Epigallocatechin-3-gallate selectively inhibits interleukin-1beta-induced activation of mitogen activated protein kinase subgroup c-Jun N-terminal kinase in human osteoarthritis chondrocytes.

Activation of mitogen activated protein kinases (MAPK) is a critical event in pro-inflammatory cytokine-induced signaling cascade in synoviocytes and chondrocytes that lead to the production of several mediators of cartilage damage in an arthritic joint. Green tea (Camellia sinensis) is a widely consumed beverage and we earlier showed that polyphenols present in green tea (GTP) inhibit the development of inflammation and cartilage damage in an animal model of arthritis. In this study we evaluated the role of epigallocatechin-3-gallate (EGCG), a green tea polyphenol which mimics its anti-inflammatory effects, in modulating the IL-1beta-induced activation of MAPK's in human chondrocytes. We discovered that EGCG inhibited the IL-1beta-induced phosphorylation of c-Jun N-terminal kinase (JNK) isoforms, accumulation of phospho-c-Jun and DNA binding activity of AP-1 in osteoarthritis (OA) chondrocytes. Also IL-1beta, but not EGCG, induced the expression of JNK p46 without modulating the expression of JNK p54 in OA chondrocytes. In immunecomplex kinase assays, EGCG completely blocked the substrate phosphorylating activity of JNK but not of p38-MAPK. EGCG had no inhibitory effect on the activation of extracellular signal-regulated kinase p44/p42 (ERKp44/p42) or p38-MAPK in OA chondrocytes. EGCG or IL-1beta did not alter the total non-phosphorylated levels of either p38-MAPK or ERKp44/p42 in OA chondrocytes. These are novel findings and indicate that EGCG may be of potential benefit in inhibiting IL-1beta-induced catabolic effects in OA chondrocytes that are dependent on JNK activity.     

IL-17, IL-1beta and TNF-alpha stimulate VEGF production by dedifferentiated chondrocytes

OBJECTIVE: To verify the involvement of proinflammatory cytokines IL-17, IL-1beta and tumor necrosis factor alpha (TNF-alpha) in cartilage vascularization by stimulating the production of vascular endothelial growth factor (VEGF) by chondrocytes isolated from patients with osteoarthritis (OA), in comparison with patients with rheumatoid arthritis (RA) and patients with femoral or humeral neck fracture (FP). DESIGN: Chondrocytes isolated from patients with OA were maintained in monolayer culture for several passages. Chondrocyte dedifferentiation was monitored by the synthesis of cathepsin B by these cells. Chondrocytes freshly isolated at each subculture (subcultures 1-3) were stimulated with IL-17, IL-1beta or TNF-alpha. Supernatants were collected, immunoassayed for the production of VEGF and cathepsin B and assayed as the source of VEGF on the VEGF sensible ECV304 cell line. The cells were used to quantify intracellular cathepsin B enzymatic activity. RESULTS: In differentiated conditions IL-1beta and TNF-alpha, but not IL-17, can inhibit the spontaneous secretion of VEGF by human OA, RA and FP chondrocytes, and IL-17 can restore the decrease in VEGF secretion caused by TNF-alpha. IL-17, together with IL-1beta and TNF-alpha, can enhance VEGF secretion to various extents by dedifferentiated OA chondrocytes. This change in effect with respect to primary culture was observable for all cytokines at the beginning of dedifferentiation, when the production of VEGF by chondrocytes had dramatically fallen and the cathepsin B synthesis had increased. The amount of VEGF induced by cytokines on dedifferentiated chondrocytes never reached the amount of VEGF produced by differentiated chondrocytes. VEGF produced by chondrocytes stimulated the ECV304 cell line proliferation. CONCLUSIONS: These results indicate that dedifferentiated OA chondrocytes secrete VEGF after stimulation with proinflammatory cytokines. This event may be responsible for neovascularization found in OA cartilage.     

Hyaluronan inhibits matrix metalloproteinase‐13 in human arthritic chondrocytes via CD44 and P38

We investigated the effects of hyaluronan (HA) on interleukin‐1β (IL‐1β)‐stimulated matrix metalloproteinase (MMP)‐13 production in human chondrocytes from patients with osteoarthritis (OA) or rheumatoid arthritis (RA). Secreted levels of MMP‐13 in conditioned media were detected by immunoblotting, while intracellular MMP‐13 synthesis in articular cartilage was evaluated by immunofluorescence microscopic analysis. Mitogen‐activated protein kinases (MAPKs), p38, extracellular signal‐regulated kinases (ERK), and c‐jun NH2‐terminal kinase (JNK) were assessed by Western blotting. IL‐1β (2 ng/ml) stimulates the secretion of MMP‐13 in both OA and RA chondrocytes. Inhibition studies using specific MAPK inhibitors revealed that IL‐1β induced MMP‐13 via p38 in both OA and RA chondrocytes. HA down‐regulates IL‐1β‐stimulated MMP‐13 and phosphorylated p38 (p‐p38) in a dose‐dependent manner (0.1, 1, 2, and 4 mg/ml). When used at 4 mg/ml, HA inhibits p‐p38 phosphorylation by more than 60%. In response to IL‐1β, RA chondrocytes express a higher level of p‐p38 than that of OA chondrocytes. Inhibition of CD44, using a blocking antibody, significantly reversed the inhibitory effect of HA on both MMP‐13 and p‐p38. Our study clearly shows that HA inhibits IL‐1β‐induced MMP‐13 via its principal receptor, CD44, and subsequent intracellular p38 MAPK signaling in OA and RA chondrocytes. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:258–264, 2011     

Expression of VEGF isoforms by epiphyseal chondrocytes during low-oxygen tension is HIF-1 alpha dependent

OBJECTIVE: To establish the role of hypoxia and HIF-1 alpha for VEGF expression of murine epiphyseal chondrocytes. To analyze the effect of hypoxia on VEGF isoform expression. MATERIALS AND METHODS: VEGF mRNA and VEGF isoform expression was investigated in epiphyses of murine newborns by in situ hybridization and real-time PCR. Further, epiphyseal chondrocytes were isolated from newborn mice with homozygous flanking of the HIF-1 alpha gene with lox-P sites. HIF-1 alpha was deleted by infection with adenovirus containing cre-recombinase. After chondrocytes reached confluency they were exposed to 0.5% or 20% oxygen, respectively. Total VEGF and VEGF isoform mRNA expression levels were measured by real-time PCR. Secreted VEGF protein was determined by ELISA. RESULTS: VEGF mRNA signals were detected in the hypertrophic zone and in the center of the proliferative zone of the murine epiphysis, which is considered to be hypoxic. Real-time PCR revealed that VEGF(120)is the dominant isoform in vivo. In cultured epiphyseal chondrocytes strongly increased VEGF gene expression levels were detected after exposure to hypoxia. Furthermore, secretion of VEGF protein was significantly enhanced under 0.5% oxygen. Remarkably, functional inactivation of HIF-1 alpha abolished the hypoxic increase of VEGF expression in chondrocytes completely. Furthermore, the soluble isoforms VEGF(120)and VEGF(164)are the most abundantly expressed splice variants in chondrocytes exposed to low oxygen levels. CONCLUSIONS: The data presented here clearly indicate that hypoxia is able to induce the synthesis of soluble VEGF isoforms by epiphyseal chondrocytes, most likely through stabilization of HIF-1 alpha. Thus it can be speculated that HIF-1 alpha is an essential prerequisite for hypoxic VEGF synthesis in the epiphysis, thereby contributing to the formation and invasion of blood vessels in long bone development.     

The mechanism of TDP‐43 gene expression on inflammatory factors and the JNK and p38 MAPK signalling pathways in ischaemic hypoxic stress dependence

In this study, the mechanism of TDP‐43 gene expression on inflammatory factors and Jun N‐terminal kinase (JNK) and p38 mitogen‐activated protein kinase (MAPK) signalling pathways in ischaemic hypoxic stress dependence was investigated. Sixty SD rats were selected and divided into the control group, the osteoarthritis (OA) model group, and the TDP‐43‐mMSCs+OA group. In the OA model group and the TDP‐43‐mMSCs+OA group, OA was established by collagenase injection. Western blotting assays were used to detect the expression of TDP‐43 in cartilage tissues of each rat. The secretion of tumour necrosis factor‐α (TNF‐α) and interleukin‐1β (IL‐1β) in the serum of rats was determined by enzyme‐linked immunosorbent assay (ELISA). The formation of cytoplasmic stress granules (SGs) and the expression of receptor for activated c‐kinase 1 (RACK1) were detected by Western blotting assays in each group of rats. The expression of MTK1 and MAPKKK phosphorylation and changes in the JNK and p38 MAPK signalling pathways were detected by Western blotting assays. Compared with the control group, the expression of TDP‐43 in the cartilage tissue of rats in the OA model group was significantly decreased. The expression of TDP‐43 in the cartilage tissue of rats in the TDP‐43‐mMSCs+OA group was significantly higher than that of the control group and the OA model group, which indicates that TDP‐43‐mMSC transplantation was successful. Enzyme‐linked immunosorbent assay results showed that the plasma TNF‐α and IL‐1β levels in the OA model group were significantly increased (P < 0.01) when compared with the control group. However, the secretion of TNF‐α and IL‐1β in the serum of the TDP‐43‐mMSCs+OA group was significantly lower than that of the model group (P < 0.01) but still higher than the control group. This indicates that overexpression of TDP‐43 reduces the inflammatory response induced by OA. Western blotting assays showed that the amount of cytoplasmic SGs in the cartilage tissue of rats in the OA model group was significantly decreased when compared with the control group. The amount of SGs in the cartilage of rats in the TDP‐43‐mMSCs+OA group was significantly higher than that of the model group. The expression of RACK1 in the cartilage tissue of rats in the OA model group was significantly higher than that of the control group. Overexpression of the TDP‐43 gene can interfere with the secretion of inflammatory factors and inhibit the activation of the JNK and p38 MAPK signalling pathways by ischaemic hypoxia stress. Thus, the molecular mechanism of chondrocytopathic lesions was reversed, which provided a new theoretical basis for the treatment of OA.     

Tacrolimus (FK506) inhibits interleukin-1β-induced angiopoietin-1, Tie-2 receptor, and vascular endothelial growth factor through down-regulation of JNK and p38 pathway in human rheumatoid fibroblast-like synoviocytes

ObjectThis study aimed to identify the regulatory effect of tacrolimus on the interleukin-1β (IL-1β)-induced expressions of angiopoietin-1 (Ang-1), Tie-2 receptor (Tie-2), and vascular endothelial growth factor (VEGF) in human rheumatoid fibroblast-like synoviocytes (FLS) and to determine the regulatory mechanism in the mitogen-activated protein kinases (MAPKs) signaling pathway.MethodsIL-1β-induced Ang-1, Tie-2, and VEGF expressions with and without tacrolimus were measured in cultured FLS using real time–polymerase chain reaction, enzyme-linked immunosorbent assay, Western blotting, and immunofluorescence staining. The effect of tacrolimus on the regulation of Ang-1, Tie-2 and VEGF expressions through the MAPK signaling pathway was identified by Western blotting and immunofluorescence staining.ResultsIL-1β appeared to induce marked expressions of Ang-1, Tie-2, and VEGF in cultured FLS. Tacrolimus significantly inhibited Ang-1, Tie-2, and VEGF mRNA and protein in cultured FLS treated with 10 ng/ml IL-1β. In addition, expressions of these angiogenic molecules were shown to involve all three of the studied MAPK signaling pathways, including ERK, JNK, and p38. However, the inhibitory effects of tacrolimus on Ang-1, Tie-2, and VEGF proteins were regulated by blocking the phosphorylations of JNK and p38 MAPK, but not that of ERK.ConclusionThis study demonstrates that tacrolimus inhibits the expressions of Ang-1, Tie-2, and VEGF by blocking the activations of the IL-1β-mediated JNK and p38 MAPK pathways in human FLS. This suggests that tacrolimus contributes to the suppression of angiogenesis in the pathogenesis of RA.     

缺氧时丝裂原激活蛋白激酶类对人肾小管上皮细胞富含半胱氨酸蛋白61基因转录活性的调控

目的 探讨丝裂原激活蛋白激酶类（MAPKs）对缺氧条件下人近端肾小管上皮细胞（HKC）中富含半胱氨酸蛋白61（Cyr61）基因转录活性的调控机制。方法 缺氧培养HKC，Northern印迹检测Cyr61mRNA表达；Western印迹检测Cyr61、p38、细胞外信号调节激酶（ERK1／2）、c—Jun—N末端蛋白激酶（JNK）以及缺氧诱导因子1c（HIF-1α）的表达。构建含有人Cyr61基因启动子的报告基因Cyr61-luc质粒，将其单独或者分别与表达活性MAPKs的质粒Ca—MEK1和Ca—MKK6共同瞬时转染HKC。通过荧光素酶活性检测观察缺氧、MAPKs抑制剂和MAPKs活性酶对Cyr61基因转录活性的调控。结果 缺氧时HKC表达cyr61、HIF-1α增高，ERK1／2、JNK、p38总量不变，而其各自的磷酸化形式均明显增加。HKC转染Cyr—luc后，p38通路抑制剂SB203580和ERK通路抑制剂PD98059显著抑制缺氧时Cyr61的转录活性，两者协同作用时抑制作用显著增强。Ca—MEK1与Cyr—luc共转染HKC后，Cyr61转录活性无改变；而Ca—MKK6与Cyr—luc共转染后，Cyr61转录活性显著增高。对缺氧培养的HKC，PD98059处理使HIF-1α和Cyr61蛋白表达显著降低；SB203580处理可显著降低Cyr61蛋白表达，但对HIF-1α无影响。结论 在HKC中，缺氧可通过p38通路直接上调Cyr61基因启动子活性，也可通过ERK1／2途径促进HIF-1α表达，间接调节Cyr61基因启动子活性。     

低氧环境对小鼠未成熟关节透明软骨细胞培养的影响

目的研究低氧和低氧模拟化合物氯化钴对小鼠未成熟关节透明软骨细胞氧感应基因和细胞表型的影响。方法经酶消化分离出小鼠未成熟关节透明软骨细胞，分别在21％氧、2％氧和150μmol/L氯化钴条件下培养一定时间。通过倒置显微镜、透射电镜和扫描电镜观察细胞形态学变化。应用定量PCR和Western Blot检测葡萄糖转运体-1（GLUT-1）、葡萄糖转运体-3（GLUT-3）、磷酸果糖激酶-1（PGK-1）和低氧诱导因子-1α（HIF—1α）的表达。应用定量PCR观察软骨细胞表型改变。应用四甲基偶氮唑盐法（MTT）检测低氧及氯化钴对软骨细胞活性的影响。用葡萄糖检测试剂盒测葡萄糖摄取量。结果不同培养条件下软骨细胞形态无明显差异。2％氧和氯化钴可增加GLUT-1、GLUT-3及PGK-1mRNA表达。2％氧和氯化钴可促进GLUT-1、GLUT-3和HIF—1α蛋白表达。低氧和氯化钴促进细胞活性，增加葡萄糖摄取并促进细胞外基质合成。结论软骨细胞能通过调节氧感应基因适应低氧环境，HIF—1α可能起关键作用。低氧能在一定程度上增加软骨细胞活性和细胞外基质合成。模拟体内氧环境培养细胞能更好地了解软骨细胞特性。     

Expression of hypoxia inducible factor-1 alpha and correlation with preoperative embolization of meningiomas

OBJECT: Vascular endothelial growth factor (VEGF) has been implicated in meningioma tumorigenesis and growth. The production of VEGF is regulated by hypoxia inducible factor-1alpha (HIF-1alpha), especially under conditions of hypoxia. In this study, the authors examine the expression of HIF-1alpha and VEGF in meningiomas, with a special emphasis on conditions of hypoxia, such as preoperative embolization, and on in vitro studies in cultured cells. METHODS: Meningiomas obtained in 142 patients were studied using immunohistochemical methods to detect HIF-1alpha and the results were correlated with the extent or lack of preoperative embolization and expression of VEGF. Primary meningioma cell cultures were established and cell culture experiments were performed using a hypoxia chamber to stimulate HIF-1alpha and VEGF production. Expression of HIF-1alpha in primary meningioma cell cultures was measured using immunoblot assays. The VEGF secretion was measured using enzyme-linked immunosorbent assay. Half of the meningiomas studied were positive for HIF-1alpha, with a strong correlation between complete embolization and HIF-1alpha expression. Most of the meningiomas studied expressed VEGF protein, and VEGF expression did not correlate with the degree of embolization. A strong correlation was found between VEGF and HIF-1alpha expression in immunohistochemical studies. Secretion of VEGF is increased by hypoxia and growth factor stimulation. In meningiomas, growth factors stimulate HIF-1alpha expression. The role of hypoxia is less clear. CONCLUSIONS: The expression of HIF-1alpha is increased by complete preoperative embolization of meningiomas. The expression of HIF-1alpha also correlates with VEGF secretion in meningiomas. Growth factor and hypoxic stimulation both contribute to VEGF control, but which is most important (or whether both are equally important) will require further studies.     

p38 MAPK and COX2 inhibition modulate human chondrocyte response to TGF-beta.

These studies compare actions of p38 MAPK inhibition and COX2 inhibition to modulate human arthritic chondrocyte responses to TGF-beta and FCS under basal and IL-1 activated conditions. Chondrocytes isolated from arthritic human femoral condyle cartilage obtained at total knee replacement were grown to 80% confluence. Proteoglycan synthesis and proliferation were measured with and without IL-1 activation in the presence and absence of growth factors and with and without inhibition of p38 MAPK or COX2 activity. Experiments to evaluate TIMP-1 production under these conditions were done using cartilage organ cultures. Neither p38 MAPK inhibitors nor COX2 inhibition affected basal proliferation. However both inhibitors enhanced the proliferative response to TGF-beta and FCS in IL-1 activated chondrocytes. TGF-beta stimulated proteoglycan synthesis was decreased by p38 MAPK inhibition, however COX2 inhibition restored the response to TGF-beta in IL-1 activated cells. In contrast, COX2 inhibition did not modulate TIMP-1 production while p38 MAPK inhibitors potentiated TGF-beta stimulated production of TIMP-1 in IL-1 activated cartilage. p38 MAPK inhibition and COX2 inhibition have unique and similar abilities to counteract some of the effects of IL-1 on human chondrocyte/cartilage metabolism. Both will partially restore the proliferative response to growth factors. p38 MAPK inhibition blunts TGF-beta stimulation of proteoglycan synthesis, but increases TIMP-1 synthesis. COX2 inhibition can restore the proteoglycan synthetic response to TGF-beta, but has no effect on cartilage TIMP-1 production. Use of these inhibitors to minimize cartilage damage in arthritic and mechanically stressed joints should reflect these characteristics.     

Tumor necrosis factor alpha can contribute to focal loss of cartilage in osteoarthritis

OBJECTIVE; To evaluate the potential for tumor necrosis factor alpha (TNFalpha)-induced focal loss of cartilage in osteoarthritic (OA) knee joints. DESIGN: Fresh cartilage from specified regions of OA joints was immunostained for TNF-receptor (R) bearing chondrocytes. Cartilage explants from the same regions were cultured with or without small amounts of TNFalpha and cumulative GAG release into supernatants measured. Concentrations of TNFalpha, p55 and p75 soluble (s) TNF-R in supernatants from cultured OA and non-arthritic (NA) synovium were measured by ELISA. RESULTS: TNF-R bearing chondrocytes were identified in OA cartilage; more specimens contained p55 TNF-R- than p75 TNF-R-bearing chondrocytes and differences in TNF-R distribution were apparent in cartilage from different regions of the same knees. TNFalpha at 5, 1, 0.5 and 0.25 ng/ml (but not 0.1 ng/ml) significantly increased glycosaminoglycans (GAG) release from cartilage explants in a dose-dependent manner. Variation in susceptibility to TNFalpha was observed in explants from different sites. TNFalpha and p75 sTNF-R, but not p55 sTNF-R, concentrations were significantly higher in OA, as compared with NA, supernatants. A significant correlation between TNFalpha and p75 sTNF-R measurements was apparent only in NA supernatants. CONCLUSIONS: Variations in chondrocyte TNF-R expression occur in OA cartilage in vivo. TNFalpha at concentrations produced by OA synovium in vitro, can degrade cartilage matrix. In most OA supernatants sTNF-R concentrations were insufficient to abrogate the effects of TNFalpha. Thus conditions exist in some OA knees for TNFalpha to contribute to focal loss of cartilage.     

Effects of hypoxia on glucose transport in primary equine chondrocytes in vitro and evidence of reduced GLUT1 gene expression in pathologic cartilage in vivo

Articular chondrocytes exist in an environment lacking in oxygen and nutrients due to the avascular nature of cartilage. The main source of metabolic energy is glucose, which is taken up by glucose transporters (GLUTs). In diseased joints, oxygen tensions and glucose availability alter as a result of inflammation and changes in vascularisation. Accordingly, in this study we examined the effects of hypoxia and the hypoxia mimetic cobalt chloride (CoCl₂) on glucose transport in equine chondrocytes and compared expression of the hypoxia responsive GLUT1 gene in normal and diseased cartilage. Monolayers of equine chondrocytes were exposed to 20% O₂, 1% O₂, CoCl₂ (75 µM), or a combination of 1% O₂ and CoCl₂. Glucose uptake was measured using 2‐deoxy‐D‐[2,6‐³H] glucose. GLUT1 protein and mRNA expression were determined by FACS analysis and qPCR, respectively. GLUT1 mRNA expression in normal and diseased cartilage was analyzed using explants derived from normal, OA, and OCD cartilage. Chondrocytes under hypoxic conditions exhibited a significantly increased glucose uptake as well as upregulated GLUT1 protein expression. GLUT1 mRNA expression significantly increased in combined hypoxia‐CoCl₂ treatment. Analysis of clinical samples indicated a significant reduction in GLUT1 mRNA in OA samples. In OCD samples GLUT1 expression also decreased but did not reach statistical significance. The increase in glucose uptake and GLUT1 expression under hypoxic conditions confirms that hypoxia alters the metabolic requirements of chondrocytes. The altered GLUT1 mRNA expression in diseased cartilage with significance in OA suggests that reduced GLUT1 may contribute to the failure of OA cartilage repair. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 529–535, 2009     

Reactive nitrogen and oxygen species in interleukin-1-mediated DNA damage associated with osteoarthritis

OBJECTIVE: Osteoarthritis (OA) is associated with increased levels of reactive nitrogen and oxygen species and pro-inflammatory cytokines, such as interleukin-1 (IL-1). Nitric oxide (NO) can mediate a number of the catabolic effects of IL-1 in articular cartilage. The aims of this study were to determine if OA cartilage shows evidence of DNA damage, and if IL-1 could induce DNA damage in non-OA cartilage by increasing NO or superoxide. METHODS: Articular chondrocytes were isolated from porcine femoral condyles and embedded in 1.2% alginate. The effects of 24h incubation with IL-1, the nitric oxide synthase 2 (NOS2)-selective inhibitor, the free radical scavenger superoxide dismutase (SOD), the NO donor NOC18, or the combined NO and peroxynitrite donor SIN-1 on DNA damage were tested, using the "comet" assay. NO production was measured using the Griess assay. The type of oxidative damage present was assessed using a modified comet assay. RESULTS: OA cartilage had significantly more DNA damage than non-OA cartilage (P<0.001). IL-1 caused an increase in DNA damage (P<0.01), which was associated with increased NO production (P<0.01). Both oxidative DNA strand breaks and base modifications of purines and pyrimidines were observed. IL-1-induced DNA damage was inhibited by an NOS2 inhibitor or by SOD (P<0.01). Furthermore, NOC18 or SIN-1 caused DNA damage (P<0.001). CONCLUSION: Our work shows chondrocytes in osteoarthritic cartilage exhibit DNA damage, and that IL-1 induces DNA damage and reactive oxygen and nitrogen species in non-OA chondrocytes in alginate.