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Objective: Synovial inflammation is one of the most characteristic events in different types of arthritis, including Osteoarthritis (OA). Emerging evidence also suggests the involvement of lipids in the regulation of inflammatory processes. The aim of this study was to elucidate the heterogeneity and spatial distribution of lipids in the OA synovial membrane and explore their putative involvement in inflammation.Method: The abundance and distribution of lipids were examined in human synovial membranes. To this end, histological cuts from this tissue were analysed by matrix-assisted laser desorption ionization - mass spectrometry imaging (MALDI-MSI). The lipidomic profile of OA synovium was characterized and compared with healthy and other forms of inflammatory arthropathies as Rheumatoid Arthritis (RA) and Psoriatic Arthritis (PsA) using principal component analysis and discriminant analysis methods. Lipid identification was undertaken by tandem MS analyses and database queries.Results: Our results reveal differential and characteristic lipidomic profiles between OA and control samples. Specifically, we unveiled that OA synovium presents elevated levels of phosphatidylcholines, fatty acids and lysophosphatidic acids and lower levels of lysophosphatidylcholines compared to control tissues. The spatial distribution of particular glycerophospholipids was also correlated with hypertrophic, inflamed or vascularized synovial areas. Compared with other inflammatory arthritis, the OA tissue showed lower amounts of phosphatidylethanolamine-based plasmalogens.Conclusions: This study provides a novel insight into the lipid profiles of synovial membrane and differences in abundance between OA and control tissues. The lipidomic alterations improves understanding of the pathogenic mechanisms of OA and may be important for its diagnosis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Summary: Objective: To study whether transforming growth factor-β1 (TGF-β1) is able to protect human chondrocytes from apoptosis and to analyze the role of phosphatases in the possible anti-apoptotic effect of TGF-β1. 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-α (TNF-α) 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-β1 significantly reduced the percentage of hypo-diploid chondrocytes, as well as the percentage of internucleosomal DNA breakage. However, in normal chondrocytes, TGF-β1 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-β1 stimulation; however in normal chondrocytes PP2A activity was significantly activated by TGF-β1. The preincubation of normal chondrocytes with TGF-β1 plus the PP2A inhibitor protein, IPP2A, reduced internucleosomal DNA breakage when compared with TGF-β1 stimulation alone. The bcl-2/bax protein ratio was significantly higher in TGF-β1 plus IPP2A preincubated normal chondrocytes than in cells stimulated with TGF-β1 alone. Conclusion: By manipulating the degree of PP2A activity, these results show the major role that PP2A plays in the outcome of TGF-β1 signal transduction. These data suggest that PP2A could be a pivotal regulator of anti-apoptotic TGF-β1-induced effects. [Copyright &y& Elsevier]

Summary: Objective: The death of chondrocytes by apoptosis is characteristic of degenerative joint diseases, such as osteoarthritis (OA). Tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) have been shown to play an important role in the development of OA. In this study we analyzed the effects of TNF-α and IL-1β 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-α (10ng/ml) or IL-1β (5ng/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; 10μM), 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 24h the percentage of apoptotic (hypodiploid) nuclei induced by TNF-α+Ro was higher than the level induced by Ro alone. The combination of IL-1β (5ng/ml) with Ro did not show a synergistic effect. A morphological analysis demonstrated that treatment with TNF-α+Ro resulted in a large number of cells with condensed nuclei and DNA fragmentation. Western blot studies indicated that IL-1β+Ro did not induce the time-dependent activation of caspase-8, -7 and -3 as seen with TNF-α+Ro. As quantified by flow cytometry, TNF-α+Ro induced a higher level of caspase-3 and -8 activation than that seen with IL-1β+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-α+Ro at 24h. Indomethacin increased the cell death induced by IL-1β+Ro; however, apoptosis induced by TNF-α+Ro was not modified by indomethacin. Conclusions: These results confirm that TNF-α and IL-1β 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. [Copyright &y& Elsevier]

Summary: 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-α (TNFα) and interleukin-1β (IL-1β) 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 (Δψ m) 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 TNFα (10ng/ml) and IL-1β (5ng/ml) for 48h significantly decreased the activity of complex I (TNFα=35% and IL-1β=35%) and the production of ATP (TNFα=18% and IL-1β=19%). Both TNFα and IL-1β 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 TNFα and IL-1β, reduced the content of proteoglycans in the extracellular matrix of normal cartilage. Conclusion: These results show that both TNFα and IL-1β 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 TNFα and IL-1β. These data could be important for understanding of the OA pathogenesis. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Summary: Objective: This study addresses the effects of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) 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-α and IL-1β differentially affected the pattern of caspase mRNA expression by human chondrocytes. TNF-α induced a gradual increase in caspase-1 and -8 mRNA levels that was not seen with IL-1β. The time sequence of caspase-3 and -7 inductions by TNF-α differs from that induced by IL-1β. Cell viability was not modified by TNF-α or IL-1β in cultured chondrocytes. Then, we employed ActD as a model to facilitate cell death. Treatment with TNF-α and ActD (TNF-α/ActD) increased cell death induced by ActD (23%). Treatment with IL-1β and ActD (IL-1β/ActD) did not modulate ActD-induced cell death. Similarly, IL-1β/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-α/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-1β/Act-D (59%). An inhibitor of caspase-8 significantly reduced only the TNF-α/ActD-induced cell death (58%). Conclusion: TNF-α and IL-1β differentially regulate the apoptotic pathway in human chondrocytes. This difference is dependent on PGE2 and caspase-8 levels. [Copyright &y& Elsevier]