Matrix metalloproteinases and aggrecanases cleave aggrecan in different zones of normal cartilage but colocalize in the development of osteoarthritic lesions in STR/ort mice.

OBJECTIVE: To map aggrecan cleavage by matrix metalloproteinases (MMPs) and aggrecanases in normal murine tibial articular cartilage (CBA strain) and in the development of spontaneous osteoarthritis (OA) in the STR/ort mouse and to assess the influence of sex hormone status on these conditions in gonadectomized STR/ort mice. METHODS: The distributions of neoepitopes of aggrecan generated by MMP (VDIPEN) and aggrecanase (NITEGE) cleavage were investigated by immunohistochemistry. RESULTS: VDIPEN neoepitope was detected mainly in the pericellular matrix of deep-zone chondrocytes in normal tibial cartilage from STR/ort and CBA mice. In early OA, VDIPEN immunostaining also localized to the pericellular matrix of chondrocytes at the site of the lesion. With increasing severity of OA lesions, VDIPEN immunostaining was also detected in the interterritorial matrix, close to the site of the lesion. In contrast, NITEGE mapped most strongly to the pericellular matrix of upper-zone chondrocytes in normal tibial cartilage. As with VDIPEN, NITEGE was strongly expressed in the pericellular matrix at the site of early OA lesions. With advancing OA, NITEGE colocalized with VDIPEN in both the pericellular and interterritorial matrices of chondrocytes adjacent to OA lesions and in those of the deep zones. Hormone status did not appear to influence the development of OA or the distribution of aggrecan neoepitopes in STR/ort mice. CONCLUSION: MMP- and aggrecanase-generated neoepitopes map predominantly to different regions in normal murine tibial cartilage. However, both groups of enzymes generate increased amounts of neoepitopes in pericellular and interterritorial matrix adjacent to histopathologic lesions of OA. Aggrecan degradation and the development of OA appear to be independent of sex hormone status in this model.     

骨关节炎兔血清中软骨寡聚基质蛋白和基质金属蛋白酶-3水平

目的 探讨血清软骨寡聚基质蛋白(COMP)与基质金属蛋白酶(MMP)-3检测应用于临床评估骨关节炎(OA)软骨病理改变的可能性.方法 伸直位石膏管型制动16只兔右后膝关节制作OA模型.以造模时间不同分为造模2周、造模6周,左后膝关节未造模故为对照组.X线影像学与病理观察模型关节的变化;评估关节软骨降解程度(OA积分);ELISA检测兔血清COMP、MMP-3水平;分析血清COMP、MMP-3水平与OA积分间的相关性.结果 (1)造模2周影像学变化较造模前不明显;造模6周兔胫骨平台边缘不光滑,关节间隙变窄,表面有毛刺样增生,胫骨平台及股骨内髁外侧可见唇样增生.(2)OA关节病变的形态学观察:造模2周兔关节软骨表面粗糙,表层裂隙;软骨细胞弥漫增多,排列紊乱;OA积分为(4.000±2.204)分.造模6周兔关节软骨表层可见较多裂隙延伸向下深达辐射层;裂隙周围可见脱水固缩坏死的软骨细胞且排列紊乱,部分成簇增生,各层结构不易分辨,有血管翳通过;OA积分为(10.620±1.408)分,与造模2周比,P=0.000.(3)造模2周兔血清COMP[(3.64±0.18)μg/L]、MMP-3[(1.99±0.81)μg/L]水平高于造模前[COMP(3.35±0.20)μg/L,MMP-3(1.61±0.71)μg/L];造模6周兔血清COMP[(3.96±0.44)μg/L]、MMP-3[(3.44±0.91)μg/L]水平高于造模前和造模2周,差异有统计学意义(P值均＜0.05).血清COMP、MMP-3水平与OA积分呈线性相关关系(r值均＞0.710,P值均小于0.05).结论 OA血清中COMP和MMP-3水平对评估OA软骨降解程度具有重要意义.

Abstract：

Objectiye To study the levels of cartilage oligomeric matrixprotein (COMP) and matrix metalloproteinase-3 (MMP-3) in the serum fluid of osteoarthritic rabbit models and their relationships with the severity of pathological changes, so as to investigate their correlation with osteoarthritis(OA). Methods The osteoarthritic animal models were get from immobilizing the right knees of 18 rabbits in full extension using plaster cast. Knee joint pathological changes of 2,6 weeks were examined for pathological severity of OA; ELISA sandwich method was used to measure the levels of COMP and MMP-3 in serum before and after modeling( at 2, 6 weeks respectively); X ray of model keens was also obtained in different period.Correlation analysis was performed to demonstrate the relationship between the levels of COMP, MMP-3 in the serum and the pathological severity of OA. Results ( 1 ) Morphological observations: immobilizing the right knees of rabbits in full extension using plaster cast was a reliable methed for osteoarthritic animal models and the typical histopathologic character was seen; the severity of osteoarthritisgradually increased with time extended. (2) The levels of COMP[(3.64 ±0. 18)μg/L], MMP-3 [(1.99 ±0. 81 ) μg/L]in the serum of 2 weeks osteoarthritic animal models were higher than those before immobilizing with plaster cast [COMP(3.35 ±0. 20) μg/L,MMP-3( 1.61 ±0. 71 ) μg/L]. The levels of COMP[(3.96 ±0. 44) μg/L],MMP-3[(3.44 ±0. 91) μg/L] of 6 weeks were much higher,with a significant difference(P ＜0.05). The levels of COMP, MMP-3 in serum had a linear correlation with the pathological severity of OA (r ＞0. 710,and P ＜ 0. 05 ). Conclusion The levels of COMP and MMP-3 in serum can help to predict and evaluate the progression of OA.     

Sexual dimorphism in the osteoarthritis of STR/ort mice may be linked to articular cytokines

BACKGROUND: STR/ort mice spontaneously develop degenerative changes of the knee joints resembling human osteoarthritis (OA), with the males being more severely affected than the females. OBJECTIVE: To analyse the early changes leading to OA by examining the articular cytokine expression and degenerative changes in STR/ort mice. METHODS: 122 STR/ort mice of both sexes aged between 2 and 15.5 months were included. Thin sections of the knees were analysed for osteoarthritic changes by haematoxylin/eosin staining. The articular cytokine expression was investigated by immunohistochemical staining using monoclonal antibodies specific for interleukin (IL)6, tumour necrosis factor alpha, transforming growth factor beta1 (TGFbeta1), IL1beta, IL4, and IL10, respectively. RESULTS: Both cartilage degeneration and articular cytokine expression differ between the sexes. The protection from cartilage degeneration in the female mice correlates with an increased expression of TGFbeta1 and IL4 at 2 months of age. CONCLUSION: The increased expression of TGFbeta1 and IL4 in young STR/ort female mice suggests that the sexual dimorphism is mediated through the articular expression of cytokines involved in cartilage metabolism.     

C57黑鼠骨关节炎模型的关节病理积分与血清及滑膜中基质金属蛋白酶-3白细胞介素-1水平的相关性研究

目的 探讨基质金属蛋白酶(MMP)-3与白细胞介素(IL)-1在骨关节炎(OA)发病中所起的作用及其用于临床判断OA早期病理变化的可能性.方法 采用C57黑鼠OA模型.在自然增龄基础上,又对小鼠进行运动负荷训练.应用形态学方法对模型关节的病理切片观察,进行OA积分评分.研究采用酶联免疫吸附试验(ELISA)的方法.检测血清及滑膜中MMP-3、IL-1的水平.采用相关性分析来检验血清和滑膜中MMP-3、IL-1水平之间的关系,以及与OA积分评分之间的相关性.结果 ①形态学观察:C57黑鼠具有自发OA特征,其OA严重程度与周龄相关,且运动负荷可加快有自发OA倾向的小鼠关节软骨病理改变.②ELISA检测:运动负荷组滑膜中MMP-3[(84±6)ng/ml]、IL-1[(48±3)ng/ml]及血清中IL-1[(38.3±5.0 mg/ml]含量高于自然增龄组[滑膜MMP-3(71±5)ng,ml;IL-1(42±3)ng/ml;血清IL-1(8.1±2.4)ng/ml],差异有统计学意义(P<0.01);血清与滑膜中MMP-3、IL-1水平、OA评分存在线性相关[r均>0.67,P均<0.01].结论 联合检测MMP-3及IL-1在OA血清中的含量,有助于疾病的诊断与病情的判断.特别是MMP-3对于OA的早期诊断具有更为重要的意义.     

A longitudinal analysis of urinary biochemical markers and bone mineral density in STR/Ort mice as a model of spontaneous osteoarthritis

Objective

To investigate the longitudinal changes both in the urinary concentrations of biochemical markers and in bone mineral density (BMD) during disease progression in the STR/Ort mouse model of osteoarthritis (OA).

Methods

Male STR/Ort mice were studied, with CBA mice used as nonarthritic controls. Radiographic evaluation and grading of the knee and measurements of urinary C‐terminal crosslinking telopeptide of type II collagen (CTX‐II), pyridinoline (Pyr), and deoxypyridinoline were performed between 8 weeks and 40 weeks of age. The BMD of the femoral shaft was measured from 20 weeks to 40 weeks of age and adjusted for body weight. Histologic evaluation and grading were performed at 40 weeks of age. STR/Ort mice were divided into 2 subgroups (STR OA and STR non‐OA) based on histologic grading.

Results

No significant differences between STR/Ort and CBA mice were observed for any biochemical marker or BMD at any time point. Urinary CTX‐II levels and BMD in the STR OA subgroup were higher than those in the STR non‐OA subgroup before radiographic changes of OA were apparent. Higher urinary Pyr levels in the STR OA subgroup were observed at the advanced stage of OA.

Conclusion

Urinary CTX‐II could be a useful marker in the early diagnosis and predicting the progression of OA, and urinary Pyr may be a potential marker to assess the severity of OA at an advanced stage. An increase in BMD prior to the establishment of radiographic OA may be related to the induction of OA.

     

Matrix metalloproteinases and aggrecanases cleave aggrecan in different zones of normal cartilage but colocalize in the development of osteoarthritic lesions in STR/ort mice

Objective

To map aggrecan cleavage by matrix metalloproteinases (MMPs) and aggrecanases in normal murine tibial articular cartilage (CBA strain) and in the development of spontaneous osteoarthritis (OA) in the STR/ort mouse and to assess the influence of sex hormone status on these conditions in gonadectomized STR/ort mice.

Methods

The distributions of neoepitopes of aggrecan generated by MMP (VDIPEN) and aggrecanase (NITEGE) cleavage were investigated by immunohistochemistry.

Results

VDIPEN neoepitope was detected mainly in the pericellular matrix of deep‐zone chondrocytes in normal tibial cartilage from STR/ort and CBA mice. In early OA, VDIPEN immunostaining also localized to the pericellular matrix of chondrocytes at the site of the lesion. With increasing severity of OA lesions, VDIPEN immunostaining was also detected in the interterritorial matrix, close to the site of the lesion. In contrast, NITEGE mapped most strongly to the pericellular matrix of upper‐zone chondrocytes in normal tibial cartilage. As with VDIPEN, NITEGE was strongly expressed in the pericellular matrix at the site of early OA lesions. With advancing OA, NITEGE colocalized with VDIPEN in both the pericellular and interterritorial matrices of chondrocytes adjacent to OA lesions and in those of the deep zones. Hormone status did not appear to influence the development of OA or the distribution of aggrecan neoepitopes in STR/ort mice.

Conclusion

MMP‐ and aggrecanase‐generated neoepitopes map predominantly to different regions in normal murine tibial cartilage. However, both groups of enzymes generate increased amounts of neoepitopes in pericellular and interterritorial matrix adjacent to histopathologic lesions of OA. Aggrecan degradation and the development of OA appear to be independent of sex hormone status in this model.

     

Osteoarthritic mice exhibit enhanced prostaglandin E2 and unchanged calcitonin gene-related peptide release in a novel isolated knee joint model

OBJECTIVE: Osteoarthritis (OA) is a painful degenerative joint disease. To assess joint nociceptor activation indirectly, we used a novel in vitro knee joint preparation and determined the release of calcitonin gene-related peptide (CGRP) and prostaglandin E2 (PGE2) in osteoarthritic mice. METHODS: We studied STR/1N mice, which spontaneously develop OA, along with CD-1 mice as controls and C57/Bl6 mice with unilateral collagenase-induced OA and C57/Bl6 control mice. The release of CGRP and PGE2 from tibial and femoral joint preparations was determined separately in vitro with enzyme immunoassays; we investigated both basal release and release induced by stimulation with capsaicin (CAP, 1 microM) or bradykinin (BK, 10 microM). RESULTS: Basal PGE2 release from femoral and tibial preparations increased by 79% and 97%, respectively, in STR/1N mice between 6 and 18 weeks of age when they developed OA, while age-matched CD-1 mice exhibited only a weak increase (23%). BK-evoked PGE2 release was significantly higher in 18-week-old STR/1N mice (931 +/- 98 pg/ml and 759 +/- 82 pg/ml from femoral and tibial preparations, respectively) than in age-matched CD-1 controls (236 +/- 38 pg/ml and 246 +/- 34 pg/ml). CAP stimulation induced a significant CGRP release, which, however, did not correlate with the temporal development of OA in STR/1N mice. Tibial but not femoral joint preparations from mice with collagenase-induced OA exhibited a significantly enhanced release upon BK stimulation compared to sham controls, while CAP-induced CGRP release did not reveal such difference. CONCLUSION: Basal and evoked PGE2 release from knee joint preparations rose while osteoarthritic alterations developed, whereas CGRP release remained unaltered. The increased PGE2 release may contribute to enhanced nociceptor sensitivity underlying chronic OA pain.     

Development and characteristics of pannus-like soft tissue in osteoarthritic articular surface in rat osteoarthritis model

OBJECTIVES: Pannus is invasive granulation tissue found on the articular cartilage having rheumatoid arthritis (RA). However, pannus-like tissue has also been found in osteoarthritis (OA). Our previous study showed that pannus-like tissue in OA (OA pannus) was frequently found in human OA samples. The purpose of the study is to investigate the development and the characteristics of OA pannus in a rat OA model. DESIGN: Ligaments of the knee joint were transected in Wister rats to induce OA. The knee joints were removed at weeks 1, 2, 4 and 6, and subjected to histological study. Samples were stained with hematoxylin and eosin (HE), Safranin-O and immuno-stained for vimentin, CD34, type II collagen and MMP-3. The whole knee joint of OA rats was implanted in SCID mice and kept for a further 3 weeks. Then the histological findings were evaluated in HE sections. RESULT: OA pannus appeared at week 2 and extend over the articular surface. OA pannus cells were positive for vimentin and/or CD34. At week 6, a part of articular surface was restored with matrix. OA pannus cells expressed MMP-3 as well as type II collagen. Histological study of rat OA knees implanted in SCID mice showed that OA pannus cells filled the joint space and invaded articular cartilage. CONCLUSIONS: The presence of OA pannus was found in a rat OA model and its features were similar to those in human OA. OA pannus had both catabolic and reparative features, and the latter feature were speculated to be dominant in the later phase of the disease under a certain environmental condition.     

Crucial role of macrophages in matrix metalloproteinase-mediated cartilage destruction during experimental osteoarthritis: involvement of matrix metalloproteinase 3

OBJECTIVE: To explore the involvement of synovial macrophages in early cartilage damage in osteoarthritis (OA), and to identify the role of matrix metalloproteinase 3 (MMP-3) in the pathology of early and late OA. METHODS: The role of synovial macrophages in MMP-mediated damage in OA was studied by depleting synovial macrophages prior to elicitation of a collagenase-induced instability model of OA. The expression of MMP in synovium and cartilage was monitored using TaqMan analysis. In spontaneous and induced OA, cartilage pathology was scored in MMP-3-knockout mice and control mice, by histologic assessment and VDIPEN staining. RESULTS: On day 14 following induction of OA, MMP-mediated neoepitopes were detected in cartilage from mice with mild experimental OA (mean +/- SD positively stained surface area 20 +/- 3.2%). Remarkably, by depleting synovial macrophages prior to induction of OA, the generation of MMP-induced neoepitopes was largely prevented (mean +/- SD positively stained surface area 5 +/- 1%; P< 0.001), indicating an important role for synovial macrophages in the occurrence of MMP-mediated cartilage damage. We observed a strong decrease in MMP-3 and MMP-9 expression in synovial but not cartilage tissue in macrophage-depleted joints. Among 2-year-old mice, spontaneous OA-like changes in the lining layer were significantly decreased in MMP-3-knockout mice compared with control mice. Even more striking was the 67% reduction in the occurrence of severe cartilage damage in MMP-3-knockout mice. In addition, MMP-mediated VDIPEN expression was significantly decreased, indicating reduced MMP-mediated cartilage breakdown. CONCLUSION: The results of this study prove that MMP-3 is involved in the generation of severe cartilage damage in murine OA. Synovial macrophages are crucial in early MMP activity and appear to mediate MMP production in synovium rather than cartilage.     

Influence of heme oxygenase 1 modulation on the progression of murine collagen-induced arthritis

OBJECTIVE: Heme oxygenase 1 (HO-1) can be induced by inflammatory mediators as an adaptive response. The objective of the present study was to determine the consequences of HO-1 modulation in the murine collagen-induced arthritis (CIA) model. METHODS: DBA/1J mice were treated with an inhibitor of HO-1, tin protoporphyrin IX (SnPP), or with an inducer of HO-1, cobalt protoporphyrin IX (CoPP), from day 22 to day 29 after CIA induction. The clinical evolution of disease was monitored visually. At the end of the experiment, joints were examined for histopathologic changes. Cytokine levels in paws were measured by enzyme-linked immunosorbent assay. Levels of HO-1, cyclooxygenase 2 (COX-2), and prostaglandin E2 (PGE2) were determined. Effects of treatments on the early phase of disease and after prophylactic administration were also assessed. RESULTS: CoPP strongly induced HO-1, resulting in the inhibition of cartilage erosion accompanied by extensive fibrosis in the joint. Levels of tumor necrosis factor alpha (TNFalpha), interleukin-2 (IL-2), and IL-10 were inhibited by CoPP, whereas levels of vascular endothelial growth factor were increased. Treatment with SnPP significantly reduced the severity of CIA, with inhibition of joint inflammation and cartilage destruction. The levels of PGE2, IL-1beta, and TNFalpha were also significantly reduced by SnPP treatment, which did not modify COX-2 protein expression. SnPP was more effective than CoPP in preventing the development of CIA (prophylactic administration). CONCLUSION: HO-1 is induced during CIA. Although overexpression of this protein causes some beneficial effects, strategies aimed at HO-1 overexpression cannot slow the progression of the chronic inflammatory disease, whereas treatment with SnPP, which inhibits HO-1, exerts prophylactic and therapeutic effects.     

Expression levels and association of gelatinases MMP-2 and MMP-9 and collagenases MMP-1 and MMP-13 with VEGF in synovial fluid of patients with arthritis

This study was performed to provide evidence, albeit indirectly, as to which matrix metalloproteinases (MMPs), among the gelatinases MMP-2 and MMP-9 and the collagenases MMP-1 and MMP-13, play a more proactive role in the angiogenic process in arthritic joint. Joint fluid was collected from 33 patients with rhuematoid arthritis (RA) and osteoarthritis (OA), and protein (MMPs and vascular endothelial growth factor (VEGF)) levels were measured by ELISA, and the association of MMPs with VEGF was evaluated in joint fluid of patients with RA or OA. The levels of collagenases (total MMP-1 and total MMP-13) and gelatinases (total MMP-2 and total MMP-9) in RA joint fluid were significantly higher than those in OA fluid. Total MMP-9 levels were significantly associated with VEGF levels in RA fluids, but not in OA fluid, while total MMP-13 levels were strongly associated with VEGF levels in both RA and OA fluid. However, total MMP-2 and total MMP-1 levels were not associated with VEGF levels in either RA or OA joint fluid. Our results indirectly suggest that in RA and OA, MMP-9 and MMP-13 may play a more important role in angiogenesis than MMP-2 and MMP-1.     

Serum matrix metalloproteinase 3 levels during treatment with sulfasalazine or combination of methotrexate and sulfasalazine in patients with early rheumatoid arthritis

OBJECTIVE: To determine the effects of treatment with sulfasalazine (SSZ) or the combination of methotrexate (MTX) and SSZ on serum matrix metalloproteinase 3 (MMP-3) levels in patients with early rheumatoid arthritis (RA). METHODS: Eighty-two patients with early RA (symptoms < 1 year and DMARD-naive at presentation) were selected who had been treated with SSZ (2000 mg/day) or with the combination of MTX (7.5-15 mg/week) and SSZ. Serum MMP-3 levels, C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), swollen joint count (SJC), tender joint count (TJC), Ritchie articular index (RAI), and the Disease Activity Score (DAS) were determined at 4 week intervals during a followup of 28 weeks for each treatment group. Response was based on clinical grounds and CRP at 12, 20, and 28 weeks. RESULTS: SSZ responders (n = 52) had lower baseline values of serum MMP-3, CRP, and ESR, compared to partial/nonresponders (n = 30), but did not differ in joint scores and DAS. In the SSZ responder group all variables decreased. In the SSZ partial/nonresponders, CRP, ESR, and SJC decreased in contrast to serum MMP-3, TJC. RAI, and DAS-3. After addition of MTX all variables decreased in 24 of the 30 patients who had shown a partial or no response taking SSZ. In the SSZ responders there was a delayed decrease in serum MMP-3 compared to CRP. CONCLUSION: Serum MMP-3 levels decrease in patients with early RA who respond to SSZ or to the combination of MTX and SSZ. In patients who respond to SSZ the changes in serum MMP-3 levels indicate a delayed response compared to CRP.     

Implication of MMP-9 and urokinase plasminogen activator (uPA) in the activation of pro-matrix metalloproteinase (MMP)-13

We examined whether the expression and activation of pro-matrix metalloproteinase (MMP)-1 varies from that of pro-MMP-13 in the joint fluid of osteoarthritis (OA) and rheumatoid arthritis (RA) patients. To do this, joint fluid was collected from 34 RA and 34 OA patients. The collagenase (pro-MMP-1 and MMP-13, total MMP-1, and MMP-13), gelatinase (total MMP-2 and MMP-9), stromelysin (total MMP-3), matrilysin (total MMP-7), uPA, and tissue inhibitor of MMP (TIMP) levels were measured by ELISA. The level of total MMP-1 in RA joint fluids was similar to that of the OA joint fluid. In contrast, the level of total MMP-13 in the RA group was significantly higher than that of the OA group. Among various MMPs (MMP-2, MMP-3, MMP-7, and MMP-9), only MMP-9 was strongly associated with total MMP-13 in both RA and OA. The level of uPA was also strongly associated with MMP-13 in RA but not OA, while the level of TIMP-1 and TIMP-2 was not significantly different between RA and OA. In conclusion, MMP-9 and uPA might be involved in the activation of pro-MMP-13 through unknown mechanisms in arthritic diseases.     

In vivo selective inhibition of mitogen-activated protein kinase kinase 1/2 in rabbit experimental osteoarthritis is associated with a reduction in the development of structural changes

OBJECTIVE: The primary aim of this study was to investigate, using an experimental rabbit model of osteoarthritis (OA), the effect of a selective mitogen-activated protein kinase kinase 1/2 (MEK-1/2) inhibitor, PD 198306, on the development of structural changes. Additional aims were to assess the effects of the inhibitor on levels of phosphorylated extracellular signal-regulated kinase 1/2 (phospho-ERK-1/2) and matrix metalloproteinase 1 (MMP-1; collagenase 1) in OA chondrocytes. METHODS: After surgical sectioning of the anterior cruciate ligament of the right knee joint, rabbits with OA were separated into 3 experimental groups: oral treatment with placebo or with PD 198306 at a therapeutic concentration of 10 mg/kg/day or 30 mg/kg/day. Each treatment started immediately after surgery. The animals were killed 8 weeks after surgery. Macroscopic and histologic studies were performed on the cartilage and synovial membrane. The levels of phospho-ERK-1/2 and MMP-1 in OA cartilage chondrocytes were evaluated by immunohistochemistry. Normal, untreated rabbits were used as controls. RESULTS: OA rabbits treated with the highest dosage of MEK-1/2 inhibitor showed decreases in the surface area (size) of cartilage macroscopic lesions (P < 0.002) and in osteophyte width on the lateral condyles (P = 0.05). Histologically, the severity of synovial inflammation (villous hyperplasia) was also reduced (P < 0.02). In cartilage from placebo-treated OA rabbits, a significantly higher percentage of chondrocytes in the superficial layer stained positive for phospho-ERK-1/2 and MMP-1 compared with normal controls. Rabbits treated with the highest dosage of PD 198306 demonstrated a significant and dose-dependent reduction in the level of phospho-ERK-1/2 and a lower level of MMP-1. CONCLUSION: This study demonstrates that, in vivo, PD 198306, a selective inhibitor of MEK-1/2, can partially decrease the development of some of the structural changes in experimental OA. This effect was associated with a reduction in the level of phospho-ERK-1/2 in OA chondrocytes, which probably explains the action of the drug.     

Matrix metalloproteinases and tissue inhibitors of metalloproteinases in synovial fluids from patients with rheumatoid arthritis or osteoarthritis

OBJECTIVE: Matrix metalloproteinases (MMPs) are expressed in joint tissues of patients with rheumatoid arthritis (RA) and osteoarthritis (OA). The objective of this study was to define the steady state levels of seven different MMPs and two tissue inhibitors of metalloproteinases (TIMPs) as well as the potential metalloproteinase activity in the synovial fluid (SF) to provide more insight into the role of MMPs in cartilage destruction in RA and OA. METHODS: Levels of MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-13, TIMP-1, and TIMP-2 in SF aspirated from knee joints of 97 patients with RA and 103 patients with OA were measured by the corresponding one step sandwich enzyme immunoassays. Proteolytic activity of MMPs in these SFs was examined in an assay using [(3)H]carboxymethylated transferrin substrate in the presence of inhibitors of serine and cysteine proteinases after activation with p-aminophenylmercuric acetate (APMA). Destruction of RA knee joints was radiographically evaluated. RESULTS: Levels of MMP-1, MMP-2, MMP-3, MMP-8, and MMP-9 were significantly higher in RA SF than in OA SF. MMP-7 and MMP-13 were detectable in more than 45% of RA SFs and in less than 20% of OA SFs, respectively. Among the MMPs examined, MMP-3 levels were extremely high compared with those of other MMPs. Direct correlations were seen between the levels of MMP-1 and MMP-3 and between those of MMP-8 and MMP-9 in RA SF. Although the levels of MMP-1 and MMP-3 increased even in the early stage of RA, those of MMP-8 and MMP-9 were low in the early stage and increased with the progression of RA. Molar ratios of the total amounts of the MMPs to those of the TIMPs were 5.2-fold higher in patients with RA than in OA, which was significant. APMA-activated metalloproteinase activity in SF showed a similar result, and a direct correlation was seen between the molar ratios and the activity in RA SF. CONCLUSIONS: Our results show that high levels of MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, and TIMP-1 are present in RA SF and suggest that once these MMPs are fully activated, they have an imbalance against TIMPs, which may contribute to the cartilage destruction in RA.     

Analysis of cartilage biomarkers in the early phases of canine experimental osteoarthritis

OBJECTIVE: To study 3 body fluids for changes in the levels of 5 biomarkers of cartilage metabolism during the early phases of experimental osteoarthritis (OA). METHODS: Twenty skeletally mature mixed-breed canines underwent unilateral surgical transection of the anterior cruciate ligament. Samples of joint fluid, serum, and urine were obtained preoperatively and just before necropsy (3 weeks or 12 weeks postoperatively). Biomarkers included 2 markers of cartilage matrix synthesis/turnover (aggrecan 846 epitope and C-propeptide of type II collagen) and 3 markers of cartilage degradation (keratan sulfate proteoglycan epitope, the collagenase-generated cleavage epitope of type II collagen [Col2-3/4C(long mono), or CIIC], and crosslinked peptides from the C-telopeptide domain of type II collagen [Col2CTx]). Significant changes in the levels of these biomarkers were determined by paired analyses. RESULTS: Joint pathology was more severe in the 12-week group compared with the 3-week group. In joint fluid, due to limited volume, only Col2-3/4C(long mono) and Col2CTx were measured. Significant elevations in the levels of both of these markers were observed in experimental joints in both the 3-week group and the 12-week group. In serum, the level of aggrecan 846 epitope was elevated at both 3 weeks and 12 weeks, the level of Col2-3/4C(long mono) was elevated at 12 weeks, and the level of Col2CTx was elevated at both 3 weeks and 12 weeks. In urine, the level of Col2-3/4C(long mono) was elevated at 12 weeks after surgery. CONCLUSION: Levels of biomarkers of intact aggrecan proteoglycan (aggrecan 846 epitope) and type II collagen degradation (Col2-3/4C(long mono) and Col2CTx) were elevated early after unilateral stifle joint injury, suggesting that these markers are sensitive and specific for early cartilage changes associated with isolated joint injury in this established model of experimental OA.     

Measurement of matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases-1 (TIMP-1) in patients with knee osteoarthritis: comparison with generalized osteoarthritis.

OBJECTIVES: To compare plasma levels of matrix metalloproteinase (MMP)-3, MMP-9 and tissue inhibitor of metalloproteinases-1 (TIMP-1) between patients with knee osteoarthritis and normal subjects, to investigate whether the degree of knee joint involvement is related to those measurements, and to compare patients with and without generalized osteoarthritis. METHODS: Eighty-three women with knee osteoarthritis (OA patients) were studied. Plasma levels of MMP-3, MMP-9 and TIMP-1 were measured by enzyme immunoassays. Knee and hand radiographs were taken of all patients. The joints of the knee and hand were graded from 0 to 4 according to Kellgren and Lawrence criteria. All OA patients were divided into a generalized OA (GOA) group (n = 37) and a knee OA (KOA) group (n = 46) according to Doherty's criteria. MMPs and TIMP were also measured in 19 normal subjects. RESULTS: Plasma levels of MMP-3 and TIMP-1 were significantly higher in OA patients than in normal subjects. In contrast, MMP-9 was lower in OA patients than in normal subjects. Plasma levels of MMP-3 and MMP-9 were not influenced by the grade of knee OA. TIMP-1 was influenced by the grade of knee OA. Plasma levels of MMP-3 were significantly elevated in GOA compared to KOA. In contrast, there were no significant differences in plasma levels of MMP-9 and TIMP-1 between GOA and KOA. CONCLUSION: Since the plasma level of MMP-3 in GOA was higher than that in KOA patients, it may be a superior indicator for whole-joint degeneration.