Valentina Calamia

Differential protein profiling of synovial fluid from rheumatoid arthritis and osteoarthritis patients using LC-MALDI TOF/TOF.

The purpose of this study was to identify those proteins relatively more abundant in the synovial fluid (SF) of patients suffering from rheumatoid arthritis (RA) and osteoarthritis (OA) using high performance liquid chromatography coupled to mass spectrometry. 20 individual SF samples from each disease were pooled into two groups (RA and OA) to reduce the contribution of extreme individual values. Prior to the proteomic analysis, samples were immunodepleted from the top 20 most abundant plasma proteins, to enrich the lower-abundance protein fractions. Then, they were subjected to protein size fractioning and in-gel digestion, followed by reversed-phase peptide separation in a nano-LC system and subsequent peptide identification by MALDI-TOF/TOF. This strategy led to the identification of 136 different proteins in SF, which is the largest number of SF proteins described up to date by proteomics. A relative quantification of the proteins between RA and OA was carried out by spectral counting analysis. In RA, our results show a greater relative abundance of proteins related to complement activation, inflammation and the immune response, such as the major matrix metalloproteinases and several neutrophil-related proteins. In OA, we detected an increase in proteins involved in the formation and remodeling of the extracellular matrix (ECM), such as fibronectin, kininogen-1, cartilage acidic protein 1 and cartilage oligomeric matrix protein. The results obtained for MMP-1, BGH3, fibronectin and gelsolin were verified by immunoblotting analyses. Some of the novel proteins identified in this work might be relevant not only for increasing knowledge on the etiopathogenesis of RA and OA processes, but also as putative disease biomarkers, as their presence in SF is a prior step to their dilution in serum. This article is part of a Special Issue entitled: Proteomics: The clinical link.

Pharmacoproteomic study of the effects of chondroitin and glucosamine sulfate on human articular chondrocytes

IntroductionChondroitin sulfate (CS) and glucosamine sulfate (GS) are symptomatic slow-acting drugs for osteoarthritis (OA) widely used in clinic. Despite their widespread use, knowledge of the specific molecular mechanisms of their action is limited. The aim of this work is to explore the utility of a pharmacoproteomic approach for the identification of specific molecules involved in the pharmacological effect of GS and CS.MethodsChondrocytes obtained from three healthy donors were treated with GS 10 mM and/or CS 200 μg/mL, and then stimulated with interleukin-1β (IL-1β) 10 ng/mL. Whole cell proteins were isolated 24 hours later and resolved by two-dimensional electrophoresis. The gels were stained with SYPRORuby. Modulated proteins were identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF/TOF) mass spectrometry. Real-time PCR and Western blot analyses were performed to validate our results.ResultsA total of 31 different proteins were altered by GS or/and CS treatment when compared to control. Regarding their predicted biological function, 35% of the proteins modulated by GS are involved in signal transduction pathways, 15% in redox and stress response, and 25% in protein synthesis and folding processes. Interestingly, CS affects mainly energy production (31%) and metabolic pathways (13%), decreasing the expression levels of ten proteins. The chaperone GRP78 was found to be remarkably increased by GS alone and in combination with CS, a fact that unveils a putative mechanism for the reported anti-inflammatory effect of GS in OA. On the other hand, the antioxidant enzyme superoxide dismutase 2 (SOD2) was significantly decreased by both drugs and synergistically by their combination, thus suggesting a drug-induced decrease of the oxidative stress caused by IL-1β in chondrocytes.ConclusionsCS and GS differentially modulate the proteomic profile of human chondrocytes. This pharmacoproteomic approach unravels the complex intracellular mechanisms that are modulated by these drugs on IL1β-stimulated human articular chondrocytes.

Metabolic Labeling of Chondrocytes for the Quantitative Analysis of the Interleukin-1-beta-mediated Modulation of Their Intracellular and Extracellular Proteomes

Chondrocytes are widely used as an in vitro model of cartilage diseases such as osteoarthritis (OA). As the unique residents of mature cartilage, they are responsible of the synthesis and release of proteins essential for a proper tissue turnover. In this work, the stable isotope labeling with amino acids in cell culture (SILAC) technique has been standardized in primary human articular chondrocytes (HACs) for quantitative proteomic analyses. Then, it has been employed to study those protein modifications caused by the proinflammatory cytokine Interleukin-1beta (IL-1β), a well-known OA mediator, in these cells. Quantitative analysis of the IL-1β-treated HACs proteome revealed a global increase in cellular chaperones concurrent with a down-regulation of the actin cytoskeleton. HACs secretome analysis led to the identification and quantification of 115 proteins and unveiled the effects of the cytokine on the cartilage extracellular matrix metabolism. Among those modulated proteins, three protein clusters were found to be remarkably increased by IL-1β: proinflammatory mediators and proteases, type VI collagen and proteins known to bind this molecule, and proteins related with the TGF-beta pathway. On the other hand, secretion of aggrecan, two vitamin K-dependent proteins, and thrombospondin, among others, was strongly reduced. Altogether, these data demonstrate the usefulness of metabolic labeling for quantitative proteomics studies in HACs, show the complementarity of intracellular proteome and secretome analyses, and provide a comprehensive study of the IL-1β-mediated effects on these cells. Proteins identified in the secretome approach have a potential use as biomarkers or therapeutic targets for OA.

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.

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.

Sequential depletion of human serum for the search of osteoarthritis biomarkers

BackgroundThe field of biomarker discovery, development and application has been the subject of intense interest and activity, especially with the recent emergence of new technologies, such as proteomics-based approaches. In proteomics, search for biomarkers in biological fluids such as human serum is a challenging issue, mainly due to the high dynamic range of proteins present in these types of samples. Methods for reducing the content of most highly abundant proteins have been developed, including immunodepletion or protein equalization. In this work, we report for the first time the combination of a chemical sequential depletion method based in two protein precipitations with acetonitrile and DTT, with a subsequent two-dimensional difference in-gel electrophoresis (2D-DIGE) analysis for the search of osteoarthritis (OA) biomarkers in human serum. The depletion method proposed is non-expensive, of easy implementation and allows fast sample throughput.ResultsFollowing this workflow, we have compared sample pools of human serum obtained from 20 OA patients and 20 healthy controls. The DIGE study led to the identification of 16 protein forms (corresponding to 14 different proteins) that were significantly (p < 0.05) altered in OA when compared to controls (8 increased and 7 decreased). Immunoblot analyses confirmed for the first time the increase of an isoform of Haptoglobin beta chain (HPT) in sera from OA patients.ConclusionsAltogether, these data demonstrate the utility of the proposed chemical sequential depletion for the analysis of sera in protein biomarker discovery approaches, exhibit the usefulness of quantitative 2D gel-based strategies for the characterization of disease-specific patterns of protein modifications, and also provide a list of OA biomarker candidates for validation.

Secretome analysis of chondroitin sulfate-treated chondrocytes reveals anti-angiogenic, anti- inflammatory and anti-catabolic properties

IntroductionChondroitin sulfate (CS) is a symptomatic slow-acting drug for osteoarthritis (OA) widely used in the clinic. The aim of this work is to find proteins whose secretion from cartilage cells under proinflammatory stimuli (IL-1β) is regulated by CS, employing a novel quantitative proteomic approach.MethodsHuman articular chondrocytes released from three normal cartilages were grown in SILAC medium. When complete incorporation of the heavy isotope was achieved, chondrocytes were stimulated with IL-1β 5 ng/ml with or without CS pretreatment (200 µg/ml). Forty-eight hours later, chondrocyte secretomes were analyzed by nano-scale liquid chromatography-mass spectrometry. Real-time PCR, western blot and immunohistochemistry analyses were employed to confirm some of the results.ResultsWe could identify 75 different proteins in the secretome of human articular chondrocytes. Eighteen of these were modulated by CS with statistical significance (six increased and 12 decreased). In normal chondrocytes stimulated with IL-1β, CS reduces inflammation directly by decreasing the presence of several complement components (CFAB, C1S, CO3, and C1R) and also indirectly by increasing proteins such as TNFα-induced protein (TSG6). TSG6 overexpression correlates with a decrease in pro-matrix metalloproteinase activation (observed in MMP1 and MMP3 levels). Finally, we observed a strong CS-dependent increase of an angiogenesis inhibitor, thrombospondin-1.ConclusionWe have generated a quantitative profile of chondrocyte extracellular protein changes driven by CS in the presence of IL-1β. We have also provided novel evidences of its anti-angiogenic, anti-inflammatory, and anti-catabolic properties. Demonstration of the anti-angiogenic action of CS might provide a novel therapeutic approach for OA targeting.

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.

Hsp90β inhibition modulates nitric oxide production and nitric oxide-induced apoptosis in human chondrocytes

BackgroundHsp90β 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 Hsp90β in key aspects of OA pathogenesis.MethodsHuman OA chondrocytes were isolated from cartilage obtained from patients undergoing joint replacement surgery, and primary cultured. Cells were stimulated with proinflammatory cytokines (IL-1β or TNF-α) and nitric oxide donors (NOC-12 or SNP). For Hsp90β 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 Hsp90β in stimulated cells.ResultsHsp90β was found to be increased by proinflammatory cytokines. Inhibition of Hsp90β by the chemicals Geldanamycin (GA) and Novobiocin (NB) caused a dose-dependent decrease of the NO production induced by IL-1β in chondrocytes, up to basal levels. Immunofluorescence analyses demonstrate that the NO donors NOC-12 and SNP also increased Hsp90β. 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.ConclusionsThe present results show how Hsp90β modulates NO production and NO-mediated cellular death in human OA chondrocytes.

Effects of intra-articular administration of glucosamine and a peptidyl-glucosamine derivative in a rabbit model of experimental osteoarthritis: a pilot study.

The aim of this pilot study was to analyze the effects of glucosamine (GlcN) and its N-acetyl-phenylalanine derivative (NAPA) in Vitamin A model of osteoarthritis (OA) in rabbits. GlcN or NAPA or saline solution was intra-articularly administered in rabbit OA knees. Histological analysis revealed that treatment with GlcN or NAPA was associated with more homogeneous chondrocyte cellularity, absence of fissures and fragmentation and more intense staining of the matrix with Alcian Blue compared to the articular surfaces of the knees treated with saline solution. Comparative in vitro study performed on rabbit primary chondrocytes revealed that GlcN and NAPA were also able to counteract the IL-1β-upregulation of genes coding for metalloproteases and inflammatory cytokines. Our preliminary in vivo and in vitro studies suggest that GlcN and NAPA could play a disease-modifying protective role in OA by an anti-catabolic effect and an anti-inflammatory activity on chondrocytes.