101 research outputs found
Análisis comparativo de células madre mesenquimales procedentes de pacientes artrósicos y sanos en diferenciación condrogénica
Comunicaciones a congreso
Glucosamine affects intracellular signalling through inhibition of mitogen-activated protein kinase phosphorylation in human chondrocytes
The aim of this study was to determine the effects of glucosamine on matrix metalloprotease (MMP) production, on mitogen-activated protein kinase (MAPK) phosphorylation, and on activator protein (AP)-1 transcription factor activation in human chondrocytes. The human immortalized cell line lbpva55 and healthy human chondrocytes (obtained from healthy donors) were subjected to challenge with 10 ng/ml IL-1β after pretreatment with 2.5 or 10 mmol/l glucosamine. MMP mRNA expression levels were evaluated using quantitative real-time PCR, and MMP protein production levels were evaluated in the culture supernatant using ELISA. MAPK phosphorylation was evaluated using Western blotting. AP-1 transcription factor activation was evaluated by measuring AP-1 DNA-binding activity. After IL-1β stimulation, levels of MMP-1, MMP-3 and MMP-13 production were markedly increased. Treatment with 2.5 and 10 mmol/l glucosamine reduced expression of these metalloproteases. MMP expression is regulated by transcription factors such as the AP-1 complex, which is activated by phosphorylated MAPKs. IL-1β stimulated phosphorylation of c-jun amino-terminal kinase, p38 MAPK and extracellular signal-regulated kinase-1/2. Glucosamine inhibited c-jun amino-terminal kinase and p38 phosphorylation, and consequently c-jun binding activity. These findings demonstrate, for the first time, that glucosamine inhibits IL-1β-stimulated MMP production in human chondrocytes by affecting MAPK phosphorylation
Búsqueda de biomarcadores séricos de artrosis mediante depleción secuencias combinada con DIGE
Comunicaciones a congreso
Evaluation of chondroprotective effect of glucosamine and chondroitin sulfate by a proteomic approach
Comunicaciones a congreso
Hypoxia conditions differentially modulate normal and osteoarthritic chondrocyte proteomes
Comunicaciones a congreso
Hsp90 beta inhibition modulates nitric oxide production and nitric oxide-induced apoptosis in human chondrocytes
Background: Hsp90 beta is a member of the Hsp90 family of protein chaperones. This family plays essential roles in the folding, maturation and activity of many proteins that are involved in signal transduction and transcriptional regulation. The role of this protein in chondrocytes is not well understood, although its increase in osteoarthritic cells has been reported. The present study aimed to explore the role of Hsp90 beta in key aspects of OA pathogenesis.
Methods: Human OA chondrocytes were isolated from cartilage obtained from patients undergoing joint replacement surgery, and primary cultured. Cells were stimulated with proinflammatory cytokines (IL-1 beta or TNF-alpha) and nitric oxide donors (NOC-12 or SNP). For Hsp90 beta inhibition, two different chemical inhibitors (Geldanamycin and Novobiocin) were employed, or siRNA transfection procedures were carried out. Gene expression was determined by real-time PCR, apoptosis was quantified by flow cytometry and ELISA, and nitric oxide (NO) production was evaluated by the Griess method. Indirect immunofluorescence assays were performed to evaluate the presence of Hsp90 beta in stimulated cells.
Results: Hsp90 beta was found to be increased by proinflammatory cytokines. Inhibition of Hsp90 beta by the chemicals Geldanamycin (GA) and Novobiocin (NB) caused a dose-dependent decrease of the NO production induced by IL-1 beta in chondrocytes, up to basal levels. Immunofluorescence analyses demonstrate that the NO donors NOC-12 and SNP also increased Hsp90 beta. Chemical inhibition or specific gene silencing of this chaperone reduced the DNA condensation and fragmentation, typical of death by apoptosis, that is induced by NO donors in chondrocytes.
Conclusions: The present results show how Hsp90 beta modulates NO production and NO-mediated cellular death in human OA chondrocytes
Alteraciones en proteínas mitocondriales de condrocitos articulares humanos descritas mediante técnicas Proteómicas
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
Estudio comparativo del secretoma de condrocitos articulares humanos analizados mediante las técnicas iTRAQ e SILAC
Comunicaciones a congreso
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