Defective chaperone-mediated autophagy is a hallmark of joint disease in patients with knee osteoarthritis.

OBJECTIVE: Defects in autophagy contribute to joint aging and Osteoarthritis (OA). Identifying specific autophagy types could be useful for developing novel treatments for OA. DESIGN: An autophagy-related gene array was performed in blood from non-OA and knee OA subjects from the Prospective Cohort of A Coruña (PROCOAC). The differential expression of candidate genes was confirmed in blood and knee cartilage and a regression analysis was performed adjusting for age and BMI. HSP90A, a chaperone mediated autophagy (CMA) marker was validated in human knee joint tissues, as well as, in mice with aging-related and surgically-induced OA. The consequences of HSP90AA1 deficiency were evaluated on OA pathogenesis. Finally, the contribution of CMA to homeostasis was studied by assessing the capacity to restore proteostasis upon ATG5-mediated macroautophagy deficiency and genetic HSP90AA1 overexpression. RESULTS: 16 autophagy-related genes were significantly down-regulated in blood from knee OA subjects. Validation studies showed that HSP90AA1 was down-regulated in blood and human OA cartilage and correlated with risk incidence of OA. Moreover, HSP90A was reduced in human OA joints tissues and with aging and OA in mice. HSP90AA1 knockdown was linked to defective macroautophagy, inflammation, oxidative stress, senescence and apoptosis. However, macroautophagy deficiency increased CMA, highlighting the CMA-macroautophagy crosstalk. Remarkably, CMA activation was sufficient to protect chondrocytes from damage. CONCLUSIONS: We show that HSP90A is a key chaperone for chondrocyte homeostasis, while defective CMA contributes to joint damage. We propose that CMA deficiency is a relevant disease mechanism and could represent a therapeutic target for OA.

Compromised autophagy precedes meniscus degeneration and cartilage damage in mice.

OBJECTIVE: Autophagy is a cellular homeostasis mechanism that facilitates normal cell function and survival. Objectives of this study were to determine associations between autophagic responses with meniscus injury, joint aging, and osteoarthritis (OA), and to establish the temporal relationship with structural changes in menisci and cartilage. METHODS: Constitutive activation of autophagy during aging was measured in GFP-LC3 transgenic reporter mice between 6 and 30 months. Meniscus injury was created by surgically destabilizing the medial meniscus (DMM) to induce posttraumatic OA in C57BL/6J mice. Levels of autophagy proteins and activation were analyzed by confocal microscopy and immunohistochemistry. Associated histopathological changes, such as cellularity, matrix staining, and structural damage, were graded in the meniscus and compared to changes in articular cartilage. RESULTS: In C57BL/6J mice, basal autophagy was lower in the meniscus than in articular cartilage. With increasing age, expression of the autophagy proteins ATG5 and LC3 was significantly reduced by 24 months. Age-related changes included abnormal Safranin-O staining and reduced cellularity, which preceded structural damage in the meniscus and articular cartilage. In mice with DMM, autophagy was induced in the meniscus while it was suppressed in cartilage. Articular cartilage exhibited the most profound changes in autophagy and structure that preceded meniscus degeneration. Systemic administration of rapamycin to mice with DMM induced autophagy activation in cartilage and reduced degenerative changes in both meniscus and cartilage. CONCLUSION: Autophagy is significantly affected in the meniscus during aging and injury and precedes structural damage. Maintenance of autophagic activity appears critical for meniscus and cartilage integrity.

Diabetes-accelerated experimental osteoarthritis is prevented by autophagy activation.

OBJECTIVE: Type 2 Diabetes (T2D) is a risk factor for osteoarthritis (OA). Autophagy, an essential homeostasis mechanism in articular cartilage, is defective in T2D and OA. However, how T2D may influence OA progression is still unknown. We aimed to determine how diabetes affects cartilage integrity and whether pharmacological activation of autophagy has efficacy in diabetic mice (db/db mice) with OA. DESIGN: Experimental OA was performed in the right knee of 9 weeks-old C57Bl/6J male mice (Lean group, N = 8) and of 9 weeks-old B6.BKS (D)-Leprdb male mice (db/db group, N = 16) by transection of medial meniscotibial and medial collateral ligaments. Left knee was employed as control knee. Rapamycin (2 mg/kg weight/day) or Vehicle (dimethyl sulfoxide) were administered intraperitoneally three times a week for 10 weeks. Histopathology of articular cartilage and synovium was evaluated by using semiquantitative scoring and synovitis grading systems, respectively. Immunohistochemistry was employed to evaluate the effect of diabetes and Rapamycin on cartilage integrity and OA biomarkers. RESULTS: Cartilage damage was increased in db/db mice compared to Lean mice after experimental OA, while no differences are observed in the control knee. Cartilage damage and synovium inflammation were reduced by Rapamycin treatment of OA-db/db mice. This protection was accompanied with a decrease in MMP-13 expression and decreased interleukin 12 (IL-12) levels. Furthermore, autophagy was increased and cartilage cellularity was maintained, suggesting that mammalian target of rapamycin (mTOR) targeting prevents joint physical harm. CONCLUSION: Our findings indicate that diabetic mice exhibit increased joint damage after experimental OA, and that autophagy activation might be an effective therapy for diabetes-accelerated OA.

Insulin decreases autophagy and leads to cartilage degradation.

OBJECTIVE: Autophagy, a key homeostasis mechanism, is defective in Osteoarthritis (OA) and Type 2 Diabetes (T2D). T2D has been proposed as a risk factor for OA. We hypothesized that diabetes impairs articular cartilage integrity by decreasing autophagy. Our objective was to investigate the effects of high glucose and insulin, characteristics of T2D, on cartilage homeostasis. METHODS: Immortalized human chondrocytes (TC28a2) and primary human chondrocytes (HC) were cultured in 25 mM or 0 mM glucose and treated with insulin (10, 100, 500 nM) for 2, 6 or 24 h. Activity of LC3-II, Akt and rpS6 was evaluated by Western blotting (WB). Human cartilage explants were cultivated with 25 mM glucose and insulin (100,1000 nM) for 24 h to evaluate histopathology. MMP-13 and IL-1ß expression was determined by immunohistochemistry and WB. Effects of Rapamycin (10 µM) were analyzed by WB. LC3 and rpS6 expression was determined by WB in chondrocytes from Healthy, Non Diabetic-OA and Diabetic-OA patients. RESULTS: Insulin downregulates autophagy by reducing LC3 II expression and increasing Akt and rpS6 phosphorylation. Loss of proteoglycans and increased MMP-13 and IL-1ß expression was observed after insulin treatment. Autophagy activation by rapamycin reversed insulin effects. Importantly, chondrocytes from diabetic-OA patients showed decreased LC3 and increased p-rpS6 expression compared to Healthy and Non-Diabetic OA patients. CONCLUSIONS: These results suggest that decreased autophagy might be a mechanism by which diabetes influences cartilage degradation. Pharmacological activation of autophagy may be an effective therapeutic approach to prevent T2D-induced cartilage damage.

Palytoxin detection and quantification using the fluorescence polarization technique.

Palytoxin (PLT) is a highly toxic nonpeptidic marine natural product, with a complex chemical structure. Its mechanism of action targets Na,K-ATPase. Fluorescence polarization (FP) is a spectroscopic technique that can be used to determine molecular interactions. It is based on exciting a fluorescent molecule with plane-polarized light and measuring the polarization degree of the emitted light. In this study, FP was used to develop a detection method based on the interaction between the Na,K-ATPase and the PLT. The Na,K-ATPase was labeled with a reactive succinimidyl esther of carboxyfluorescein, and the FP of protein-dye conjugate was measured when the amount of PLT in the medium was modified. The assay protocol was first developed using ouabain as a binding molecule. The final result was a straight line that correlates FP units and PLT concentration. Within this line the PLT equivalents in a natural sample can be quantified. A selective cleaning procedure to mussel samples and dinoflagellates cultures was also developed to avoid the matrix effect. The LOQ (limit of quantification) of the method is 10nM and the LOD (limit of detection) is 2 nM. This new PLT detection method is easier, faster, and more reliable than the other methods described to date.

Anti-apoptotic effect of transforming growth factor-beta1 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.

Differential effects of tumor necrosis factor-alpha and interleukin-1beta on cell death in human articular chondrocytes.

OBJECTIVE: The death of chondrocytes by apoptosis is characteristic of degenerative joint diseases, such as osteoarthritis (OA). Tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) have been shown to play an important role in the development of OA. In this study we analyzed the effects of TNF-alpha and IL-1beta on cell death in normal human chondrocytes. METHODS: Normal human chondrocytes were isolated from knee cartilage obtained at autopsy from 30 adult cadaveric donors. The cells were stimulated with TNF-alpha (10 ng/ml) or IL-1beta (5 ng/ml) in the presence or absence of Ro 31-8220 (Ro: a structurally related analog of bisindolylmaleimide that inhibits mitogen-activated protein kinase phosphatase 1 [MKP-1]) (Ro; 10 microM), an MKP-1 inhibitor, which induces apoptosis in chondrocytes. Apoptosis was evaluated by flow cytometry (propidium iodide) and nuclear morphology was evaluated with 4',6'-dianidino-2-phenylindole dihydrochloride. The expressions of caspase-8, -7 and -3 and Bcl-2 were analyzed by Western blot and the activation of caspase-3 and -8 was measured by flow cytometry. Prostaglandin E2 (PGE2) was evaluated by enzyme-linked immunosorbent assay. RESULTS: At 24 h the percentage of apoptotic (hypodiploid) nuclei induced by TNF-alpha+Ro was higher than the level induced by Ro alone. The combination of IL-1beta (5 ng/ml) with Ro did not show a synergistic effect. A morphological analysis demonstrated that treatment with TNF-alpha+Ro resulted in a large number of cells with condensed nuclei and DNA fragmentation. Western blot studies indicated that IL-1beta+Ro did not induce the time-dependent activation of caspase-8, -7 and -3 as seen with TNF-alpha+Ro. As quantified by flow cytometry, TNF-alpha+Ro induced a higher level of caspase-3 and -8 activation than that seen with IL-1beta+Ro. Pre-incubation for 2h with caspase inhibitors for caspase-3, -7, -8 and pan-caspase significantly decreased the hypodiploid DNA peak induced by treatment with TNF-alpha+Ro at 24 h. Indomethacin increased the cell death induced by IL-1beta+Ro; however, apoptosis induced by TNF-alpha+Ro was not modified by indomethacin. CONCLUSIONS: These results confirm that TNF-alpha and IL-1beta regulate apoptosis differently in this human chondrocyte model and that the differing effects of these cytokines are PGE2-independent. Indomethacin potentiates the effect of IL-1 on cell death and this may explain the reported effect of indomethacin on the progression of joint destruction.

Mitochondrial activity is modulated by TNFalpha and IL-1beta in normal human chondrocyte cells.

OBJECTIVE: Pro-inflammatory cytokines play an important role in osteoarthritis (OA). In osteoarthritic cartilage, chondrocytes exhibit an alteration in mitochondrial activity. This study analyzes the effect of tumor necrosis factor-alpha (TNFalpha) and interleukin-1beta (IL-1beta) on the mitochondrial activity of normal human chondrocytes. MATERIALS AND METHODS: Mitochondrial function was evaluated by analyzing the activities of respiratory chain enzyme complexes and citrate synthase, as well as by mitochondrial membrane potential (Deltapsim) and adenosine triphosphate (ATP) synthesis. Bcl-2 family mRNA expression and protein synthesis were analyzed by RNase protection assay (RPA) and Western-blot, respectively. Cell viability was analyzed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and apoptosis by 4', 6-diamidino-2-phenylindole dihydrochloride (DAPI) stain. Glycosaminoglycans were quantified in supernatant by a dimethyl-methylene blue binding assay. RESULTS: Compared to basal cells, stimulation with TNFalpha (10 ng/ml) and IL-1beta (5 ng/ml) for 48 h significantly decreased the activity of complex I (TNFalpha=35% and IL-1beta=35%) and the production of ATP (TNFalpha=18% and IL-1beta=19%). Both TNFalpha and IL-1beta caused a definitive time-dependent decrease in the red/green fluorescence ratio in chondrocytes, indicating depolarization of the mitochondria. Both cytokines induced mRNA expression and protein synthesis of the Bcl-2 family. Rotenone, an inhibitor of complex I, caused a significant reduction of the red/green ratio, but it did not reduce the viability of the chondrocytes. Rotenone also increased Bcl-2 mRNA expression and protein synthesis. Finally, rotenone as well as TNFalpha and IL-1beta, reduced the content of proteoglycans in the extracellular matrix of normal cartilage. CONCLUSION: These results show that both TNFalpha and IL-1beta regulate mitochondrial function in human articular chondrocytes. Furthermore, the inhibition of complex I by both cytokines could play a key role in cartilage degradation induced by TNFalpha and IL-1beta. These data could be important for understanding of the OA pathogenesis.

Cytokines, tumor necrosis factor-alpha and interleukin-1beta, differentially regulate apoptosis in osteoarthritis cultured human chondrocytes.

OBJECTIVE: This study addresses the effects of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) on cell death in human chondrocytes. METHODS: Osteoarthritis (OA) human chondrocytes stimulated with Actinomycin-D (ActD) were used as a cellular apoptotic model. Caspase family mRNA expression and protein synthesis were analyzed by the ribonuclease protection assay and Western-blot, respectively. Cell viability and apoptosis were evaluated using the 3-[4,5-dimethylthiazol-2yl] 2,5-diphenyl tetrazolium bromide (MTT) assay and flow cytometry, respectively. Prostaglandin E2 (PGE2) and nitric oxide (NO) were evaluated by enzyme-linked immunosorbent assay (ELISA) and the Griess method, respectively. RESULTS: TNF-alpha and IL-1beta differentially affected the pattern of caspase mRNA expression by human chondrocytes. TNF-alpha induced a gradual increase in caspase-1 and -8 mRNA levels that was not seen with IL-1beta. The time sequence of caspase-3 and -7 inductions by TNF-alpha differs from that induced by IL-1beta. Cell viability was not modified by TNF-alpha or IL-1beta in cultured chondrocytes. Then, we employed ActD as a model to facilitate cell death. Treatment with TNF-alpha and ActD (TNF-alpha/ActD) increased cell death induced by ActD (23%). Treatment with IL-1beta and ActD (IL-1beta/ActD) did not modulate ActD-induced cell death. Similarly, IL-1beta/ActD did not induce an increase in the activation of caspase-3 and -7 and poly (ADP-ribose) polymerase (PARP) cleavage observed by the incubation with TNF-alpha/ActD. These different effects were not due to bcl-2 or mcl-1 levels. Inhibition of PGE2 synthesis by indomethacin increased the cell death induced by IL-1beta/Act-D (59%). An inhibitor of caspase-8 significantly reduced only the TNF-alpha/ActD-induced cell death (58%). CONCLUSION: TNF-alpha and IL-1beta differentially regulate the apoptotic pathway in human chondrocytes. This difference is dependent on PGE2 and caspase-8 levels.

Phosphatase-1 and -2A inhibition modulates apoptosis in human osteoarthritis chondrocytes independently of nitric oxide production.

OBJECTIVE: To characterise the role of phosphatase-1 and -2A (PP1/2A) in the modulation of apoptosis in human osteoarthritis (OA) chondrocytes. METHODS: Human OA chondrocytes were isolated from cartilage obtained from the femoral heads of patients undergoing joint replacement surgery. Cell viability was evaluated by MTT assay. Apoptosis was quantified by ELISA, which measures DNA fragmentation. Nitric oxide (NO) production was evaluated by the Greiss method, and inducible nitric oxide synthase (iNOS) protein synthesis was studied by western blotting. RESULTS: Inhibition of PP1/2A by the specific inhibitor okadaic acid (OKA) dose and time dependently caused a reduction of cell viability (OKA at 50 nmol/l: a reduction to 60% and 43% at 48 and 72 hours, respectively). Genomic DNA from chondrocytes treated with OKA at 50 and 100 nmol/l for 48 hours displayed increased internucleosomal DNA fragmentation by 11 and 13 fields, respectively. Light microscopy and DAPI studies showed that OKA induced DNA condensation and fragmentation, typical of death by apoptosis. The caspase inhibitors Z-VAD-FMK and Z-DEVD-FMK increased cell viability, reduced by OKA at 50 nmol/l to 87% and 73%, respectively. OKA did not increase iNOS protein synthesis or NO production. CONCLUSION: PP1/2A modulate apoptosis in human OA chondrocytes; this is independent of NO production but dependent on caspases.

Effect of nitric oxide on mitochondrial respiratory activity of human articular chondrocytes.

OBJECTIVE: To investigate the effect of nitric oxide (NO) on mitochondrial activity and its relation with the apoptosis of human articular chondrocytes. MATERIALS AND METHODS: Mitochondrial function was evaluated by analysing respiratory chain enzyme complexes, citrate synthase (CS) activities, and mitochondrial membrane potential (Delta psi m). The activities of the mitochondrial respiratory chain (MRC) complexes (complex I: NADH CoQ(1) reductase, complex II: succinate dehydrogenase, complex III: ubiquinol cytochrome c reductase, complex IV: cytochrome c oxidase) and CS were measured in human articular chondrocytes isolated from normal cartilage. The Delta psi m was measured by 5,5',6,6'-tetracholoro-1,1',3,3'-tetraethylbenzimidazole carbocyanide iodide (JC-1) using flow cytometry. Apoptosis was analysed by flow cytometry. The mRNA expression of caspases was analysed by ribonuclease protection analysis and the detection of protein synthesis by western blotting. Sodium nitroprusside (SNP) was used as an NO compound donor. RESULTS: SNP at concentrations higher than 0.5 mmol/l for 24 hours induced cellular changes characteristic of apoptosis. SNP elicited mRNA expression of caspase-3 and caspase-7 and down regulated bcl-2 synthesis in a dose and time dependent manner. Furthermore, 0.5 mM SNP induced depolarisation of the mitochondrial membrane at 5, 12, and 24 hours. Analysis of the MRC showed that at 5 hours, 0.5 mM SNP reduced the activity of complex IV by 33%. The individual inhibition of mitochondrial complex IV with azide modified the Delta psi m and induced apoptosis. CONCLUSIONS: This study suggests that the effect of NO on chondrocyte survival is mediated by its effect on complex IV of the MRC.

Fisiopatología de la artrosis: ¿cuál es la actualidad? / Physiopathology of arthrosis: what is the state of the art?

La artrosis (OA) es una patología degenerativa de las articulaciones que se caracteriza por la degradación del cartílago articular hialino. Su progresión es lenta y tiene una etiología múltiple que implica el envejecimiento, la obesidad y la influencia genética como algunos de los factores que favorecen el desarrollo de la OA. En su fase final refleja una insuficiencia de los procesos de reparación del cartílago, resultando en la degradación de la matriz extracellular, muerte del condrocito (por apoptosis) y pérdida total de la integridad del cartílago. El condrocito es el único tipo celular presente en el cartílago maduro y causante de la reparación del tejido dañado. Sin embargo, el desarrollo de esta patología no sólo afecta al cartílago, sino a toda la estructura articular, incluyendo el hueso subcondral y el tejido sinovial. En esta revisión se evaluaran estos cambios desde el punto de vista molecular y celular y se revelará la complejidad de esta patología que incluye múltiples cambios en la estructura articular (AU)