M.J. López-Armada

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%).

Mitochondrial dysfunction increases inflammatory responsiveness to cytokines in normal human chondrocytes

OBJECTIVE Alterations in mitochondria play a key role in the pathogenesis of osteoarthritis (OA). The role of inflammation in the progression of OA has also acquired important new dimensions. This study was undertaken to evaluate the potential role of mitochondrial dysfunction in increasing the inflammatory response of normal human chondrocytes to cytokines. METHODS Mitochondrial dysfunction was induced by commonly used inhibitors. Interleukin-1β (IL-1β) and tumor necrosis factor α (TNFα) were used as inflammatory mediators. IL-8 and cyclooxygenase 2 (COX-2) protein and messenger RNA (mRNA) expression and prostaglandin E(2) (PGE(2) ) levels were assessed. The chemotactic activity of neutrophils was assayed. Additionally, inhibitors of reactive oxygen species (ROS) and NF-κB were used to identify possible inflammatory response pathways induced by mitochondrial dysfunction, and the effects of the natural antioxidant resveratrol were tested. RESULTS Pretreatment with antimycin A or oligomycin (inhibitors of mitochondrial respiratory chain complexes III and V, respectively) triggered a strong potentiation of IL-1β-induced IL-8 mRNA and protein expression (mean ± SEM at 18 hours 5,932 ± 1,995 pg/50,000 cells for IL-1β alone versus 16,241 ± 5,843 pg/50,000 cells for antimycin A plus IL-1β and 20,087 ± 5,407 pg/50,000 cells for oligomycin plus IL-1β; P < 0.05). Similar results were observed with TNFα or when expression of the inflammatory mediator COX-2 or PGE(2) production was assessed. Mitochondrial dysfunction increased the chemotactic activity induced by cytokines, and ROS and NF-κB inhibitors decreased the production of IL-8. Resveratrol significantly reduced the inflammatory response. CONCLUSION Our findings indicate that mitochondrial dysfunction could amplify the responsiveness to cytokine-induced chondrocyte inflammation through ROS production and NF-κB activation. This pathway might lead to the impairment of cartilage and joint function in OA.

Mitochondrial dysfunction activates cyclooxygenase 2 expression in cultured normal human chondrocytes.

OBJECTIVE Mitochondrial alterations play a key role in the pathogenesis of osteoarthritis (OA). This study evaluated a potential role of mitochondrial respiratory chain (MRC) dysfunction in the inflammatory response of normal human chondrocytes. METHODS Commonly used inhibitors of the MRC were utilized to induce mitochondrial dysfunction in normal human chondrocytes. Levels of prostaglandin E(2) (PGE(2)) protein and expression of cyclooxygenase 2 (COX-2) and COX-1 messenger RNA (mRNA) and protein were analyzed. To identify the underlying mechanisms responsible for PGE(2) liberation, reactive oxygen species (ROS) were measured. Inhibitors of ROS, including vitamin E, and inhibitors of mitochondrial Ca(2+) and NF-kappaB were used to test their effects on the MRC. RESULTS Antimycin A and oligomycin (inhibitors of mitochondrial complexes III and V, respectively) significantly increased the levels of PGE(2) (mean +/- SEM 505 +/- 132 pg/50,000 cells and 288 +/- 104 pg/50,000 cells, respectively, at 24 hours versus a basal level of 29 +/- 9 pg/50,000 cells; P < 0.05) and increased the expression of COX-2 at both the mRNA and protein levels. Expression of COX-1 did not show any modulation with either inhibitor. Further experiments revealed that antimycin A and oligomycin induced a marked increase in the levels of ROS. Production of PGE(2) and expression of COX-2 protein were inhibited by antioxidants, vitamin E, and mitochondrial Ca(2+) and NF-kappaB inhibitors. The response to blockers of mitochondrial Ca(2+) movement showed that ROS production was dependent on mitochondrial Ca(2+) accumulation. CONCLUSION These results strongly suggest that, in human chondrocytes, the inhibition of complexes III and V of the MRC induces an inflammatory response, which could be especially relevant in relation to PGE(2) production via mitochondrial Ca(2+) exchange, ROS production, and NF-kappaB activation. These data may prove valuable for a better understanding of the participation of mitochondria in the pathogenesis of OA.

Decreased metalloproteinase production as a response to mechanical pressure in human cartilage: a mechanism for homeostatic regulation

Articular cartilage is optimised for bearing mechanical loads. Chondrocytes are the only cells present in mature cartilage and are responsible for the synthesis and integrity of the extracellular matrix. Appropriate joint loads stimulate chondrocytes to maintain healthy cartilage with a concrete protein composition according to loading demands. In contrast, inappropriate loads alter the composition of cartilage, leading to osteoarthritis (OA). Matrix metalloproteinases (MMPs) are involved in degradation of cartilage matrix components and have been implicated in OA, but their role in loading response is unclear. With this study, we aimed to elucidate the role of MMP-1 and MMP-3 in cartilage composition in response to mechanical load and to analyse the differences in aggrecan and type II collagen content in articular cartilage from maximum- and minimum-weight-bearing regions of human healthy and OA hips. In parallel, we analyse the apoptosis of chondrocytes in maximal and minimal load areas. Because human femoral heads are subjected to different loads at defined sites, both areas were obtained from the same hip and subsequently evaluated for differences in aggrecan, type II collagen, MMP-1, and MMP-3 content (enzyme-linked immunosorbent assay) and gene expression (real-time polymerase chain reaction) and for chondrocyte apoptosis (flow cytometry, bcl-2 Western blot, and mitochondrial membrane potential analysis). The results showed that the load reduced the MMP-1 and MMP-3 synthesis (p < 0.05) in healthy but not in OA cartilage. No significant differences between pressure areas were found for aggrecan and type II collagen gene expression levels. However, a trend toward significance, in the aggrecan/collagen II ratio, was found for healthy hips (p = 0.057) upon comparison of pressure areas (loaded areas > non-loaded areas). Moreover, compared with normal cartilage, OA cartilage showed a 10- to 20-fold lower ratio of aggrecan to type II collagen, suggesting that the balance between the major structural proteins is crucial to the integrity and function of the tissue. Alternatively, no differences in apoptosis levels between loading areas were found – evidence that mechanical load regulates cartilage matrix composition but does not affect chondrocyte viability. The results suggest that MMPs play a key role in regulating the balance of structural proteins of the articular cartilage matrix according to local mechanical demands.

Xeno‐implantation of pig chondrocytes into rabbit to treat localized articular cartilage defects: an animal model

Articular cartilage has only a limited ability to regenerate. The transplantation of autologous chondrocytes is currently used to treat focal defects in human articular cartilage, although use of organs, tissues, or cells from different species is being investigated as an alternative treatment. The object of this study was to use xeno‐transplantation of cultured pig chondrocytes for the repair of rabbit chondral defects, and to analyze the significance of tissue rejection in this animal model. Partial chondral defects, including removal of cartilage tissue and a part of the subchondral bone, were created in the lateral femoral condyles of 30 adult New Zealand White rabbits. A periosteal flap was sutured to the native cartilage with the cambium layer facing the defect. As a control, culture medium was injected into the defect void of one group of rabbits while in a treatment group, chondrocytes, isolated from normal femoral pig cartilage, were injected into the defect void. All rabbits were killed by 24 weeks. Macroscopic changes of the cartilage were analyzed using Mankins score. The distal femoral portion was studied histologically using hematoxylin and eosin, alcian blue, toluidine blue, and Masons trichrome. Pig cells and pig genetic material were detected in the neo‐synthesized tissue by immunohistochemical detection of SLA‐II‐DQ and polymerase chain reaction analysis of the gene SLA‐II‐DQB. The synovial membrane was studied histologically by hematoxylin and eosin staining. In the control group, on average, less than 25 percent of the chondral defect was filled. The repair tissue had an irregular surface with few cells similar to chondrocytes or fibroblasts and a minimal formation of extracellular matrix. In the treatment group, the chondral defect was approximately 90 percent filled with good integration between the neo‐synthesized cartilage and the native cartilage. The repair tissue had a smooth surface with cells similar to chondrocytes and a hyaline‐like extracellular matrix. The neo‐synthesized cartilage was morphologically similar to hyaline cartilage. Importantly, there were no signs of graft‐vs.‐host rejections or infiltration by immune cells. In the neo‐synthesized tissue, pig genetic material was detected in 27 ± 5 percent of all cells. These cells containing pig genetic material were distributed throughout the neo‐synthesized cartilage. We conclude that the xeno‐transplantation of chondrocytes could be an alternative method for the repair of articular cartilage defects.

Proteomic analysis by two-dimensional electrophoresis to identify the normal human chondrocyte proteome stimulated by tumor necrosis factor α and interleukin-1β

OBJECTIVE To determine the intracellular proteome of normal human chondrocytes stimulated with interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha) and to ascertain differences in the protein expression patterns of these 2 cytokines. METHODS Normal human knee cartilage chondrocytes were incubated for 48 hours without stimulation or stimulated with IL-1beta (5 ng/ml) or with TNFalpha (10 ng/ml). For each culture condition, protein extracts from 4 normal subjects were pooled and resolved using 2-dimensional electrophoresis. Protein spots were visualized with Sypro stain, and qualitative and quantitative analyses were performed using PDQuest software. Protein spots were then identified by mass spectrometry, using matrix-assisted laser desorption ionization-time-of-flight/time-of-flight technology. RESULTS We identified 37 spots by mass spectrometry (MS) or by MS/MS, corresponding to 35 different proteins. In IL-1beta-stimulated chondrocytes, IL-1beta was found to modulate 22 proteins, as compared with unstimulated chondrocytes. All of these proteins except connective tissue growth factor (CCND2) were up-regulated. Proteins involved in cellular metabolism and energy (23%) that were up-regulated or induced by IL-1beta included nicotinamide phosphoribosyltransferase, long-chain fatty acid-coenzyme A ligase 4, delta-aminolevulinic acid dehydratase, triosephosphate isomerase, and an isoform of glyceraldehyde-3-phosphate dehydrogenase. In TNFalpha-stimulated chondrocytes, TNFalpha was found to modulate 20 proteins, as compared with unstimulated chondrocytes. All of these except chitinase 3-like 1 (cartilage glycoprotein 39), proteasome activator complex subunit 2, and G3PDH, were up-regulated. Eighteen proteins were differently modulated by IL-1beta and TNFalpha. Of these, 45% were related to metabolism. CONCLUSION IL-1beta and TNFalpha induce different profiles of intracellular protein expression in healthy human chondrocytes. Most of the proteins that are differently regulated are proteins that are implicated in the generation of cellular energy and in glycolysis.

Effect of nitric oxide on mitochondrial activity of human synovial cells

BackgroundNitric oxide (NO) is a messenger implicated in the destruction and inflammation of joint tissues. Cartilage and synovial membrane from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) have high levels of NO. NO is known to modulate various cellular pathways and, thus, inhibit the activity of the mitochondrial respiratory chain (MRC) of chondrocytes and induce the generation of reactive oxygen species (ROS) and cell death in multiple cell types. For these reasons, and because of the importance of the synovial membrane in development of OA pathology, we investigated the effects of NO on survival, mitochondrial function, and activity of fibroblastic human OA synovial cells.MethodsHuman OA synovia were obtained from eight patients undergoing hip joint replacement. Sodium nitroprusside (SNP) was used as a NO donor compound and cell viability was evaluated by MTT assays. Mitochondrial function was evaluated by analyzing the mitochondrial membrane potential (Δψm) with flow cytometry using the fluorofore DePsipher. ATP levels were measured by luminescence assays, and the activities of the respiratory chain complexes (complex I: NADH CoQ1 reductase, complex II: succinate dehydrogenase, complex III: ubiquinol-cytochrome c reductase, complex IV: cytochrome c oxidase) and citrate synthase (CS) were measured by enzymatic assay. Protein expression analyses were performed by western blot.ResultsSNP at a concentration of 0.5 mM induced cell death, shown by the MTT method at different time points. The percentages of viable cells at 24, 48 and 72 hours were 86.11 ± 4.9%, 74.31 ± 3.35%, and 43.88 ± 1.43%, respectively, compared to the basal level of 100% (*p < 0.05). SNP at 0.5 mM induced depolarization of the mitochondrial membrane at 12 hours with a decrease in the ratio of polarized cells (basal = 2.48 ± 0.28; SNP 0.5 mM = 1.57 ± 0.11; *p < 0.01). The time course analyses of treatment with SNP at 0.5 mM demonstrated that treatment reliably and significantly reduced intracellular ATP production (68.34 ± 14.3% vs. basal = 100% at 6 hours; *p < 0.05). The analysis of the MRC at 48 hours showed that SNP at 0.5 mM increased the activity of complexes I (basal = 36.47 ± 3.92 mol/min/mg protein, SNP 0.5 mM = 58.08 ± 6.46 mol/min/mg protein; *p < 0.05) and III (basal = 63.87 ± 6.93 mol/min/mg protein, SNP 0.5 mM = 109.15 ± 30.37 mol/min/mg protein; *p < 0.05) but reduced CS activity (basal = 105.06 ± 10.72 mol/min/mg protein, SNP at 0.5 mM = 66.88 ± 6.08 mol/min/mg protein.; *p < 0.05), indicating a decrease in mitochondrial mass. Finally, SNP regulated the expression of proteins related to the cellular cycle; the NO donor decreased bcl-2, mcl-1 and procaspase-3 protein expression.ConclusionsThis study suggests that NO reduces the survival of OA synoviocytes by regulating mitochondrial functionality, as well as the proteins controlling the cell cycle.

Mitochondrial dysfunction promotes and aggravates the inflammatory response in normal human synoviocytes

OBJECTIVES In RA, synoviocytes cause increased oxidative stress, leading to mitochondrial alterations that may participate in the pathogenesis of RA. Here we investigated whether mitochondrial dysfunction induces inflammatory responses in cultured normal human synoviocytes, a hallmark of RA. METHODS Mitochondrial dysfunction was induced with the inhibitor oligomycin. The effects of mitochondrial dysfunction on cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2) and IL-8 expression; cellular and mitochondrial reactive oxygen species (ROS) production; nuclear factor-κB (NF-κB) activation and p65 translocation were studied. ROS scavengers (N-acetylcysteine and mitoTEMPO) and an NF-κB inhibitor (BAY-117085) were used to investigate the pathways involved. The natural anti-inflammatory antioxidant resveratrol was also tested. RESULTS Mitochondrial dysfunction per se significantly stimulated mitochondrial ROS production as well as low-grade expressions of COX-2, PGE2 and IL-8. Interestingly, mitochondrial dysfunction induced by pretreatment of synoviocytes with oligomycin synergized with IL-1β to increase the expression of these inflammatory mediators. The inflammatory effects of mitochondrial damage appeared to be dependent on ROS production and NF-κB activation since the inflammatory response was counteracted by both N-acetylcysteine and mitoTEMPO and it was also reduced by BAY-117085. Antimycin A and paraquat (inhibitors of mitochondrial function) also induced inflammatory responses. Furthermore, resveratrol significantly reduced the inflammatory response by decreasing ROS production and NF-κB activation. CONCLUSION These data suggest that mitochondrial dysfunction could induce an inflammatory response in normal human synoviocytes and sensitize these cells, causing a significant amplification of the inflammatory response induced by IL-1β. Resveratrol may represent a promising strategy in controlling the synovial inflammatory response.

Anti-apoptotic effect of transforming growth factor-β1 on human articular chondrocytes: role of protein phosphatase 2A

OBJECTIVE To study whether transforming growth factor-beta1 (TGF-beta1) is able to protect human chondrocytes from apoptosis and to analyze the role of phosphatases in the possible anti-apoptotic effect of TGF-beta1. METHODS Cartilage was obtained from patients with osteoarthritis (OA) who were undergoing joint replacement; normal cartilage was obtained from cadavers who had no history of joint disease. Chondrocytes stimulated with tumor necrosis factor-alpha (TNF-alpha) plus Ro 31-8220 (a specific inhibitor of mitogen-activated kinase phosphatase-1 - MKP-1) were employed as an in vitro model of apoptosis. Apoptosis was assessed by flow cytometry and a cell death immunoassay. Protein phosphatase 2A (PP2A) activity was estimated by measuring the absorbance of a molybdate:malachite green:phosphate reaction complex. MKP-1, bcl-2 and bax expressions were quantified by western blot. RESULTS In OA cells, TGF-beta1 significantly reduced the percentage of hypo-diploid chondrocytes, as well as the percentage of internucleosomal DNA breakage. However, in normal chondrocytes, TGF-beta1 did not reduce apoptosis, as assessed by both the percentage of hypo-diploid chondrocytes and internucleosomal DNA breakage. MKP-1 expression did not show significant modulation in OA or normal chondrocytes. However, PP2A activity was differentially modulated in normal and OA chondrocytes. In OA chondrocytes, PP2A activity was not altered by TGF-beta1 stimulation; however in normal chondrocytes PP2A activity was significantly activated by TGF-beta1. The preincubation of normal chondrocytes with TGF-beta1 plus the PP2A inhibitor protein, IPP2A, reduced internucleosomal DNA breakage when compared with TGF-beta1 stimulation alone. The bcl-2/bax protein ratio was significantly higher in TGF-beta1 plus IPP2A preincubated normal chondrocytes than in cells stimulated with TGF-beta1 alone. CONCLUSION By manipulating the degree of PP2A activity, these results show the major role that PP2A plays in the outcome of TGF-beta1 signal transduction. These data suggest that PP2A could be a pivotal regulator of anti-apoptotic TGF-beta1-induced effects.

Clinical significance of high levels of soluble tumour necrosis factor-α receptor-2 produced by alternative splicing in rheumatoid arthritis: a longitudinal prospective cohort study

OBJECTIVES We investigated whether serum levels of an alternatively spliced soluble (s)TNF receptor-2 (DS-TNFR2) affected the clinical response to anti-TNF-α therapy, classical DMARDs or radiological evidence of disease progression in patients with RA. METHODS We included 116 patients with RA. Cohort 1: 52 DMARD-naïve early RA patients [mean (s.d.) disease duration 8.5 (6.2) months] who started gold salts and MTX therapies. Cohort 2: 64 MTX-resistant established RA patients [144 (107) months] who started infliximab therapy. We evaluated the European League Against Rheumatism (EULAR) response to therapy and the serum levels of DS-TNFR2, sTNFR2 and ACPAs at baseline and at 12 months. In Cohort 1, radiological progression and levels of MMP-1 were also determined. RESULTS In Cohort 1, 40% of patients had high baseline levels (HL > 50 ng/ml) of DS-TNFR2 with significantly higher RF and ACPA levels than patients with normal levels (NL ≤ 50 ng/ml) of DS-TNFR2. The EULAR response to DMARDs was similar in HL and NL patients. Radiographic progression was observed in 23.5% of all patients after 12 months. In Cohort 2, 26.6% of patients had HL of DS-TNFR2 with significantly higher RF and ACPA levels than patients with NLs. The EULAR response from 6 to 30 weeks was prolonged in the HL group compared with the NL group. CONCLUSIONS Patients with HL of DS-TNFR2 maintained a prolonged therapeutic response to anti-TNF-α therapy and had proportionally less radiographic progression compared with patients with NLs.