Prognostic model to predict the incidence of radiographic knee osteoarthritis.

OBJECTIVE: Early diagnosis of knee osteoarthritis (KOA) in asymptomatic stages is essential for the timely management of patients using preventative strategies. We develop and validate a prognostic model useful for predicting the incidence of radiographic KOA (rKOA) in non-radiographic osteoarthritic subjects and stratify individuals at high risk of developing the disease. METHODS: Subjects without radiographic signs of KOA according to the Kellgren and Lawrence (KL) classification scale (KL=0 in both knees) were enrolled in the OA initiative (OAI) cohort and the Prospective Cohort of A Coruña (PROCOAC). Prognostic models were developed to predict rKOA incidence during a 96-month follow-up period among OAI participants based on clinical variables and serum levels of the candidate protein biomarkers APOA1, APOA4, ZA2G and A2AP. The predictive capability of the biomarkers was assessed based on area under the curve (AUC), and internal validation was performed to correct for overfitting. A nomogram was plotted based on the regression parameters. Model performance was externally validated in the PROCOAC. RESULTS: 282 participants from the OAI were included in the development dataset. The model built with demographic, anthropometric and clinical data (age, sex, body mass index and WOMAC pain score) showed an AUC=0.702 for predicting rKOA incidence during the follow-up. The inclusion of ZA2G, A2AP and APOA1 data significantly improved the model's sensitivity and predictive performance (AUC=0.831). The simplest model, including only clinical covariates and ZA2G and A2AP serum levels, achieved an AUC=0.826. Both models were internally cross-validated. Predictive performance was externally validated in an independent dataset of 100 individuals from the PROCOAC (AUC=0.713). CONCLUSION: A novel prognostic model based on common clinical variables and protein biomarkers was developed and externally validated to predict rKOA incidence over a 96-month period in individuals without any radiographic signs of disease. The resulting nomogram is a useful tool for stratifying high-risk populations and could potentially lead to personalised medicine strategies for treating OA.

An Atlas of the Knee Joint Proteins and Their Role in Osteoarthritis Defined by Literature Mining.

Osteoarthritis (OA) is the most prevalent rheumatic pathology. However, OA is not simply a process of wear and tear affecting articular cartilage but rather a disease of the entire joint. One of the most common locations of OA is the knee. Knee tissues have been studied using molecular strategies, generating a large amount of complex data. As one of the goals of the Rheumatic and Autoimmune Diseases initiative of the Human Proteome Project, we applied a text-mining strategy to publicly available literature to collect relevant information and generate a systematically organized overview of the proteins most closely related to the different knee components. To this end, the PubPular literature-mining software was employed to identify protein-topic relationships and extract the most frequently cited proteins associated with the different knee joint components and OA. The text-mining approach searched over eight million articles in PubMed up to November 2022. Proteins associated with the six most representative knee components (articular cartilage, subchondral bone, synovial membrane, synovial fluid, meniscus, and cruciate ligament) were retrieved and ranked by their relevance to the tissue and OA. Gene ontology analyses showed the biological functions of these proteins. This study provided a systematic and prioritized description of knee-component proteins most frequently cited as associated with OA. The study also explored the relationship of these proteins to OA and identified the processes most relevant to proper knee function and OA pathophysiology.

A meta-analysis and a functional study support the influence of mtDNA variant m.16519C on the risk of rapid progression of knee osteoarthritis.

OBJECTIVES: To identify mitochondrial DNA (mtDNA) genetic variants associated with the risk of rapid progression of knee osteoarthritis (OA) and to characterise their functional significance using a cellular model of transmitochondrial cybrids. METHODS: Three prospective cohorts contributed participants. The osteoarthritis initiative (OAI) included 1095 subjects, the Cohort Hip and Cohort Knee included 373 and 326 came from the PROspective Cohort of Osteoarthritis from A Coruña. mtDNA variants were screened in an initial subset of 450 subjects from the OAI by in-depth sequencing of mtDNA. A meta-analysis of the three cohorts was performed. A model of cybrids was constructed to study the functional consequences of harbouring the risk mtDNA variant by assessing: mtDNA copy number, mitochondrial biosynthesis, mitochondrial fission and fusion, mitochondrial reactive oxygen species (ROS), oxidative stress, autophagy and a whole transcriptome analysis by RNA-sequencing. RESULTS: mtDNA variant m.16519C is over-represented in rapid progressors (combined OR 1.546; 95% CI 1.163 to 2.054; p=0.0027). Cybrids with this variant show increased mtDNA copy number and decreased mitochondrial biosynthesis; they produce higher amounts of mitochondrial ROS, are less resistant to oxidative stress, show a lower expression of the mitochondrial fission-related gene fission mitochondrial 1 and an impairment of autophagic flux. In addition, its presence modulates the transcriptome of cybrids, especially in terms of inflammation, where interleukin 6 emerges as one of the most differentially expressed genes. CONCLUSIONS: The presence of the mtDNA variant m.16519C increases the risk of rapid progression of knee OA. Among the most modulated biological processes associated with this variant, inflammation and negative regulation of cellular process stand out. The design of therapies based on the maintenance of mitochondrial function is recommended.

Association of the serological status of rheumatoid arthritis patients with two circulating protein biomarkers: A useful tool for precision medicine strategies.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation of the joints and presence of systemic autoantibodies, with a great clinical and molecular heterogeneity. Rheumatoid Factor (RF) and anti-citrullinated protein antibodies (ACPA) are routinely used for the diagnosis of RA. However, additional serological markers are needed to improve the clinical management of this disease, allowing for better patient stratification and the desirable application of precision medicine strategies. In the present study, we investigated those systemic molecular changes that are associated with the RF and ACPA status of RA patients. To achieve this objective, we followed a proteomic biomarker pipeline from the discovery phase to validation. First, we performed an iTRAQ-based quantitative proteomic experiment on serum samples from the RA cohort of the Hospital of Santiago de Compostela (CHUS). In this discovery phase, serum samples from the CHUS cohort were pooled according to their RF/ACPA status. Shotgun analysis revealed that, in comparison with the double negative group (RF-/ACPA-), the abundance of 12 proteins was altered in the RF+/ACPA+ pool, 16 in the RF+/ACPA- pool and 10 in the RF-/ACPA+ pool. Vitamin D binding protein and haptoglobin were the unique proteins increased in all the comparisons. For the verification phase, 80 samples from the same cohort were analyzed individually. To this end, we developed a Multiple Reaction Monitoring (MRM) method that was employed in a comprehensive targeted analysis with the aim of verifying the results obtained in the discovery phase. Thirty-one peptides belonging to 12 proteins associated with RF and/or ACPA status were quantified by MRM. In a final validation phase, the serum levels of alpha-1-acid glycoprotein 1 (A1AG1), haptoglobin (HPT) and retinol-binding protein 4 (RET4) were measured by immunoassays in the RA cohort of the Hospital of A Coruña (HUAC). The increase of two of these putative biomarkers in the double seropositive group was validated in 260 patients from this cohort (p = 0.009 A1AG1; p = 0.003 HPT). The increased level of A1AG1 showed association with RF rather than ACPA (p = 0.023), whereas HPT showed association with ACPA rather than RF (p = 0.013). Altogether, this study has allowed a further classification of the RA seropositive patients into two novel clusters: RF+A1AG+ and ACPA+HPT+. The determination of A1AG1 and HPT in serum would provide novel information useful for RA patient stratification, which could facilitate the effective implementation of personalized medicine in routine clinical practice.

Efficacy, safety and cost-effectiveness of methotrexate, adalimumab or their combination in non-infectious non-anterior uveitis: a protocol for a multicentre, randomised, parallel three arms, active-controlled, phase III open label with blinded outcome assessment study.

INTRODUCTION: Non-infectious uveitis include a heterogeneous group of sight-threatening and incapacitating conditions. Their correct management sometimes requires the use of immunosuppressive drugs (ISDs), prescribed in monotherapy or in combination. Several observational studies showed that the use of ISDs in combination could be more effective than and as safe as their use in monotherapy. However, a direct comparison between these two treatment strategies has not been carried out yet. METHODS AND ANALYSIS: The Combination THerapy with mEthotrexate and adalImumAb for uveitis (CoTHEIA) study is a phase III, multicentre, prospective, randomised, single-blinded with masked outcome assessment, parallel three arms with 1:1:1 allocation, active-controlled, superiority study design, comparing the efficacy, safety and cost-effectiveness of methotrexate, adalimumab or their combination in non-infectious non-anterior uveitis. We aim to recruit 192 subjects. The duration of the treatment and follow-up will last up to 52 weeks, plus 70 days follow-up with no treatment. The complete and maintained resolution of the ocular inflammation will be assessed by masked evaluators (primary outcome). In addition to other secondary measurements of efficacy (quality of life, visual acuity and costs) and safety, we will identify subjects' subgroups with different treatment responses by developing prediction models based on machine learning techniques using genetic and proteomic biomarkers. ETHICS AND DISSEMINATION: The protocol, annexes and informed consent forms were approved by the Reference Clinical Research Ethic Committee at the Hospital Clínico San Carlos (Madrid, Spain) and the Spanish Agency for Medicines and Health Products. We will elaborate a dissemination plan including production of materials adapted to several formats to communicate the clinical trial progress and findings to a broad group of stakeholders. The promoter will be the only access to the participant-level data, although it can be shared within the legal situation. TRIAL REGISTRATION NUMBER: 2020-000130-18; NCT04798755.

Proteomic Analysis of Synovial Fibroblasts and Articular Chondrocytes Co-Cultures Reveals Valuable VIP-Modulated Inflammatory and Degradative Proteins in Osteoarthritis.

Osteoarthritis (OA) is the most common musculoskeletal disorder causing a great disability and a reduction in the quality of life. In OA, articular chondrocytes (AC) and synovial fibroblasts (SF) release innate-derived immune mediators that initiate and perpetuate inflammation, inducing cartilage extracellular matrix (ECM) degradation. Given the lack of therapies for the treatment of OA, in this study, we explore biomarkers that enable the development of new therapeutical approaches. We analyze the set of secreted proteins in AC and SF co-cultures by stable isotope labeling with amino acids (SILAC). We describe, for the first time, 115 proteins detected in SF-AC co-cultures stimulated by fibronectin fragments (Fn-fs). We also study the role of the vasoactive intestinal peptide (VIP) in this secretome, providing new proteins involved in the main events of OA, confirmed by ELISA and multiplex analyses. VIP decreases proteins involved in the inflammatory process (CHI3L1, PTX3), complement activation (C1r, C3), and cartilage ECM degradation (DCN, CTSB and MMP2), key events in the initiation and progression of OA. Our results support the anti-inflammatory and anti-catabolic properties of VIP in rheumatic diseases and provide potential new targets for OA treatment.

Targeted phospholipidomic analysis of synovial fluid as a tool for osteoarthritis deep phenotyping.

Objective: The aim of this study was to carry out a targeted phospholipidomic analysis on synovial fluid (SF) from patients with different grades of osteoarthritis (OA) and controls, in order to search for specific phospholipid profiles that may be useful for the deep phenotyping of this disease. Design: Multiple reaction monitoring-mass spectrometry (MRM/MS) was applied to explore the potential phospholipidomic differences in the SF of knee OA patients (n â€‹= â€‹15) (subclassified into early- and late-stage OA) and non-OA controls (n â€‹= â€‹4). Multivariate statistical analyses conducted by partial least squares discriminant analysis (PLS-DA) and hierarchical clustering analysis (HCA) were performed to identify significantly altered phospholipids in OA, characterize phospholipidomic profiles associated with the radiographic stage of the disease and describe potential endotypes at early stages. Results: Significant discrimination of phospholipid profiles between non-OA controls and the early- and late-stage OA groups were found by PLS-DA and HCA. Compared to SF from non-OA controls, OA patients showed higher levels of most quantified phospholipid species, including phosphatidylcholines (PC), phosphatidylserines and phosphatidylinositols. Furthermore, several PC species showed significant differences in abundance between the two OA subgroups and were negatively correlated with cartilage damage. Finally, two distinct endotypes of early-stage OA were identified based on the phospholipidomic profile of SF. Conclusions: Our data provides a novel insight into the phospholipid profiles of OA synovial fluid, revealing specific alterations associated with the radiographic stage of the disease. This targeted phospholipidomic profiling also facilitated the characterization of two different OA endotypes at early stages of the disease.

Integrative Metabolic Pathway Analysis Reveals Novel Therapeutic Targets in Osteoarthritis.

In osteoarthritis (OA), impairment of cartilage regeneration can be related to a defective chondrogenic differentiation of mesenchymal stromal cells (MSCs). Therefore, understanding the proteomic- and metabolomic-associated molecular events during the chondrogenesis of MSCs could provide alternative targets for therapeutic intervention. Here, a SILAC-based proteomic analysis identified 43 proteins related with metabolic pathways whose abundance was significantly altered during the chondrogenesis of OA human bone marrow MSCs (hBMSCs). Then, the level and distribution of metabolites was analyzed in these cells and healthy controls by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), leading to the recognition of characteristic metabolomic profiles at the early stages of differentiation. Finally, integrative pathway analysis showed that UDP-glucuronic acid synthesis and amino sugar metabolism were downregulated in OA hBMSCs during chondrogenesis compared with healthy cells. Alterations in these metabolic pathways may disturb the production of hyaluronic acid (HA) and other relevant cartilage extracellular matrix (ECM) components. This work provides a novel integrative insight into the molecular alterations of osteoarthritic MSCs and potential therapeutic targets for OA drug development through the enhancement of chondrogenesis.

Predictive modeling of therapeutic response to chondroitin sulfate/glucosamine hydrochloride in knee osteoarthritis.

BACKGROUND: In the present study, we explored potential protein biomarkers useful to predict the therapeutic response of knee osteoarthritis (KOA) patients treated with pharmaceutical grade Chondroitin sulfate/Glucosamine hydrochloride (CS+GH; Droglican, Bioiberica), in order to optimize therapeutic outcomes. METHODS: A shotgun proteomic analysis by iTRAQ labelling and liquid chromatography-mass spectrometry (LC-MS/MS) was performed using sera from 40 patients enrolled in the Multicentre Osteoarthritis interVEntion trial with Sysadoa (MOVES). The panel of proteins potentially useful to predict KOA patient's response was clinically validated in the whole MOVES cohort at baseline (n = 506) using commercially available enzyme-linked immunosorbent assays kits. Logistic regression models and receiver-operating-characteristics (ROC) curves were used to analyze the contribution of these proteins to our prediction models of symptomatic drug response in KOA. RESULTS: In the discovery phase of the study, a panel of six putative predictive biomarkers of response to CS+GH (APOA2, APOA4, APOH, ITIH1, C4BPa and ORM2) were identified by shotgun proteomics. Data are available via ProteomeXchange with identifier PXD012444. In the verification phase, the panel was verified in a larger set of KOA patients (n = 262). Finally, ITIH1 and ORM2 were qualified by a blind test in the whole MOVES cohort at baseline. The combination of these biomarkers with clinical variables predict the patients' response to CS+GH with a specificity of 79.5% and a sensitivity of 77.1%. CONCLUSIONS: Combining clinical and analytical parameters, we identified one biomarker that could accurately predict KOA patients' response to CS+GH treatment. Its use would allow an increase in response rates and safety for the patients suffering KOA.

Analysis of Endogenous Peptides Released from Osteoarthritic Cartilage Unravels Novel Pathogenic Markers.

Osteoarthritis (OA) is a pathology characterized by the loss of articular cartilage. In this study, we performed a peptidomic strategy to identify endogenous peptides (neopeptides) that are released from human osteoarthritic tissue, which may serve as disease markers. With this aim, secretomes of osteoarthritic and healthy articular cartilages obtained from knee and hip were analyzed by shotgun peptidomics. This discovery step led to the identification of 1175 different peptides, corresponding to 101 proteins, as products of the physiological or pathological turnover of cartilage extracellular matrix. Then, a targeted multiple reaction monitoring-mass spectrometry method was developed to quantify the panel of best marker candidates on a larger set of samples (n = 62). Statistical analyses were performed to evaluate the significance of the observed differences and the ability of the neopeptides to classify the tissue. Eight of them were differentially abundant in the media from wounded zones of OA cartilage compared with the healthy tissue (p < 0.05). Three neopeptides belonging to Clusterin and one from Cartilage Oligomeric Matrix Protein showed a disease-dependent decrease specifically in hip OA, whereas two from Prolargin (PRELP) and one from Cartilage Intermediate Layer Protein 1 were significantly increased in samples from knee OA. The release of one peptide from PRELP showed the best metrics for tissue classification (AUC = 0.834). The present study reveals specific neopeptides that are differentially released from knee or hip human osteoarthritic cartilage compared with healthy tissue. This evidences the intervention of characteristic pathogenic pathways in OA and provides a novel panel of peptidic candidates for biomarker development.

Proteomic Characterization of Epithelial-Like Extracellular Vesicles in Advanced Endometrial Cancer.

Endometrial cancer (EC) is the most frequent gynecological cancer. Tumor dissemination affecting ∼20% of EC patients is characterized at the primary carcinoma by epithelial-to-mesenchymal transition (EMT) associated with myometrial infiltration. At distant sites, the interaction of circulating tumor cells (CTCs) with the microenvironment is crucial for metastatic colonization, with a participation of the extracellular vesicles (EVs). We comprehensively approached these primary and secondary sites to study the impact of tumor EVs on the metastatic efficiency of CTCs in EC. Tumor EVs in circulation reproduce the epithelial phenotype predominant in the primary carcinoma, whereas CTCs are characterized by an EMT phenotype. We modeled this EMT-related clinical scenario in the Hec1A endometrial cell line and characterized the epithelial-like EVs in circulation by SILAC proteome analysis. The identification of proteins involved in cell-cell and cell-matrix interaction and binding, together with in vitro evidence of an improved adhesion of CTC to a functionalized endothelium, suggests a contribution of the epithelial-like EVs in the homing of CTCs at metastatic sites. Accordingly, adhesion protein LGALS3BP was found to be significantly enriched in circulating EVs from a cohort of EC patients with a high risk of recurrence by targeted proteomics (multiple reaction monitoring), highlighting its potential in liquid biopsy in EC.

In vitro comprehensive analysis of VA692 a new chemical entity for the treatment of osteoarthritis.

Selective cyclooxigenase (COX)-2 inhibitors were developed to prevent traditional non-steroidal anti-inflammatory drugs (tNSAIDs) gastro-intestinal adverse effects. VA692, a recently disclosed selective COX-2 inhibitor, structurally related to well-known marketed coxibs, showed anti-inflammatory, and anti-nociceptive properties. The aim of this study was to analyze the anti-inflammatory effect of VA692, in comparison with celecoxib. At this purpose we evaluated the pro-inflammatory cytokines and anti-oxidant enzymes gene expression, apoptosis and ROS production, and PGE2 release in chondrocytes (both primary cultures and immortalized T/C-28a2 cell line) treated with the two drugs. Furthermore, a proteomic analysis has been performed in T/C-28a2 cell line to evaluate modifications in their proteomic profile following drug treatment in presence of IL-1ß. Our results demonstrated the anti-inflammatory effect of the novel synthesized VA692, and confirmed those of celecoxib, in counteracting the stimulus of IL-1ß in both osteoarthritic (OA) chondrocytes and T/C-28a2 cell line. Furthermore, the data underlined the possible anti-apoptotic and anti-oxidant role of VA692, implying its regulation in superoxide anion production as indicated by the modulation of anti-oxidant enzymes. The proteomic analysis provides new information about the effect of VA692 on human T/C-28a2 intracellular proteome, demonstrating the usefulness of this approach in the identification and quantifications of several proteins. Modulation of some proteins such as Hsp90 and SOD by VA692 could explain its role in the therapeutic approach of OA. Based on our results, we can affirm that VA692 has more beneficial effect compared with celecoxib particularly regarding the modulation of oxidant/anti-oxidant system and proteome profile of human articular chondrocytes.

Proteomic Analysis of the E3 Ubiquitin-Ligase Hakai Highlights a Role in Plasticity of the Cytoskeleton Dynamics and in the Proteasome System.

Carcinoma, the most common type of cancer, arises from epithelial cells. The transition from adenoma to carcinoma is associated with the loss of E-cadherin and, in consequence, the disruption of cell-cell contacts. E-cadherin is a tumor suppressor, and it is down-regulated during epithelial-to-mesenchymal transition (EMT); indeed, its loss is a predictor of poor prognosis. Hakai is an E3 ubiquitin-ligase protein that mediates E-cadherin ubiquitination, endocytosis and finally degradation, leading the alterations of cell-cell contacts. Although E-cadherin is the most established substrate for Hakai activity, other regulated molecular targets for Hakai may be involved in cancer cell plasticity during tumor progression. In this work we employed an iTRAQ approach to explore novel molecular pathways involved in Hakai-driven EMT during tumor progression. Our results show that Hakai may have an important influence on cytoskeleton-related proteins, extracellular exosome-associated proteins, RNA-related proteins and proteins involved in metabolism. Moreover, a profound decreased expression in several proteasome subunits during Hakai-driven EMT was highlighted. Since proteasome inhibitors are becoming increasingly used in cancer treatment, our findings suggest that the E3 ubiquitin-ligase, such as Hakai, may be a better target than proteasome for using novel specific inhibitors in tumor subtypes that follow EMT.

Multiplexed mass spectrometry monitoring of biomarker candidates for osteoarthritis.

The methods currently available for the diagnosis and monitoring of osteoarthritis (OA) are very limited and lack sensitivity. Being the most prevalent rheumatic disease, one of the most disabling pathologies worldwide and currently untreatable, there is a considerable interest pointed in the verification of specific biological markers for improving its diagnosis and disease progression studies. Considering the remarkable development of targeted proteomics methodologies in the frame of the Human Proteome Project, the aim of this work was to develop and apply a MRM-based method for the multiplexed analysis of a panel of 6 biomarker candidates for OA encoded by the Chromosome 16, and another 8 proteins identified in previous shotgun studies as related with this pathology, in specimens derived from the human joint and serum. The method, targeting 35 different peptides, was applied to samples from human articular chondrocytes, healthy and osteoarthritic cartilage, synovial fluid and serum. Subsequently, a verification analysis of the biomarker value of these proteins was performed by single point measurements on a set of 116 serum samples, leading to the identification of increased amounts of Haptoglobin and von Willebrand Factor in OA patients. Altogether, the present work provides a tool for the multiplexed monitoring of 14 biomarker candidates for OA, and verifies for the first time the increased amount of two of these circulating markers in patients diagnosed with this disease. SIGNIFICANCE: We have developed an MRM method for the identification and relative quantification of a panel of 14 protein biomarker candidates for osteoarthritis. This method has been applied to analyze human articular chondrocytes, articular cartilage, synovial fluid, and finally a collection of 116 serum samples from healthy controls and patients suffering different degrees of osteoarthritis, in order to verify the biomarker usefulness of the candidates. HPT and VWF were validated as increased in OA patients.

Identification of Factors Produced and Secreted by Mesenchymal Stromal Cells with the SILAC Method.

Mesenchymal stromal cells (MSCs) secrete a large variety of proteins and factors, which shape the secretome. These proteins participate in multiple cellular functions, including the promotion of regenerative processes in the damaged tissue. Secretomes derived from either undifferentiated MSCs or these cells undergoing osteogenic, chondrogenic, or adipogenic differentiation have been characterized using different liquid chromatography tandem mass spectrometry (LC-MS/MS)-based quantitative proteomic approaches. In this chapter, we describe the use of the Stable Isotope Labeling by Amino Acids in Cell culture (SILAC) strategy for the identification and relative quantification of the mesenchymal stromal cell secretome, specifically during chondrogenesis.

Secretome analysis of human articular chondrocytes unravels catabolic effects of nicotine on the joint.

PURPOSE: Osteoarthritis (OA) is a degenerative joint pathology characterized by articular cartilage degradation that lacks from efficient therapy. Since previous epidemiological data show a high controversy regarding the role of smoking in OA, we aimed to evaluate the effects of nicotine (the most physiologically active compound of tobacco) on the joint. EXPERIMENTAL DESIGN: Secretome analyses, based on metabolic labeling followed by LC-MALDI-TOF/TOF analysis, were carried out using an in vitro model of articular inflammation (primary human articular chondrocytes treated with interleukin-1ß), and also on osteoarthritic cells. ELISA and Western blot assays were performed to verify some of the results. RESULTS: Nineteen proteins were altered by nicotine in the model of articular inflammation, including several cytokines and proteases. We confirmed the increased secretion by nicotine of matrix metalloproteinase 1 and two proposed markers of OA, fibronectin, and chitinase 3-like protein 1. Finally, four components of the extracellular matrix of cartilage were decreased by nicotine in OA chondrocytes. CONCLUSIONS AND CLINICAL RELEVANCE: Our data contribute to a better understanding of the molecular mechanisms that are modulated by nicotine in cartilage cells, suggesting a negative effect of this drug on the joint.

Lessons from the proteomic study of osteoarthritis.

Osteoarthritis is the most common rheumatic pathology and one of the leading causes of disability worldwide. It is a very complex disease whose etiopathogenesis is not fully understood. Furthermore, there are serious limitations for its management, since it lacks specific and sensitive biomarkers for early diagnosis, prognosis and therapeutic monitoring. Proteomic approaches performed in the last few decades have contributed to the knowledge on the molecular mechanisms that participate in this pathology and they have also led to interesting panels of putative biomarker candidates. In the next few years, further efforts should be made for translating these findings into the clinical routines. It is expected that targeted proteomics strategies will be highly valuable for the verification and qualification of biomarkers of osteoarthritis.

The Spanish biology/disease initiative within the human proteome project: Application to rheumatic diseases.

The Spanish Chromosome 16 consortium is integrated in the global initiative Human Proteome Project, which aims to develop an entire map of the proteins encoded following a gene-centric strategy (C-HPP) in order to make progress in the understanding of human biology in health and disease (B/D-HPP). Chromosome 16 contains many genes encoding proteins involved in the development of a broad range of diseases, which have a significant impact on the health care system. The Spanish HPP consortium has developed a B/D platform with five programs focused on selected medical areas: cancer, obesity, cardiovascular, infectious and rheumatic diseases. Each of these areas has a clinical leader associated to a proteomic investigator with the responsibility to get a comprehensive understanding of the proteins encoded by Chromosome 16 genes. Proteomics strategies have enabled great advances in the area of rheumatic diseases, particularly in osteoarthritis, with studies performed on joint cells, tissues and fluids. BIOLOGICAL SIGNIFICANCE: In this manuscript we describe how the Spanish HPP-16 consortium has developed a B/D platform with five programs focused on selected medical areas: cancer, obesity, cardiovascular, infectious and rheumatic diseases. Each of these areas has a clinical leader associated to a proteomic investigator with the responsibility to get a comprehensive understanding of the proteins encoded by Chromosome 16 genes. We show how the Proteomic strategy has enabled great advances in the area of rheumatic diseases, particularly in osteoarthritis, with studies performed on joint cells, tissues and fluids. This article is part of a Special Issue entitled: HUPO 2014.

Quantitative proteomic profiling of human articular cartilage degradation in osteoarthritis.

Osteoarthritis (OA) is the most common rheumatic pathology and is characterized primarily by articular cartilage degradation. Despite its high prevalence, there is no effective therapy to slow disease progression or regenerate the damaged tissue. Therefore, new diagnostic and monitoring tests for OA are urgently needed, which would also promote the development of alternative therapeutic strategies. In the present study, we have performed an iTRAQ-based quantitative proteomic analysis of secretomes from healthy human articular cartilage explants, comparing their protein profile to those from unwounded (early disease) and wounded (advanced disease) zones of osteoarthritic tissue. This strategy allowed us to identify a panel of 76 proteins that are distinctively released by the diseased tissue. Clustering analysis allowed the classification of proteins according to their different profile of release from cartilage. Among these proteins, the altered release of osteoprotegerin (decreased in OA) and periostin (increased in OA), both involved in bone remodelling processes, was verified in further analyses. Moreover, periostin was also increased in the synovial fluid of OA patients. Altogether, the present work provides a novel insight into the mechanisms of human cartilage degradation and a number of new cartilage-characteristic proteins with possible biomarker value for early diagnosis and prognosis of OA.

A pharmacoproteomic study confirms the synergistic effect of chondroitin sulfate and glucosamine.

Osteoarthritis (OA) is the most common age-related rheumatic disease. Chondrocytes play a primary role in mediating cartilage destruction and extracellular matrix (ECM) breakdown, which are main features of the OA joint. Quantitative proteomics technologies are demonstrating a very interesting power for studying the molecular effects of some drugs currently used to treat OA patients, such as chondroitin sulfate (CS) and glucosamine (GlcN). In this work, we employed the iTRAQ (isobaric tags for relative and absolute quantitation) technique to assess the effect of CS and GlcN, both alone and in combination, in modifying cartilage ECM metabolism by the analysis of OA chondrocytes secretome. 186 different proteins secreted by the treated OA chondrocytes were identified. 36 of them presented statistically significant differences (p ≤ 0.05) between untreated and treated samples: 32 were increased and 4 decreased. The synergistic chondroprotective effect of CS and GlcN, firstly reported by our group at the intracellular level, is now demonstrated also at the extracellular level.