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Biomarkers of cartilage and surrounding joint tissue

The identification and clinical demonstration of efficacy and safety of osteo- and chondro-protective drugs are met with certain difficulties. During the last few decades, the pharmaceutical industry has, in the field of rheumatology, experienced disappointments associated with the development of disease modification. Today, the vast amount of patients suffering from serious, chronic joint diseases can only be offered treatments aimed at improving symptoms, such as pain and acute inflammation, and are not aimed at protecting the joint tissue. This huge, unmet medical need has been the driver behind the development of improved analytical techniques allowing better and more efficient clinical trial design, implementation and analysis. With this review, we aim to provide a brief and general overview of biochemical markers of joint tissue, with special focus on neoepitopes. Furthermore, we highlight recent studies applying biochemical markers in joint degenerative diseases. These disorders, including osteoarthritis, rheumatoid arthritis and spondyloarthropathies, are the most predominant disorders in Europe and the USA, and have enormous socioeconomical impact.

Anne S Siebuhr1, Yi He1, Natasja S Gudmann1, Aurelie Gram1, Cecilie F Kjelgaard-Petersen1, Per Qvist1, Morten A Karsdal1 & Anne C Bay-Jensen*,1 Nordic Bioscience, Biomarkers & Research, Herlev Hovedgade 207, Herlev DK-2730, Denmark *Author for correspondence: acbj@ nordicbioscience.com 1

Keywords:  arthritis • biochemical marker • collagen • extracellular matrix • neoepitope • rheumatic disease • spondyloarthropathy • synovial joint

Medical need for biomarkers in disease profiling & drug development According to the US FDA, biomarkers should, to a higher degree, be implemented in drug development and clinical trials to improve decision-making on dosing, treatment time, trial design options, risk/benefit and transfer knowledge to new labels (i.e., application). Less than 10% of all drug candidates that enter preclinical studies will complete clinical development and be submitted for regulatory approval. The reason for the low number pass-through drugs is that many drug candidates fail due to toxicity issues either in preclinical or clinical phases. By implementing biomarkers for screening of drug candidates at earlier time points (e.g., in vitro and/or preclinically), potential safety issues can be addressed in advance and hence increase the drug development efficiency, and possibly

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reduce the overall cost. In addition, screening using biomarkers could facilitate the identification of drug candidates that would be excluded from the clinical trials due to safety concerns in preclinical studies. Further­more, safety and efficacy issues could be disclosed earlier [1] . Different types of biomarkers are needed for different stages of drug development: biomarkers that describe disease progression, which correlate with known clinical indices (e.g., a gold standard); biomarkers of efficacy in both known and unknown biological mechanisms associated with clinical outcome; and biomarkers that are predictive of clinical outcome (surrogate end point). On the basis of established biomarkers – that is, markers that reflect different physiological or metabolic processes, subjects in preclinical or clinical trial can be characterized and profiled, and the timeline of treatment can be

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Review  Siebuhr, He, Gudmann et al. assessed. Normally, a clinical end point is established on the basis of the test subject’s health status, functionality and survival. With the use of biomarkers, a surrogate end point can be set, which ideally can predict the response to a drug (e.g., benefits and/or side effects). Furthermore, to facilitate drug development, biomarkers can be used for monitoring whether a new candidate drug hits its target, alters biological mechanisms and affects the disease in question. At development phase, the drug candidate can be excluded if there is no effect or it can be used for identification of additional targets, if it had other than the expected effect (labeling extension) [2] . Moreover, the ability of a biomarker to predict the preclinical effects may enable more drugs to enter clinical trials and lead to d­evelopment of valuable pharmaceuticals. The primary objective of using biomarkers in drug development and patient managment is to provide the best evidence for rejecting the null hypothesis of no treatment effect and thereby demonstrate the efficacy and safety of a drug. Since biomarkers are considered as objective measures of biological events of pathological or pharmacological events, the probability of type I (false positive) and type II (false negative) errors become lower. At present, neoepitope biomarkers can to some degree identify patients in the general population; however, focus should be put into the study of the heterogeneity of the disease population because the number of type I and II errors is still high in ­clinical  trials. Joint tissue biology: in health & sickness Cartilage matrix

Healthy cartilage is composed of four zones; the superficial zone, transitional zone, deep zone and calcified zone. The superficial zone has a fine network of collagen fibrils and small, almost inactive, elongated chondrocytes (the only cell type in cartilage) in parallel to the articular surface. The transitional zone contains round active chondrocytes and has larger collagen fibrils starting a transition from parallel to columnar orientation. The complete columnar orientation is reached in the deep zone [3] . The calcified zone, located immediately below the tidemark, works as a mechanical buffer between the uncalcified cartilage and s­ubchondral bone [4] . The major proteins of the extracellular matrix (ECM) of cartilage are aggrecan and type II collagen, along with small amounts of type IX, X and XI collagens [3,5] . Aggrecan is a proteoclycan with highly polarized glycosaminoglycan side chains that increase the osmolarity of the cartilage. Consequently, approximately 75% of cartilage consists of water, which provides protection from loading pressure. The collagen

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network’s primary function is to ensure that aggrecan is kept in place [6] . Chondrocytes are sparsely distributed and responsible for maintaining homeostasis in the ECM since there is no innervation or v­ascularization in the cartilage [3,5] . In the early stages of degenerative joint disease, the cartilage loses the superficial zone as well as the organized structure. This leads to fibrillation as the disease progresses. As for the chondrocytes, they start to proliferate and become hypertrophic before they turn into apoptotic cells (the same pattern is seen in endochondral ossification) [6,7] . In the hypertrophic state, the cells secrete proteins such as matrix metalloproteinase (MMP)-13, which degrade collagen and VEGF, which start neovascularization. Chondrocytes have also been associated with secretion of type X collagen [8,9] . In the pathogenesis, aggrecan is degraded by A disintegrin and metalloproteinase with thrombospondin motifs 4 and 5, thereby compromising the ECM [5] . Together, these factors contribute to the loss of cartilage leading to sclerosis and may in the earlier stages be responsible for edema by increasing the water content of the ­cartilage by as much as 6% [6,10] . Bone matrix

Bone is a dynamic tissue, which constantly changes in response to changes in load or metabolic demands [11] . During healthy physiological stages in adults, the turnover changes as bone resorption is replaced by bone formation in a process termed ‘remodeling’ [12] . Fatigued or damaged bone is in this way replaced and the metabolic demands of calcium and phosphorus are kept at homeostasis [13] . On a cellular level. bones contain osteoblasts, osteoclasts, bone lining cells and osteocytes. Osteoblasts are the bone-forming cells, whereas osteoclasts are in charge of bone resorption [14] . Bone-lining cells and osteocytes play different roles in controlling the bone turnover process [13,15–17] . The ECM is composed of organic matrix (20–40%), inorganic matrix (50–70%) and water (5–10%). The composition of the ECM is, together with the macroscopic shape, a determinant for the strength and elasticity of the bone. The inorganic matrix is made up of hydroxyapatite crystals consisting of calcium and phosphates [18] . In total, 90% of the organic part of the bone matrix is composed of collagen type I. The remaining 10% is a mixture of glycoproteins such as proteoglycans, osteonectin and other proteins (osteocalcin and osteopontin). Type I collagen is a heterotrimeric molecule consisting of two α1 chains and one α2 chain that constitute highly organized fibrils, which gives a scaffold with tensile strength for osteoblasts to deposit minerals [19] . Differentiation and activation of osteoclasts are exceedingly dependent on the receptor activator

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Biomarkers of cartilage & surrounding joint tissue 

NF-κB ligand (RANKL)/RANK system [20] . RANKL was originally identified to be produced by activated T cells, but it can also be expressed in osteoblasts and bone marrow stromal cells [21] . Another key factor in this system is osteoprotegerin (OPG), which specifically binds to RANKL and inhibits RANKL activity by preventing its binding to its receptor RANK [20,22–24] . In pathological stages, the RANKL/OPG ratio may change [25] . An increase of RANKL and consequently a higher level of bone resorption will occur as a consequence of either hormonal change or as a side effect of medication (e.g., glucocorticoids); both are common causes of osteoporosis [26–28] . The increase may also be induced by inflammatory cytokines (e.g., TNF-α, IL-1 and IL-6) as in case of rheumatoid arthritis (RA) [29] , as well as in other inflammatory diseases [30–32] . In other diseases, bone turnover favors bone formation as seen locally in osteoarthritis (OA)-affected joints, when the disease progresses [33] . This way, the subchondral bone t­hickens and becomes sclerotic [34] . Connective tissue of the joint

Several connective tissues are connected to the synovial joint: tendon, ligament, joint capsule and synovial membrane. This review will only cover the synovial membrane as it is the most studied within degenerative joint diseases. The synovial membrane is a thin mesenchymal structure that encloses the joint cavity and consists of two distinctively different layers; the intimae and subintimae [35] . The intimae is a uniform layer that is two to four cell layers thick and consists of synovial fibroblast and synovial macrophages, but increases to 10–12 cell layers with arthritis. The cells of the intimae are imbedded in fine fibrillar ultrastructure of type III, IV, V and VI collagen, and little type I collagen. The subintimae is the actual physical membrane and the supportive stroma for the intimae layer [35,36] . It is relatively rich in type I collagen and contains vessels, but it is relative acellular, featuring synovial fibroblasts and few infiltrating cells. With inflammatory arthritis, infiltration of immune cells leads to formation of inflammation of the synovial membrane – that is, synovitis, which further leads to massive changes within the synovial membrane and surrounding tissues. The increased number of cells is accompanied by hyperplasia of the lining cells, which is identified as thickening of the membrane. This thickening is more pronounced in RA than OA [37,38] . Patients with inflammatory joint diseases have increased levels of TNF-α and IL-1β in the synovial fluid, mainly produced by the synovial macrophages [39,40] . These cytokines stimulate synovial fibroblasts to produce proteases and additional proinflammatory cytokines [41] . Elevated levels of MMP-1, MMP-3 and

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MMP-10 increases the invasive potential of the synovial fibroblast [42] , which enables pannus to invade the articular cartilage [38] . Furthermore, the synovial membrane is highly vascularized with inflammatory arthritis compared with healthy individuals, which is a result of increased angiogenic activity related to increased levels of VEGF [43,44] . Biochemical markers of joint diseases Several biomarkers are available for joint degenerative diseases; some have been validated in several clinical trials, whereas others yet need to be investigated further. Tables 1–4 provide an overview of biochemical markers, which have a neoepitope origin. Neoepitopes are epitopes that are derived by specific pathological processes. The best described neoepitopes are those that are generated by proteolytical action on the ECM (Figure 1) . Proinflammatory cytokines and factors are often the main drivers of degenerative connective tissue diseases, such as OA and RA. The measurement of neoepitopes can be described as protein fingerprinting because these signals are derived through specific pathological processes [45] . Rheumatoid arthritis

RA is one of the most common autoimmune diseases and is estimated to affect 0.5–1% of the world population [141] . It will usually target diarthrodial joints [142] , where the synovial membrane becomes inflamed and will potentially invade and destruct articular cartilage and the underlying bone [141] , leading to joint damage and impaired function [143–145] . During the last decade, there has been increased focus on identifying and treating RA early to prevent joint disability. Thus, there is need for the development of more sensitive and specific diagnostic and prognostic tests in order to select the right treatment for the right patient [146] . So far, imaging techniques provide a diagnostic tool and are traditionally applied to view damages that have already occurred. Traditional parameters of inflammation such as erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) are usually evaluated, but it is not specific for the disease [118] . Alternative options are therefore explored. This section aims to give an overview of different biochemical markers, which are used to determine disease stage, diagnosis and ­prediction of progression of RA. CRP is, as mentioned, widely used as a marker of inflammation. This gives an estimate of the cytokine production in RA and there has been a documented link between high levels of CRP and increased risk of radiological progression [147] . However, some biologics such as tocilizumab (a humanized monoclonal antibody against the IL-6 receptor) has been found

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Table 1. Assays developed towards collagens. Target molecule Assay

Short description of assay

Type I collagen

CTX-I

Bone resorption; a sandwich ELISA using monoclonal antibodies F1103 and F12 both binding to a cathepsin K-derived C-telopeptide neoepitope, EKAHD-β-GGR, where D-β-G denotes an isomerized linkage between D and G

Ref.

Type I collagen

NTX-I

Bone resorption; EIA detecting a fragment of the N-telopeptide of type I collagen

[50]

Type I collagen

ICTP

MMP-mediated type I collagen-type degradation; RIA detecting a fragment of the C-telopeptide of type I collagen

[51,52]

Type I collagen

C1M

MMP-mediated type I collagen degradation; this MMP-generated neoepitope is degraded by cathepsins, indicating that this marker is a biomarker of connective tissue type I collagen

Type I collagen

P1NP

Collagen formation; type I collagen formation in tissues other than bone

Type II collagen

PIIANP

Collagen formation; PIIANP is an alternative RNA splicing form and can be expressed by adult articular chondrocytes in OA cartilage; a competitive polyclonal antibody-based ELISA recognizes the additional 69-aa cysteine-rich domain in the N-propeptide

Type II collagen

PIINP

Collagen formation; PIINP excludes extra 69-aa cysteine-rich domain in the N-propeptide and is expressed by normal adult articular chondrocytes

[58]

Type II collagen

PIINP

Collagen formation; a competitive ELISA based on a monoclonal antibody mF7504 recognizing GPQGPAGEQGPRGDR in the propeptide

[59]

Type II collagen

PIICP

Collagen formation; PIICP is a commercially available RIA

Type II collagen

9A4/5109 Cartilage degradation; the collagenase-derived neoepitope GEGAAGPSGAEGPPGPQG775 containing the carboxyl terminus of the long threequarter fragment; the monoclonal antibody 5109 detects the sequence ‘EGAAGPS’, while another monoclonal antibody 9A4 recognizes the sequence ‘GPPGPQG’

Type II collagen

CTX-II

Cartilage degradation; a competition ELISA using the monoclonal antibody F4601 recognizing the C-telopeptide neoepitope EKGPDP; however, van Spil et al. recently argued that urinary CTX-II had unique relations with bone markers compared with other cartilage markers and may reflect bone rather than cartilage metabolism

Type II collagen

TIINE

Cartilage degradation; an LC–MS/MS assay using the monoclonal antibody 5109 (see above) to affinity purify fragments subjected to MS/MS, detects a collagenase-derived 45-mer containing the carboxyl terminus of the long three-quarter fragment

[68,69]

Type II collagen

HELIX-II

Cartilage degradation; a competitive ELISA using polyclonal rabbit antibodies recognizing the neoepitope 622ERGETGPP*GTS632, where P* denotes hydroxyproline’ however, a recent publication has highlighted that this sequence does not occur in type II collagen, whereas type III collagen may be one of the candidate sources

[70,71]

Type II collagen

C2M

Cartilage degradation; a competitive ELISA measuring an metalloproteinase-derived type II collagen neoepitope

Type II collagen

Coll2-1

Cartilage degradation; immunoassays specific for a peptide of the α-helical region of type II collagen 108HRGYPGLDG116

[73–77]

Type II collagen

Coll2-1 NO2

Cartilage degradation; immunoassays specific for Coll-2 nitrated form 108HRGY(NO2) PGLDG116

[73–77]

Type II collagen

C2C

Cartilage degradation; EIA using a monoclonal antibody recognizing the carboxyl terminus of the three-quarter fragment of the degraded α1 (II) chain

[46–49]

[53]

[54] [55–57]

[60–62] [63]

[64–67]

[72]

[78,79]

aa: Amino acid; C1M: MMP-mediated degradation fragments of type I collagen; C2M: MMP-mediated degradation fragments of type II collagen; C3M: MMP-mediated degradation fragments of type III collagen; C4M: MMP-mediated degradation fragments of type IV collagen; C5M: MMP-mediated degradation fragments of type V collagen; C6M: MMP-mediated degradation fragments of type VI collagen; CTX-I: C-terminal telopeptide of type I collagen; CTX-II: C-terminal telopeptide of type II collagen; EIA: Enzyme immunoassay; HELIX-II: Type II collagen helical peptide; ICTP: Cross-linked carboxyterminal telopeptide of type I collagen; LC–MS/MS: Liquid chromatography–tandem mass spectrometry; MMP: Matrix metalloproteinase; MS/MS: Tandem mass spectrometry; NTX-I: N-telopeptide of type I collagen; OA: Osteoarthritis; P1NP: Procollagen type 1 N-terminal propeptide; P4NP 7S: N-terminal propeptides of type IV collagen 7S domain; PIIANP: N-terminal type IIA procollagen propeptide; PIICP: C-terminal propeptide of type II collagen; PIINP: N-terminal propeptide of type II collagen; RIA: Radioimmunoassay; TIINE: Collagen type II neoepitope.

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Biomarkers of cartilage & surrounding joint tissue 

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Table 1. Assays developed towards collagens (cont.). Target molecule Assay

Short description of assay

Type II collagen

Collagen degradation; EIA using a rabbit polyclonal antibody that bind to the C-terminal (COL2-3/4C [short]), a neoepitope generated by cleavage of native human type II collagen by collagenases; this assay has cross-reactivity to type I collagen

[80]

Type III collagen C3M

MMP-mediated type III collagen degradation; a competitive ELISA measuring an metalloproteinase-derived type II collagen neoepitope

[81]

Type III procollagen

PIIINP

Collagen type III formation; RIA using polyclonal antibodies recognizing the propeptide of collagen type III

[82]

Type III procollagen

Pro-C3

Collagen type III formation; a neoepitope-specific competitive ELISA towards the N-terminal propeptide of type III collagen

[83]

Type IV collagen P4NP 7S

Type IV collagen formation; a competitive ELISA measuring a propeptide of type II collagen neoepitope

[84]

Type IV collagen C4M

MMP-mediated type IV collagen degradation; a competitive ELISA measuring an metalloproteinase-derived type II collagen neoepitope

[85]

Type V collagen

MMP-mediated type V collagen degradation; a competitive ELISA measuring an metalloproteinase-derived type II collagen neoepitope

[86]

Cartilage degradation; MMP-mediated type VI collagen degradation; a competitive ELISA measuring an metalloproteinase-derived type II collagen neoepitope

[87]

C1, C2

C5M

Type VI collagen C6M

Ref.

aa: Amino acid; C1M: MMP-mediated degradation fragments of type I collagen; C2M: MMP-mediated degradation fragments of type II collagen; C3M: MMP-mediated degradation fragments of type III collagen; C4M: MMP-mediated degradation fragments of type IV collagen; C5M: MMP-mediated degradation fragments of type V collagen; C6M: MMP-mediated degradation fragments of type VI collagen; CTX-I: C-terminal telopeptide of type I collagen; CTX-II: C-terminal telopeptide of type II collagen; EIA: Enzyme immunoassay; HELIX-II: Type II collagen helical peptide; ICTP: Cross-linked carboxyterminal telopeptide of type I collagen; LC–MS/MS: Liquid chromatography–tandem mass spectrometry; MMP: Matrix metalloproteinase; MS/MS: Tandem mass spectrometry; NTX-I: N-telopeptide of type I collagen; OA: Osteoarthritis; P1NP: Procollagen type 1 N-terminal propeptide; P4NP 7S: N-terminal propeptides of type IV collagen 7S domain; PIIANP: N-terminal type IIA procollagen propeptide; PIICP: C-terminal propeptide of type II collagen; PIINP: N-terminal propeptide of type II collagen; RIA: Radioimmunoassay; TIINE: Collagen type II neoepitope.

to lower CRP to normal levels in almost all patients and the efficacy would be overrated if other parameters were not adjusted as well [148] . Rheumatoid factor (RF) is also widely used and established as a diagnostic marker. It is an autoantibody directed against the Fc region of human immunoglobulins. Deposits of RF associated with IgG occur in several tissues of RA patients including the synovium of the joints, where it promotes inflammation, resulting in tissue damages [149] . The accuracy of RF for diagnosing RA was determined to be 78% in a cohort study of 238 patients [150] . Another study included 9712 individuals of the general Danish population, who were followed for 17–19 years. From this cohort, it was revealed that elevated RF levels led to a 26-times higher long-term risk of developing RA and a 10-year absolute risk of 32% RA [151] . Studies support the correlation between the levels of RF and RA diagnosis as well as the prognosis of RA [152–154] . Alhough it should be noted that one major disadvantage with RF includes that it is relative unspecific for RA, as it is also associated with other autoimmune diseases [151] . RF may also be found in other infectious diseases, such as hepatitis [155] . Since there has been an established correlation between antibodies that recognize citrullinated (a

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specific type of post-translational modifications of proteins, where arginine is enzymatically converted to citrulline) proteins have become an attractive target for biomarkers of RA [156] , and the development of assays that measures these autoantibodies was initiated decades ago [157] . Anti-citrullinated protein antibodies (ACPAs) are likely to be involved in RA in approximately 70% of incidents and they are often detectable years before onset of the disease [158] . It has become standard procedure to classify patients into those who present ACPAs and those who do not. The clinical pictures are similar for both classifications in the early phases of the disease, but at follow-up time points, ACPA positive individuals have more swollen joints and more severe radiographic damages [159] . Several studies have found that urinary levels of C-terminal telopeptide of type II collagen (CTX-II) were shown to be correlated to joint erosions [160,161] . CTX-II has also revealed a predictive value in other clinical studies, when RA patients received either methotrexate (MTX) or etanercept (a fully-human, soluble TNF receptor fusion protein) [162] , in a cohort where conventional disease-modifying antirheumatic drugs were applied [118] and in a cohort where conventional disease-modifying antirheumatic drugs were applied

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Table 2. Assays developed towards extracellular matrix proteins that are not collagens. Extracellular matrix protein Biomarker

Description of the biomarker measure

Aggrecan

KS/OA-1

Cartilage degradation; sandwich ELISA using a monoclonal antibody to KS and a monoclonal antibody, OA-1, to the N-terminal sequence (ARGSVIL) generated by aggrecanase

Ref.

Aggrecan

CS846

Cartilage turnover; a monoclonal antibody, 846, detecting a epitope on chondroitin sulfate was applied for assay development. This assay may be confined to the most intact and recently synthesized aggrecan molecular

Aggrecan

342-G2

Cartilage degradation; sandwich ELISA using a monoclonal antibody, F-78, as capture antibody; AF-28 recognizes the FFGVG neoepitope generated by MMP cleavage

[95,96]

Aggrecan

G1/G2

Cartilage turnover; sandwich ELISA employing a monoclonal antibody F-78 both as a capturing and detecting antibody for detection of intact aggrecan; the ELISA can measure all aggrecan fragments carrying both G1 and G2

[95,96]

Aggrecan

NITEGE-373 Cartilage degradation; competitive ELISA using monoclonal antibody for detecting aggrecanase mediated aggrecan fragment carrying neoepitope NITEGE-373

[96]

Aggrecan

374-ARGSV Cartilage degradation; competitive ELISA using monoclonal antibody for detecting aggrecanase derived aggrecan fragment carrying neoepitope 374-ARGSV

[96]

Biglycan

BGM

Biglycan degradation; competitive ELISA using a neoepitope specific monoclonal antibody for measuring an MMP-9- and -12-mediated biglycan neoepitope

[97]

Cartilage oligomeric protein

COMP

Cartilage turnover; an inhibition ELISA assay using a monoclonal antibody against COMP; meanwhile, a commercial sandwich ELISA has been described using two monoclonal antibodies recognizes different antigenic determinants

Elastin

ELNM

ECM remodeling; MMP-mediated elastin degradation; a competitive ELISA named ‘ELN-441’, specifically recognizes MMP-9- and -12-generated elastin neoepitopes

Human glycoprotein 39/chondrex

High serum Cartilage turnover; detecting a 40-kDa fragment of glycoprotein 39 YKL-40 Commercial sandwich ELISA available

[88]

[89–94]

[98–103]

[104]

[105,106]

Hyaluronic acid/hyaluronan Serum HA

Cartilage turnover; hyaluronic acid can be measured by a competitive [107–109] ELISA, which is based on the aggregation of hyaluronate with cartilage proteoglycan monomers, followed by a binding of a monoclonal antibody to the KS on the proteoglycan

Matrilin-3

MATN3

Cartilage turnover; a competitive ELISA developed to measure MATN3 levels in synovial fluids

Osteocalcin

OC

Bone formation; numerous assays available

[110]

BGM: Matrix metalloproteinase-degraded biglycan; COMP: Cartilage oligomeric matrix protein; ECM: Extracellular matrix; HA: Hyluronen acid; KS: Keratan sulfate; MATN3: Matrilin 3; MMP: Matrix metalloproteinase; OC: Osteocalcin; PYD: Pyridinoline. [118] and in a cohort where patients were either receiving

prednisolone or MTX in combiantion with sulfasalazine or with sulfasalazine alone [162] . This suggests that the prediction of erosive changes can be applied independent of the subpopulation and treatment strategy. Levels of cartilage oligomeric matrix protein (COMP) have been found to be of relevance in RA as well. COMPs that reflect cartilage turnover have been found to be indicative of bone erosions in RA. Several studies have found that RA patients with a high level

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in serum correlates with detectable radiographic erosions [163,164] . Just as there has been found a positive correlation between COMP and the level of CRP and MMP-3 [163] . Markers of bone degradation are worth considering when evaluating the disease stage of RA. Cross-linked carboxyterminal telopeptide of type I collagen (ICTP) has been identified to be the most relevant measure for bone destruction in RA [165] . Bone degradation generated by osteoclast-induced MMP cleavage of type I

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Biomarkers of cartilage & surrounding joint tissue 

collagen [51] seems to be a dominant part of inflammatory bone diseases including RA [120] . Degradation markers that measure fragments degraded by cathepsin K during bone resorption include C-terminal telopeptide of type I collagen (CTX-I) and N-terminal telopeptide of the type I collagen (NTX) [166,51,167] . These markers are able to detect an increase of bone resorption even before structural changes become visible radiographically [168–170] . Whether they are applicable for RA is up for discussion. Some studies suggest that CTX-I provides predicative knowledge about the disease progression [162,171] , whereas other studies concluded that there was only weak or no association with CTX-I and prediction of radiographic progression [161] . MMP-mediated degradation fragments of type I collagen (C1M) is another marker of MMP-mediated type I collagen degradation. This neoepitope marker has shown to be a promising prognostic marker and a predictor of treatment efficacy in a placebo-controlled study of almost 600 patients treated with tocilizumab [172] . C1M is different from the other type I collagen markers, as it is not a measure of bone degradation, but a marker of connective tissue destruction. This is due to the C1M fragment being destroyed by catheptin K (secreted by osteoclasts during bone resorption). Other markers, which have been suggested to be of prognostic value in RA, although they are not specific for the disease, include: MMP-3 [118] , MMP-1 [173] , RANKL [174] , C2C (type II collagen degradation) [175] , C1,2C (type I and II collagen degradation)  [175] , tis-

sue inhibitor of metalloproteinases-1 (aggrecan) [175] .

[118]

Review

and CS846

Ankylosing spondylitis

Ankylosing spondylitis (AS) is characterized by inflammatory spinal pain and occasionally by peripheral joint and swelling of entheses. The current measure of disease progression is the Modified Stoke AS Spine Score and for the assessment of disease activity, the Ankylosing Spondylitis Disease Activity Score or Bath Ankylosing Spondylitis Disease Activity Index is used. However, no serological biomarker is routinely used in AS diagnosis. As described above, several biomarkers used in other joint diseases have been investigated in tAS, such as CRP and MMP-3. CRP has shown to be elevated with AS in a cohort analysis [176–178] and MMP-3 has shown to be associated with disease activity [179–183] and predicting responders to biologics [184] . CRP has furthermore been shown to be a biomarker of treatment efficacy in trials with biologics [132,179–180,184–185] . Acute phase reactants other than CRP have also been studied, such as serum amyloidP (SAP) and serum amyloid-A (SAA), where SAA has been found to be related to disease activity, indicating this as a valuable indicator of disease activity alongside MMP-3 [186] . Both SAP and SAA have been found to be possible markers of treatment efficacy in ­anti-TNF-α t­rials  [176,184] . Biomarkers of tissue destruction have been intensively investigated in AS, as many of the markers have

Table 3. Assays developed towards proteolytic enzymes. Matrix degrading enzyme

Biomarker

Description of the biomarker measure

Ref.

ADAMTS-4

acADAMTS-4

A competitive ELISA specifically measures the active form of the protease utilizing a monoclonal antibody toward the N-terminus of active ADAMTS-4 exposed after the prodomain is cleaved off 

[111]

MMP-1

MMP-1

Total MMP-1; numerous assays are available

[112–114]

MMP-3

MMP-3

Total MMP-3; numerous assays are available

[115]

MMP-8

MMP-8

Total MMP-8; numerous assays are available

[116]

MMP-9

MMP-9

Total MMP-9; numerous assays are available

MMP-13

MMP-13

Total MMP-13; indicates cartilage degradation and hypertrophy; numerous assays are available

[118,119]

Macrophage

TRAcP5a

Serum TRAcP5a has no relationship to bone metabolism, but is a measure of activated macrophages and chronic inflammation; biotinylated anti-TRACP antibodies were used to immobilize serum TRACP isoforms; TRACP activity was measured using 4-nitrophenyl phosphate as a substrate

[120,121]

Osteoclast

TRAcP5b

Osteoclast number; monoclonal antibody to TRAcP5b that is specific for osteoclasts, but not their activity

[117]

[120,122–124]

acADAMTS-4: Active A disintegrin and metalloproteinase with thrombospondin motif 4; ADAMTS-4: A disintegrin and metalloproteinase with thrombospondin motifs 4; MMP: Matrix metalloproteinase; TRACP: Tartrate-resistant acid phosphatase; TRAcP5a: Serum tartrate-resistant acid phosphatase 5a; TRAcP5b: Serum tartrate-resistant acid phosphatase 5b.

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Table 4. Assays developed towards molecules other than extracellular matrix proteins, proteases and proteins containing post-translational modifications. Modified protein

Biomarker

Description of the biomarker measure

Ref.

CCP

Anti-CCP

Cartilage turnover; ELISA for testing anti-CCP antibody; there are anti-CCP2 (generation 2) IgG2, anti-CCP3 IgG3 and anti-CCP3 IgA antibody

[125–128]

Citrullinated vimentin

VICM

Activated macrophages; a competitive ELISA using a monoclonal antibody against an MMP-derived and citrullinated vimentin neoeptiope

[129,130]

Deamidated cartilage oligomeric protein

D-COMP

Cartilage turnover; an ELISA was developed using a monoclonal antibody 6-1A12, which can recognize deamidated Asp64 COMP and show specificity for a particular joint site

Dickkopf-related protein 1

DKK-1

Regulator of the Wnt pathway; DKK-1 is one of the most studied Wnt inhibitor

C-creative protein

High-sensitivity CRP Acute inflammation; an assay to detect small (0.2–10 mg/l) changes in magnitude of inflammation

C-creative protein

CRPM

Chronic tissue inflammation; MMP-mediated CRP degradation, indicating chronic tissue inflammation

[131]

[132–137] [138,139] [140]

CCP: Cyclic citrullinated peptide; COMP: Cartilage oligomeric matrix protein; CRP: C-reactive protein; CRPM: Matrix metalloproteinase-mediated C-reactive protein; D-COMP: Deaminated COMP; DKK: Dickkopf-related protein 1; MMP: Matrix metalloproteinase; VICM: Citrullinated and matrix metalloproteinase-degraded vimentin.

shown usefulness in related arthritides: for bone turnover: CTX-I, Propeptide of type I collagen (PINP) and osteocalcin (OC); and for cartilage turnover: CTX-II and COMP. CTX-I is slightly elevated in AS compared with healthy [132,187] , and CTX-I and OC have shown to be associated with disease duration [188] , disease activity and treatment efficacy [184,189–190] . However, a single study found that CTX-I was not a marker of treatment efficacy [132] , illustrating that further investigation of the biomarker in relation to AS is needed. The association between CTX-I and OC indicates the skewed turnover of bone with AS. Bone formation has also been investigated with PINP in trials of anti-TNF-α treatments. PINP was increased after 3 months and until 2 years [184,189] , and was at baseline correlated to disease duration [188] . Moreover, the level of PINP and OC were lower in responders to treatment compared with nonresponders and PINP was a strong predictor of response after 14 weeks of treatment, with an odds ratio (OR) of 7.2 [184] . Investigation of biomarkers of cartilage turnover has found that CTX-II is associated with disease activity [181] and correlated with inflammation (CRP) [187] . It is debated whether CTX-II is associated with radiographic progression, as one study shows an association [187] and another shows no association [191] . C2M, a marker of MMP-mediated type II collagen degradation, was found to be of potential use as a prognostic and diagnostic marker in AS [129,192] . However, combining C2M with C3M and C6M increased the diagnostic value of C2M. Other MMP-mediated collagen

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degradation fragments, C1M, C4M and C5M, are elevated with AS [86,192] . COMP has shown to be correlated with inflammation [191] and radiographic damage at baseline [185] . Lastly, total aggrecan (CS846) has shown to be sensitive to change in disease activity and responders to treatment had an increased baseline of the marker [180] . Recent discoveries indicate that autoantibodies may be involved in AS pathology [129,193] . Bodnar et al. studied a small cohort of 43 patients and 44 healthy controls, and found that anti-mutated citrullinated vimentin was increased in AS patients compared with controls. However, only 37% was positive for antianti-mutated citrullinated vimentin [193] . In an unrelated study in our laboratory, a monoclonal antibody was used to detect citrullinated and MMP-degraded vimentin, and it was found that citrullinated and MMP-degraded vimentin may be of prognostic value in AS [129] . Psoriatic arthritis

Psoriatic arthritis (PsA) is a chronic and inflammatory arthritis occurring in a subgroup of psoriasis patients. The disease manifestations share many similarities with those found in RA. However, the most pronounced difference between PsA and RA is the bone formation in PsA that is absent in RA. Psoriasis affects approximately 2% of the adult population, but only 30% of these get PsA [194,195] . PsA is associated with inflammatory joint destruction as in RA, but the damage and disability is less pronounced or equal to the

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Biomarkers of cartilage & surrounding joint tissue 

damage and disability found in RA. Many of the biomarkers and treatments used in RA have been investigated in PsA, but the number of clinical studies of the effect of the treatments, based on quantification of soluble biomarkers, is limited. In a study of the TNF-α inhibitor therapy golimumab, MMP-3, CRP and SAP were significantly decreased at week 14 in patients receiving treatment compared with placebo. Moreover, baseline or 4-week levels of CRP and MMP-3 were prognostic of a good outcome of golimumab treatment as early as at week 14 [196] . In another small study of 24 patients, MMP-3 was significantly decreased (p < 0.005) with a fully human monoclonal antibody with a high specificity for TNF-α (adalimumab), and another TNF-α inhibitor [197] treatment in an immunological study of biopsies from the same 24 patients showed a strong correlation between reduction in MMP-3 and clinical improvement [198] . In addition, MMP-3 has shown the ability to distinguish PsA from psoriasis [199] . As PsA is an inflammatory disorder, CRP and ESR have been investigated as biomarkers of the disease. CRP has shown to be a biomarker that can differentiate between PsA and psoriasis even though CRP is elevated in both diseases [199] . Van Kuijk et al. have shown that CRP and ESR were significantly decreased (p < 0.003) with adalimumab treatment after only 4 weeks of treatment. However, MMP-3 was suggested to be superior as a biomarker compared with CRP and ESR, as it MMP-3 may reflect a change in tissue damage instead of systemic inflammation. Moreover, not all PsA patients have elevated CRP levels, making MMP-3 a global PsA biomarker [197] . Other biomarkers that have shown to differentiate between PsA and psoriasis include OPG and the CPII:C2C ratio. The increased level of OPG may indicate the presence of bone formation and the CPII:C2C ratio indicates increased cartilage turnover [199] . Other tissue-derived biomarkers have been studied in PsA to investigate the effect of treatment. None of the tissuespecific marker levels investigated (OC, PINP, NTX, ICTP, CPII, C2C or COMP) were altered in 4 or 12 weeks of adalimumab treatment, although there was a trend towards a decrease in NTX (p = 0.078) and CPII (p = 0.053) levels [197] . Chandran et al. have reported in a cross-sectional study a significant lowering (p = 0.036) of C2C in PsA patients on MTX tr­eatment [199] . Juvenile idiopathic arthritis

Juvenile idiopathic arthritis (JIA), also known as juvenile RA, is a disease affecting children. It is the most common form of arthritis in children and adolescents. The prevalence is approximately 0.1–0.2% and it

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Review

involves an inflammatory process that, if not treated, will eventually lead to bone and cartilage damage. Several studies have shown that inflammatory-related cytokines and proteases are elevated in JIA patients, such as TNF-α, IL-6, IL-12, MMP-3, MMP-1 and IL-17. Therefore, most studies of treatments for JIA are A

Proinflammatory molecules IL-17 IL-6

TNF

IL-1

TGF

PDGF Membrane

Receptor

Extracellular matrix degrading proteinases MMPs ADAMTs Cathepsins

ITAM, SYK, JAC, PI3, RAS and RAC, among others Intracellular signaling P38, P44, AKT and JNK, among others

DNA

B Extracellular matrix Proteolytical enzyme

Extracellular matrix molecule

End product is the protein fingerprint of the tissue

Figure 1. The inflammatory burden of elevated cytokines and growth factors leads to degradation of the extracellular matrix. (A) Connective tissue diseases are often regulated by proinflammatory cytokines, which initiate a downstream pathway ultimately leading to the expression and release of proteolytical enzymes, such a MMPs. (B) These enzymes degrade the extracellular matrix resulting in the release of protein fragments, which can be described as protein fingerprint and neoepitopes, because they are the end product of a given pathological event. ADAMT: A disintegrin and metalloproteinase with thrombospondin motif; MMP: Matrix metalloproteinase.

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Review  Siebuhr, He, Gudmann et al. based on treating the inflammatory component. Treatment efficacy studies therefore investigate the usage of biomarkers related to inflammation. Other bio­markers briefly investigated in JIA are COMP, aggrecan, ICTP and OC. The level of COMP is ambiguous in literature as COMP has been found to be elevated with JIA [200] , but in other studies, found to be significantly decreased with JIA [201–203] . COMP concentration may prove to be a marker that indicates growth impairment in systemic JIA. Aggrecan fragments in circulation have been shown to be elevated with JIA [204] . ICTP and OC have shown to be slightly decreased with JIA and may i­ndicate altered bone turnover in JIA [205] . Several studies have investigated the acute phase proteins CRP, SAP and SAA. Ling et al. found that these biomarkers could discriminate JIA patients with active and patients with inactive disease [206] . SAA has been shown to be decreased in responders to treatment, indicating a predictive power of SAA [207] . Moreover, SAA has been shown to be a more sensitive biomarker than ESR and CRP in JIA [208] . ESR has been found to be correlated to joint count [209] , and ESR and CRP have been shown to be correlated to the presence of synovitis [200] . CRP is furthermore predictive of treatment efficacy with MTX plus infliximab [210] . In a study only investigating the effect of MTX on CRP, CRP was found to be significantly decreased after 2 weeks, but elevated at 14 weeks of treatment compared with controls [211] . Furthermore, CRP has also shown to be predictive of responders (patients fulfilling ACR50) to MTX at baseline [212] . Not only has CRP shown to be an early prognostic biomarker with MTX, but MMP-3 and IL-1 have also shown to be early ­prognostic biomarkers [211,212] . Osteoarthritis

OA is a common joint degenerative disease affecting the tissue of the joint. Cartilage, as one of the major compartments of joint, has attracted much attention. The turnover of cartilage is normally maintained by a balance between catabolic and anabolic processes, but in the case of disease pathologies, the rate of cartilage degradation exceeds the rate of formation, resulting in a net loss of cartilage matrix [213] . It is impossible to discuss all biomarkers studied in OA in detail and we are therefore focusing on the recent publications of b­iomarkers in clinical settings. The urinary biomarker of CTX-II is a neoepitope ELISA [64] . Urinary CTX-II is a widely used biomarker for cartilage degradation and has been shown to be highly elevated in OA patients, as well as being prognostic for disease development. Christgau et al. reported that OA patients had a twofold significant increase in CTX-II, which was found to be associated

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with cartilage degradation visualized by x-ray [64] . Urinary CTX-II levels has also been shown to be associated with rapidly destructive hip OA [65] . However, some circadian variation (

Biomarkers of cartilage and surrounding joint tissue.

The identification and clinical demonstration of efficacy and safety of osteo- and chondro-protective drugs are met with certain difficulties. During ...
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