RESUMEN
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.
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Cartílago Articular , Osteoartritis de la Rodilla , Humanos , Cartílago Articular/metabolismo , Articulación de la Rodilla/metabolismo , Articulación de la Rodilla/patología , Osteoartritis de la Rodilla/metabolismoRESUMEN
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.
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Osteoartritis de la Rodilla , Humanos , Osteoartritis de la Rodilla/diagnóstico por imagen , Osteoartritis de la Rodilla/epidemiología , Pronóstico , Estudios Prospectivos , Incidencia , Articulación de la Rodilla , Biomarcadores , Progresión de la EnfermedadRESUMEN
OBJECTIVE: Osteoarthritis (OA) is a complex disease involving contributions from both local joint tissues and systemic sources. Patient characteristics, encompassing sociodemographic and clinical variables, are intricately linked with OA rendering its understanding challenging. Technological advancements have allowed for a comprehensive analysis of transcripts, proteomes and metabolomes in OA tissues/fluids through omic analyses. The objective of this review is to highlight the advancements achieved by omic studies in enhancing our understanding of OA pathogenesis over the last three decades. DESIGN: We conducted an extensive literature search focusing on transcriptomics, proteomics and metabolomics within the context of OA. Specifically, we explore how these technologies have identified individual transcripts, proteins, and metabolites, as well as distinctive endotype signatures from various body tissues or fluids of OA patients, including insights at the single-cell level, to advance our understanding of this highly complex disease. RESULTS: Omic studies reveal the description of numerous individual molecules and molecular patterns within OA-associated tissues and fluids. This includes the identification of specific cell (sub)types and associated pathways that contribute to disease mechanisms. However, there remains a necessity to further advance these technologies to delineate the spatial organization of cellular subtypes and molecular patterns within OA-afflicted tissues. CONCLUSIONS: Leveraging a multi-omics approach that integrates datasets from diverse molecular detection technologies, combined with patients' clinical and sociodemographic features, and molecular and regulatory networks, holds promise for identifying unique patient endophenotypes. This holistic approach can illuminate the heterogeneity among OA patients and, in turn, facilitate the development of tailored therapeutic interventions.
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Osteoartritis , Proteómica , Humanos , Metabolómica , Perfilación de la Expresión Génica , Proteoma , Osteoartritis/genética , Osteoartritis/metabolismoRESUMEN
OBJECTIVE: Osteoarthritis (OA) is the most prevalent musculoskeletal disease affecting articulating joint tissues, resulting in local and systemic changes that contribute to increased pain and reduced function. Diverse technological advancements have culminated in the advent of high throughput "omic" technologies, enabling identification of comprehensive changes in molecular mediators associated with the disease. Amongst these technologies, genomics and epigenomics - including methylomics and miRNomics, have emerged as important tools to aid our biological understanding of disease. DESIGN: In this narrative review, we selected articles discussing advancements and applications of these technologies to OA biology and pathology. We discuss how genomics, deoxyribonucleic acid (DNA) methylomics, and miRNomics have uncovered disease-related molecular markers in the local and systemic tissues or fluids of OA patients. RESULTS: Genomics investigations into the genetic links of OA, including using genome-wide association studies, have evolved to identify 100+ genetic susceptibility markers of OA. Epigenomic investigations of gene methylation status have identified the importance of methylation to OA-related catabolic gene expression. Furthermore, miRNomic studies have identified key microRNA signatures in various tissues and fluids related to OA disease. CONCLUSIONS: Sharing of standardized, well-annotated omic datasets in curated repositories will be key to enhancing statistical power to detect smaller and targetable changes in the biological signatures underlying OA pathogenesis. Additionally, continued technological developments and analysis methods, including using computational molecular and regulatory networks, are likely to facilitate improved detection of disease-relevant targets, in-turn, supporting precision medicine approaches and new treatment strategies for OA.
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Metilación de ADN , Epigenómica , Genómica , Osteoartritis , Humanos , Osteoartritis/genética , Estudio de Asociación del Genoma Completo , MicroARNs/genética , Predisposición Genética a la EnfermedadRESUMEN
OBJECTIVES: To discover autoantibodies to non-modified proteins associated with the presence/absence of anticitrullinated protein antibodies (ACPA) in rheumatoid arthritis (RA). METHODS: The autoantibody repertoire of 80 ACPA negative and 80 ACPA positive RA subjects from the Swedish population-based Epidemiological Investigation of RA (EIRA) cohort was screened using a suspension bead array built on protein fragments earlier described as autoimmunity targets. Four autoantibodies positive in the initial screening were validated in another set of EIRA samples containing 317 ACPA-positive, 302 ACPA-negative and 372 age- and sex-matched controls. The relationship between the four autoantibodies and lung abnormalities on high-resolution computed tomography (HRTC) was examined in 93 early RA patients from LURA cohort. Association between the autoantibodies, smoking and MHC class II alleles was assessed by logistic regression analysis. RESULTS: : Anti-ANOS1 and anti-MURC IgG levels were associated with ACPA-positive status (OR = 3.02; 95% CI 1.87-4.89; and OR = 1.86; 95% CI 1.16-2.97, respectively) and increased in ACPA-positive patients compared with controls. Anti-ANOS1 IgG was associated with smoking habit (OR = 2.11; 95% CI 1.22-3.69) and anti-MURC IgG with the presence of the MHC class II "shared-epitope" genes (OR = 1.95; 95% CI 1.11-3.46). Anti-TSPYL4 IgG was associated with ACPA-negative (OR = 0.41; 95% CI 0.19-0.89). Anti-TSPYL4 IgG and anti-MAP2K6 IgG levels were increased in the ACPA-negative patients compared with controls. Presence of anti-MAP2K6 IgG and anti-TSPYL4 IgG correlated negatively with HRCT-defined lung abnormalities. CONCLUSIONS: These four autoantibodies may be useful in diagnostics and in predicting clinical phenotypes of RA.
RESUMEN
Rheumatic diseases are high prevalence pathologies with different etiology and evolution and low sensitivity in clinical diagnosis. Therefore, it is necessary to develop an early diagnosis method which allows personalized treatment, depending on the specific pathology. The biology/disease initiative, at Human Proteome Project, is an integrative approach to identify relevant proteins in the human proteome associated with pathologies. A previously reported literature data mining analysis, which identified proteins related to osteoarthritis (OA), rheumatoid arthritis (RA), and psoriatic arthritis (PSA) was used to establish a systematic prioritization of potential biomarkers candidates for further evaluation by functional proteomics studies. The aim was to study the protein profile of serum samples from patients with rheumatic diseases such as OA, RA, and PSA. To achieve this goal, customized antibody microarrays (containing 151 antibodies targeting 121 specific proteins) were used to identify biomarkers related to early and specific diagnosis in a screening of 960 serum samples (nondepleted) (OA, n = 480; RA, n = 192; PSA, n = 288). This functional proteomics screening has allowed the determination of a panel (30 serum proteins) as potential biomarkers for these rheumatic diseases, displaying receiver operating characteristics curves with area under the curve values of 80-90%.
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Artritis Psoriásica , Artritis Reumatoide , Osteoartritis , Enfermedades Reumáticas , Humanos , Proteoma , Artritis Reumatoide/metabolismo , Osteoartritis/diagnóstico , Enfermedades Reumáticas/diagnóstico , Biomarcadores , Artritis Psoriásica/diagnósticoRESUMEN
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.
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ADN Mitocondrial , Osteoartritis de la Rodilla , Humanos , ADN Mitocondrial/genética , Osteoartritis de la Rodilla/genética , Especies Reactivas de Oxígeno , Estudios Prospectivos , Mitocondrias/genética , Inflamación/metabolismoRESUMEN
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.
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Redes y Vías Metabólicas , Terapia Molecular Dirigida , Osteoartritis/tratamiento farmacológico , Osteoartritis/metabolismo , Estudios de Casos y Controles , Condrogénesis , Humanos , Células Madre Mesenquimatosas/metabolismo , Metaboloma , Vía de Pentosa Fosfato , Uridina Difosfato Ácido Glucurónico/biosíntesisRESUMEN
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.
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Biomarcadores/metabolismo , Cartílago Articular/citología , Osteoartritis de la Cadera/metabolismo , Osteoartritis de la Rodilla/metabolismo , Péptidos/metabolismo , Proteómica/métodos , Anciano , Anciano de 80 o más Años , Cartílago Articular/metabolismo , Cartílago Articular/patología , Estudios de Casos y Controles , Células Cultivadas , Cromatografía Liquida , Medios de Cultivo Condicionados/química , Matriz Extracelular/metabolismo , Femenino , Humanos , Masculino , Especificidad de Órganos , Osteoartritis de la Cadera/patología , Osteoartritis de la Rodilla/patología , Espectrometría de Masas en TándemRESUMEN
A steady increase in the incidence of osteoarthritis and other rheumatic diseases has been observed in recent decades, including autoimmune conditions such as rheumatoid arthritis, spondyloarthropathies, systemic lupus erythematosus, systemic sclerosis, and Sjögren's syndrome. Rheumatic and autoimmune diseases (RADs) are characterized by the inflammation of joints, muscles, or other connective tissues. In addition to often experiencing debilitating mobility and pain, RAD patients are also at a higher risk of suffering comorbidities such as cardiovascular or infectious events. Given the socioeconomic impact of RADs, broad research efforts have been dedicated to these diseases worldwide. In the present work, we applied literature mining platforms to identify "popular" proteins closely related to RADs. The platform is based on publicly available literature. The results not only will enable the systematic prioritization of candidates to perform targeted proteomics studies but also may lead to a greater insight into the key pathogenic processes of these disorders.
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Enfermedades Autoinmunes/metabolismo , Proteínas/metabolismo , Proteoma , Enfermedades Reumáticas/metabolismo , Artritis Reumatoide/metabolismo , Minería de Datos , Humanos , Osteoartritis/metabolismoRESUMEN
OBJECTIVE: To find autoantibodies (AAbs) in serum that could be useful to predict incidence of radiographic knee osteoarthritis (KOA). DESIGN: A Nucleic-acid Programmable Protein Arrays (NAPPA) platform was used to screen AAbs against 2125 human proteins in sera at baseline from participants free of radiographic KOA belonging to the incidence and non-exposed subcohorts of the Osteoarthritis Initiative (OAI) who developed or not, radiographic KOA during a follow-up period of 96 months. NAPPA-ELISA were performed to analyse reactivity against methionine adenosyltransferase two beta (MAT2ß) and verify the results in 327 participants from the same subcohorts. The association of MAT2ß-AAb levels with KOA incidence was assessed by combining several robust biostatistics analysis (logistic regression, Receiver Operating Characteristic and Kaplan-Meier curves). The proposed prognostic model was replicated in samples from the progression subcohort of the OAI. RESULTS: In the screening phase, six AAbs were found significantly different at baseline in samples from incident compared with non-incident participants. In the verification phase, high levels of MAT2ß-AAb were significantly associated with the future incidence of KOA and with an earlier development of the disease. The incorporation of this AAb in a clinical model for the prognosis of incident radiographic KOA significantly improved the identification/classification of patients who will develop the disorder. The usefulness of the model to predict radiographic KOA was confirmed on a different OAI subcohort. CONCLUSIONS: The measurement of AAbs against MAT2ß in serum might be highly useful to improve the prediction of OA development, and also to estimate the time to incidence.
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Autoanticuerpos/sangre , Diagnóstico Precoz , Articulación de la Rodilla/diagnóstico por imagen , Osteoartritis de la Rodilla/diagnóstico , Autoanticuerpos/inmunología , Biomarcadores/sangre , Estudios de Casos y Controles , Progresión de la Enfermedad , Ensayo de Inmunoadsorción Enzimática , Femenino , Estudios de Seguimiento , Humanos , Incidencia , Masculino , Persona de Mediana Edad , Osteoartritis de la Rodilla/sangre , Osteoartritis de la Rodilla/epidemiología , Valor Predictivo de las Pruebas , Curva ROC , Radiografía , España/epidemiologíaRESUMEN
PURPOSE OF REVIEW: 'Omics' technologies developed for the massive analysis of the major biologically relevant molecules (genes, proteins, metabolites) have been applied to the study of osteoarthritis (OA) for more than a decade. RECENT FINDINGS: 'Omics' studies have undoubtedly contributed to increase the knowledge on pathogenic processes related with OA and have provided hundreds to thousands of molecules that might have a putative biomarker utility for this disease. SUMMARY: This review describes the most recent 'omics' studies in OA research, their conclusions, and discuss those remaining challenges. Still many validation studies must be performed in large and well-characterized cohorts for the translation of the findings from 'omics' strategies to clinical applications. The development of tools for the intelligent integration of 'omics' data with clinical and imaging information is also mandatory to take full profit of the work that has been already performed.
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Osteoartritis/genética , Proteínas/genética , Biomarcadores/metabolismo , Estudio de Asociación del Genoma Completo , Genómica , Humanos , Metabolómica , Osteoartritis/metabolismo , Proteínas/metabolismo , ProteómicaRESUMEN
Osteoarthritis (OA) is one of the most prevalent articular diseases. The identification of proteins closely associated with the diagnosis, progression, prognosis, and treatment response is dramatically required for this pathology. In this work, differential serum protein profiles have been identified in OA and rheumatoid arthritis (RA) by antibody arrays containing 151 antibodies against 121 antigens in a cohort of 36 samples. Then the identified differential serum protein profiles have been validated in a larger cohort of 282 samples. The overall immunoreactivity is higher in the pathological situations in comparison with the controls. Several proteins have been identified as biomarker candidates for OA and RA. Most of these biomarker candidates are proteins related to inflammatory response, lipid metabolism, or bone and extracellular matrix formation, degradation, or remodeling.
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Artritis Reumatoide/diagnóstico , Biomarcadores/sangre , Osteoartritis/diagnóstico , Análisis por Matrices de Proteínas/métodos , Anticuerpos , Artritis Reumatoide/sangre , Artritis Reumatoide/patología , Estudios de Casos y Controles , Progresión de la Enfermedad , Humanos , Osteoartritis/sangre , Osteoartritis/patología , Pronóstico , Resultado del TratamientoRESUMEN
INTRODUCTION: The heterogeneity of Rheumatoid Arthritis (RA) and the absence of clinical tests accurate enough to identify the early stages of this disease have hampered its management. Therefore, proteomics research is increasingly focused on the discovery of novel biological markers, which would not only be able make an early diagnosis, but also to gain insight into the different pathological mechanisms underlying the heterogeneity of RA and also to stratify patients, which is critical to enabling effective treatments. Areas covered: The proteomic approaches that have been utilised to provide knowledge about RA pathogenesis, and to identify biomarkers for RA diagnosis, prognosis, disease monitoring and prediction of response to therapy, are summarized. Expert commentary: Although each proteomic study is unique in its design, all of them have contributed to the understanding of RA pathogenesis and the discovery of promising biomarkers for patient stratification, which would improve clinical care of RA patients. Still, efforts need to be made to validate these findings and translate them into clinical practice.
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Artritis Reumatoide/metabolismo , Técnicas de Diagnóstico Molecular/métodos , Proteoma/metabolismo , Proteómica/métodos , Artritis Reumatoide/diagnóstico , Artritis Reumatoide/etiología , Biomarcadores/química , Biomarcadores/metabolismo , Humanos , Proteoma/químicaRESUMEN
Mass spectrometry imaging (MSI) is a technique used to visualize the spatial distribution of biomolecules such as peptides, proteins, lipids or other organic compounds by their molecular masses. Among the different MSI strategies, MALDI-MSI provides a sensitive and label-free approach for imaging of a wide variety of protein or peptide biomarkers from the surface of tissue sections, being currently used in an increasing number of biomedical applications such as biomarker discovery and tissue classification. In the field of rheumatology, MALDI-MSI has been applied to date for the analysis of joint tissues such as synovial membrane or cartilage. This review summarizes the studies and key achievements obtained using MALDI-MSI to increase understanding on rheumatic pathologies and to describe potential diagnostic or prognostic biomarkers of these diseases. This article is part of a Special Issue entitled: MALDI Imaging, edited by Dr. Corinna Henkel and Prof. Peter Hoffmann.
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Enfermedades Reumáticas/diagnóstico , Enfermedades Reumáticas/patología , Animales , Biomarcadores/metabolismo , Cartílago/metabolismo , Cartílago/patología , Humanos , Pronóstico , Enfermedades Reumáticas/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Membrana Sinovial/metabolismo , Membrana Sinovial/patologíaRESUMEN
Mesenchymal stem cells (MSC) are an interesting alternative for cell-based therapy of cartilage defects attributable to their capacity to differentiate toward chondrocytes in the process termed chondrogenesis. The metabolism of lipids has recently been associated with the modulation of chondrogenesis and also with the development of pathologies related to cartilage degeneration. Information about the distribution and modulation of lipids during chondrogenesis could provide a panel of putative chondrogenic markers. Thus, the discovery of new lipid chondrogenic markers could be highly valuable for improving MSC-based cartilage therapies. In this work, MS imaging was used to characterize the spatial distribution of lipids in human bone marrow MSCs during the first steps of chondrogenic differentiation. The analysis of MSC micromasses at days 2 and 14 of chondrogenesis by MALDI-MSI led to the identification of 20 different lipid species, including fatty acids, sphingolipids, and phospholipids. Phosphocholine, several sphingomyelins, and phosphatidylcholines were found to increase during the undifferentiated chondrogenic stage. A particularly detected lipid profile was verified by TOF secondary ion MS. Using this technology, a higher intensity of phosphocholine-related ions was observed in the peripheral region of the micromasses collected at day 14.
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Condrogénesis/fisiología , Biología Computacional/métodos , Lípidos/análisis , Espectrometría de Masas/métodos , Células Madre Mesenquimatosas/citología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Biomarcadores/análisis , Biomarcadores/química , Células de la Médula Ósea/química , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Cabeza Femoral/citología , Humanos , Lípidos/química , Células Madre Mesenquimatosas/química , Células Madre Mesenquimatosas/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/análisis , Fosfotransferasas (Aceptor de Grupo Alcohol)/químicaRESUMEN
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.
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Biomarcadores/metabolismo , Osteoartritis/metabolismo , Proteómica , Humanos , Osteoartritis/diagnóstico , Osteoartritis/fisiopatología , PronósticoRESUMEN
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.
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Cartílago Articular/metabolismo , Osteoartritis/metabolismo , Proteoma/metabolismo , Proteómica/métodos , Anciano , Anciano de 80 o más Años , Biomarcadores/metabolismo , Western Blotting , Cartílago Articular/patología , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Cromatografía Liquida , Femenino , Humanos , Masculino , Persona de Mediana Edad , Análisis Multivariante , Osteoartritis/diagnóstico , Osteoartritis/genética , Osteoprotegerina/genética , Osteoprotegerina/metabolismo , Proteoma/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Líquido Sinovial/metabolismo , Espectrometría de Masas en Tándem , Técnicas de Cultivo de TejidosRESUMEN
Human mesenchymal stem cells (hMSCs) can be triggered to differentiate toward chondrocytes and thus harbor great therapeutic potential for the repair of cartilage defects in osteoarthritis (OA) and other articular diseases. However, the molecular mechanisms underlying the chondrogenesis process are still in part unknown. In this work, we followed a double-stable isotope labeling by amino acids in cell culture (SILAC) strategy to evaluate the quantitative modulation of the secretome of stem cells isolated from bone marrow (hBMSCs) during the first steps of their chondrogenic differentiation. Analysis by LC-ESI-MS/MS led to the identification of 221 proteins with a reported extracellular localization. Most of them were characteristic of cartilage extracellular matrix, and 34 showed statistically significant quantitative alterations during chondrogenesis. These include, among others, cartilage markers such as Proteoglycan 4 or COMP, anticatabolic markers (TIMP1), reported markers of cartilage development (Versican), and a suggested marker of chondrogenesis, CRAC1. Altogether, our work demonstrates the usefulness of secretome analysis for understanding the mechanisms responsible for cartilage matrix formation, and it reports a panel of extracellular markers potentially useful for the evaluation of tissue development in cell therapy- or tissue engineering-based approaches for cartilage repair.
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Diferenciación Celular , Condrocitos/citología , Células Madre Mesenquimatosas/metabolismo , Western Blotting , Células Cultivadas , Cromatografía Liquida , Electroforesis en Gel de Poliacrilamida , Humanos , Células Madre Mesenquimatosas/citología , Proteínas/química , Proteínas/metabolismo , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
We tested a semiautomated protocol for the proper storage and conservation in a hospital biobank of tryptic peptide extracts coming from samples with low and high protein complexity for subsequent mass spectrometry analysis. Low-complexity samples (serum albumin, serotransferrin. and alpha-S1-casein) were loaded in replicates in SDS-PAGE and subjected to standard in-gel trypsin digestion. For LC-MALDI-TOF/TOF analysis, purified ß-galactosidase and human serum samples were in-solution digested following standard procedures and desalted with C18 stage-tips. In both cases, peptides extracts were aliquoted in individually 2D coded tubes, vacuum-dried, barcode-read, and stored in an automated -20 °C freezer in the Biobank facility. Samples were kept dried at -20 °C until the corresponding time-point of analysis, then reconstituted in the proper buffer and analyzed by either MALDI-TOF/TOF (peptide fingerprinting and MS/MS) or LC-MALDI-TOF/TOF following a highly reproducible pattern to ensure the reproducibility of the results. Protein identification was done with either Mascot or Protein Pilot as search engines using constant parameters. Over a period of 1 year we checked six different time points at days 0, 7, 30, 90, 180, and 365. We compared MS and MS/MS protein score, number of identified peptides, and coverage of the identified proteins. In the low complexity samples, the number of peptides detected gradually decreased over time, especially affecting the MS score. However, two of the three proteins - serum albumin and serotransferrin - were identified by both PMF and MS/MS at day 90. By day 180, only MS/MS identification in some replicates was possible. By LC-MS/MS, ß-galactosidase and the most abundant serum proteins were identified with good scores at all time points even by day 365, with no detectable peptide loss or decrease in the fragmentation efficiency, although a progressive decrease in peptide intensity indicates that detection of low abundant proteins could not be optimal after very long periods of time. Our results encourage us to use the biobank facility in the future for long-term storage - up to 3 months - of dried peptide extracts.