Inhibition of histone deacetylases antagonized FGF2 and IL-1β effects on MMP expression in human articular chondrocytes

Fibroblast growth factor-2 (FGF2) and interleukin-1β (IL-1β) stimulate the expression of matrix metalloproteinases (MMPs) in articular chondrocytes, which may contribute to cartilage degradation and development of osteoarthritis. Histone deacetylases (HDACs) have recently been implicated in the regulation of MMP gene expression. To investigate the functional involvement of HDACs in the signaling pathway of FGF2 and IL-1β, we examined the effects of HDAC inhibition on activities of FGF2 or IL-1β on gene expression of MMP-1, MMP-3, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS5), collagen type II, and aggrecan. Human articular chondrocyte cultures were treated with FGF2 or IL-1β in the presence or absence of HDAC inhibitor (trichostatin A, TSA). Gene expression levels after treatments were assessed using quantitative real time PCR. Results showed that FGF2 and IL-1β both increased MMP-1 and -13 expression, while IL-1β also increased MMP-3 mRNA levels. These effects were attenuated in the presence of TSA in a dose dependent manner. In contrast to the effects on MMPs, FGF2 decreased mRNA levels of ADAMTS-5, which was not affected by HDAC inhibition. FGF2, IL-1β, and TSA inhibited expression of aggrecan, while TSA also decreased mRNA levels of collagen type II. These findings showed that HDAC inhibition antagonized FGF2 and IL-1β induced MMP expression. Combination of FGF2 and the HDAC inhibitor decreases both anabolic and catabolic genes, which may slow the cartilage turnover and be beneficial for maintaining cartilage integrity.     

The matrix metalloproteinase/tissue inhibitor of matrix metalloproteinase profile in colorectal polyp cancers

Aims:  The matrix metalloproteinase (MMP)/tissue inhibitor of metalloproteinase (TIMP) system has a major role in tumour invasion and metastasis. Roles in pathways involved in early tumour development are also being identified for this system, and the aim of this study was to define the expression profile of the major MMPs and TIMPs in colorectal polyp cancers.
Methods and results:  The expression and cellular localization of individual MMPs and TIMPs was determined in colorectal polyp cancers by immunohistochemistry. All the MMPs and TIMPs showed immunoreactivity in carcinomatous epithelium. MMP1 ( P  < 0.001), MMP2 ( P  = 0.003), MMP3 ( P  = 0.004), TIMP1 ( P  = 0.01) and TIMP2 ( P  < 0.001) showed significant increases in immunoreactivity in carcinomatous epithelium compared with adenomatous epithelium. MMP7 showed immunoreactivity in carcinomatous epithelium, but showed no immunoreactivity in either normal epithelium or adenomatous epithelium. MMP and TIMP expression was limited in normal epithelium to MMP1, MMP2 and TIMP3.
Conclusions:  This study defines the expression profile of MMPs and TIMPs in colorectal polyp cancers and shows that the increased expression of MMPs and TIMPs occurs at an early stage of colorectal neoplasia. It provides evidence to support the hypothesis that these molecules have a key involvement in the early stages of tumour development.     

Early Cartilage Degeneration in a Rat Experimental Model of Developmental Dysplasia of the Hip

Osteoarthritis (OA) is a common long-term complication of developmental dysplasia of the hip (DDH) that is associated with a higher incidence of OA. In addition, the age of onset of OA in DDH patients is significantly younger than in the general population. In order to investigate the early degeneration in DDH cartilage, we used a rat DDH model that was established by the straight-leg swaddling position. The hips were isolated from the DDH model rats and an untreated control group at postnatal weeks 2, 4, 6, and 8. Histology and proteoglycan levels were observed in articular cartilage using Safranin O staining. Biomarkers of cartilage degeneration, including type X collagen and matrix metalloproteinase (MMP)-13, were assessed using immunohistochemistry and quantitative real-time polymerase chain reaction. In addition, expressions of ADAMTS-4 and ADAMTS-5 were studied using quantitative real-time polymerase chain reaction at different ages. DDH rats showed decreased proteoglycans and derangement of chondrocytes when compared with the control group. Collagen X and MMP-13 expressions were higher in the superficial zone of DDH rats than in that of controls (p < 0.05), and the increase was age-dependent. mRNA expression of Collagen X and MMP-13 showed similar results (p < 0.05). A significant increase in mRNA expression of ADAMTS-5 was found in the DDH model cartilage at 8 weeks (p < 0.05). However, no change was observed in ADAMTS-4 expression. This study shows that degenerative cartilage changes occur at an early stage in the rat DDH model and become aggravated with age.     

创伤性关节炎软骨中基质金属蛋白酶13及组织特异性抑制剂1的表达*

背景：关节软骨损伤后继发性退变机制仍不十分清楚。近年研究发现关节软骨细胞外基质合成与降解失衡是造成软骨变性的重要原因之一,其中基质金属蛋白酶可能起决定性的作用。 目的：观察基质金属蛋白酶13及其组织特异性抑制剂1在创伤性关节炎中的表达及与软骨退变的关系。 方法：将新西兰兔分别用骨水泥制作的模具从1.33 kg,46 cm高度和0.43 kg,20 cm高度进行自由落体撞击制备轻型和重型撞击创伤性关节炎兔模型。 结果与结论：苏木精-伊红染色和免疫组织化学染色结果显示,基质金属蛋白酶13和组织特异性抑制剂1在两组均增高(P < 0.05)。组织特异性抑制剂1在重型撞击创伤性关节炎模型兔软骨组织中的分泌明显高于轻型撞击组(P < 0.05)。说明关节软骨在创伤后,基质金属蛋白酶13及组织特异性抑制剂1表达上调,共同作用促进了关节软骨退变,其中组织特异性抑制剂1的过量表达可能是造成关节软骨进一步损害的原因之一。     

Platelet-derived growth factor and transforming growth factor-β modulate the expression of matrix metalloproteinases and migratory function of human airway smooth muscle cells

Background Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinase (TIMPs) have been suggested to be involved in the pathogenesis of asthma. Their expression in airway smooth muscle (ASM) cells could be involved in collagen turnover and migration of these cells and thus may contribute to airway remodelling.
Objective To examine the effect of pro-fibrotic growth factors TGF-β and platelet-derived growth factor (PDGF) on the expression of MMPs/TIMPs in cultured human ASM cells and to examine the role of MMP in the migration of ASM cells.
Methods ASM cells were stimulated with TGF-β and/or PDGF. Expression and activity of MMP-1, MMP-2, MMP-3, TIMP-1 and TIMP-2 were evaluated by quantitative RT-PCR, Western blot and zymography. Modified Boyden-chamber migration assay was performed to investigate the effect of secreted MMP-3 and TIMP-1 on ASM-cell migration.
Results PDGF strongly up-regulated the expression of MMP-1 at mRNA and protein levels. PDGF, when combined with TGF-β, caused synergistic up-regulation of MMP-3. TIMP-1 was additively up-regulated by TGF-β and PDGF. These growth factors had no effect on the expression of MMP-2 and TIMP-2. U0126, an extracellular signal-regulated kinase (ERK) pathway inhibitor, inhibited the up-regulation of MMP-1 by PDGF. The synergistic/additive up-regulation of MMP-3 and TIMP-1 was inhibited by U0126 and SB431542, a Smad pathway inhibitor. Supernatant from ASM cells in which MMP-3 production was knocked down by RNA interference showed a decreased migratory effect on ASM cells, whereas supernatant from cells with suppressed TIMP-1 expression resulted in increased migration.
Conclusion Our results suggest that PDGF with/without TGF-β could facilitate migration of ASM cells by modification of MMP–TIMP balance through the ERK pathway.     

Serum matrix metalloproteinase and tissue inhibitor of metalloproteinase concentrations in infertile women achieved pregnancy following IVF-ET

PROBLEM: Matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase (TIMP) play important roles throughout various stages of pregnancy, including embryo implantation, trophoblastic invasion, placentation in early gestation, and cervical dilatation in later gestation, and feto-maternal membrane lysis. It would be beneficial if assessment of serum concentrations of MMP and TIMP could predict successful implantation following in vitro fertilization-embryo transfer (IVF-ET). This study was performed to compare serum MMP and TIMP concentrations between patients with and without the establishment of pregnancy following ET. METHOD OF STUDY: Ten patients who conceived and 10 patients who did not after IVF-ET were entered in the present study. Only intra-uterine single pregnancies with uneventful courses to term were included in the study subjects. Blood samples were obtained at 7, 14 and 21 days after oocyte retrieval. Serum concentrations of MMP-1, MMP-2, MMP-3, and TIMP-1 were measured using enzyme-linked immunosorbent assay. These variables were compared with estradiol (E(2)), progesterone (P(4)), and betahCG levels in the patients' sera. Clinical pregnancies were diagnosed only when fetal heartbeat was visualized on ultrasound. RESULTS: There were no significant differences in serum MMP concentrations between the pregnant group and the non-pregnant group. However, serum TIMP-1 concentrations on Days 14 and 21 in the pregnant group were significantly higher than those in non-pregnant group [Day 14: 223.1 +/- 11.9 versus 177.5 +/- 20.6 ng/mL (P = 0.004); Day 21: 215.4 +/- 27.8 versus 181.5 +/- 27.4 ng/mL (P = 0.03)]. Serum TIMP-1 concentrations were also correlated with serum E(2) and P(4) levels (P < 0.0001), but not with those of the MMPs. None of MMP nor TIMP-1 were correlated with serum betahCG level. CONCLUSIONS: It was demonstrated that the patients who successfully conceived after IVF-ET showed significantly higher levels of TIMP-1 at 14 and 21 days after IVF-ET, but not at day 7; further work will be required to determine if serum TIMP-1 can be used to improve prediction of pregnancy outcome in these patients.     

基质金属蛋白酶MMP-2、MMP-9及其抑制因子TIMP-1、TIMP-2在子宫内膜异位症血清中的表达及临床意义

目的探讨基质金属蛋白酶MMP-2、MMP-9及其抑制因子TIMP-1、TIMP-2在子宫内膜异位症（EMs）血清中的表达及临床意义。方法采用双抗体夹心酶联免疫吸附法（ELISA）检测55例EMs患者和30例对照组血清中删P-2,MMP-9、TIMP-1和TIMP-2水平。结果BMs组血清中MMP-2和MMP-9水平显著高于对照组,TIMP-1和TIMP-2水平显著低于时照组（P〈0．05）。Ⅲ-Ⅳ期血清MMP-2和MMP-9水平显著高于Ⅰ-Ⅱ期和对照组,TIMP-1和TIMP-2水平显著低于Ⅰ-Ⅱ期和对照组（P〈0．05）。结论MMP-2、MMP-9与Elds发生和发展相关,TIMP-1、TIMP-2对MMP-2、MMP-9水平调节有重要作用。     

膝关节软骨中基质金属蛋白酶13及其组织抑制剂1的表达

背景：基质金属蛋白酶组织抑制剂1是与基质金属蛋白酶13相对应的拮抗剂,两者间表达水平和功能活性的平衡对细胞外基质的代谢状态起着重要作用,但在DH豚鼠骨关节炎发生发展过程中,两者表达水平,尤其两者表达水平比值的变化尚不明确。 目的：探讨不同月龄DH豚鼠关节软骨中基质金属蛋白酶13、基质金属蛋白酶组织抑制剂1表达比值的变化及其与DH豚鼠增龄性原发骨性关节炎发病过程中软骨退变程度的关系。 方法：选取2,4,8,12月龄雌性DH各6只,取膝关节观察关节软骨的大体形态后常规脱钙、包埋、制作石蜡切片,用于VG染色行组织学观察,采用Mankin评分系统定量分析关节软骨退变情况,采用免疫组织化学方法检测膝关节软骨中基质金属蛋白酶13、基质金属蛋白酶组织抑制剂1表达情况,应用 Image pro-Plus 6.0软件对免疫组织化学阳性蛋白表达情况进行积分吸光度计算。线性回归分析判断Mankin评分与基质金属蛋白酶13/基质金属蛋白酶组织抑制剂1比值的相关性。 结果与结论：DH豚鼠2月龄膝关节软骨无关节炎表现,4 月龄出现轻度软骨退变,并随月龄增加进行性加重,Mankin评分随月龄增加逐渐增高,各组之间的差异均有显著性意义(P < 0.05)。基质金属蛋白酶13、基质金属蛋白酶组织抑制剂1的表达均随月龄进行性增加,两者比值与Mankin评分呈正相关性(P < 0.05)。结果提示DH豚鼠4月龄出现膝关节软骨退行性变化,随月龄增加而进行性加重,其病理改变与基质金属蛋白酶13/基质金属蛋白酶组织抑制剂1表达失衡有关。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：     

Differential expression of ADAMTS-1, -4, -5 and TIMP-3 in rat spinal cord at different stages of acute experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is an animal model of inflammatory demyelination, a pathological event common to multiple sclerosis (MS). During CNS inflammation there are alterations in the extracellular matrix (ECM). A Disintegrin and Metalloproteinase with Thrombospondin motifs (ADAMTS)-1, -4 and -5 are proteases present in the CNS, which are able to cleave the aggregating chondroitin sulphate proteoglycans, aggrecan, phosphacan, neurocan and brevican. It is therefore important to investigate changes in their expression in different stages of EAE induction. We have investigated expression of ADAMTS-1, -4, -5 and tissue inhibitor of metalloproteinase (TIMP)-3, by real-time RT-PCR. We have also examined protein expression of ADAMTS-1, -4 and -5 by western blotting and immunocytochemistry in spinal cord from animals at different stages of disease progression. Our study demonstrated a decrease in ADAMTS-4 mRNA and protein expression. TIMP-3 was decreased at the mRNA level although protein levels were increased in diseased animals compared to controls. Our study identifies changes in ADAMTS expression during the course of CNS inflammation which may contribute to ECM degradation and disease progression.     

Differential expression of matrix metalloproteinase (MMP)-2, MMP-9 and tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 in non-melanoma skin cancer: implications for tumour progression

AIMS: To investigate the expression of matrix metalloproteinase (MMP)-2, MMP-9, and tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 in non-melanoma skin cancer (NMSC) and to compare their expression between different tumour types and with clinicopathological factors. METHODS AND RESULTS: A study of 11 normal skin, 29 Bowen's disease (BD), 40 squamous cell carcinoma (SCC) and 38 basal cell carcinoma (BCC) samples for MMP-2, MMP-9, TIMP-1 and TIMP-2 expression was carried out using immunohistochemistry and in situ hybridization. The expression of all metalloproteinases was greater in tumours than in normal skin. MMP-2 and MMP-9 expression was more extensive in the stroma of SCC than of BCC or BD. TIMP-1 expression was greater in the stroma of BCC than of SCC or BD and TIMP-2 expression was greater in the stroma of SCC than of BD. There was a correlation between increased metalloproteinase expression and depth of lesion (MMP-2 and TIMP-2), inflammation (MMP-2, MMP-9, TIMP-1 and TIMP-2) and microvessel density (MMP-2, MMP-9 and TIMP-2). CONCLUSIONS: MMP-2, MMP-9, TIMP-1 and TIMP-2 play an important role in the pathogenesis of non-melanoma skin cancer, but differ significantly in their expression levels between the tumour types examined. The immunoexpression of these proteins may be useful indicators of cutaneous cancer invasion and progression.     

Gene expression and matrix proteinase activity in osteochondrosis dessicans

Introduction Osteochondrosis dessicans (OCD) is a disorder of unknown aetiology where often a fragment of cartilage and subchondral bone separates from the articular surface. Previous studies have shown histological changes in glycosaminoglycan content in OCD cartilage compared to normal cartilage ( Koch et al. 1997 ). It has also been shown in equine OCD cartilage that there is excessive degradation of type‐II collagen compared to normal cartilage ( Laverty et al. 2002 ). The present study was undertaken to examine the gene expression in human OCD cartilage compared to its normal autologous articular cartilage and human osteoarthritic (OA) cartilage. Methods Cartilage from five OCD patients (18–34 years) was obtained at the time of surgery. Pieces of cartilage were either snap‐frozen (in preparation for RNA isolation) or the proteoglycans extracted with 4 m GuHCl. Total RNA was isolated from the cartilage using RNeasy minicolumns and reagents (Qiagen) according to the manufacturer's protocol. RT‐PCR was performed using an RNA PCR kit (Perkin‐Elmer) using a number of oligonucleotide primers. GuHCl‐extracted proteoglycan fragments were analysed using Western blotting with a number of antibodies to aggrecan metabolites, collagen metabolites and the small leucine‐rich proteoglycans. Results and discussion When OCD cartilage was compared to normal and human OA cartilage, there was an increase in aggrecan, collagen type‐II and collagen type‐X RNA expression. There was no change in RNA expression of link protein or type‐I collagen. The RNA expression of the aggrecanases (ADAMTS enzymes) was also different in the three different cartilage samples. Neither ADAMTS‐1, ‐4 or ‐5 was present in the normal cartilage. In contrast, in the OCD cartilage, there was expression of both ADAMTS‐1 and ‐4, whereas in the OA cartilage, there was expression of ADAMTS‐4 and ‐5. In the case of MMP RNA expression, MMP‐3 was decreased and MMP‐13 increased in OCD cartilage compared to both normal and OA samples. In addition, the expression of all three TIMP isoforms was increased in the OCD cartilage. Although inflammatory components are not expected in OCD pathology, expressions of inflammatory mediators such as COX‐2, IL‐1‐α and TNF‐α were all increased in the OCD cartilage when compared to normal, but expression of these mRNAs in the OA cartilage was higher. Analysis of proteoglycan fragments in the OCD cartilage by Western blotting showed the presence of aggrecan fragments containing the G1 domain, interglobular domain and the C‐terminal neoepitope generated by aggrecanase cleavage. There was also immunoreactivity for biglycan and link protein. Conclusion These results suggest that the phenotypic expression of chondrocytes at the site of the OCD lesion are markedly different from ‘normal’ articular cartilage and also pathological OA cartilage. Interestingly, the expression patterns of matrix proteinases and their natural inhibitors were also markedly different in OCD cartilage, again suggesting that there are specific biochemical expression patterns in OCD pathology, which may potentially be biomarkers of the disease process. Further studies are necessary to elucidate how the differences in gene expression and matrix protease activity may be involved in the aetiology of OCD.     

Effects of high mobility group box protein-1, interleukin-1β, and interleukin-6 on cartilage matrix metabolism in three-dimensional equine chondrocyte cultures

The effects of high mobility group box protein (HMGB)-1, interleukin (IL)-1β, and IL-6 on equine articular chondrocytes were investigated, with emphasis on detecting differences between anatomical sites exposed to different loading in vivo, using three-dimensional (3D) cell cultures established with chondrocytes from dorsal radial facet (DRF, highly loaded) and palmar condyle (PC, less loaded) of the third carpal bone (C3). Expression of important genes involved in cartilage metabolism, presence of glycosaminoglycans and cartilage oligomeric matrix protein (COMP) in pellets, and concentrations of matrix metalloproteinase (MMP)-13 and aggrecan epitope CS 846 were evaluated. Compared to controls, IL-1β treatment increased gene expression of versican, matrix-degrading enzymes, and tissue inhibitor of metalloproteinase (TIMP)-1, and decreased aggrecan and collagen type I and type II expression. In addition, IL-1β-treated pellets showed decreased safranin O staining and increased COMP immunostaining and MMP-13 concentrations in culture supernatants. Effects of IL-6 and HMGB-1 on gene expression were variable, although upregulation of Sry-related high-mobility group box 9 (Sox9) was often present and statistically increased in HMGB-1-treated pellets. Response to cytokines rarely differed between DRF and PC pellets. Thus, site-associated cartilage deterioration in equine carpal osteoarthritis (OA) is not explained by topographically different responses to inflammatory mediators. Differences in gene expressions of structural matrix proteins in untreated DRF and PC pellets were noted in the youngest horses, which may indicate differences in the chondrocytes potential to produce matrix in vivo. Overall, a strong catabolic response was induced by IL-1β, whereas slight anabolic effects were induced by IL-6 and HMGB-1.     

Expression and correlation of matrix metalloproteinase-7 and interleukin-15 in human osteoarthritis

Objectives: To investigate the expression and correlation of matrix metalloproteinase (MMP)-7 and interleukin (IL)-15 in human osteoarthritis (OA). Methods: From October 2013 to December 2014, 30 patients with OA were enrolled. In addition, anther 30 patients with simple meniscus injury were collected as a control group. There were no significant differences in age and gender between the two groups. Articular cartilage tissue was obtained from both OA patients and control group patients. Protein, mRNA, and serum expression levels of MMP-7 and IL-15 in the both two groups were determined by immunohistochemical (IHC), in situ hybridization, and enzyme-linked immunosorbent assay (ELISA) assay, respectively. Additionally, correlation between MMP-7 and IL-15 expression level in cartilage tissue and serum was assessed using Pearson correlation analysis. Results: Protein, mRNA, and serum expression levels of MMP-7 and IL-15 in patients with OA were all significantly increased in OA patients compared with the control group. Besides, there were strong positive relationships between articular MMP-7 level and serum MMP-7 level (R² = 0.573, P = 0.018), between articular IL-15 level and serum IL-15 level (R² = 0.861, P = 0.023), and between serum IL-15 level and serum MMP-7 level (R² = 0.602, P = 0.012). Conclusion: These results suggest that MMP-7 and IL-15 might play important roles in the pathogenesis of OA, and IL-15 and MMP-7 has positive correlation in OA.     

Effects of high mobility group box protein-1, interleukin-1β, and interleukin-6 on cartilage matrix metabolism in three-dimensional equine chondrocyte cultures

The effects of high mobility group box protein (HMGB)-1, interleukin (IL)-1β, and IL-6 on equine articular chondrocytes were investigated, with emphasis on detecting differences between anatomical sites exposed to different loading in vivo, using three-dimensional (3D) cell cultures established with chondrocytes from dorsal radial facet (DRF, highly loaded) and palmar condyle (PC, less loaded) of the third carpal bone (C3). Expression of important genes involved in cartilage metabolism, presence of glycosaminoglycans and cartilage oligomeric matrix protein (COMP) in pellets, and concentrations of matrix metalloproteinase (MMP)-13 and aggrecan epitope CS 846 were evaluated. Compared to controls, IL-1β treatment increased gene expression of versican, matrix-degrading enzymes, and tissue inhibitor of metalloproteinase (TIMP)-1, and decreased aggrecan and collagen type I and type II expression. In addition, IL-1β-treated pellets showed decreased safranin O staining and increased COMP immunostaining and MMP-13 concentrations in culture supernatants. Effects of IL-6 and HMGB-1 on gene expression were variable, although upregulation of Sry-related high-mobility group box 9 (Sox9) was often present and statistically increased in HMGB-1-treated pellets. Response to cytokines rarely differed between DRF and PC pellets. Thus, site-associated cartilage deterioration in equine carpal osteoarthritis (OA) is not explained by topographically different responses to inflammatory mediators. Differences in gene expressions of structural matrix proteins in untreated DRF and PC pellets were noted in the youngest horses, which may indicate differences in the chondrocytes potential to produce matrix in vivo. Overall, a strong catabolic response was induced by IL-1β, whereas slight anabolic effects were induced by IL-6 and HMGB-1.     

Up-regulation of MMP-8 and MMP-9 activity in the BALB/c mouse spinal cord correlates with the severity of experimental autoimmune encephalomyelitis

Induction of EAE can be inhibited or repressed by administration of soluble metalloproteinase inhibitors. We studied the matrix metalloproteinase (MMP) and their tissue inhibitor (TIMP) expression pattern in experimental autoimmune encephalomyelitis (EAE) of the resistant Th2 prone BALB/c mouse, where the disease can be induced with ultrasound-emulsified antigen/adjuvant (son-ag), but not with conventional technique (syr-ag). We found highly elevated expression of MMP-8 (neutrophil collagenase) mRNA and protein in diseased son-ag challenged mice, colocalizing to neutrophil infiltrates found in brain and extensively in the spinal cord submeningeal space. MMP-8 expression has not been found previously in sensitive mouse strains. The infiltrates stained positive also for MMP-9 protein, and brain homogenates from corresponding mice showed MMP-9 activity during overt disease (days 12-16 post-immunization). TIMP-1 gene expression could be detected in CNS samples from diseased son-ag challenged mice but not in syr-ag or control mice, and the TIMP-1 protein colocalized with GFAP-staining. In contrast, in syr-ag mice both TIMP-2 and TIMP-3 gene expression in the spinal cords was elevated. The results show that sonication, but not extrusion, creates an adjuvant formula potent in activating the matrix metalloproteinase cascade similar to sensitive mouse strains, strongly implicating their role in EAE induction in this Th2 prone strain. The study provides the basis for establishment of MMP-specific therapy in this model.     

基质金属蛋白酶-2、金属蛋白酶组织抑制因子-2表达与滑膜肉瘤转移、肿瘤血管生成的关系及其预后意义

目的探讨滑膜肉瘤肿瘤细胞中基质金属蛋白酶-2（MMP-2）和金属蛋白酶组织抑制因子-2（TIMP-2）的表达情况及其预后意义。方法采用免疫组织化学SP法检测72例滑膜肉瘤肿瘤细胞中MMP-2、TIMP-2的表达情况,收集每例临床病理学参数并统计生存率、以CD31标记检测微血管密度（MVD）并分析与生存率的关系。结果（1）MMP-2、TIMP-2的表达阳性率分别为84．7％（61／72）和83．3％（60／72）,二者表达强度呈负相关关系（r=-0．290,P=0．013）。（2）有转移病例出现MMP-2高表达和TIMP-2低表达的比例明显高于无转移病例（分别P=0．010,P=0．002）。（3）MMP-2高表达病例的MVD明显大于其低表达者（P=0．005）,TIMP-2高表达病例的MVD明显小于其低表达者（P=0．048）。（4）单因素和多因素生存分析均显示TIMP-2低表达与患者预后不良有关（分别P=0．002,P=0．016）。结论MMP-2和TIMP-2表达异常可能与滑膜肉瘤的转移及肿瘤血管生成有关,TIMP-2低表达可能提示患者预后不良。     

In situ hybridization studies of metalloproteinases 2 and 9 and TIMP-1 and TIMP-2 expression in human prostate cancer

The expression of MMP-2, MMP-9, TIMP-1, TIMP-2, and the urokinase receptor were examined in fetal and normal prostate tissues, benign prostatic hyperplasia and prostate cancer (n=117). In situ hybridization with digoxigenin-labeled oligonucleotide probes demonstrated that TIMP-1 and TIMP-2 were expressed at elevated levels in the stroma of Gleason sum 5 tissues, whereas MMP-2 and MMP-9 were expressed at relatively low levels. In higher Gleason sum tissues (GS 8-10), TIMP-1 and TIMP-2 were not expressed, whereas MMP-2 and MMP-9 were intensely expressed. Furthermore, TIMP-1 and TIMP-2 expression was high in organ-confined specimens (OC, n=43), somewhat lower in specimens with capsular penetration (CP, n=29), and low or negative in samples with surgical margin/seminal vesicle (M/SV, n=17) and lymph node (LN, n=13) involvement. In contrast, MMP-2 and MMP-9 expression was low in the OC tissues; and noticeably higher in CP, M/SV, and LN specimens. Finally, correlation of TIMP and MMP expression with GS and pathological stage versus cure rate further revealed that a high percentage of organ-confined, GS 5 specimens expressing TIMP and little MMP were cured. In comparison, few of the GS 7-10 patients with capsular penetration and expressing MMP and little TIMP were cured. The data suggest that TIMP-1 (and TIMP-2) and MMP-2 (and MMP-9) are independent predictors of outcome.     

Interleukin-4 downregulates the cyclic tensile stress-induced matrix metalloproteinases-13 and cathepsin B expression by rat normal chondrocytes

Mechanical stress plays a key role in the pathogenesis of cartilage destruction seen in osteoarthritis (OA). We investigated the effect of cyclic tensile stress (CTS) on the anabolic and catabolic gene expression of rat cultured normal chondrocytes using the Flexercell strain unit. The effects of interleukin (IL)-4, a chondroprotective cytokine, on the changes in gene expression induced by CTS were also investigated. CTS (7% elongation at 0.5 Hz) for 24 h did not affect the expression of aggrecan and type II collagen, whereas CTS significantly upregulated matrix metalloproteinase (MMP)-13 and cathepsin B mRNA expression by chondrocytes. IL-1beta expression was also signifi cantly upregulated by CTS up to 12 h. The upregulation of MMP-13 was observed at 3 h, which was earlier than that of IL-1beta. Furthermore, pre-treatment with IL-4 (10 ng/ml) suppressed both MMP-13 and cathepsin B induction by mechanical stress, as well as CTS-induced IL-1beta expression. Our results suggest that IL-4 might have a therapeutic value in the treatment of OA by downregulation of mechanical stress-induced MMP-13 and cathepsin B expression by chondrocytes.