Cristina Ruiz-Romero

The role of mitochondria in osteoarthritis

Mitochondria are important regulators of cellular function and survival that may have a key role in aging-related diseases. Mitochondrial DNA (mtDNA) mutations and oxidative stresses are known to contribute to aging-related changes. Osteoarthritis (OA) is an aging-associated rheumatic disease characterized by articular cartilage degradation and elevated chondrocyte mortality. Articular cartilage chondrocytes survive and maintain tissue integrity in an avascular, low-oxygen environment. Recent ex vivo studies have reported mitochondrial dysfunction in human OA chondrocytes, and analyses of mitochondrial electron transport chain activity in these cells show decreased activity of Complexes I, II and III compared to normal chondrocytes. This mitochondrial dysfunction may affect several pathways that have been implicated in cartilage degradation, including oxidative stress, defective chondrocyte biosynthesis and growth responses, increased cytokine-induced chondrocyte inflammation and matrix catabolism, cartilage matrix calcification, and increased chondrocyte apoptosis. Mitochondrial dysfunction in OA chondrocytes may derive from somatic mutations in the mtDNA or from the direct effects of proinflammatory mediators such as cytokines, prostaglandins, reactive oxygen species and nitric oxide. Polymorphisms in mtDNA may become useful as biomarkers for the diagnosis and prognosis of OA, and modulation of serum biomarkers by mtDNA haplogroups supports the concept that mtDNA haplogroups may define specific OA phenotypes in the complex OA process.

Mitochondrial dysregulation of osteoarthritic human articular chondrocytes analyzed by proteomics: a decrease in mitochondrial superoxide dismutase points to a redox imbalance.

Mitochondria are involved in many cellular processes; mitochondrial dysfunctions have been associated with apoptosis, aging, and a number of pathological conditions, including osteoarthritis (OA). Mitochondrial proteins are attractive targets for the study of metabolism of the chondrocyte, the unique cell type present in mature cartilage, and its role in tissue degradation. Using a proteomics approach based on two-dimensional DIGE and MALDI-TOF/TOF mass spectrometric identification of mitochondria- enriched protein fractions from human articular chondrocytes, we analyzed mitochondrial protein changes that are characteristic of OA chondrocytes. A total of 73 protein forms were unambiguously identified as significantly altered in OA; 23 of them have been previously described as mitochondrial. An extensive statistical and cluster analysis of the data revealed a mitochondrial protein profile characteristic for OA. This pattern includes alterations in energy production, maintenance of mitochondrial membrane integrity, and free radical detoxification. Real time PCR, Western blot, and immunohistofluorescence assays confirmed a significant decrease of the major mitochondrial antioxidant protein manganese-superoxide dismutase (SOD2) in the superficial layer of OA cartilage. As possible outputs for this antioxidant deficiency, we found an increase of intracellular reactive oxygen species generation in OA chondrocytes and also verified an OA-dependent increase in the mitochondrial tumor necrosis factor-α receptor-associated protein 1 (TRAP1), a chaperone with a reported reactive oxygen species antagonist role. Our results describe the differences between the mitochondrial protein profiles of normal and OA chondrocytes, demonstrating that mitochondrial dysregulation occurs in cartilage cells during OA and highlighting redox imbalance as a key factor in OA pathogenesis.

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.

Proteomic analysis of human osteoarthritic chondrocytes reveals protein changes in stress and glycolysis

Osteoarthritis (OA) is characterized by cartilage degradation. The chondrocyte is the only cell type present in mature cartilage, and it is important in the control of cartilage integrity. The aim of this study was to analyze, by a proteomic approach, the changes that are characteristic of OA chondrocytes, and to identify new OA‐related proteins. Chondrocytes were isolated from the cartilage of ten OA patients undergoing joint replacement and ten donors with no history of joint disease. Whole‐cell proteins were resolved by 2‐DE and stained with SYPRO Ruby. Protein expression patterns of 2‐DE gels from OA and normal chondrocyte proteins were analyzed with PDQuest 7.3.1 software. OA‐related proteins were identified by MALDI‐TOF or MALDI‐TOF/TOF MS. The results were validated for ANXA1, GSTO1, GRP78, and HSP90β in cells by Western blotting and in tissue cartilage by immunohistochemistry. Results showed an average of 700 protein spots that were present in the 2‐DE gels. Compared to normal chondrocytes, 19 protein spots were found to be significantly increased in OA cells (ratio OA:N≥2.0, p<0.05), whereas nine were decreased in OA chondrocytes (ratio OA:N≤0.5, p<0.05). Three stress response proteins were increased (HSP90β, GRP78, and GRP94) and three proteins involved in glycolysis were decreased (enolase, glyceraldehyde 3‐phosphate dehydrogenase, and fructose biphosphate aldolase). Functionally, almost all proteins could be classified as proteins involved in cellular metabolism (33%), structure (21%), or protein targeting (21%).

Identification of a panel of novel serum osteoarthritis biomarkers.

Osteoarthritis (OA) is the most common rheumatic pathology. Because currently available diagnostic methods are limited and lack sensitivity, the identification of new specific biological markers for OA has become a focus. The purpose of this study was to identify novel protein biomarkers for moderate and severe OA in serum. Sera were obtained from 50 moderate OA patients, 50 severe OA patients, and 50 nonsymptomatic controls. Serum protein levels were analyzed using isobaric tags for relative and absolute quantitation (iTRAQ) and matrix-assisted laser desorption/ionization (MALDI)-TOF/TOF mass spectrometry. We identified 349 different proteins in the sera, 262 of which could be quantified by calculation of their iTRAQ ratios. Three sets of proteins were significantly (p < 0.05) changed in OA samples compared to controls. Of these, 6 were modulated only in moderate OA, 13 only in severe OA and 7 in both degrees. Although some of these proteins, such as cartilage oligomeric matrix protein, have a previously reported putative biomarker value for OA, most are novel biomarker candidates for the disease. These include some complement components, lipoproteins, von Willebrand factor, tetranectin, and lumican. The specificity and selectivity of these candidates need to be validated before new molecular diagnostic or prognostic tests for OA can be developed.

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.

A comparison of depletion versus equalization for reducing high-abundance proteins in human serum

In this work three methods to diminish the content of most highly abundant proteins in human serum have been studied and compared. Protein depletion with ACN or DTT and protein equalization with the ProteoMiner™ (PM) have been assessed by 1‐D gel electrophoresis and MS. After treatment 5, 18 and 9 major proteins within the 20 most abundant proteins in serum were identified for the ACN, DTT and PM methods, respectively. The ACN method was efficient for depleting high molecular weight proteins, over 75 KDa, resulting in 10±4% (n=3) of the total protein content remaining in the depleted serum. In addition, 75% of the proteins belonging to the group of the 20 most abundant proteins were not detected, making this depletion strategy a cheap alternative to expensive commercial tools regularly used for removing high abundance proteins from serum. The ACN extract was found rich in apolipoproteins. The dithithreitol method promotes the precipitation of proteins rich in disulfide bonds, mainly albumin, with 73±7% (n=3) of the total protein content remaining in the depleted serum, which was found rich in immunoglobulins. The PM method compresses the dynamic range of the serum proteins, rendering an extract containing 16±2% (n=3) of the total initial protein content. The extract was found to be rich in both apolipoproteins and immunoglobulins. As a general rule the DTT and PM methods provide a compression of the dynamic range of serum protein concentrations while the ACN method allows an effective depletion of the protein fraction above 72 KDa.

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.

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.