髋关节骨关节炎与股骨颈骨折患者滑液对去分化软骨细胞VEGF表达的影响

目的 研究髋关节骨关节炎(osteoarthritis,OA)及股骨颈骨折(femoral neck fracture,FNF)患者软骨细胞血管内皮生长因子(vascular endothelial growth factor,VEGF)表达,同时对比分析OA滑膜液及创伤后滑膜液对去分化软骨细胞分泌VEGF的影响,从而探索炎性因子致去分化软骨细胞异常的VEGF表达变化.方法 取12例髋关节OA与8例FNF患者的软骨进行HE和番红O-固绿染色后行Mankin评分.将OA组及FNF组软骨组织分别粉碎后富集软骨细胞进行单层培养,取细胞换液时遗弃的上清液作为检测标本,以cathepsin B作为检测OA组软骨细胞去分化程度的指标,并且动态检测VEGF水平,并各培养基于传代培养时分别加入OA滑膜液、DMEM及创伤后滑膜液以观察其对软骨细胞VEGF分泌的影响.数据统计分析使用SPSS 11.0统计软件.结果 OA组软骨Mankin评分明显较骨折组升高,随着培养时间延长软骨细胞逐渐丧失其原有细胞形态并伴显著的cathepsin B上调和VEGF下凋;OA滑膜液可促使软骨细胞特别是OA组软骨细胞上调分泌VEGF,但刺激所产生的VEGF较原代培养初期明显减少,而FNF滑膜液对VEGF上调影响不大.结论 OA软骨损害Mankin评分与原代培养初期VEGF成正相关;软骨细胞体外原代培养后迅速表现为去分化特性;OA较FNF滑膜液可以促使软骨细胞特别是OA组细胞表达更多的VEGF,提示OA滑膜液参与病程进展,并且OA组软骨细胞病理表型更倾向于表达较多VEGF;而FNF滑膜液则相对有利于维持软骨细胞的原有表型.     

SH关节腔内注射治疗兔膝关节炎的实验研究

[目的]探讨透明质酸钠（sodium hyaluronate，SH）关节腔内注射治疗膝骨性关节炎（OA）的效果。[方法]24只兔子建立骨关节炎动物模型，随机分OA组、SH组、对照组，观察3组软骨、滑膜细胞病理切片及软骨MMP-1免疫组化，进行软骨Mankin＇s评分，以及检测血液和关节液的IL-1含量的效果。[结果]SH组可见软骨破坏减轻，滑膜纤维增生减少，Mankin＇s评分有明显改善（P〈0．05）；血、膝关节滑液IL-1浓度降低（P〈0．05），但关节软骨中MMP表达仍然活跃。[结论]SH能减少滑液中炎性介质IL-1的分泌，从而减轻滑膜炎症，缓解对软骨细胞的破坏，延缓OA进展，但仍阻止不了OA进展。     

Caspase-1表达和软骨细胞凋亡在骨关节炎的意义

目的观察骨关节炎（osteoarthritis,OA）关节软骨标本的Caspase-1表达和软骨细胞凋亡的程度,初步探讨两者的相关性和与OA病变程度的关系。方法选取2007年3月至2007年11月进行关节置换的OA患者的关节软骨标本26例作为OA组,男8例,女18例;髋20例,膝6例;年龄52～81岁,平均60岁。选取截肢的正常关节软骨标本10例作为对照组,男7例,女3例;年龄16～45岁,平均40岁;均排除结构性破坏。按改良Mankin病理评分进行分级,将标本分成正常软骨组0～1分（A组）即对照组;OA软骨轻度退变组2～5分（B组）、OA软骨中度退变组6～9分（C组）和OA软骨重度退变组10～14分（D组）,B～D组即OA组。分别采用HE染色和番红O/固绿染色后,采用免疫组化和TUNEL检测法,检测软骨细胞中Caspase-1的表达和凋亡细胞阳性率。结果 （1）对照组Caspase-1表达低于OA组;Caspase-1的表达与Mankin评分呈正相关;（2）对照组的凋亡细胞阳性率低于OA组的凋亡细胞阳性率;软骨凋亡细胞阳性率与Mankin病理评分呈正相关;（3）A～D组Caspase-1的表达和凋亡细胞阳性率不存在相关性。结论 （1）Caspase-1与OA发生发展有关,能反应OA软骨的病变进展程度;（2）软骨细胞凋亡是OA的可能发病机理,其凋亡率可能影响OA病程进展。     

独活挥发油灌胃对兔膝骨关节炎的保护作用及其机制

目的探讨独活挥发油(VOOA)灌胃对兔膝骨关节炎(OA)的保护作用及其机制。方法 6月龄日本大耳白兔24只,采用右膝关节前交叉韧带切断术(ACLT)建立兔膝OA模型,随机分为4组:模型组(OA组)、假手术组(Sham组)、盐水组(ACLT+NS组)和VOOA组(ACLT+VOOA组),每组6只。ACLT+VOOA组术后予以VOOA 0.2 ml/(kg.d)灌胃,ACLT+NS组给予等量的生理盐水。8周后处死动物,采用肉眼观察各组软骨及滑膜组织,病理切片行HE染色并进行软骨Mankin评分;检测血清及关节液内白细胞介素1(IL-1)、生长转化因子β(TGF-β)的含量;免疫组织化学法测定软骨中IL-1、TGF-β的含量。结果 ACLT+VOOA组可见软骨破坏减轻,滑膜纤维增生减少,Mankin评分低于ACLT+NS组及OA组,差异有统计学意义(P<0.05);血清、膝关节滑液IL-1含量低于ACLT+NS组及OA组,差异有统计学意义(P<0.05);TGF-β表达量高于ACLT+NS组、OA组及Sham组,差异有统计学意义(P<0.05)。结论 VOOA灌胃能够部分阻止兔OA关节软骨的退变,其机制可能为减少滑液中炎性介质IL-1的分泌、促进TGF-β的分泌,从而减轻滑膜炎症、缓解对软骨细胞的破坏。     

5氟脲嘧啶关节腔注射治疗兔膝骨性关节炎的实验研究

[目的]评价5氟脲嘧啶（5-Fluorouracil，5Fu）关节腔注射治疗兔膝骨性关节炎（osteoarthritis，OA）的疗效。[方法]24只兔子制成骨关节炎模型随机分成OA组、5Fu组和对照组，OA组立即处死，5Fu组按5Fu2ms／kg关节腔注射，每周1次连续4次，对照组注射等量生理盐水，最后1次治疗后1周处死。观察3组滑膜组织的光镜、电镜改变及软骨的光镜、MMP-1免疫组化改变，比较软骨Mankin＇s评分及关节液中IL-1的浓度。[结果]5Fu组可见软骨破坏减轻，滑膜炎症明显抑制，Mankin’s评分明显改善（P〈0．01）；关节液IL-1浓度降低（P〈0．05），关节软骨中MMP-1表达减弱。[结论]5Fu关节腔内注射能抑制滑膜炎症，缓解软骨的破坏。     

软骨形态发生蛋白1诱导犬真皮成纤维细胞向软骨细胞表型分化的实验研究

目的探讨软骨形态发生蛋白1（Cartilage-derived morphogenetic protein-1,CDMP1）诱导体外培养的犬真皮成纤维细胞（Dermal fibroblasts,DFs）向软骨细胞表型分化的可行性。方法应用CDMP1细胞诱导液对体外扩增至第4代的犬DFs进行诱导培养,观察细胞的形态变化,以免疫荧光染色和RT-PCR检测细胞中软骨特异蛋白的表达情况。结果诱导培养6 d后,实验组部分DFs呈星形或多角形,免疫组化染色显示有软骨特异的II型胶原分泌,RT-PCR检测也表明有Ⅱ型胶原、aggrecan和SOX9表达。但是诱导培养10 d后,均转为阴性。结论 CDMP1可以诱导犬DFs向软骨细胞表型分化,有可能成为组织工程化纤维软骨的种子细胞。     

兔关节软骨细胞聚集培养的生物学性状观察

目的 观察聚集培养软骨细胞生物学性状的变化,为软骨细胞移植建立合适的体外培养方法。方法2001年11月至2004年6月酶消化法分离成年兔关节软骨细胞,分别低密度单层培养和高密度聚集培养,组化及免疫组化法观察细胞表型变化。结果低密度培养时,前3代细胞增殖迅速,但很快去分化,3代以后增殖缓慢,细胞表型大部丢失;聚集培养时,软骨细胞去分化速度减缓;传3代后细胞聚集培养,细胞表型部分恢复。结论聚集培养利于维持软骨细胞表型,原代细胞聚集培养或传代培养后聚集培养是较好的获取大量优良软骨细胞的培养方式。     

补肾活血方对大鼠膝骨关节炎滑膜细胞β-catenin、MMP-7的影响

目的:观察Wnt经典信号通路中β-catenin在大鼠膝骨关节炎滑膜细胞胞核中的表达,并观察补肾活血方对大鼠膝骨关节炎(OA)膝关节滑膜细胞胞核中β-catenin和滑膜细胞MMP-7的调控作用。方法:采用Hulth法建立大鼠膝关节骨性关节炎模型,胶原酶消化法原代培养正常滑膜细胞与OA滑膜细胞。将所培养滑膜细胞分为正常组、OA模型组和补肾活血方组,用补肾活血方含药血清作用OA滑膜细胞48h后,采用Western Blot法检测滑膜细胞中MMP-7、胞核β-catenin的蛋白表达变化;采用酶联免疫吸附法(ELISA)检测滑膜细胞上清液中MMP-7表达变化。结果:OA滑膜细胞培养成功,WesternBlot结果显示OA滑膜细胞中MMP-7、胞核β-catenin表达均明显高于正常滑膜细胞,补肾活血方对OA滑膜细胞中MMP-7、胞核β-catenin表达有明显下调作用;ELISA结果显示OA滑膜细胞上清液中MMP-7表达明显高于正常滑膜细胞上清液,补肾活血方对其有明显下调作用。结论:①β-catenin在OA滑膜细胞胞核中表达升高,表明Wnt经典信号通路在大鼠膝骨关节炎滑膜中是激活的。②MMP-7在OA滑膜细胞和上清液中表达升高,印证了MMP-7是Wnt/β-catenin信号通路下游基因。③滑膜细胞Wnt/β-catenin信号通路的活化、MMP-7的表达升高可能是导致关节软骨降解,促进骨性关节炎形成的机制之一。④补肾活血方对Wnt/β-catenin信号通路的调控作用是其抑制软骨降解,促进软骨修复,治疗骨性关节炎的可能机制之一。     

藻酸钠微球三维立体培养恢复去分化软骨细胞表型的实验研究

[目的] 研究在连续体外单层培养条件下不同接种密度对大鼠关节软骨细胞分化状态的影响,并探讨藻酸钠微球三维立体培养恢复去分化关节软骨细胞表型的调节机制.[方法]以正常密度(3×10<,4>/cm<'2>)或低密度(3×10<'2>/cm<'2>)分别连续体外单层传代培养大鼠膝关节软骨细胞使其去分化,RT-PCR检测Ⅰ、Ⅱ型胶原和聚集蛋白聚糖mRNA的表达以确定其分化状态.取正常密度培养时的去分化软骨细胞,藻酸钠微球包被4周以恢复其表型,在该立体培养过程中将特异的SIRT1抑制剂-EX-527加入到培养基中抑制其表达,甲苯胺兰染色微球中软骨细胞分泌的细胞外基质,Western blot检测各种条件下SIRT1和Sox-9的表达.[结果] 当以正常密度连续体外单层培养时,软骨细胞在第4代发生去分化,低密度时于第1代即明显去分化,此时SIRT1和Sox9表达明显降低.藻酸钠微球三维立体培养能显著地增强去分化软骨细胞中Ⅱ型胶原、聚集蛋白聚镛mRNA以及SIRT1、Sox9蛋白的表达,同时降低Ⅰ型胶原mRNA的表达.EX-527不仅限制藻酸钠微球中的软骨细胞周围细胞外基质的大量生成而且还抑制了SIRT1和Sox9的表达.[结论]关节软骨细胞连续体外单层培养的去分化与细胞接种密度有关,低密度培养加速去分化过程.藻酸钠微球三维立体培养恢复去分化关节软骨细胞表型的作用可能是通过细胞-细胞、细胞-细胞外基质间的相互作用,从而激活SIRT1的表达,继而增强Sox9的转录活性来实现.     

骨关节炎早中期关节软骨YKL-40表达与软骨细胞凋亡的关系

背景：软骨细胞凋亡是骨关节炎（osteoarthritis,OA）发病过程中重要的病理学特征。YKL-40是壳质酶蛋白家族的一种糖蛋白,但不具有壳质酶活性,在关节炎软骨、滑膜、巨噬细胞等均有表达,可能与炎症的状态、组织重塑等功能有关。YKL-40在OA早中期中的作用尚有待研究。目的：探讨OA早中期关节软骨YKL-40表达与软骨细胞凋亡率（apoptosisindex,AI）的关系。方法：通过前交叉韧带切断术（anterior cruciate ligament transaction,ACLT）建立SD大鼠膝关节OA模型,组织学评估软骨退变程度,采用改良Mankin评分系统进行评估,免疫组织化学法检测YKL-40的表达情况及软骨细胞AI,观察两个指标的表达特点,分析两者在此病变过程中的关系。结果：随造模时间的延长,软骨出现退变并逐渐加重,YKL-40的表达与软骨细胞AI呈正相关。结论：软骨细胞凋亡是OA早中期的重要事件,YKL-40可能为OA早中期病理过程中软骨细胞凋亡的重要影响因子。     

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.     

Metabolic characteristics of in vitro cultured human chondrocytes in relation to the histopathologic grade of osteoarthritis

Isolated human chondrocytes derived from healthy and osteoarthritic (OA) cartilage were cultured in high density in a newly designed microculture system. The severity of OA was graded according to a modified histopathologic score originally described by Mankin et al. Chondrocytes from adult patients with OA showed 35S-sulphate and 3H-thymidine incorporation in vitro, which increased with severity of the disease through Mankin 11-12. Incorporation rapidly declined after Mankin 11-12. Both matrix synthesis and cell proliferation were strongly reduced in the severe grades of OA. Histologic examination of the newly formed cartilage was only possible if the chondrocytes were derived from less severe grades of OA. Microscopy showed healthy chondrocytes surrounded by newly synthesized matrix, which stained well with specific dyes, indicating the ability of the cells to synthesize normal matrix components. The phenotype of human articular chondrocytes, derived from different grades of OA, was maintained in a high-density culture system. The data suggest dysregulation of the cell metabolism in OA cartilage. The increased cell metabolism was directly related to the histopathologic grade of OA.     

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.     

Osteoarthritis synovial fluid activates pro-inflammatory cytokines in primary human chondrocytes

Purpose

Two of the most common joint diseases are rheumatoid arthritis (RA) and osteoarthritis (OA). Cartilage degradation and erosions are important pathogenetic mechanisms in both joint diseases and have presently gained increasing interest. The aim of the present study was to investigate the effects of the synovial fluid environment of OA patients in comparison with synovial fluids of RA patients on human chondrocytes in vitro.

Methods

Primary human chondrocytes were incubated in synovial fluids gained from patients with OA or RA. The detection of vital cell numbers was determined by histology and by using the Casy Cell Counter System. Cytokine and chemokine secretion was determined by a multiplex suspension array.

Results

Microscopic analysis showed altered cell morphology and cell shrinkage following incubation with synovial fluid of RA patients. Detection of vital cells showed a highly significant decrease of vital chondrocyte when treated with RA synovial fluids in comparison with OA synovial fluids. An active secretion of cytokines such as vascular endothelial growth factor (VEGF) of chondrocytes treated with OA synovial fluids was observed.

Conclusions

Significantly increased levels of various cytokines in synovial fluids of RA, and surprisingly of OA, patients were shown. Activation of pro-inflammatory cytokines of human chondrocytes by synovial fluids of OA patient supports a pro-inflammatory process in the pathogenesis of OA.     

Expression analysis of different collagens and cytokines in cartilage cells derived from arthrotic hip and knee joints

AIM: Osteoarthritis (OA) is characterized by an irreversible destruction of articular cartilage. This is associated with a multiplicity of factors, causing an increased catabolic metabolism in cartilage. However, the prevalence of the OA is very variable in different joints. Therefore , we conducted a comparative analysis of chondrocytes derived from knee and hip joints with respect to their expression of inflammatory factors, such as IL-1beta, IL-1beta-receptorantagonist, iNOS, components of cartilage matrix (collagen I, II, and VI) as well as vimentin. METHODS: Different cytokines and proteins were detected by immune-histochemical staining of cartilage samples ex vivo. Further, chondrocytes were isolated from OA knee and hip joints, expanded in vitro and gene expression patterns were investigated by quantitative RT-PCR. RESULTS: Chondrocytes from knee and hip joints of OA patients express collagenes I, II and VI, IL-1beta and IL-1beta-RA, iNOS as well as Vimentin. A significant difference in gene expression patterns was not found in chondrocytes from the hip joints versus the knee joint ex vivo or in primary culture cells in vitro. However, in vitro the expression of type I collagen exceeded the expression of type II collagen. The IL-1beta-expression was high ex vivo, remained low during primary culture but was significantly elevated after primary culture in hip chondrocytes. CONCLUSION: Osteoarthritic gene expression patterns in cells derived from hip or knee joints ex vivo and in primary culture were not significantly different. We conclude that the rather frequent occurrence of OA in these joints in comparison to the ankle joint may be associated with a close physiological relation of cells in these joints. However, future studies which will include ankle cartilage must be investigated in further detail.     

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.     

膝关节骨关节炎滑液和软骨骨桥蛋白水平与其病变程度的相关性

目的 探讨膝关节骨关节炎(osteoarthritis,OA)患者滑液和关节软骨骨桥蛋白(osteopontin,OPN)水平及其与病变严重程度的关系.方法 随机选取50例膝关节OA患者[男15例,女35例;年龄48～81岁,平均(61.8±7.4)岁]和10名健康对照者[男4例,女6例;年龄59～68岁,平均年龄(63.2±6.0)岁]作为研究对象.采用Mankin评分评价疾病严重程度,Kellgren-Lawrence标准进行放射学分级,酶联免疫吸附法测定关节滑液OPN水平,免疫组化方法测定关节软骨OPN光密度值.结果 OA患者与对照者相比,关节滑液OPN水平[(4519.60±1830.37)pg/ml:(1179.70±303.39)pg/ml)和关节软骨OPN光密度值[(0.60±-0.06):(0.43±0.07)]均明显升高.关节滑液OPN水平与关节软骨OPN表达呈正相关(r=0.411,P=0.003).关节滑液OPN水平与OA病变严重程度(KL分级)呈正相关(r=0.581,P＜0.001).关节软骨OPN表达与OA病变严重程度(Mankin评分)呈正相关(r=0.675,P＜0.001).结论 关节滑液和关节软骨OPN水平与病变严重程度相关.     

Relationship of plasma and synovial fluid vascular endothelial growth factor with radiographic severity in primary knee osteoarthritis

Purpose

Recent evidence suggests that angiogenesis and inflammation contribute to the development and progression of osteoarthritis (OA). The purpose of this study was to investigate vascular endothelial growth factor (VEGF) levels in plasma and synovial fluid of patients with knee OA and to determine the relationship of VEGF levels with disease severity in knee OA.

Methods

A total of 100 subjects were enrolled in this study (80 knee OA patients and 20 healthy controls). Plasma and synovial fluid VEGF levels were analysed using enzyme-linked immunosorbent assay. VEGF expressions in synovial membrane and articular cartilage samples were assessed using immunohistochemistry.

Results

VEGF level in synovial fluid of knee OA patients was tenfold higher than that in paired plasma (P < 0.001). Both plasma and synovial fluid VEGF exhibited a positive correlation with radiographic severity (r = 0.454 and r = 0.727, P < 0.001, respectively). VEGF expression was highly detectable in synovial lining cells and articular chondrocytes of knee OA patients.

Conclusions

VEGF levels in both plasma and synovial fluid were positively correlated with the severity of knee OA. Therefore, VEGF may be useful for monitoring OA severity and could play a substantial role in the development and progression of knee OA.     

Production of cartilage oligomeric matrix protein (COMP) by cultured human dermal and synovial fibroblasts

OBJECTIVE: Cartilage oligomeric matrix protein (COMP) is a large disulfide-linked pentameric protein. Each of its five subunits is approximately 100,000 Da in molecular weight. COMP was originally identified and characterized in cartilage and it has been considered a marker of cartilage metabolism because it is currently thought not to be present in other joint tissues, except for tendon. To confirm the tissue specificity of COMP expression we examined cultured human dermal fibroblasts, human foreskin fibroblasts, and normal human synovial cells for the synthesis of COMP in culture. METHOD: Normal synovial cells and normal human dermal foreskin fibroblasts were isolated from the corresponding tissues by sequential enzymatic digestions and cultured in media containing 10% fetal bovine serum until confluent. During the final 24 h of culture, the cells were labeled with 35S-methionine and 35S-cysteine in serum- and cysteine/methionine-free medium. The newly synthesized COMP molecules were immunoprecipitated from the culture media with a COMP-specific polyclonal antiserum, or with monoclonal antibodies or affinity-purified COMP antibodies. The immunoprecipitated COMP was analyzed by electrophoresis in 5.5% polyacrylamide gels. For other experiments, synovial cells cultured from the synovium of patients with rheumatoid arthritis (RA) and osteoarthritis (OA) were similarly examined. RESULTS: A comparison of the amounts of COMP produced by each cell type (corrected for the DNA content) revealed that synovial cells produced > or = 9 times more COMP than chondrocytes or dermal fibroblasts. COMP could be easily detected by immunoprecipitation in all cell types. Electrophoretic analysis revealed a distinct band with an apparent MW of 115-120 kDa in samples from each of the three cell types, regardless of the antibody used. COMP expression in cultures of synoviocytes derived from OA and RA patients showed that OA and RA synovial cells produced similar amounts of monomeric COMP of identical size to those COMP monomers produced by normal synovial cells. The addition of TGF-beta to these cultures resulted in an increase in COMP production in normal, OA and RA synovial cells (45, 116 and 115% respectively). CONCLUSION: These studies demonstrate that substantial amounts of COMP are produced by several mesenchymal cells including synoviocytes and dermal fibroblasts. These findings raise important concerns regarding the utility of measurements of COMP levels in serum or in synovial fluid as markers of articular cartilage degradation because of the likelihood that a substantial proportion of COMP or COMP fragments present in serum or synovial fluid may be produced by cells other than articular chondrocytes.