Selected reaction monitoring assays in mesenchymal stem cells from osteoarthritis patients

Osteoarthritis (OA) is considered the most prevalent form of arthritis. The aim of this study was to verify potential protein OA biomarkers by applying Selected Reaction Monitoring (SRM) assays to protein extracts obtained from Bone Marrow-Mesenchymal Stem Cells (BM-MSCs) isolated from OA patients.BM aspirates were obtained from the femoral channel of OA patients at the time of surgery and from the femoral channel of hip fracture subjects without OA during hip joint replacement surgery for the treatment of subcapital fracture.SRM results verified the differential expression of several protein biomarkers in BM-MSCs from OA patients.     

Clinical validation of an immunoaffinity LC-MS/MS assay for the quantification of a collagen type II neoepitope peptide: A biomarker of matrix metalloproteinase activity and osteoarthritis in human urine

The degradation of type II collagen has been associated with the pathology of osteoarthritis (OA). Matrix metalloproteinases (MMPs) are enzymes that are responsible for catalyzing the degradation of collagen and, therefore, are pursued as potential targets for the treatment of OA. Collagen-derived peptides identified as a reflection of in vivo MMP activity have been investigated as target biomarkers of MMP activity as well as potential biomarkers of OA disease state and/or progression. An immunoaffinity liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay developed for the quantification of the most abundant urinary type II collagen neoepitope (uTIINE) peptide, a 45-mer with 5 HO-proline residues resulting from MMP-13-catalyzed degradation, was validated for clinical use. Validation experiments were designed with attention to specific challenges related to quantification of endogenous analytes. The validated method is sensitive, selective, accurate (<15% relative error) and precise (<15% coefficient of variation) over a linear range of 0.156-7.50 ng/ml. Sample stability and inter- and intrasubject variability were evaluated in the urine of normal and OA populations. The method was applied to analyze human urine samples from clinical studies investigating the utility of uTIINE as a potential biomarker for OA.     

骨性关节炎生物学标志物研究进展

骨性关节炎(oseteoarthritis,OA)是一种随着年龄增长发病率明显升高的退行性变,常累及脊柱、髋、膝等人体负重关节,以关节缓慢发展的疼痛、肿胀,伴功能障碍为临床表现,主要有滑膜增生、软骨破坏、软骨下骨骨化及骨赘形成等一系列病理表现。OA对人类的健康和生活质量影响很大,随着老龄化社会的到来,本病的发病率日趋升高,其研究已成为医学领域中的重要课题。目前,OA的早期诊断、病变监测和有效防治仍是骨科领域亟待解决的疑难问题。随着分子生物学的发展和研究手段的提高,许多研究者都在试图寻找用于临床评价OA的生物学标志物。本文将就OA研究中所使用的主要标志物进行综述,为深入研究OA提供方便。     

Biomarkers in osteoarthritis

Biomarkers aid the study of osteoarthritis (OA) in a number of different ways. In this article we summarise briefly their multiple uses and reflect on how the study reported in a previous edition of Arthritis Research & Therapy should promote further investigation of cartilage oligomeric matrix protein (COMP). COMP is foremost among hitherto investigated biomarkers and is most consistently shown to predict knee OA progression. Precisely what role it plays in OA pathogenesis remains unclear and elucidating this may be key to defining, and then targeting, the cellular pathways involved in OA.     

骨关节炎相关生物标志物

骨关节炎（osteoarthritis）是关节软骨进展的退化性疾病,并累及周围组织结构的病变,是致老年人伤残主要原因之一。目前,以临床表现和影像学诊断为主,缺乏早期检测和预后评估的有效方法。生物标志物的检查是具有前景的研究方向,在关节软骨结构改变之前,各种生物标志物代谢发生变化,其能帮助诊断和预测骨关节炎的发生发展及其预后。然而,生物标志物在临床诊断和治疗相关的应用仍需加以证实。通过广泛查阅近年有关骨关节炎相关分子生物诊断的相关文献,有助于了解生物标志物对于骨关节炎的早期诊断意义和临床应用前景。本文就关节软骨、骨和滑膜等不同组织类型相关的生物标志物进行综述。     

Application of biomarkers in the clinical development of new drugs for chondroprotection in destructive joint diseases: a review

Emerging evidence supports the concept that biochemical markers are clinically useful non-invasive diagnostic tools for the monitoring of changes in cartilage turnover in patients with destructive joint diseases such as osteoarthritis (OA) and rheumatoid arthritis (RA). Epidemiological studies demonstrated that measurements of different degradation products of proteins in the extracellular matrix of hyaline cartilage in urine or serum samples are (1) increased in OA or RA patients compared with healthy individuals, (2) correlate with disease activity, and (3) are predictive for the rate of changes in radiographic measures of cartilage loss. The present review provides an updated list of available biomarkers and summarize the research data arguing for their clinical utility. In addition, it addresses the question whether or not the monitoring of biomarkers during different treatment modalities could be a useful approach to characterize the chondro-protective effects of approved and candidate drugs. Finally, it briefly reviews the in vitro/ex vivo experimental settings — isolated chondrocyte cultures and articular cartilage explants — that can assist in the verification of novel markers, but also studies assessing direct effects of drug candidates on chondrocytes. Collectively, biomarkers may acquire a function as established efficacy parameters in the clinical development of novel chondro-protective agents.     

Application of biomarkers in the clinical development of new drugs for chondroprotection in destructive joint diseases: a review

Emerging evidence supports the concept that biochemical markers are clinically useful non-invasive diagnostic tools for the monitoring of changes in cartilage turnover in patients with destructive joint diseases such as osteoarthritis (OA) and rheumatoid arthritis (RA). Epidemiological studies demonstrated that measurements of different degradation products of proteins in the extracellular matrix of hyaline cartilage in urine or serum samples are (1) increased in OA or RA patients compared with healthy individuals, (2) correlate with disease activity, and (3) are predictive for the rate of changes in radiographic measures of cartilage loss. The present review provides an updated list of available biomarkers and summarize the research data arguing for their clinical utility. In addition, it addresses the question whether or not the monitoring of biomarkers during different treatment modalities could be a useful approach to characterize the chondro-protective effects of approved and candidate drugs. Finally, it briefly reviews the in vitro/ex vivo experimental settings — isolated chondrocyte cultures and articular cartilage explants — that can assist in the verification of novel markers, but also studies assessing direct effects of drug candidates on chondrocytes. Collectively, biomarkers may acquire a function as established efficacy parameters in the clinical development of novel chondro-protective agents.     

Sex differences in magnetic resonance imaging-based biomarkers and in those of joint metabolism

Sex differences in the prevalence, incidence, and severity of osteoarthritis (OA) have long been known. Some differences in the evaluation of this issue across studies may be related to differences in study design, sampling, study size, study populations, targeted joint sites, and definitions of OA. This report highlights recent studies of sex differences in individual joint components imaged by magnetic resonance imaging and in systemic biomarkers of joint metabolism. Particularly important are those studies that examine this issue in young unaffected adults and children before the development of disease. Despite some variation across studies, women appear for the most part to have a thinner and more reduced volume of cartilage in the knee than men, and this may occur from early childhood. It is not clear whether women have a more accelerated rate of cartilage volume loss than men. Few data exist on sex differences in systemic biomarkers of joint metabolism. In these studies, it is critically important to characterize the total body burden of OA and the presence of comorbid conditions likely to influence a given biomarker. Lastly, future research should dovetail studies of sex differences in imaging and biochemical biomarkers with genetics to maximize insight into the mechanisms behind observed sex differences.     

Evaluation of a pretreatment method for two-dimensional gel electrophoresis of synovial fluid using cartilage oligomeric matrix protein as a marker

Osteoarthritis (OA) is the most common rheumatic pathology. One of the major objectives of OA research is the development of early diagnostic strategies such as those using proteomic technology. Synovial fluid (SF) in OA patients is a potential source of biomarkers for OA. The efficient and reliable preparation of SF proteomes is a critical step towards biomarker discovery. In this study, we have optimized a pretreatment method for two-dimensional gel electrophoresis (2DE) separation of the SF proteome, by enriching low-abundance proteins and simultaneously removing hyaluronic acid, albumin, and IgG. SF samples pretreated using this optimized method were then evaluated by 1DE and 2DE separation followed by immunodetection of cartilage oligomeric matrix protein (COMP), a known OA biomarker, and by the identification of 3 proteins (apolipoprotein, haptoglobin precursor, and fibrinogen D fragment) that are related to joint diseases.     

Large-scale gene expression profiles, differentially represented in osteoarthritic synovium of the knee joint using cDNA microarray technology

Osteoarthritis (OA) is one of the most common age-related chronic disorders of articular cartilage, joints and bone tissue. Diagnosis of OA commonly depends on clinical and radiographic findings. However, changes in cartilage associated with the early stage of OA cannot be detected using radiographs, because significant cartilage degeneration must occur before radiographic findings show alterations of the appearance of cartilage. To identify new biomarkers of OA, we analysed gene expression profiles of synovium from 43 patients with OA, ten patients with rheumatoid arthritis (RA), and eight non-OA/non-RA patients using a novel cDNA microarray chip. We identified 21 genes with simultaneous significant differences in expression between OA and non-OA/non-RA groups and between OA and RA groups. Linear discriminant analysis showed that the three groups could be well separated using those 21 genes. Statistical analysis also revealed that several of the 21 genes were associated with disease progression and clinical presentation. The graphical modelling method indicated that some of the 21 genes are significantly associated with a particular clinical presentation, suggesting biological relationships among those genes. This is the first report of the use of cDNA microarray technology to create large-scale gene expression profiles differentially expressed in situ in OA synovium of the knee joint.     

Identification of a panel of novel serum osteoarthritis biomarkers

Osteoarthritis (OA) is the most common rheumatic pathology. Because currently available diagnostic methods are limited and lack sensitivity, the identification of new specific biological markers for OA has become a focus. The purpose of this study was to identify novel protein biomarkers for moderate and severe OA in serum. Sera were obtained from 50 moderate OA patients, 50 severe OA patients, and 50 nonsymptomatic controls. Serum protein levels were analyzed using isobaric tags for relative and absolute quantitation (iTRAQ) and matrix-assisted laser desorption/ionization (MALDI)-TOF/TOF mass spectrometry. We identified 349 different proteins in the sera, 262 of which could be quantified by calculation of their iTRAQ ratios. Three sets of proteins were significantly (p < 0.05) changed in OA samples compared to controls. Of these, 6 were modulated only in moderate OA, 13 only in severe OA and 7 in both degrees. Although some of these proteins, such as cartilage oligomeric matrix protein, have a previously reported putative biomarker value for OA, most are novel biomarker candidates for the disease. These include some complement components, lipoproteins, von Willebrand factor, tetranectin, and lumican. The specificity and selectivity of these candidates need to be validated before new molecular diagnostic or prognostic tests for OA can be developed.     

Discovery and development of a type II collagen neoepitope (TIINE) biomarker for matrix metalloproteinase activity: from in vitro to in vivo

Destruction of cartilage by matrix metalloproteinases (MMPs) plays a significant role in the pathology of osteoarthritis (OA). A translatable biomarker of MMP activity would enable development of MMP inhibitors for the treatment of OA and potentially the improved diagnosis of OA. A directed approach to identifying specific MMP cleavage products as potential biomarkers has been undertaken. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to identify peptides generated by MMP-driven degradation of human articular cartilage (HAC) in vivo. It was shown that a 45-mer peptide fragment of collagen type II with five hydroxyprolines (OH) can be selectively produced by the activity of collagenase, an enzyme purported to be involved in the pathology of OA. This 45-mer is the most abundant neoepitope peptide found in biological fluids such as urine and synovial fluid. An immunoaffinity LC-MS/MS assay has been developed to quantify collagen type II neoepitope peptides as biomarkers of collagenase modulation. The lower limit of quantification for this assay was established to be 0.035 nM. The assay was used to measure the levels of collagen type II peptides in the urine of both clinical (healthy human subjects) and preclinical species. The urinary levels of the most abundant peptides are reported for rat, rabbit, guinea pig, dog, and healthy human adult subjects. The utility of this peptide to monitor collagenase activity in vivo has been demonstrated through its detailed characterization in HAC explants as well as in the urine of human and other preclinical species.     

Large-scale gene expression profiles,differentially represented in osteoarthritic synovium of the knee joint using cDNA microarray technology

Osteoarthritis (OA) is one of the most common age-related chronic disorders of articular cartilage, joints and bone tissue. Diagnosis of OA commonly depends on clinical and radiographic findings. However, changes in cartilage associated with the early stage of OA cannot be detected using radiographs, because significant cartilage degeneration must occur before radiographic findings show alterations of the appearance of cartilage. To identify new biomarkers of OA, we analysed gene expression profiles of synovium from 43 patients with OA, ten patients with rheumatoid arthritis (RA), and eight non-OA/non-RA patients using a novel cDNA microarray chip. We identified 21 genes with simultaneous significant differences in expression between OA and non-OA/non-RA groups and between OA and RA groups. Linear discriminant analysis showed that the three groups could be well separated using those 21 genes. Statistical analysis also revealed that several of the 21 genes were associated with disease progression and clinical presentation. The graphical modelling method indicated that some of the 21 genes are significantly associated with a particular clinical presentation, suggesting biological relationships among those genes. This is the first report of the use of cDNA microarray technology to create large-scale gene expression profiles differentially expressed in situ in OA synovium of the knee joint.     

Most recent developments in strategies to reduce the progression of structural changes in osteoarthritis: today and tomorrow

Osteoarthritis (OA), the most common of all arthritic conditions, is a social and financial burden to all nations. The most recent research has significantly advanced our understanding of the cause of OA and risk factors associated with it. These findings have provided useful information that has helped in the daily management of patients with OA. Some preventative measures and a number of therapeutic agents and drugs are available, which may help to reduce the progression of OA in certain patients. Moreover, the most recent progress in research has significantly enhanced our knowledge of the factors involved in the development of the disease and of the mechanisms responsible for its progression. This has allowed identification of several new therapeutic targets in a number of pathophysiological pathways. Consequently, the field is opening up to a new era in which drugs and agents that can specifically block important mechanisms responsible for the structural changes that occur in OA can be brought into development and eventually into clinical trials.     

瘦素对骨关节炎影响的研究进展

瘦素(Leptin)是脂肪因子的一种,可以在骨关节炎(OA)患者的血浆、滑液、软骨中被检测到。OA是一种最常见的关节疾病,其可以发生于全身的多个关节,以骨质和滑膜组织新陈代谢的改变、关节软骨的破坏和由此引起的关节软骨的减少为特征。瘦素是一种由肥胖(0b)基因编码的一个小的非糖基化肽激素。最开始,瘦素仅仅被认为是一种脂肪细胞源性的小分子(16KD),在下丘脑中枢水平作为一个饱感因子介导食物摄入量减少,并增加能量的消耗。现在已经发现,瘦素在机体内可发挥多种生理作用,并与OA病情有关。本文通过对瘦素与OA、软骨、肥胖、生物标志物、脂联素等之间的联系做一综述,以了解瘦素与OA之间的联系,为OA的治疗方面的进一步研究提供帮助。     

Sequential depletion of human serum for the search of osteoarthritis biomarkers

ABSTRACT: BACKGROUND: The field of biomarker discovery, development and application has been the subject of intense interest and activity, especially with the recent emergence of new technologies, such as proteomics-based approaches. In proteomics, search for biomarkers in biological fluids such as human serum is a challenging issue, mainly due to the high dynamic range of proteins present in these types of samples. Methods for reducing the content of most highly abundant proteins have been developed, including immunodepletion or protein equalization. In this work, we report for the first time the combination of a chemical sequential depletion method based in two protein precipitations with acetonitrile and DTT, with a subsequent two-dimensional difference in-gel electrophoresis (2D-DIGE) analysis for the search of osteoarthritis (OA) biomarkers in human serum. The depletion method proposed is non-expensive, of easy implementation and allows fast sample throughput. RESULTS: Following this workflow, we have compared sample pools of human serum obtained from 20 OA patients and 20 healthy controls. The DIGE study led to the identification of 16 protein forms (corresponding to 14 different proteins) that were significantly (p < 0.05) altered in OA when compared to controls (8 increased and 7 decreased). Immunoblot analyses confirmed for the first time the increase of an isoform of Haptoglobin beta chain (HPT) in sera from OA patients. CONCLUSIONS: Altogether, these data demonstrate the utility of the proposed chemical sequential depletion for the analysis of sera in protein biomarker discovery approaches, exhibit the usefulness of quantitative 2D gel-based strategies for the characterization of disease-specific patterns of protein modifications, and also provide a list of OA biomarker candidates for validation.     

Salivary and serum procalcitonin and C-reactive protein as biomarkers of periodontitis in United States veterans with osteoarthritis or rheumatoid arthritis

Serum procalcitonin (ProCT) is elevated in response to bacterial infections, whereas high sensitivity C-reactive protein (hsCRP) is a nonspecific inflammatory marker that is increased by excess adipose tissue. We examined the efficacy of ProCT and hsCRP as biomarkers of periodontitis in the saliva and serum of patients with arthritis, which is characterized by variable levels of systemic inflammation that potentially can confound the interpretation of inflammatory biomarkers. Blood and unstimulated whole saliva were collected from 33 patients with rheumatoid arthritis (RA) and 50 with osteoarthritis (OA). Periodontal status was assessed by full mouth examination and patients were categorized as having no/mild, moderate or severe periodontitis by standard parameters. Salivary and serum ProCT and hsCRP concentrations were compared. BMI, diabetes, anti-inflammatory medications and smoking status were ascertained from the patient records. Differences between OA and RA in proportionate numbers of patients were compared for race, gender, diabetes, adiposity and smoking status. Serum ProCT was significantly higher in arthritis patients with moderate to severe and severe periodontitis compared with no/mild periodontitis patients. There were no significant differences in salivary ProCT or salivary or serum hsCRP in RA patients related to periodontitis category. Most of the OA and RA patients were middle aged or older, 28.9% were diabetic, 78.3% were overweight or obese, and slightly more than half were either current or past smokers. The OA and RA groups differed by race, but not gender; blacks and males were predominant in both groups. The OA and RA groups did not differ in terms of controlled or uncontrolled diabetes, smoking status or BMI. The RA patients had been prescribed more anti-inflammatory medication than the OA patients. Our results demonstrate that circulating ProCT is a more discriminative biomarker for periodontitis than serum hsCRP in patients with underlying arthritis. Any elevation in salivary and serum hsCRP due to periodontitis apparently was overshadowed by differences among these patients in factors that influence CRP, such as the extent of inflammation between RA and OA, the extent of adipose tissue, the use of anti- inflammatory medications and smoking status. Although our study showed no differences in salivary ProCT related to severity of periodontitis, this biomarker also may be useful with further refinement.     

Proteomics of osteoarthritic chondrocytes and cartilage

Osteoarthritis (OA) is characterized by irreversible destruction of the articular cartilage. OA affects more than 100 million individuals worldwide and has a major impact on patients' quality of life. The lack of effective therapy that prevents, inhibits or reverses the progress of OA often leaves only the option of surgical interventions. Thus, identification of the factors that contribute to OA pathogenesis is necessary for better understanding of OA pathobiology and discovery of effective therapies. Recent proteomic studies have been conducted to identify pathological mediators and biomarkers of OA, which have pinpointed novel pathways involved in cartilage degeneration. This article summarizes the recent findings, compares major techniques used in OA proteomics and discusses key proteins in OA and their potential use as therapeutic targets.     

Proteomics of osteoarthritic chondrocytes and cartilage

Osteoarthritis (OA) is characterized by irreversible destruction of the articular cartilage. OA affects more than 100 million individuals worldwide and has a major impact on patients’ quality of life. The lack of effective therapy that prevents, inhibits or reverses the progress of OA often leaves only the option of surgical interventions. Thus, identification of the factors that contribute to OA pathogenesis is necessary for better understanding of OA pathobiology and discovery of effective therapies. Recent proteomic studies have been conducted to identify pathological mediators and biomarkers of OA, which have pinpointed novel pathways involved in cartilage degeneration. This article summarizes the recent findings, compares major techniques used in OA proteomics and discusses key proteins in OA and their potential use as therapeutic targets.     

Progression Analysis and Stage Discovery in Continuous Physiological Processes Using Image Computing

We propose an image computing-based method for quantitative analysis of continuous physiological processes that can be sensed by medical imaging and demonstrate its application to the analysis of morphological alterations of the bone structure, which correlate with the progression of osteoarthritis (OA). The purpose of the analysis is to quantitatively estimate OA progression in a fashion that can assist in understanding the pathophysiology of the disease. Ultimately, the texture analysis will be able to provide an alternative OA scoring method, which can potentially reflect the progression of the disease in a more direct fashion compared to the existing clinically utilized classification schemes based on radiology. This method can be useful not just for studying the nature of OA, but also for developing and testing the effect of drugs and treatments. While in this paper we demonstrate the application of the method to osteoarthritis, its generality makes it suitable for the analysis of other progressive clinical conditions that can be diagnosed and prognosed by using medical imaging.