Autophagy Activation by Resveratrol Reduces Severity of Experimental Rheumatoid Arthritis.

SCOPE: Previous work reported that dietary supplementation with resveratrol lowers synovial hyperplasia, inflammatory and oxidative damage in an antigen-induced arthritis (AIA) model. Here, it is investigated whether resveratrol can regulate the abnormal synovial proliferation by inducing autophagy and controlling the associated inflammatory response. METHODS AND RESULTS: Animals treated with resveratrol 8 weeks before AIA induction show the highest significant signal for microtubule-associated protein 1 light chain 3 by confocal microscopy. Besides, resveratrol significantly reduces p62 expression, but it does not increase the signal of beclin-1. Also, active caspase-3 expression, as well as poly(ADP-ribose) polymerase, is upregulated in the AIA group, and is significantly reduced in resveratrol-treated AIA group. Resveratrol also mitigates angiopoietin-1 and vascular endothelial growth factor signals. Finally, resveratrol significantly reduces the serum levels of IL-1ß, C reactive protein, and prostaglandin E2, as well as nuclear factor κB synovial tissue expression, which shows a significant correlation with p62 expression. CONCLUSION: Dietary supplementation with resveratrol induces the noncanonical autophagy pathway and limits the cross-talk with inflammation, which in consequence modulates the synovial hyperplasia. Preventive strategies that incorporate dietary intervention with resveratrol may offer a potential therapeutic alternative to drugs to influence the risk of rheumatoid arthritis and influence its course.

The mitochondrial inhibitor oligomycin induces an inflammatory response in the rat knee joint.

BACKGROUND: Recent findings support a connection between mitochondrial dysfunction and activation of inflammatory pathways in articular cells. This study investigates in vivo in an acute model whether intra-articular administration of oligomycin, an inhibitor of mitochondrial function, induces an oxidative and inflammatory response in rat knee joints. METHODS: Oligomycin was injected into the rat left knee joint on days 0, 2, and 5 before joint tissues were obtained on day 6. The right knee joint served as control. Results were evaluated by macroscopy and histopathology and by measuring cellular and mitochondrial reactive oxygen species (ROS), 4-hydroxy-2-nonenal (4-HNE, a marker of lipid peroxidation), nuclear factor erythroid 2-related factor 2 (Nrf2), and CD68 (macrophages) and chemokine levels. The marker of mitochondrial mass COX-IV was also evaluated. RESULTS: The macroscopic findings showed significantly greater swelling in oligomycin-injected knees than in control knees. Likewise, the histological score of synovial damage was also increased significantly. Immunohistochemical studies showed high expression of IL-8, coinciding with a marked infiltration of polymorphonuclears and CD68+ cells in the synovium. Mitochondrial mass was increased in the synovium of oligomycin-injected joints, as well as cellular and mitochondrial ROS production, and 4-HNE. Relatedly, expression of the oxidative stress-related transcription factor Nrf2 was also increased. As expected, no histological differences were observed in the cartilage; however, cytokine-induced neutrophil chemoattractant-1 mRNA and protein expression were up-regulated in this tissue. CONCLUSIONS: Mitochondrial failure in the joint is able to reproduce the oxidative and inflammatory status observed in arthritic joints.

A pathogenetic role for endothelin-1 in peritoneal dialysis-associated fibrosis.

In patients undergoing peritoneal dialysis (PD), chronic exposure to nonphysiologic PD fluids elicits low-grade peritoneal inflammation, leading to fibrosis and angiogenesis. Phenotype conversion of mesothelial cells into myofibroblasts, the so-called mesothelial-to-mesenchymal transition (MMT), significantly contributes to the peritoneal dysfunction related to PD. A number of factors have been described to induce MMT in vitro and in vivo, of which TGF-ß1 is probably the most important. The vasoconstrictor peptide endothelin-1 (ET-1) is a transcriptional target of TGF-ß1 and mediates excessive scarring and fibrosis in several tissues. This work studied the contribution of ET-1 to the development of peritoneal damage and failure in a mouse model of PD. ET-1 and its receptors were expressed in the peritoneal membrane and upregulated on PD fluid exposure. Administration of an ET receptor antagonist, either bosentan or macitentan, markedly attenuated PD-induced MMT, fibrosis, angiogenesis, and peritoneal functional decline. Adenovirus-mediated overexpression of ET-1 induced MMT in human mesothelial cells in vitro and promoted the early cellular events associated with peritoneal dysfunction in vivo. Notably, TGF-ß1-blocking peptides prevented these actions of ET-1. Furthermore, a positive reciprocal relationship was observed between ET-1 expression and TGF-ß1 expression in human mesothelial cells. These results strongly support a role for an ET-1/TGF-ß1 axis as an inducer of MMT and subsequent peritoneal damage and fibrosis, and they highlight ET-1 as a potential therapeutic target in the treatment of PD-associated dysfunction.

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-1ß (IL-1ß) 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-1ß-induced IL-8 mRNA and protein expression (mean ± SEM at 18 hours 5,932 ± 1,995 pg/50,000 cells for IL-1ß alone versus 16,241 ± 5,843 pg/50,000 cells for antimycin A plus IL-1ß and 20,087 ± 5,407 pg/50,000 cells for oligomycin plus IL-1ß; 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.

Effect of nitric oxide on mitochondrial activity of human synovial cells.

BACKGROUND: Nitric 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. METHODS: Human 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. RESULTS: SNP 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. CONCLUSIONS: This study suggests that NO reduces the survival of OA synoviocytes by regulating mitochondrial functionality, as well as the proteins controlling the cell cycle.

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

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.

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.