Considerations for understanding protein measurements: Identification of formation, degradation and more pathological relevant epitopes.

OBJECTIVES: There is a need for precision medicine and an unspoken promise of an optimal approach for identification of the right patients for value-based medicine based on big data. However, there may be a misconception that measurement of proteins is more valuable than measurement of fewer selected biomarkers. In population-based research, variation may be somewhat eliminated by quantity. However, this fascination of numbers may limit the attention to and understanding of the single. This review highlights that protein measurements (with collagens as examples) may mean different things depending on the targeted epitope - formation or degradation of tissues, and even signaling potential of proteins. DESIGN AND METHODS: PubMed was searched for collagen, neo-epitope, biomarkers. RESULTS: Ample examples of assays with specific epitopes, either pathological such as HbA1c, or domain specific such as pro-peptides, which total protein arrays would not have identified were evident. CONCLUSIONS: We suggest that big data may be considered as the funnel of data points, in which most important parameters will be selected. If the technical precision is low or the biological accuracy is limited, and we include suboptimal quality of biomarkers, disguised as big data, we may not be able to fulfill the promise of helping patients searching for the optimal treatment. Alternatively, if the technical precision of the total protein quantification is high, but we miss the functional domains with the most considerable biological meaning, we miss the most important and valuable information of a given protein. This review highlights that measurements of the same protein in different ways may provide completely different meanings. We need to understand the pathological importance of each epitope quantified to maximize protein measurements.

Elevated ectodomain of type 23 collagen is a novel biomarker of the intestinal epithelium to monitor disease activity in ulcerative colitis and Crohn's disease.

BACKGROUND: Impaired intestinal epithelial barrier is highly affected in inflammatory bowel disease. Transmembrane collagens connecting the epithelial cells to the extracellular matrix have an important role in epithelial cell homeostasis. Thus, we sought to determine whether the transmembrane type 23 collagen could serve as a surrogate marker for disease activity in patients with Crohn's disease and ulcerative colitis. METHODS: We developed an enzyme-linked immunosorbent assay to detect the ectodomain of type 23 collagen (PRO-C23) in serum, followed by evaluation of its levels in both acute and chronic dextran sulphate sodium colitis models in rats and human inflammatory bowel disease cohorts. Serum from 44 Crohn's disease and 29 ulcerative colitis patients with active and inactive disease was included. RESULTS: In the acute and chronic dextran sulphate sodium-induced rat colitis model, the PRO-C23 serum levels were significantly increased after colitis and returned to normal levels after disease remission. Serum levels of PRO-C23 were elevated in Crohn's disease (p < 0.05) and ulcerative colitis (p < 0.001) patients with active disease compared to healthy donors. PRO-C23 differentiated healthy donors from ulcerative colitis (area under the curve [AUC]: 0.81, p = 0.0009) and Crohn's disease (AUC: 0.70, p = 0.0124). PRO-C23 differentiated ulcerative colitis patients with active disease from those in remission (AUC: 0.75, p = 0.0219) and Crohn's disease patients with active disease from those in remission (AUC: 0.68, p = 0.05). CONCLUSION: PRO-C23 was elevated in rats with active colitis, and inflammatory bowel disease patients with active disease. Therefore, PRO-C23 may be used as a surrogate marker for monitoring disease activity in ulcerative colitis and Crohn's disease.

The intestinal tissue homeostasis - the role of extracellular matrix remodeling in inflammatory bowel disease.

Introduction: Extracellular matrix (ECM) remodeling of the intestinal tissue is important in inflammatory bowel disease (IBD) due to the extensive mucosal remodeling. There are still gaps in our knowledge as to how ECM remodeling is related to intestinal epithelium homeostasis and healing of the intestinal mucosa.Areas covered: The aim of this review is to highlight the importance of the ECM in relation to the pathogenesis of IBD, while addressing basement membrane and interstitial matrix remodeling, and the processes of wound healing of the intestinal tissue in IBD.Expert opinion: In IBD, basement membrane remodeling may reflect the integrity of the intestinal epithelial-cell homeostasis. The interstitial matrix remodeling is associated with deep inflammation such as the transmural inflammation as seen in fistulas and intestinal fibrosis leading to fibrostenotic strictures, in patients with CD. The interplay between wound healing processes and ECM remodeling also affects the tissue homeostasis in IBD. The interstitial matrix, produced by fibroblasts, holds a very different biology as compared to the epithelial basement membrane in IBD. In combination with integration of wound healing, quantifying the interplay between damage and repair to these sub compartments may provide essential information in IBD patient profiling, mucosal healing and disease management.

Potential diagnostic value of a type X collagen neo-epitope biomarker for knee osteoarthritis.

OBJECTIVE: Phenotypic changes of chondrocytes toward hypertrophy might be fundamental in the pathogenesis of osteoarthritis (OA), of which type X collagen (Col10) is a well-known marker. The purpose was to develop a specific immunoassay for blood quantification of a newly identified neo-epitope of type Col10 to assess its diagnostic value for radiographic knee OA. METHODS: A neo-epitope of Col10 was identified in urine samples from OA patients. A monoclonal antibody against the neo-epitope was produced in Balb/C mice. The enzyme responsible for the cleavage was identified. Immunohistochemical detection of this neo-epitope was performed on human OA cartilage. An immunoassay (Col10neo) was developed and quantified in two clinical studies: the C4Pain-003 and the NYU OA progression study. Receiver operating characteristic curve (ROC) curve analysis was carried out to evaluate the discriminative power of Col10neo between OA and rheumatoid arthritis (RA). RESULTS: A neo-epitope specific mAb was produced. The Cathepsin K-generated neo-epitope was localized to the pericellular matrix of chondrocytes, while its presence was extended and more prominent in superficial fibrillation in the cartilage with advanced degradation. In the C4Pain study, a higher level of Col10neo was seen in subjects with greater KL grade. The group of the highest tertile of Col10neo included more subjects with KL3-4. In the NYU study, Col10neo was statistically higher in OA than control or RA. ROC curve analysis revealed area under the curve was 0.88 (95% CI 0.81-0.94). CONCLUSION: Our findings indicate that Col10neo linked to hypertrophic chondrocytes could be used as a diagnostic biochemical marker for knee OA.

A fragment of SPARC reflecting increased collagen affinity shows pathological relevance in lung cancer - implications of a new collagen chaperone function of SPARC.

The matricellular protein SPARC (secreted proteome acidic and rich in cysteine) is known to bind collagens and regulate fibrillogenesis. Cleavage of SPARC at a single peptide bond, increases the affinity for collagens up to 20-fold. To investigate if this specific cleavage has pathological relevance in fibrotic disorders, we developed a competitive ELISA targeting the generated neo-epitope on the released fragment and quantified it in serum from patients with lung cancer, idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD) and healthy subjects. Furthermore, the ability of SPARC to protect fibrillar collagens from proteolytic degradation was investigated in vitro, potentially adding a new collagen chaperone function to SPARC. The fragment was significantly elevated in lung cancer patients when compared to healthy subjects measured in a discovery cohort (p = 0.0005) and a validation cohort (p < 0.0001). No significant difference was observed for IPF and COPD patients compared to healthy subjects. When recombinant SPARC was incubated with type I or type III collagen and matrix metalloproteinase-9, collagen degradation was completely inhibited. Together, these data suggest that cleavage of SPARC at a specific site, which modulates collagen binding, is a physiological mechanism increased during pathogenesis of lung cancer. Furthermore, inhibition of fibrillar collagen degradation by SPARC adds a new chaperone function to SPARC which may play additional roles in the contribution to increased collagen deposition leading to a pro-fibrotic and tumorigenic environment.

The good and the bad collagens of fibrosis - Their role in signaling and organ function.

Usually the dense extracellular structure in fibrotic tissues is described as extracellular matrix (ECM) or simply as collagen. However, fibrosis is not just fibrosis, which is already exemplified by the variant morphological characteristics of fibrosis due to viral versus cholestatic, autoimmune or toxic liver injury, with reticular, chicken wire and bridging fibrosis. Importantly, the overall composition of the ECM, especially the relative amounts of the many types of collagens, which represent the most abundant ECM molecules and which centrally modulate cellular functions and physiological processes, changes dramatically during fibrosis progression. We hypothesize that there are good and bad collagens in fibrosis and that a change of location alone may change the function from good to bad. Whereas basement membrane collagen type IV anchors epithelial and other cells in a polarized manner, the interstitial fibroblast collagens type I and III do not provide directional information. In addition, feedback loops from biologically active degradation products of some collagens are examples of the importance of having the right collagen at the right place and at the right time controlling cell function, proliferation, matrix production and fate. Examples are the interstitial collagen type VI and basement membrane collagen type XVIII. Their carboxyterminal propeptides serve as an adipose tissue hormone, endotrophin, and as a regulator of angiogenesis, endostatin, respectively. We provide an overview of the 28 known collagen types and propose that the molecular composition of the ECM in fibrosis needs careful attention to assess its impact on organ function and its potential to progress or reverse. Consequently, to adequately assess fibrosis and to design optimal antifibrotic therapies, we need to dissect the molecular entity of fibrosis for the molecular composition and spatial distribution of collagens and the associated ECM.

Serological biomarkers detect active joint destruction and inflammation in patients with haemophilic arthropathy.

INTRODUCTION: Progressive arthropathy caused by recurrent joint bleeds is a severe complication in haemophilia. AIM: We investigated whether biomarkers of cartilage and bone degradation, and inflammation were altered in haemophilia patients and whether these biomarkers could identify haemophilia patients with arthropathy. METHODS: Serum from 35 haemophilia patients with varying degrees of arthropathy and 43 age- and gender-matched control subjects were analysed. Biomarkers of cartilage degradation (C2M, COMP, CTX-II, ADAMTS5), cartilage formation (PRO-C2), bone formation (PINP), bone resorption (CTX-I) and inflammation (hsCRP, CRPM) were measured by ELISA. Arthropathy was assessed by radiological evaluation (Pettersson score) and physical examination (Gilbert score). RESULTS: In patients with haemophilia, cartilage degradation, measured by C2M, CTX-II and COMP, was increased by 25% (P < 0.05) compared with control subjects. Levels of the cartilage degradation enzyme, ADAMTS5, were 10% lower in haemophilia patients (P < 0.05). Bone formation (PINP) was reduced by 25% (P < 0.05) in haemophilia patients, whereas bone resorption (CTX-I) was increased by 30% (P < 0.001). Acute inflammation (hsCRP) was increased by 50% (P < 0.01), whereas chronic inflammation (CRPM) was decreased by 25% (P < 0.0001). The hsCRP/CRPM ratio was 60% higher (P < 0.001) in haemophilia patients relative to control subjects. A biomarker panel combining C2M, CRPM, and ADAMTS5 could distinguish haemophilia patients from control subjects with 85.3% accuracy (P < 0.0001). We found no strong correlation between biomarkers and radiological and physical examination of the joint. CONCLUSION: Biomarkers detect increased cartilage and bone degradation, and altered inflammatory activity in haemophilia patients with arthropathy. These biomarkers could potentially be used to identify patients with progressing joint disease.

Altered collagen turnover in factor VIII-deficient rats with hemophilic arthropathy identifies potential novel serological biomarkers in hemophilia.

Essentials Joint bleeding in hemophilia may induce significant remodeling of the extracellular matrix. Biomarkers of collagen turnover were investigated in a F8-/- rat model of hemophilic arthropathy. Biomarkers of cartilage degradation increased significantly during development of arthropathy. Basement membrane and interstitial matrix turnover changed significantly following hemarthrosis. SUMMARY: Background Hemophilic arthropathy is a severe complication of hemophilia. It is caused by recurrent bleeding into joint cavities, which leads to synovial inflammation, fibrosis, cartilage degradation and bone remodeling. Extracellular matrix remodeling of affected tissues is a hallmark of these pathological processes. Objectives The aim of this study was to use serological biomarkers of collagen turnover to evaluate extracellular matrix remodeling in a factor VIII-deficient rat model of hemophilic arthropathy. Methods F8-/- rats and wild-type littermate controls were subjected to repeated knee bleeds induced by needle puncture on days 0 and 14. Development of arthropathy was confirmed by histology after termination on day 28. Serum samples were collected at baseline and throughout the study and analyzed for biomarkers of collagen turnover, including collagens of the basement membrane (type IV collagen), the interstitial matrix (collagen types III, V and VI) and cartilage (type II collagen). Results In F8-/- rats, induced knee bleeding and subsequent development of arthropathy caused significant alterations in collagen turnover, measured as changes in serological biomarkers of basement membrane turnover, interstitial matrix turnover and cartilage degradation. Biomarkers of type II collagen degradation correlated significantly with cartilage degradation and degree of arthropathy. Hemophilic rats had a 50% higher turnover of the basement membrane than wild-type littermates at baseline. Conclusions Joint bleeding and hemophilic arthropathy cause changes in turnover of extracellular matrix collagens in hemophilic rats. Biomarkers of collagen turnover may be used to monitor joint bleeding and development of blood-induced joint disease in hemophilia.

Collagen-mediated hemostasis.

Collagens mediate essential hemostasis by maintaining the integrity and stability of the vascular wall. Imbalanced turnover of collagens by uncontrolled formation and/or degradation may result in pathologic conditions such as fibrosis. Thickening of the vessel wall because of accumulation of collagens may lead to arterial occlusion or thrombosis. Thinning of the wall because of collagen degradation or deficiency may lead to rupture of the vessel wall or aneurysm. Preventing excessive hemorrhage or thrombosis relies on collagen-mediated actions. Von Willebrand factor, integrins and glycoprotein VI, as well as clotting factors, can bind collagen to restore normal hemostasis after trauma. This review outlines the essential roles of collagens in mediating hemostasis, with a focus on collagens types I, III, IV, VI, XV, and XVIII.

Syndecan proteoglycan contributions to cytoskeletal organization and contractility.

Cells exert tension on the extracellular matrix through specific receptors that link to the actin cytoskeleton. The best characterized are the integrins, which, when activated and clustered, can link to the extracellular matrix at specialized adhesion zones, known as focal contacts or focal adhesions. However, other transmembrane receptors can also localize there, including one transmembrane proteoglycan, syndecan-4. This heparan sulfate proteoglycan can also link directly to the cytoskeleton through alpha-actinin, and can signal through protein kinase C. In turn, the pathway leads to RhoA and Rho kinases that control actomyosin contractility. Syndecan-4 may, therefore, be a sensor of tension exerted on the matrix. These processes are described here, their significance being potential roles in wound contraction, tumor-stroma interactions, fibrosis and the regulation of motility.