Identificación de nuevos biomarcadores de la condrogénesis de células madre mesenquimales de pacientes con artrosis mediante técnicas proteómicas

[Resumen]La capacidad de diferenciación de las células madre mesenquimales (MSCs) hacia condrocitos, en un proceso conocido como condrogénesis, concede a estas células un gran potencial terapéutico para la reparación de los defectos del cartílago característicos de enfermedades reumáticas como la artrosis. Sin embargo los mecanismos moleculares que participan en este proceso de diferenciación no son del todo conocidos. las técnicas proteómicas posibilitan analizar globalmente la actividad celular, ya que permiten obtener perfiles proteicos que proporcionan Información sobre las conexiones existentes entre las proteínas y las rutas o procesos celulares en las que participan. En este trabajo se han empleado diferentes metodologías proteómicas para estudiar los mecanismos moleculares que participan en la diferenciación condrogénica de las MSCs obtenidas de médula ósea, con el fin de identificar nuevos marcadores condrogénicos que puedan emplearse en la monitorización de terapias celulares encaminadas a la reparación del cartílago. Inicialmente se estandarizó el marcaje metabólico SILAC en MSCs aisladas de médula ósea para llevar a cabo los estudios proteómicos cuantitativos. Una vez optimizado este método, su aplicación nos permitió describir las proteínas intracelulares cuya abundancia se altera durante el proceso de diferenciación de las MSCs. Los niveles de expresión de 65 proteínas se modularon significativamente entre los dos estadios celulares analizados (indiferenciado (día 2) y diferenciado (día 14)). La mayor parte de estas proteínas estaban involucradas en el metabolismo celular y en la glucólisis. En base a este trabajo, decidimos realizar otro estudio en el que aplicamos el doble marcaje SILAC seguido de un análisis nanolC-ESI-MS para identificar y cuantificar las proteínas secretadas por las MSCs a los dlas 2 y 14 del proceso condrogénico. Mediante esta estrategia, se detectaron cambios significativos en la abundancia de 34 proteínas entre los dos tiempos analizados. la mayor parte de las protelnas aumentadas a día 14 son proteínas relacionadas con la matriz extracelular del cartílago, y entre ellas validamos mediante inmunoensayo el incremento de proteoglicano 4 (PRG4)y TIMP1 como potenciales biomarcadores de diferenciación condrogénica.Finalmente, se comprobó la utilidad de las técnicas de imagen mediante espectrometría de masas (MS Imaging) para la caracterización de lipldos involucrados en el proceso de diferenciación. En este último estudio demostramos que las MSCs metabolizan la esfingomielina durante la diferenciación condrogénica, V que la fosfocolina V sus derivados son posibles marcadores de indiferenciación de MSCs[Resumo] A capacidad e de diferenciación das células nai mesenquimais (MSCs) cara os condrocitos, nun proceso coñecido como condroxénese, outorga a estas célulasFinalmente, se comprobó la utilidad de las técnicas de imagen mediante espectrometría de masas (MS Imaging) para la caracterización de lipldos involucrados en el proceso de diferenciación. En este último estudio demostramos que las MSCs metabolizan la esfingomielina durante la diferenciación condrogénica, V que la fosfocolina V sus derivados son posibles marcadores de indiferenciación de MSCs. un gran potencial terapéutico para a reparación dos defectos da cartilaxe característicos de enfermidades reumáticas como a artrose. Non obstante, os mecanismos moleculares que participan neste proceso de diferenciación non son de todo coñecidos. As técnicas proteómicas posibilitan analizar globalmente a actividade celular, xa que permiten obter perfís proteicos que proporcionan información sobre as conexións existentes entre as prote(nas e as rutas ou ,procesos celulares nos que participan. Neste traballo empregáronse diferentes metodoloxías proteómicas para estudar os mecanismos moleculares que participan na diferenciación condroxénica das MSCs obtidas da medula ósea, ca fin de identificar novos marcadores condroxénicos que poidan empregarse na monitorización de terapias celulares encamiñadas á reparación da cartilaxe . Inicialmente estandarizouse a marcaxe metabólica SILAC nas MSCs iIIadas de medula ósea para levar a cabo os estudos proteómicos cuantitativos. Unha vez optimizado este método, a súa aplicación permitiunos describir as proteínas intracelulares cuxa abundancia altérase durante o proceso de diferenciación das MSCs. Os niveis de expresión de 65 proteínas moduláronse significativamente entre os dous estadios celulares analizados (indiferenciado (dia 2) e diferenciado (día 14)). A maior parte destas proteínas estaban implicadas no metabolismo celular e na glucólise. En base a este traballo, decidimos realizar outro estudo no que aplicamos a doble marcaxe SILAC seguido da análise nanoLC-ESI-MS para identificar e cuantificar as proteínas secretas palas MSCs ós días 2 e 14 do proceso de condroxénese. Mediante esta estratexia, detectáronse cambios significativos na abundancia de 34 proteínas entre os dous tempos analizados. A maior parte das proteínas aumentadas a día 14 son proteínas relacionadas coa matriz extracelular da cartilaxe, e entre elas validamos mediante inmunoensaios o incremento do proteoglicano 4 (PRG4) e TIMPl como potenciais biomarcadores da diferenciación condroxénica. Finalmente, comprobouse a utilidade das técnicas de imaxen mediante espectrometrla de masas (MS Imaging) para a caracterización de ¡¡pidos involucrados no proceso de diferenciación. Neste último estudo demostramos que as MSCs metabolizan a esfingomielina durante a diferenciación condroxénica, e que a fosfocoJina e os seus derivados son posibles marcadores de indiferenciación das MSCs.[Abstract]The differentiation capacity of mesenchvmal stem cells (MSCs) towards chondrocytes, in a process called chondrogenesis, provides them a great therapeutic potential for the repalr of cartJlage defects in rheumatic diseases such as osteoarthritis (OA). However, the molecular mechanisms underlving the chondrogenesis process are still in part unknown. Proteomic tools enable us to globallv analvze cellular activitv, obtaining proteomic profiles that provide us information about the present connections between proteins and ceJlular pathwavs. In this work, we have emploved different proteomic strategies in order to elucidate the molecular mechanisms involved in the chondrogenesis process of bone marrow MSCs, with the purpose of identify new chondrogenic markers useful for molecular monitorization in cell therapv-based approaches for cartllage repair. Firstly, we standardized SILAC labeling in bone marrow MSCs in order to carry out quantitatlve proteomic experiments. Once the SILAC method was optimized, its application aJlowed us to describe the modulations of intraceJlular proteins during the chondrogenic differentiation of MSCs. 65 proteins exhibited a significant modulation of their levels between the two different stages of chondrogenic differentiation (daV 2 vs. dav 14). Most of these proteins were involved in cellular metabolism and glvcolvsis. Next, we emploved a double-SILAC strategy, followed bV nanoLC-ESI·MS analysis, to Identify and quantify the extraceJlular proteins of M5Cs at davs 2 and 14 of chondrogenesis. Using this methodology, we found alterations in the abundance of 34 proteins between the two time points evaluated. Most of the proteins increased at day 14 were cartilage-specific ECM proteins. Proteoglvcan 4 (PRG4) and TIMP1 were validated bV immunoassavs so both have a valuable potential use as biomarkers of chondrogenic differentiation. Flnallv, we confirmed the utilltV of mass spectrometry imaging (MSI) for the characterization of the lipids involved in chondrogenesis. In this studv, we confirmed that M5Cs mobilize the sphingomvelin during their differentiation toward chondrocytes and the phosphocholine-related lipids could be markers of the undifferentiated stage

Targeted Phospholipidomic Analysis of Synovial Fluid as a Tool for Osteoarthritis Deep Phenotyping

[Abstract] 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 ​= ​15) (subclassified into early- and late-stage OA) and non-OA controls (n ​= ​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.This work is supported by grants from Fondo Investigación Sanitaria-Spain (PI16/02124, PI17/00404, PI19/01206, PI20/00793 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 is also supported by AE CICA-INIBIC (ED431E 2018/03) and grants IN607A 2017/11, IN607A 2021/7 and IN607D 2020/10 from Axencia Galega de Innovacion - Xunta de Galicia. The Biomedical Research Networking Center (CIBER) is an initiative from Instituto de Salud Carlos III (ISCIII). The Proteomics Unit of GIR belongs to ProteoRed, PRB3- ISCIII (PT17/0019/0014)Xunta de Galicia; ED431E 2018/03Xunta de Galicia; IN607A 2017/11Xunta de Galicia; IN607A 2021/7Xunta de Galicia; IN607D 2020/1

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

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

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