Intrajoint comparisons of gene expression patterns in human osteoarthritis suggest a change in chondrocyte phenotype.

Osteoarthritis (OA) is a degenerative cartilage disease with varying degrees of severity within a given joint. The purpose of this study was to define a sampling procedure for comparing human minimal and advanced OA cartilage in the same patient and to determine basic patterns of gene expression in these regions. A specific hypothesis under study was that the expression level of Bcl-2 would correlate with Sox9 and aggrecan mRNA expression in vivo as has been demonstrated in vitro. Femoral condylar advanced OA cartilage was located within 1cm of overt lesions, and minimal cartilage was taken from areas with no obvious surface defects. Histological sections were scored for disease severity and chondroitin sulfate and hydroxyproline content was determined. The expression level of nine specific genes (aggrecan, collagen type II, Bcl-2, Sox9, Link protein, osteopontin, and MMP-13, -3, and -9) was determined by quantitative real time PCR. The scores for fibrillation, chondrocyte cloning, and proteoglycan depletion were significantly different between advanced and minimal OA cartilage. The advanced OA cartilage had significantly less chondroitin sulfate than the minimal OA cartilage. Osteopontin mRNA expression showed a 3.6-fold increase in advanced compared to minimal OA cartilage. In contrast, the level of mRNA coding for aggrecan, link protein, Bcl-2, Sox9 and MMP-3, -9, -13 were all decreased in advanced compared to minimal cartilage in the majority of the patients studied. Collagen type II mRNA expression displayed a wide-range of variation. A statistically significant correlation was observed both between Bcl-2 and Sox9 mRNA level, and between Bcl-2 and aggrecan mRNA expression. The patient matched comparison of minimal and advanced OA cartilage revealed differences in cellular and tissue characteristics, and changes in gene expression that may be involved in OA progression. In addition, Bcl-2 may also play a role in regulating the expression of aggrecan through Sox9 in vivo as well as in vitro.     

Osteoblasts from the sclerotic subchondral bone downregulate aggrecan but upregulate metalloproteinases expression by chondrocytes. This effect is mimicked by interleukin-6, -1beta and oncostatin M pre-treated non-sclerotic osteoblasts

OBJECTIVE: To determine the effects of osteoarthritic (OA) subchondral osteoblasts on the metabolism of human OA chondrocytes in alginate beads. METHODS: Human chondrocytes were isolated from OA cartilage and cultured in alginate beads for 4 days in the absence or in the presence of osteoblasts isolated from non-sclerotic (N) or sclerotic (SC) zones of human OA subchondral bone in monolayer (co-culture system). Before co-culture, osteoblasts were incubated for 72 h with or without 1.7ng/ml interleukin (IL)-1beta, 100 ng/ml IL-6 with its soluble receptor (50 ng/ml) or 10 ng/ml oncostatin M (OSM). Aggrecan (AGG) and matrix metalloproteases (MMP)-3 and -13 mRNA levels in chondrocytes were quantified by real-time polymerase chain reaction. AGG production was assayed by a specific enzyme amplified sensitivity immunoassay. RESULTS: SC, but not N, osteoblasts induced a significant inhibition of AGG production and AGG gene expression by human OA chondrocytes in alginate beads, and significantly increased MMP-3 and MMP-13 gene expression by chondrocytes. When they were pre-incubated with IL-1beta, IL-6 or OSM, N osteoblasts inhibited AGG synthesis and increased MMP-3 and -13 gene expression by chondrocytes in alginate beads in a same order of magnitude as SC osteoblasts. CONCLUSIONS: These results demonstrate that SC OA subchondral osteoblasts could contribute to cartilage degradation by stimulating chondrocytes to produce more MMP and also by inhibiting AGG synthesis.     

Contribution of interleukin 17 to human cartilage degradation and synovial inflammation in osteoarthritis

OBJECTIVE: To compare the effect of interleukin (IL)-17, IL-1beta and TNF-alpha on chemokine production by human chondrocytes and synovial fibroblasts isolated from patients with osteoarthritis (OA). The expression of IL-1beta mRNA by OA chondrocytes was also assessed, as well as the presence and expression of IL-17 receptor (IL-17R) in OA chondrocytes and synovial fibroblasts after stimulation with IL-17, IL-1beta and TNF-alpha. DESIGN: Synovial fibroblasts and chondrocytes isolated from patients with OA were stimulated in vitro with IL-17, IL-1beta or TNF-alpha. Supernatants were collected and immunoassayed for the presence of IL-8, GRO-alpha (CXC chemokines) and MCP-1, RANTES (CC chemokines). The cells were used to detect the presence of IL-17R and the expression of IL-17R mRNA. Stimulated chondrocytes were also used to detect IL-1beta production and mRNA expression. RESULTS: IL-17 upregulated the release of IL-8 and GRO-alpha both by synovial fibroblasts and chondrocytes, and the release of MCP-1 only by chondrocytes. IL-17 was a weaker stimulator than IL-1beta and TNF-alpha, except for GRO-alpha release which was maximally upregulated by IL-1beta, less by IL-17 and minimally by TNF-alpha. When compared to IL-1beta, IL-17 was more active on chondrocytes than on fibroblasts. In chondrocytes the expression of IL-1beta mRNA was enhanced by IL-17 and TNF-alpha, with a maximum level reached by IL-1beta. IL-17 and TNF-alpha stimulated IL-1beta release in few subjects. Neither IL-17, IL-1beta nor TNF-alpha modulated the presence of IL-17R and the expression of IL-17R mRNA. CONCLUSIONS: These data suggest that IL-17 could contribute to cartilage breakdown and synovial infiltration in OA by inducing both the release of chemokines by chondrocytes and synovial fibroblasts and, in a less extent, the synthesis of IL-1beta by chondrocytes.     

Diacerhein and rhein reduce the ICE-induced IL-1beta and IL-18 activation in human osteoarthritic cartilage

OBJECTIVE: IL-1beta plays a fundamental role in osteoarthritis (OA) pathophysiology and cartilage destruction. Targeting the activation mechanism of this cytokine appears to be important as a therapeutic approach. As the interleukin-1 converting enzyme (ICE) is the physiologic modulator of the production of active IL-1beta, we investigated the effect of diacerhein and its active metabolite rhein used in the treatment of OA patients, on the enzyme expression and synthesis on human OA cartilage. Further, we looked at the effect of both drugs on the production of the active form of IL-1beta and IL-18. METHODS: The expression and synthesis of ICE were investigated on human OA cartilage explants using in-situ hybridization and immunohistochemical methods, respectively. The effect of the drugs on ICE OA chondrocytes was also determined by Northern blotting and a specific ELISA assay. Furthermore, the effect of both drugs on the level of active IL-1beta and IL-18 was examined by immunohistochemistry. RESULTS: Data showed that diacerhein and rhein have no true effect on reducing total ICE mRNA by both Northern blotting analysis and in-situ hybridization. A marked and statistically significant decrease was, however, found for protein production. ELISA showed a reduction of 31% (P< 0.04) for diacerhein and 50% (P< 0.02) for rhein. The drugs' immunohistological cell score reduction was similar to data from the ELISA, and a statistical significant reduction of ICE production was found at both superficial and deep zones of the cartilage. IL-1beta and IL-18 were both preferentially produced in chondrocytes of the superficial zone. For each of these cytokines, both drugs demonstrated a statistically significant decrease in this zone. A marked decrease was also noted in the deep zone, but statistical significance was reached only for rhein. CONCLUSION: These results provide a novel regulatory mechanism by which diacerhein and rhein could exert a down-regulation on IL-1's effect on OA cartilage.     

硫酸镁与氯化钠溶液灌洗对创伤性关节炎软骨损伤修复的影响

目的通过股骨髁间钻孔构建兔创伤性关节炎(PTOA)模型,比较相同渗透压下(400 mOsm/L)硫酸镁与氯化钠溶液持续灌洗对PTOA软骨损伤修复的影响。方法对18只新西兰白兔采用股骨髁间钻孔构建PTOA模型,随机分为PTOA组、氯化钠组和硫酸镁组,每组6只。ELISA检测关节腔积液白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)、Ⅱ型胶原蛋白(CollagenⅡ)的表达,qPCR检测滑膜组织IL-1β、TNF-α、金属蛋白酶-3(MMP-3)基因表达。行软骨切片染色组织学观察及骨关节炎(OA)评分。结果关节腔积液IL-1β、TNF-α、CollagenⅡ分泌硫酸镁组少于PTOA组和氯化钠组,滑膜组织IL-1β、TNF-α和MMP-3 mRNA水平硫酸镁组低于PTOA组和氯化钠组;软骨组织切片OA评分硫酸镁组低于PTOA组和氯化钠组,差异均有统计学意义(P<0.05)。结论与相同渗透压下氯化钠溶液相比,硫酸镁溶液有利于减轻关节内炎症,减少CollagenⅡ及蛋白聚糖丢失,对软骨退变起到保护作用,有利于延缓PTOA病情进展。     

Relationship between body habitus and joint leptin levels in a knee osteoarthritis population

Synovial fluid (SF) leptin has been shown to have an association with cartilage degeneration. Our objective was to examine the relationship between different measures of body habitus and SF leptin levels in an end‐stage knee osteoarthritis (OA) population. Sixty consecutive patients with knee OA were surveyed prior to surgery for demographic data. Body habitus was assessed with the body mass index (BMI), waist circumference (WC), and waist–hip ratio (WHR). SF and serum samples were analyzed for leptin and adiponectin using specific ELISA. Nonparametric correlations and linear regression modeling was used to identify the relationship between the measures of body habitus and SF leptin levels. Females had greater levels of leptin than males in both the serum and SF. Significant correlations were found between SF leptin levels and BMI and WC (R² 0.44 and 0.38, respectively; p < 0.05). Regression modeling showed that female gender and WC were independent predictors of a greater SF leptin level independent of age, BMI, and presence of diabetes (p < 0.05). WC may be a more accurate measure of body habitus than BMI in the relationship between the metabolic effects of adipose tissue and OA. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:329–333, 2010     

Differential induction and regulation of matrix metalloproteinases in osteoarthritic tissue and fluid synovial fibroblasts

OBJECTIVES: To investigate the secretion profiles of matrix metalloproteinases (MMP) and their inhibitors (TIMP) in synovial fluid-derived fibroblasts and to compare them with those of tissue-derived fibroblasts. METHODS: Fibroblast cultures established from synovial tissues (TSC) and fluids (FSC) of the same OA patients were stimulated with tumor necrosis factor(TNF)-alpha, interleukin(IL)-1alpha, IL-1beta, IL-6 and a combination of TNFalpha and IL-1beta. Cocultures of fibroblasts and cartilage were stimulated either with the cytokine combination or with osteoarthritic synovial fluid. Secretion of MMP-1, MMP-3, MMP-8, MMP-13, TIMP-1, and TIMP-2 was measured by enzyme-linked immunosorbent assay. Gelatin zymography and immunoblotting were performed to demonstrate enzyme activity. RESULTS: TNFalpha, IL-1alpha, and IL-1beta led to marked increases in MMP-1 and MMP-3 release (up to 4.2-fold and 547-fold, respectively) by synovial fibroblasts, whereas secretion of MMP-13 was induced by concomitant administration of TNFalpha and IL-1beta. Expression of intracellular MMP-8 was stimulated by cytokines, but adhesion of synovial fibroblasts to cartilage was required for the release. Throughout the study, significantly higher levels of secreted MMPs were observed in stimulated FSC compared to TSC cultures. Furthermore, increases in MMP secretion were not accompanied by increases in secreted TIMP-1 and TIMP-2, resulting in marked imbalances between enzyme and inhibitor levels. CONCLUSIONS: The results provide strong evidence for a significant impact of synovial-derived MMPs on cartilage destruction in OA. In this context, fibroblasts present in the synovial fluid appeared to play an outstanding role.     

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     

RelA is required for IL-1beta stimulation of Matrix Metalloproteinase-1 expression in chondrocytes

OBJECTIVE: Interleukin-1beta (IL-1beta) stimulates collagenase-1 (Matrix Metalloproteinase-1 (MMP-1)) expression in articular chondrocytes, leading to cleavage of type II collagen and irreversible cartilage degradation. The nuclear factor-kappa B (NF-kappaB) pathway is potently activated in IL-1beta-stimulated cells and has been implicated as an intermediate in MMP-1 gene expression. However, the roles of individual NF-kappaB family members during IL-1beta-induced MMP-1 gene expression have not been defined. RESULTS: To address the relationship between the NF-kappaB pathway and MMP-1 gene activation in chondrocytes, primary cultured human articular chondrocyte cultures (HAC) and SW-1353 cells were stimulated with IL-1beta over a 24-h time course and MMP-1, NF-kappaB1, NF-kappaB2 and RelA gene expression was assayed. IL-1beta-induced MMP-1 expression was comparable in HAC and SW-1353 cells both temporally and quantitatively. MMP-1 gene expression was mirrored by increases in NF-kappaB gene expression, and inhibition of NF-kappaB nuclear translocation with dominant-negative IkappaBalpha reduced IL-1beta-dependent MMP-1 gene expression. IL-1beta activated the NF-kappaB pathway in chondrocytes, both through phosphorylation and transient degradation of IkappaBalpha, as well as through sustained phosphorylation of RelA. Small inhibitory RNAs (siRNA) specific for RelA resulted in significant reduction of MMP-1 mRNA, whereas siRNA for NF-kappaB1 and NF-kappaB2 augmented IL-1beta-induced MMP-1 expression. CONCLUSIONS: Our data demonstrate that IL-1beta activation of the NF-kappaB pathway is required for IL-1beta induction of MMP-1 in chondrocytes and that RelA can work independently of NF-kappaB1 or NF-kappaB2 to activate this gene expression program.     

Anti-interleukin-1 effects of diacerein and rhein in human osteoarthritic synovial tissue and cartilage cultures.

OBJECTIVE: The etiology of osteoarthritis (OA) is still a matter of debate. Several factors are known to be involved in the destruction of the articular cartilage. Interleukin-1 (IL-1) plays an important role in the pathogenesis of osteoarthritis (OA) either directly or through the stimulation of catabolic factors, such as nitric oxide (NO). The objective of this study was to evaluate the effect of diacerein, a new anti-OA agent and its active metabolite, rhein, on the production and function of IL-1beta, nitric oxide (NO) and receptor agonist (IL-1ra) in human OA cartilage and synovial tissue cultures. DESIGN: Synovial tissue and cartilage derived from OA patients were kept in culture for 48-72 hours in the presence of 1 microg/ml of lipopolysaccharide (LPS) with or without diacerein (10(-7)-10(-5) M), rhein (10(-7)-10(-5) M) and hydrocortisone (5 microg/ml). IL-1beta, IL-1ra, NO productions and 35S uptake were measured in culture media. In some experiments the resulting supernatants from synovial tissue cultures were added to cartilage. RESULTS: Diacerein and rhein, as well as hydrocortisone, significantly inhibited LPS-induced IL-1beta production by synovial tissue and cartilage. They also significantly reversed the inhibitory effect of LPS on cartilage 35S uptake. Culture media from synovial tissue containing LPS+diacerein (10(-6) M) or +rhein (10(-6) M) had a significantly less inhibitory effect on cartilage synthesis than culture media containing LPS only. Diacerein and rhein decreased NO release in synovial tissue and cartilage media and increased IL-1ra levels in cartilage culture media. CONCLUSION: An inhibitory effect of diacerein and rhein at therapeutic concentrations on both IL-1beta secretion and function in human synovial tissue and cartilage is suggested. Diacerein and rhein effects on NO production by LPS-stimulated OA synovial tissue and cartilage may both contribute and elucidate their anti-OA properties.     

Glucosamine sulfate modulates the levels of aggrecan and matrix metalloproteinase-3 synthesized by cultured human osteoarthritis articular chondrocytes

OBJECTIVE: The functional integrity of articular cartilage is determined by a balance between chondrocyte biosynthesis of extracellular matrix and its degradation. In osteoarthritis (OA), the balance is disturbed by an increase in matrix degradative enzymes and a decrease in biosynthesis of constitutive extracellular matrix molecules, such as collagen type II and aggrecan. In this study, we examined the effects of the sulfate salt of glucosamine (GS) on the mRNA and protein levels of the proteoglycan aggrecan and on the activity of matrix metalloproteinase (MMP)-3 in cultured human OA articular chondrocytes. DESIGN: Freshly isolated chondrocytes were obtained from knee cartilage of patients with OA. Levels of aggrecan and MMP-3 were determined in culture media by employing Western blots after incubation with GS at concentrations ranging from 0.2 to 200 microM. Zymography (casein) was performed to confirm that effects observed at the protein level were reflected at the level of enzymatic activity. Northern hybridizations were used to examine effects of GS on levels of aggrecan and MMP-3 mRNA. Glycosaminoglycan (GAG) assays were performed on the cell layers to determine levels of cell-associated GAG component of proteoglycans. RESULTS: Treatment of OA chondrocytes with GS (1.0-150 microM) resulted in a dose-dependent increase in aggrecan core protein levels, which reached 120% at 150 microM GS. These effects appeared to be due to increased expression of the corresponding gene as indicated by an increase in aggrecan mRNA levels in response to GS. MMP-3 levels decreased (18-65%) as determined by Western blots. Reduction of MMP-3 protein was accompanied by a parallel reduction in enzymatic activity. GS caused a dose-dependent increase (25-140%) in cell-associated GAG content. Chondrocytes obtained from 40% of OA patients failed to respond to GS. CONCLUSIONS: The results indicate that GS can stimulate mRNA and protein levels of aggrecan core protein and, at the same time, inhibit production and enzymatic activity of matrix-degrading MMP-3 in chondrocytes from OA articular cartilage. These results provide a cogent molecular mechanism to support clinical observations suggesting that GS may have a beneficial effect in the prevention of articular cartilage loss in some patients with OA.     

The effects of sodium hyaluronate on mRNA expressions of matrix metalloproteinase-1,-3 and tissue inhibitor of metalloproteinase-1 in cartilage and synovium of traumatic osteoarthritis model

teoarthritis (OA)isadegenerativeprocessofjointscharacterizedbyprogressivedeteriorationanderosionofcartilage .TraumaticOAcausedbyjointsinjuryandoperationiscommonlyobserved .Intra articularinjectionofsodiumhyaluronate (HA )isnowwidelyusedintreatmentofOA .It…