Distinct signaling pathways are involved in hypoxia- and IL-1-induced VEGF expression in human articular chondrocytes.

Recent data suggest that vascular endothelial growth factor (VEGF) is involved in the pathogenesis of osteoarthritis (OA). Cartilage is an avascular tissue, leading to a low cartilage O2 level. Thus, in a variety of pathologic or physiologic conditions, VEGF is partly regulated by hypoxic stress. The implications of hypoxia for VEGF expression by OA chondrocytes, however, are not known. We investigated the regulatory system of VEGF in OA chondrocytes under hypoxic conditions. Chondrocytes were obtained from articular cartilage of patients with OA. Cells were cultured and then incubated under hypoxic (95% N2, 5% CO2) or normoxic conditions, with or without interleukin (IL)-1 (10 ng/mL) stimulation. The mitogen activated protein kinase (MAPK) inhibitors were also used. VEGF levels in the culture supernatants were measured using an enzyme-linked immunosorbent assay. Western blot analysis was used to examine the expression of hypoxia inducible factor (HIF)-1alpha. Hypoxia significantly increased VEGF levels (p<0.05). Hypoxia-induced VEGF secretion was abolished by p38MAPK inhibitor, but not by JNK inhibitor. In contrast, IL-1-induced VEGF secretion was blocked by JNK inhibitor, and not by p38MAPK inhibitor. Both hypoxia and IL-1-induced HIF-1alpha were attenuated by p38 MAPK and JNK inhibitors. We demonstrate that hypoxia and IL-1 induce VEGF production in chondrocytes through distinct MAPK signaling pathways, indicating that VEGF is induced in a HIF-1-dependent or -independent manner in chondrocytes.     

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     

Localization of bone morphogenetic protein-2 in human osteoarthritic cartilage and osteophyte

OBJECTIVES: To examine the localization of bone morphogenetic protein (BMP)-2 mRNA and protein in human osteoarthritic (OA) articular cartilage and osteophyte. DESIGN: Five normal, four growing and 14 OA human cartilage samples, graded histomorphologically by Mankin Score, were studied by in situ hybridization and immunohistochemistry for the expression of BMP-2. RESULTS: BMP-2 mRNA was present in chondrocytes in neonatal growing articular cartilage, but was scarcely present in normal adult articular cartilage. In OA articular cartilage, BMP-2 mRNA and protein were detected in both clustering and individual chondrocytes in moderately or severely damaged OA cartilage. In moderately damaged OA cartilage, BMP-2 mRNA was localized in both upper and middle zone chondrocytes, but was not detected in deep layer chondrocytes. In severely damaged OA cartilage, cellular localization of BMP-2 mRNA was extended to the deep zone. In the area of osteophyte formation, BMP-2 mRNA was intensely localized in fibroblastic mesenchymal cells, fibrochondrocytes, chondrocytes and osteoblasts in newly formed osteophytic tissue. The pattern of BMP-2/4 immunolocalization was associated with that of mRNA localization. CONCLUSIONS: BMP-2 mRNA and BMP-2/4 were detected in cells appearing in OA tissues. BMP-2 was localized in cells of degenerating cartilage as well as osteophytic tissue. Given the negative localization of BMP-2 in normal adult articular cartilage, BMP-2 might be involved in the regenerating and anabolic activities of OA cells, which respond to cartilage damage occurring in osteoarthritis.     

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.     

骨关节炎软骨细胞凋亡调控基因的研究

目的　比较分析正常人及老年性骨关节炎患者软骨细胞bax和bcl 2的表达及细胞凋亡状况。　方法　取 9例骨关节炎患者的关节软骨做实验标本 ,以 6例无骨关节炎病史的意外死亡者关节软骨作为正常对照 ;采用逆转录 /聚合酶链反应 (RT PCR)方法检测bax和bcl 2mRNA表达 ,免疫组化检测bax和bcl 2蛋白 ;应用TUNEL方法进行凋亡细胞原位检测。　结果　骨关节炎患者和正常对照软骨细胞都能表达bax和bcl 2mRNA ;骨关节炎关节软骨细胞baxmRNA表达量较正常对照显著增高 (P <0 0 1) ,bcl 2mRNA表达量也高于正常对照组 (P <0 0 5 ) ,两组间bax/bcl 2表达量的比值差异无显著性意义 (P >0 0 5 ) ;免疫组化可检测到相应表达水平的蛋白 ;骨关节炎软骨细胞凋亡 (4%～ 14% )多于正常对照 (0～ 2 % )。　结论　软骨细胞凋亡受bax和bcl 2共同调节 ;bax和bcl 2的共同调节结果可能是OA患者软骨细胞凋亡增加 ,但凋亡率不高、病理过程进展缓慢的一个重要的原因     

Sustained hypoxia enhances chondrocyte matrix synthesis

Articular cartilage is an avascular tissue with chondrocytes in the deeper zones existing under conditions of sustained hypoxia. Using a hypoxic chamber to provide controlled hypoxia, this study was performed to determine whether sustained hypoxia enhances the production of cartilage matrix proteins. Freshly isolated primary bovine articular chondrocytes were encapsulated in three-dimensional alginate beads and maintained at 2% oxygen with media changes using media pre-equilibrated to 2% oxygen. Immunolocalization of HIF-1α was performed to verify hypoxic conditions. Sustained hypoxia resulted in an increase in proteoglycan synthesis after only 1 day, as measured by ³⁵S-sulfate incorporation. This increase was maintained for the duration of the 17 day study. After 17 days of hypoxic culture, increases in total type II collagen and COL2A1 gene expression were probed by indirect immunofluorescence, type II collagen ELISA, and real-time qPCR; in addition, increased glycosaminoglycan deposition was observed as determined by chemical analysis. These studies show that sustained hypoxia enhances articular chondrocyte matrix synthesis and viability in three-dimensional alginate culture. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 793–799, 2009     

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.     

Influence of osteoarthritis grade on molecular signature of human cartilage

Articular chondrocytes maintain cartilage matrix turnover and have the capacity for anabolic and catabolic activities that can be influenced by injury and disease. This study tested the hypothesis that catabolic genes are upregulated with regional osteoarthritis (OA) disease severity within a joint. With IRB approval, specimens of knee cartilage obtained as discarded tissues from subjects undergoing arthroplasty were partitioned for each subject by OA disease severity and evaluated for gene expression by RT‐PCR. There was regional OA grade‐associated upregulation of expected inflammatory mediators TNF‐α, TNF receptors, IFN‐γ, and interleukins as well as genes encoding proteolytic enzymes, including Adamts‐5 and MMPs. Osteoclast‐related genes, cathepsin K, tartrate‐resistant acid phosphatase (TRAP), RANKL, RANK, M‐CSF, and c‐fms, but not osteoprotegerin, were induced in advanced grades. In vitro treatment of normal human chondrocytes with interleukin‐1β upregulated similar genes; this provides evidence that chondrocytes per se can be the source of osteoclast‐related factors. Immunohistochemical staining showed that RANK‐ and RANKL‐positive cells were abundant in advanced grades, especially in chondrocyte clusters. This suggests a possible autocrine mechanism by which an osteoclast phenotype is induced in articular chondrocytes. In sum, these studies identified gene expression signatures in human OA cartilage based upon regional disease severity within a joint. There was an effect of OA Grade on expression of osteoclastic lytic enzymes and regulatory factors in human articular chondrocytes. Induction of an osteoclast‐like phenotype in chondrocytes may be part of OA progression and suggests specific therapeutic approaches. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:454–462, 2016.     

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.     

Human cartilage glycoprotein 39 (HC gp-39) mRNA expression in adult and fetal chondrocytes, osteoblasts and osteocytes by in-situ hybridization

OBJECTIVE: To examine the expression pattern of human cartilage glycoprotein 39 (HC gp-39) mRNA in human cartilage and bone. DESIGN: In-situ hybridization analysis was used to examine the expression pattern of human cartilage glycoprotein 39 (HC gp-39) mRNA in adult human osteoarthritic articular cartilage from various stages of disease, as well as in human osteophytic tissue and in human fetal bone. RESULTS: In cartilage from patients with mild osteoarthritic cartilage degeneration, HC gp-39 was expressed at moderate to high levels only in chondrocytes of the superficial zone. In advanced OA cartilage, cloning chondrocytes of the superficial zone expressed high levels of HC gp-39 and chondrocytes of the mid- and deep zones were also positive. HC gp-39 was undetectable in the chondrocytes of normal articular cartilage. In osteophytic tissue, the expression of HC gp-39 mRNA was intense in flattened, end-stage osteoblasts and in primary osteocytes in both endochondral and intramembranous bone formation. Proliferating osteoblasts expressed low to moderate levels. Notably, mature osteocytes were negative for HC gp-39 expression. Chondrocytes in the secondary ossification center of developing fetal cartilage demonstrated high expression while growth plate and mineralized cartilage chondrocytes had lower expression. Osteoblasts at sites of endochondral and intramembranous bone formation were positive for expression of HC gp-39. CONCLUSIONS: The stage-specific expression of HC gp-39 in fetal development and adult remodelling bone and cartilage provides evidence for a specific functional or structural role for HC gp-39 in bone and cartilage tissue. HC gp-39 is expressed in diseased human osteoarthritic cartilage and osteophyte, but not in non-diseased tissue, and its distribution within the tissue changes as disease progresses. OA is characterized not only by cartilage degeneration, but by increased subchondral bone formation and osteophytosis. The results from this study indicate that the increased HC gp-39 expression in OA serum and synovial fluid may reflect not only cartilage degeneration but increased osteogenesis.     

FrzB-2: a human secreted frizzled-related protein with a potential role in chondrocyte apoptosis

OBJECTIVE: To characterize a novel secreted frizzled-related protein (SFRP) and determine its tissue distribution at the mRNA and protein level. METHODS: The FrzB-2 gene was identified by expressed sequence tag (EST) analysis of human tissue-derived libraries. Tissue distribution of FrzB-2 mRNA was determined by Northern blot analysis and in situ hybridization. FrzB-2 protein reactivity was localized in human OA articular cartilage by immunocytochemistry, using a polyclonal antibody against a peptide sequence unique to FrzB-2. Apoptosis was detected in articular cartilage sections using Tunel staining. RESULTS: ESTs corresponding to FrzB-2 were found in osteoblast, chondrosarcoma, osteosarcoma, osteoclastoma and synovial fibroblast libraries. FrzB-2 mRNA is expressed in a number of tissues and cell types including bone-related cells and tissues such as primary human osteoblasts and osteoclastoma. In situ hybridization studies showed strong FrzB-2 mRNA expression in human chondrocytes in human osteoarthritic (OA) cartilage but negligible levels in normal cartilage chondrocytes. The FrzB-2 cDNA encodes a secreted 40 kDa protein consisting of 346 amino acids. FrzB-2 is 92. 5% identical to the rat orthologue, DDC-4, which has been shown to be associated with physiological apoptosis. FrzB-2 protein was selectively detected in human OA articular cartilage by immunocytochemistry, using a polyclonal antibody. Consistent with its potential role in apoptosis, positive FrzB-2 staining and Tunel positive nuclei staining were detected in chondrocyte clones in sections of human OA cartilage. CONCLUSION: These data suggest that FrzB-2 may play a role in apoptosis and that the expression of this protein may be important in the pathogenesis of human OA.     

Prevalence and consequences of musculoskeletal symptoms in symphony orchestra musicians vary by gender: a cross-sectional study

Background

Tenascin-C (TN-C) is an extracellular matrix glycoprotein that is involved in tissue injury and repair processes. We analyzed TN-C expression in normal and osteoarthritic (OA) human cartilage, and evaluated its capacity to induce inflammatory and catabolic mediators in chondrocytes in vitro. The effect of TN-C on proteoglycan loss from articular cartilage in culture was also assessed.

Methods

TN-C in culture media, cartilage extracts, and synovial fluid of human and animal joints was quantified using a sandwich ELISA and/or analyzed by Western immunoblotting. mRNA expression of TN-C and aggrecanases were analyzed by Taqman assays. Human and bovine primary chondrocytes and/or explant culture systems were utilized to study TN-C induced inflammatory or catabolic mediators and proteoglycan loss. Total proteoglycan and aggrecanase -generated ARG-aggrecan fragments were quantified in human and rat synovial fluids by ELISA.

Results

TN-C protein and mRNA expression were significantly upregulated in OA cartilage with a concomitant elevation of TN-C levels in the synovial fluid of OA patients. IL-1 enhanced TN-C expression in articular cartilage. Addition of TN-C induced IL-6, PGE₂, and nitrate release and upregulated ADAMTS4 mRNA in cultured primary human and bovine chondrocytes. TN-C treatment resulted in an increased loss of proteoglycan from cartilage explants in culture. A correlation was observed between TN-C and aggrecanase generated ARG-aggrecan fragment levels in the synovial fluid of human OA joints and in the lavage of rat joints that underwent surgical induction of OA.

Conclusions

TN-C expression in the knee cartilage and TN-C levels measured in the synovial fluid are significantly enhanced in OA patients. Our findings suggest that the elevated levels of TN-C could induce inflammatory mediators and promote matrix degradation in OA joints.     

Homeostasis of the extracellular matrix of normal and osteoarthritic human articular cartilage chondrocytes in vitro

OBJECTIVE: In normal articular cartilage cells, the IGFRI/insulin-like growth factor 1 (IGF-1) autocrine pathway was shown to overrule the catabolic effects of the IL-1/IL-1RI pathway by up-regulation of the IL-1RII decoy receptor. The activity of the IGF-1/IGFR1 and IL-1/IL-1R pathways, and of the IL-1RII control mechanism in the synthesis and turnover of the extracellular matrix (ECM) by chondrocytes from normal and osteoarthritic (OA) articular cartilage was compared in order to identify possible therapeutic targets of this disease. METHODS: Phenotypically stable human articular cartilage cells were obtained from normal and OA cartilage of the same knee showing focal OA. The cells were cultured in alginate beads over 1 week to re-establish the intracellular cytokine and growth factors, to reexpress the respective plasma membrane receptors and to reach equilibrium in accumulated cell-associated matrix (CAM) compounds. Following liberation of the cells from the alginate beads, the levels of cell-associated matrix (CAM) aggrecan, type II collagen and fibronectin, of intracellular IGF-1, IL-1alpha and beta and of their respective plasma membrane-bound receptors, IGFR1, IL-1RI and the decoy receptor IL-1RII, were assayed using flow cytometry. RESULTS: Coordinated production and accumulation of CAM aggrecan and type II collagen under the effect of the IGFR1/IGF-1 autocrine pathway-as documented for chondrocytes from healthy controls-was absent when the chondrocytes had been obtained from OA joints. When compared with cells obtained from normal tissues, chondrocytes from fibrillated OA cartilage expressed significantly higher intracellular IGF-1 levels and plasma membrane-bound IGFR1. At the same time, significantly higher intracellular IL-1alpha and beta levels and upregulated plasma membrane-bound IL-1RI were observed. Plasma membrane-bound IL-1RII decoy receptor was downregulated in OA chondrocytes. The levels of CAM aggrecan, type II collagen and fibronectin were significantly reduced in the chondrocytes obtained from pathological tissue. CONCLUSION: Paired analysis of normal and OA chondrocytes from the same knee joint has shown an enhanced capacity of chondrocytes from OA cartilage to produce ECM macromolecules. However, the same cells have increased catabolic signalling pathways. As a consequence of this increased IL-1 activity and the reduced amounts of IL-1RII decoy receptor, less of the produced ECM macromolecules may persist in the CAM of the OA chondrocytes.