomarker targets, as they are more specific for the tissue of origin compared to their intact origins [10]. This novel class of biochemical markers, referred to as protein fingerprints [11], measuring protein degradation or protein modifications rather than intact proteins, may provide additional clinical information. Recently several different protein degradation or formation markers have been developed [12], [13], [14], [15], [16], [17], suggesting the emerging protein fingerprint technology of importance for pathological accuracy.

Endopeptidases, such as matrix metalloproteinases (MMPs), play a major role in the degradation of ECM proteins such as collagens and proteoglycans [18], [19], [20]. In particular, in connective tissue diseases such as fibrosis, MMP-2 and -9 have been shown to be highly up-regulated [21], [22], [23]. Recently, neoepitope-based biochemical markers found in urine and serum have received increased attention for their diagnostic and prognostic potential [10]. In particular for slow progressing diseases, such as osteoporosis and osteoarthritis, bone resorption and cartilage degradation markers, based on type I and II collagen degradation products (protein fingerprints) respectively, have been studied extensively [24].

Type V collagen has been documented to be critically important for the formation of collagen fibrils [25], exemplified by an almost virtual lack of collagen fibril formation in the COL5A1^−/− mice. In alignment, the heterozygous mice were associated with a 50% reduction in fibril number and dermal collagen content. This indicates a central role for type V collagen in the life dependent regulation of fibrillogenesis, suggesting this collagen type to be of pivotal interest in many connective diseases. However, there is still a conceptual lack of understanding of the role of type V collagen turnover in connective diseases that may in part be due to the technical inabilities for investigation of type V collagen degradation and turnover. Interestingly, very recently a limited diverse set of proteins was found to bind type V collagen, beginning to elucidate the molecular function of this molecule in more details, of which MMP-2 was one of them [26]. In addition to the molecular characterization, more evidence is emerging towards the fact that type V collagen directly affects different cellular phenotypes by inducing dynamic motility and other cellular activities, suggesting that this protein may be more than a passive component of the ECM [27], [28]. In direct support of this, we recently described a very strong correlation to liver fibrosis with the formation of type V collagen in two separate animal models of liver fibrosis [16], suggesting a central role of type V collagen formation in excessive tissue turnover.

Type V collagen is a fibril-forming collagen, together with types I, II, III and XI [29], and is formed as heterotrimers of three different α-chains (α1, α2, α3). It typically forms heterofibrils with type I and III collagens and contributes to the organic bone matrix, corneal stroma and the interstitial matrix of muscles, liver, lungs and placenta [30]. Type V collagen mutation results in a range of connective tissue diseases, of which the Ehlers–Danlos syndrome (EDS) was the best described. EDS is a heterogeneous group of heritable disorders characterized by joint hypermobility and skin changes (e.g., hyperextensibility, thinness and fragility). The disease can be divided into different subtypes, EDS types I and II. EDS types I and II are characterized by atrophic scars, skin hyperextensibility and joint laxity. It is evident that both subtypes result from mutations in the COL5A1 and COL5A2 genes that encode two of the polypeptide chains of type V collagen [25], [31], [32], [33]. This highlights the importance of type V collagen in connective tissue diseases.

The aims of this study were 1) to develop an enzyme-linked immunosorbent assay (ELISA) capable of detecting a fragment of type V collagen generated by MMP-2 and MMP-9 and 2) to evaluate the use of this assay as biomarker for AS.