Búsqueda de biomarcadores de artrosis mediante técnicas proteómicas

[Resumen] La Artrosis (OA) es la enfermedad reumática más frecuente e invalidante a nivel mundial. Se caracteriza principalmente por una degradación progresiva del cartílago articular pero también por cambios en el hueso subcondral y la inflamación de la membrana sinovial. Pese a su elevada prevalencia, los métodos de diagnóstico actuales de la OA son poco sensibles y no existe tratamiento eficaz frente a la enfermedad. Estas limitaciones han originado un considerable interés por encontrar marcadores biológicos específicos que reflejen las variaciones cuantitativas y dinámicas de la remodelación de la articulación durante la artrosis. Teniendo en cuenta que el proceso de OA implica diferentes tejidos y complejos procesos biológicos, la estrategia diagnóstica más prometedora podría ser la combinación del estudio de diferentes biomarcadores con el fin de facilitar el diagnóstico precoz, evaluar la progresión de la enfermedad y posibilitar la monitorización de terapias alternativas. Así, en esta tesis se han empleado diferentes técnicas proteómicas de expresión diferencial basadas en espectrometría de masas y en arrays de proteínas para abordar el estudio de la búsqueda de potenciales biomarcadores de artrosis. En el primer trabajo presentado, se analizó el perfil de degradación del cartílago artrósico con la finalidad de identificar potenciales marcadores tempranos y de progresión de la enfermedad. Para ello, se utilizaron muestras de diferentes zonas del cartílago artrósico humano y también cartílago sano. En este estudio, se identificó un panel de 76 proteínas que estaban diferencialmente liberadas por el cartílago artrósico al compararlo con el cartílago sano. A continuación, se validó por inmunodetección el potencial de dos proteínas como marcadores de progresión de la degradación del cartílago artrósico. En el segundo trabajo, se utilizaron muestras de líquido sinovial de pacientes con OA y pacientes con artritis reumatoide (AR), con el objetivo de caracterizar el perfil proteico del líquido sinovial en la OA. Posteriormente, se validaron por inmunodetección los resultados obtenidos para 4 de las proteínas cuantificadas por espectrometría de masas. A continuación, utilizamos arrays de proteínas para buscar potenciales biomarcadores séricos de la OA. Así, en el tercer trabajo, se utilizaron muestras de suero de pacientes con artrosis, artritis reumatoide y controles sanos con el objetivo de identificar autoanticuerpos con potencial biomarcador de la artrosis empleando arrays de antígenos. En este trabajo, detectamos que los niveles de inmunorreactividad frente a 7 antígenos permitían distinguir los pacientes artrósicos de los individuos sanos mientras que los niveles de inmunorreactividad frente a otros 7 antígenos, permitían distinguir entre los pacientes con OA y con AR. Por último, se construyeron arrays de anticuerpos en suspensión con el fin de identificar, en un gran número de muestras, un panel de proteínas en el suero con potencial biomarcador para el diagnóstico de la artrosis. Esta metodología se utilizó para analizar muestras de suero de pacientes con diferentes enfermedades reumáticas (artrosis, artritis reumatoide y artritis psoriásica) e individuos sanos. Los resultados obtenidos proporcionaron un perfil proteico con potencial biomarcador para el diagnóstico de la artrosis de rodilla.[Resumo] A artrose (OA) é a enfermidade reumática mais frecuente e invalidante a nivel mundial. Caracterízase principalmente por unha degradación progresiva da cartilaxe articular pero tamén por cambios no óso subcondral e a inflamación da membrana sinovial. Pese a súa prevalencia, os métodos actuais de diagnose da OA son pouco sensibles e non existe tratamento eficaz frente a enfermidade. Estas limitacións orixinaron un considerable interés por encontrar marcadores biolóxicos específicos que reflexen variacións cuantitativas e dinámicas da remodelación da articulación durante a OA. Tendo en conta que o proceso artrósico implica a diferentes texidos e complexos procesos biolóxicos, a estratexia diagnóstica mais prometedora podería ser a combinación do estudio de diferentes biomarcadores coa finalidade de facilitar a diagnose temperá, evaluar a progresión da enfermidade e posibilitar a monitorización de terapias alternativas. Así, nesta tese se empregaron diferentes técnicas proteómicas de expresión diferencial basadas en espectrometría de masas e en arrays de proteínas para abordar o estudo da búsqueda de biomarcadores de artrose. No primeiro traballo presentado, se analizou o perfil de degradación proteica da cartilaxe artrósica co obxectivo de indentificar potenciais marcadores temperáns e de progresión da enfermidade. Para isto, se utilizaron mostras de diferentes zonas da cartilaxe artrósica humana e tamén cartilaxe sana. En este estudio, se identificou un panel de 76 proteínas que estaban diferencialmente liberadas pola cartilaxe artrósica en comparación coa cartilaxe sana. A continuación, validamos por inmunodetección o potencial de dúas proteínas como marcadores da degradación progresiva da cartilaxe artrósica. No segundo traballo, se empregaron mostras de líquido sinovial de pacientes con artrose e pacientes con artritis reumatoide (AR), co obxectivo de caracterizar o perfil proteico do líquido sinovial na OA. Posteriormente, se validaron por inmunodetección os resultados obtidos para dúas das proteínas cuantificadas por espectrometría de masas. A continuación, utilizamos arrays de proteínas para buscar potenciais biomarcadores séricos da OA. Así, no terceiro traballo, se utilizaron mostras de soro de pacientes con OA, pacientes con AR e individuos sanos para buscar autoanticorpos con potencial biomarcador da artrose empregando arrays de antíxenos. Neste traballo, detectamos que os niveis de inmurreactividade frente a 7 antíxenos permitían distinguir entre pacientes con OA e individuos sanos, mentres que os niveis de inmurreactividade frente a outros 7 antíxenos permitían distinguir entre pacientes con OA e AR. Por último, se construíron arrays de anticorpos en suspensión coa finalidade de identificar, nun gran número de mostras, un panel de proteínas no soro con potencial biomarcador para a diagnose da artrose. Así, se analizaron mostras de soro de doentes con diferentes enfermidades reumáticas (artrose, artritis reumatoide e artritis psoriásica) e individuos sanos. Os resultados obtidos proporcionaron un perfil proteico con potencial biomarcador para a diagnose da artrose de xeonllo.[Abstract] Osteoarthritis (OA) is the most common and disabling rheumatic disease worldwide. It is characterized by the progressive loss of cartilage, subchondral bone remodelling and synovial inflammation. Despite its high prevalence, current diagnosis methods are poor sensitive and there is no efficient treatment for the disease. These limitations have prompted a considerable interest in finding specific biological markers to reflect quantitative and dynamic joint changes that occur in OA. Taking in account that OA process involves different tissues and complex biological process, the most promising strategy for early diagnosis and monitoring of the disease, could be the combination of the study of different biomarkers. Therefore, in this thesis, different proteomic technologies based on mass spectrometry and protein arrays were employed to address the research of potential OA biomarkers. Firstly, we aimed to study the protein profile of OA cartilage degradation to find potential early and progression OA biomarkers. For this, we used OA and healthy human cartilage explants. A panel of 76 differentially secreted proteins from OA cartilage compared to normal cartilage was found. Then, two proteins with potential value as progression biomarkers were validated. Next, we used synovial fluid from OA patients and rheumatoid arthritis (RA) patients to find a characteristic protein profile of OA process using mass spectrometry. Four modulated proteins were validated. Then we used protein arrays to find potential serum OA biomarkers. Therefore, antigen arrays were used to search for serum autoantibodies in OA comparing the autoantibody profile from OA patients, healthy donors as well as RA patients. We observed immunoreactivity levels towards 7 antigens allowed to distinguish between OA patients and healthy controls, whereas the immunoreactivity towards other 7 antigens enables to discriminate between OA and RA patients. Finally, we used antibody suspension bead arrays to identify, in a large set of samples, a panel of serum proteins with potential value for OA diagnosis. This methodology was used to analyse serum samples from patients with different rheumatic diseases (OA, RA and psoriatic arthritis) and healthy donors. The results obtained provide a serum protein profile with a great potential for knee OA diagnosis

O programa STEmbach na Área de Bioquímica e Bioloxía Molecular da Facultade de Ciencias

Colección: Contextos Universitarios Transformadores (CUT). Número 7[Resumo] O programa do bacharelato de excelencia en Ciencias e Tecnoloxía, STEMbach, é un programa da Xunta de Galicia, orientado a alumnos de primeiro e segundo de Bacharelato cuxo obxetivo é fomentar as ciencias entre os estudantes, baseándose no pensamento científico e a través dun plantexamento eminentemente práctico. O papel da Universidade e brindar aos alumnos a posibilidade de realizar proxectos científicos nas instalacións da UDC dirixidos por Profesores especialistas nas diversas materias, achegándoos, dun modo realista, ao mundo da investigación científica. A Área de Bioquímica e Bioloxía Molecular da Facultade de Ciencias participou nas tres últimas edicións, desenvolvendo seis proxectos distintos onde participaron catro IES, públicos e concertados e un total de 14 alumnos. Aínda que polo momento o número de estudantes que participaron nos proxectos STEMbach é relativamente baixo, as enquisas realizadas mostran que para a maioría o programa é útil e interesante, sobre todo a parte experimental realizada na UDC, destacando que lles gustaría dedicarlle máis tempo. Pola sua banda, os profesores de bacharelato manifestaron a sua preferencia polo establecemento dunha colaboración estable entre o seu centro e o proxecto da UDC seleccionado, repetindo o mesmo ano tras ano, e así poder axustar mellor a parte teórica á práctica.[Abstract] The program of the High School degree with excellence recognition in Science and Technology, STEMbach is a program of the Xunta de Galicia, aimed at first and second year high school students to promote science among students, based on scientific thinking and through an eminently practical approach. The role of the University is to provide students the possibility of carrying out scientific projects at UDC directed by specialist Professors in several subjects, bringing students closer to the world of scientific research in a realistic way. The Area of Biochemistry and Molecular Biology of the Faculty of Sciences has participated in the last three editions, developing six different projects where four IES, public and private and 14 students have participated. Although the number of students who participated in the STEMbach projects is relatively low, surveys show that for most students the program is useful and interesting, especially the experimental part performed at UDC, emphasizing that they would like to spend more time at laboraroy. On the other hand, the high school teachers expressed their preference for the establishment of a stable collaboration between their center and the selected UDC project, repeating the same project year after year, allowing adjust the theoretical part to practice.http://hdl.handle.net/2183/3249

A Clinical Model Including Protein Biomarkers Predicts Radiographic Knee Osteoarthritis: A Prospective Study Using Data From the Osteoarthritis Initiative

The Osteoarthritis Initiative (OAI) is a public-private partnership comprised of five contracts (N01-AR-2-2258; N01-AR-2-2259; N01-AR-2-2260; N01-AR-2-2261; N01-AR-2-2262) funded by the National Institutes of Health, a branch of the Department of Health and Human Services, and conducted by the OAI Study Investigators. Private funding partners include Pfizer, Inc.; Novartis Pharmaceuticals Corporation; Merck Research Laboratories; and GlaxoSmithKline. Private sector funding for the OAI is managed by the Foundation for the National Institutes of Health. This work has been supported by grants from Fondo Investigación Sanitaria (PI16/02124, PI17/00404, PI19/01206, DTS17/00200, CIBER-CB06/01/0040 and RETIC-RIER-RD16/0012/0002), integrated in the National Plan for Scientific Program, Development and Technological Innovation 2013–2016 and funded by the ISCIII-General Subdirection of Assessment and Promotion of Research-European Regional Development Fund (FEDER) “A way of making Europe”. This study has been also supported by grants IN607A2017/11, IN607D2020/10 and AE CICA-INIBIC (ED431E 2018/03) from Xunta de Galicia. The Proteomics Unit of GIR belongs to ProteoRed, PRB3- ISCIII (PT17/0019/0014). L.L. is supported by Xunta de Galicia (IN606B-2016/2005) and Contrato Sara Borrell (CD19/00229) Fondo de Investigación Sanitaria, ISCIII. I.R.P. is supported by Contrato Miguel Servet-II Fondo de Investigación Sanitaria (CPII17/00026). This work was also supported by the KTH Center for Applied Precision Medicine funded by the Ehrling Persson foundation, as well as the Human Protein Atlas project funded by Knut and Alice Wallenberg FoundationXunta de Galicia; IN607A2017/11Xunta de Galicia; IN607D2020/10Xunta de Galicia; ED431E 2018/03Xunta de Galicia; IN606B-2016/200

Analysis of endogenous peptides released from osteoarthritic articular cartilage unravels novel pathogenic markers

[Abstract] 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.Instituto de Salud Carlos III; PI14/01707Instituto de Salud Carlos III; PI16/02124Instituto de Salud Carlos III; PI17/00404Instituto de Salud Carlos III; CIBER-CB06/01/0040Instituto de Salud Carlos III; DTS17/00200Instituto de Salud Carlos III; RETIC-RIER-RD16/0012/0002Instituto de Salud Carlos III; PT17/0019/001

Discovery of circulating proteins associated to knee radiographic osteoarthritis

[Abstract] Currently there are no sufficiently sensitive biomarkers able to reflect changes in joint remodelling during osteoarthritis (OA). In this work, we took an affinity proteomic approach to profile serum samples for proteins that could serve as indicators for the diagnosis of radiographic knee OA. Antibody suspension bead arrays were applied to analyze serum samples from patients with OA (n = 273), control subjects (n = 76) and patients with rheumatoid arthritis (RA, n = 244). For verification, a focused bead array was built and applied to an independent set of serum samples from patients with OA (n = 188), control individuals (n = 83) and RA (n = 168) patients. A linear regression analysis adjusting for sex, age and body mass index (BMI) revealed that three proteins were significantly elevated (P < 0.05) in serum from OA patients compared to controls: C3, ITIH1 and S100A6. A panel consisting of these three proteins had an area under the curve of 0.82 for the classification of OA and control samples. Moreover, C3 and ITIH1 levels were also found to be significantly elevated (P < 0.05) in OA patients compared to RA patients. Upon validation in additional study sets, the alterations of these three candidate serum biomarker proteins could support the diagnosis of radiographic knee OA.Instituto de Salud Carlos III; PI-16/02124Instituto de Salud Carlos III; PI-14/01707Instituto de Salud Carlos III; PI-12/00329Instituto de Salud Carlos III; CIBER-CB06/01/0040Instituto de Salud Carlos III; RETIC-RIER-RD12/0009/001

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

[Abstract] 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.Insituto de Salud Carlos III; PI14/01707Instituto de Salud Carlos III; PI16/02124Insituto de Salud Carlos III; PI17/00404Instituto de Salud Carlos III; DTS17/00200Instituto de Salud Carlos III; CIBER-CB06/01/0040Insituto de Salud Carlos III; RETIC-RIER-RD16/0012/000

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

[Abstract] 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.Instituto de Salud Carlos III; CIBER-CB06/01/0040Instituto de Salud Carlos III; PI11/02397Instituto de Salud Carlos III; PI12/00329Instituto de Salud Carlos III; RETIC-RIER-RD12/0009/0018Ministerio de Ciencia e Innovación; PLE2009-0144Xunta de Galicia; 10 PXIB 310153 P

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

[Abstract] 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 serum anti-centromere protein F antibodies with clinical response to infliximab in patients with rheumatoid arthritis: a prospective study

[Abstract] Background: One-third of rheumatoid arthritis (RA) patients demonstrate no clinical improvement after receiving tumor necrosis factor inhibitors (TNFi). The presence of serum autoantibodies is a hallmark in RA and may provide information on future response to treatment. The aim of this prospective study was to search for novel serum autoantibodies useful to predict clinical response to TNFi. Methods: The autoantibody repertoire was profiled on RA patients treated with TNFi as a first line of biologic therapy (N = 185), who were recruited in three independent cohorts. The presence and levels of autoantibodies in serum at baseline were analysed in association with the clinical response after 24 weeks follow-up. A multiplex bead array built using antigens selected from an initial untargeted screening was employed to identify the autoantibodies on a discovery cohort (N = 50) and to verify and validate the results on verification (N = 61) and validation (N = 74) cohorts. Non-parametric tests, meta-analysis and Receiver Operating Curves (ROC) were performed in order to assess the clinical relevance of the observed findings. Results: Novel autoantibodies were associated with the clinical response to TNFi, showing different reactivity profiles among the different TNFi. The baseline levels of IgG antibodies against Centromere protein F (CENPF), a protein related to cell proliferation, were significantly (p<0.05) increased in responders (N = 111) to infliximab (IFX) compared to non-responders (N = 44). The addition of anti-CENPF antibodies to demographic and clinical variables (age, sex, DAS28-ESR) resulted in the best model to discriminate responders, showing an area under the curve (AUC) of 0.756 (95% CI [0.639-0.874], p = 0.001). A further meta-analysis demonstrated the significant association of anti-CENPF levels with the patient's subsequent response to IFX, showing a standardized mean difference (SMD) of -0.65 (95% CI [-1.02;-0. 27], p = 0.018). Conclusions: Our study reveals for the first time the potential of circulating anti-CENPF antibodies to predict the clinical response to IFX before starting the treatment. This finding could be potentially useful to guide therapeutic decisions and may lead to further studies focusing on the role of CENPF on RA pathology.Instituto de Salud Carlos III; PI14/01707Instituto de Salud Carlos III; PI16/02124Instituto de Salud Carlos III; PI17/00404Instituto de Salud Carlos III; PI19/01206Instituto de Salud Carlos III; CIBER-CB06/01/0040Instituto de Salud Carlos III; RETIC-RIER-RD16/0012/0002Instituto de Salud Carlos III; PRB3-ISCIII-PT17/0019/0014)