Stromelysin, gelatinase A and TIMP-1 in prosthetic interface tissue: a role for macrophages in tissue remodelling

Aseptic loosening of prosthetic components is the most important long-term complication of total joint replacement. To investigate the underlying destructive mechanisms, periprosthetic tissues from both well-fixed and loosened sites from six patients, undergoing surgery for aseptic loosening of knee or hip prostheses, were analysed in detail by immunohistochemical methods for the presence, of matrix metalloproteinases and tissue inhibitor of metalloproteinases-1 (TIMP-1). The tissues contained small numbers of cells positive for either collagenase, stromelysin, gelatinase A or TIMP-1; these were randomly distributed, neither specifically next to the bone interface nor to wear particles, and the number of positive cells did not correlate with macroscopic observations at operation. Gelatinase A was co-localized in cells with prolyl-4-hydroxylase, an enzyme involved in collagen synthesis. The predominant cell type in these tissues was shown to be the macrophage by the use of cell marker antibodies. Dual localization was not technically possible but the results strongly suggest that monocyte/macrophages were the primary source of gelatinase A and TIMP-1. Stromelysin was immunolocalized on connective tissue matrix in four patients, and gelatinase A in one patient, and were also observed in tissues in which there was no evidence of cellular synthesis of these enzymes. This suggests that secretion had taken place previously, resulting in enzyme bound to matrix for some time. Taken together, these data indicate that localized focal connective tissue remodelling occurs in periprosthetic tissues from both well fixed and loosened sites.     

Inhibitory activity on matrix metalloproteinases and cell-proliferating activity of tissue inhibitor of metalloproteinases-1 (TIMP-1)-contrastive difference between human and bovine TIMP-1s on mouse cell proliferation

Either human or bovine TIMP-1 inhibited matrix metalloproteinase (MMP) activities, such as collagenolytic, gelatinolytic and caseinolytic, expressed by mouse cells as well as those expressed by human cells. Bovine TIMP-1 stimulated the proliferation of both human and mouse cells, but human TIMP-1 only proliferate human cells and not mouse cells indicating that the cell-proliferating activity has more strict species specificity than MMP inhibitory activity. All the results from [(3)H]thymidine incorporation, the phosphorylation of tyrosine-containing cellular proteins and extracellular-signal-regulated protein kinases supported the cell-proliferating properties of both human and bovine TIMP-1s. Human TIMP-1 seems not to bind to TIMP-1 receptors on mouse cells.     

Matrix metalloproteinases MMP-7, MMP-9 and their tissue inhibitor TIMP-1: expression in chronic sinusitis vs nasal polyposis

BACKGROUND: Nasal polyps (NP) are characterized by pseudocyst formation, whereas the mucosa in chronic sinusitis (CS) only shows a limited oedema. Matrix metalloproteinases (MMPs) are a family of endopeptidases able to degrade the extracellular matrix. Differences in histological features between CS and NP might be related to the respective expression of MMPs and their tissue inhibitors (TIMPs). OBJECTIVE: The aim of this study was to investigate MMP-7, MMP-9 and TIMP-1 proteins in NP and CS in comparison with normal mucosa. METHODS: Nasal samples, obtained from controls (n = 10), from NP (n = 8) and from CS (n = 10), were analysed by immunohistochemistry and enzyme-linked immunosorbent assay (ELISA). RESULTS: In NP, compared with controls, staining for MMP-9 and MMP-7 appeared in blood vessels. Matrix metalloproteinase-9-positive inflammatory cells could be detected in increased numbers in pseudocyst formations. Concentrations of MMP-9 protein was found significantly increased in both CS and NP compared with controls, while MMP-7 was significantly increased in NP compared with controls and CS. Tissue inhibitors of metalloproteinase-1 protein was significantly increased in CS and NP when compared with controls. CONCLUSIONS: Chronic sinusitis and NP show different pattern of MMP-7/-9 and TIMP-1 expression. We suggest that this difference in regulation of enzymes is related to the respective tissue remodelling observed in both diseases.     

Role of matrix metalloproteinases and their tissue inhibitor of metalloproteinases in myxomatous change of cardiac floppy valves

To clarify the underlying cause of myxomatous changes in cardiac floppy valves, the expression of the matrix metalloproteinases (MMP) and the tissue inhibitors of metalloproteinases (TIMP) was investigated in cardiac valves. Valves were obtained from nine patients with floppy valves, from 13 patients with other valvular disease types, and from four patients with normal valves. Immunohistochemical analyses for MMP-2, MMP-9, TIMP-1, and TIMP-2, and gelatin zymography for MMP-2 and MMP-9 were performed. Compared with the spongiosa of normal valves, the myxomatous area of floppy valves had stronger immunohistochemical reaction to MMP-2 and MMP-9, and weaker reaction to TIMP-2. Activated MMP-2 and MMP-9 were detected in eight out of nine cases of floppy valves. Activated MMP-2 was detected at low levels in two cases of normal valves showing mild expansion of the spongiosa without macroscopic floppiness. The ratio of active/total MMP-2 and MMP-9 increased in floppy valves compared with normal valves. These results suggest that the imbalance between MMP and TIMP and the increased activity of MMP-2 and MMP-9 may correlate with myxomatous changes observed in floppy valves. Valves with a slight myxomatous change and activated MMP-2 may develop into floppy valves with increases in the activity of MMP.     

Regulation of matrix metalloproteinases and their inhibitor genes in lipopolysaccharide-induced endotoxemia in mice

An imbalance between matrix metalloproteinases (MMPs) and inhibitors of MMPs (TIMPs) may contribute to tissue destruction that is found in various inflammatory disorders. To determine in an in vivo experimental setting whether the inflammatory reaction in the course of lipopolysaccharide (LPS)-induced endotoxemia causes an altered balance in the MMP/TIMP system, we analyzed the expression of a number of MMP and TIMP genes as well as MMP enzymatic activity in the liver, kidney, spleen, and brain at various time points after systemic injection of different doses of LPS in mice. Injection of sublethal doses of LPS led to an organ- and time-specific pattern of up-regulation of several MMP genes and the TIMP-1 gene in the liver, spleen, and kidney, whereas in the brain only TIMP-1 was induced. Injection of a lethal dose of LPS caused similar but more prolonged expression of these MMP genes as well as the induction of additional MMP genes in all organs. In LPS-treated mice in situ hybridization revealed collagenase 3 gene induction in cells resembling macrophages whereas TIMP-1 RNA was detected predominantly in parenchymal cells. Finally, gelatin zymography revealed increased gelatinolytic activity in all organs after LPS treatment. These observations highlight a dramatic shift in favor of increased expression of the MMP genes over the TIMP genes during LPS-induced endotoxemia, and suggest that MMPs may contribute to the development of organ damage in endotoxemia.     

Expression of matrix metalloproteinases and their inhibitors in human brain tumors

Sixty human brain tumors, classified according to the New World Health Organization (WHO) classification including, grade I schwannomas, meningiomas and pilocytic astrocytomas, grade II astrocytomas, grade III anaplastic astrocytomas, grade IV glioblastomas, grade III anaplastic oligodendrogliomas and grade IV glioblastomas and lung and melanoma metastases were analyzed for the expression of three matrix metalloproteinases (MMPs), two tissue inhibitors of MMPs (TIMPs) and for MMP activity. Some correlation was found between MMP expression and the degree of malignancy. Western blotting analysis revealed a more uniform pattern of distribution of MMP-2 (gelatinase A) than of MMP-9 (gelatinase B) and MMP-12 (metalloelastase) among tumors. MMP-9 levels were found to be significantly higher in grade III anaplastic astrocytomas and anaplastic oligodendrogliomas than those in grade I schwannomas and meningiomas. Anaplastic astrocytomas and Grade IV glioblastomas expressed significantly higher levels MMP-12 than grade I meningiomas. All sixty tumors showed a similar pattern of activity in zymography, proMMP-9 being the major species detected. Interestingly, TIMP-1 and TIMP-2 expression levels were especially low in tumors of grade II and grade III but significantly higher in tumors of grade I, particularly in schwannomas. Taken together, these data suggest that: 1) a balance between MMPs and TIMPs has an important role to play in human brain tumors; 2) TIMP expression may be valuable markers for tumor malignancy. This revised version was published online in July 2006 with corrections to the Cover Date.     

柚皮甙干预鼠颅顶骨破骨细胞的形成及功能

背景：柚皮甙能诱导骨形态发生蛋白2的基因表达,促进成骨细胞系的增殖和分化。体外细胞实验提示柚皮甙有抑制破骨细胞的形成及抗骨质疏松的作用。 目的：建立含有柚皮甙的鼠颅顶骨培养模型,观察不同剂量下柚皮甙对破骨细胞增殖分化的影响。 方法：实验选取出生4 d的SD乳鼠颅顶骨,在分别含有0,1,10,100 mg/L 的柚皮甙的培养基中培养,于培养的第1,3,7,10天测定柚皮甙对鼠颅顶骨中破骨细胞标志酶抗酒石酸酸性磷酸酶阳性细胞数及培养基中钙离子浓度的影响。 结果与结论：第1天各组间抗酒石酸酸性磷酸酶阳性细胞数及培养基中钙离子浓度无明显变化,第3,7天各组间抗酒石酸酸性磷酸酶阳性细胞数随柚皮甙质量浓度增加而减少,而培养基中钙离子浓度变化明显增加,到第10天这种变化趋势最为明显。说明柚皮甙能影响鼠颅顶骨中抗酒石酸酸性磷酸酶阳性细胞数量及其功能,并且这种作用成明显的时间剂量相关性。提示柚皮甙不仅能促进成骨细胞的增殖,同时也能减少破骨细胞的分化或加速其凋亡。     

1, 25二羟基胆钙化醇诱导大鼠骨髓单核细胞向破骨细胞转化的机制

目的:探讨1,25二羟基胆钙化醇(1,25(OH)2D3)诱导大鼠骨髓单核细胞向破骨细胞转化时明胶酶表达及其参与骨陷窝形成机制。方法:分离乳鼠骨髓内细胞,诱导生成破骨样细胞。姬姆莎、抗酒石酸酸性磷酸酶(TRAP)染色鉴定。扫描电镜观察诱导出的细胞贴附于骨片上形成的骨陷窝,明胶酶谱检测细胞培养液中明胶酶表达水平。结果:单核细胞经1,25(OH)2D3诱导,第9日生成大量的破骨样细胞。姬姆莎染色显示出多核(≥3个),TRAP染色阳性,扫描电镜观察破骨细胞在骨片上培养时产生骨陷窝。1,25(OH)2D3组基质金属蛋白酶2(MMP-2)表达水平增加显著。结论:1,25(OH)2D3诱导单核细胞产生大量破骨细胞。参与骨陷窝形成的明胶酶是MMP-2。这可能是破骨细胞通过某些机制,促进其他非破骨细胞分泌有活性的MMP-2参与噬骨。     

Therapeutic ionizing radiation induced bone loss: a review of in vivo and in vitro findings

Radiation therapy is one of the routine treatment modalities for cancer patients. Ionizing radiation (IR) can induce bone loss, and consequently increases the risk of fractures with delayed and nonunion of the bone in the cancer patients who receive radiotherapy. The orchestrated bone remodeling can be disrupted due to the affected behaviors of bone cells, including bone mesenchymal stem cells (BMSCs), osteoblasts and osteoclasts. BMSCs and osteoblasts are relatively radioresistant compared with osteoclasts and its progenitors. Owing to different radiosensitivities of bone cells, unbalanced bone remodeling caused by IR is closely associated with the dose absorbed. For doses less than 2 Gy, osteoclastogenesis and adipogenesis by BMSCs are enhanced, while there are limited effects on osteoblasts. High doses (>10 Gy) induce disrupted architecture of bone, which is usually related to decreased osteogenic potential. In this review, studies elucidating the biological effects of IR on bone cells (BMSCs, osteoblasts and osteoclasts) are summarized. Several potential preventions and therapies are also proposed.     

Distribution patterns of matrix metalloproteinase (MMP)-2 and -9 and their inhibitors (TIMP-1 and TIMP-2) in the human decidua during early pregnancy

Early human trophoblast shows dramatic invasive properties during early pregnancy. A tightly-regulated activation of matrix metalloproteinases (MMPs) is considered to be of critical importance for the control of trophoblast invasion. The aim of the present study was to determine MMP-2, MMP-9, TIMP-1 and TIMP-2 protein expression in decidual endometrium during the first trimester of pregnancy (22-42 days post coitus) with special attention to their expression patterns in endometrial compartments. Cytokeratin staining applied to adjacent sections was used to identify epithelial and trophoblast cells. We observed that MMP-2, particularly in the fourth week, appeared to be expressed more strongly in extravillous trophoblasts (EVTs) and vascular endothelial cells in the first trimester of pregnancy. Therefore, MMP-2 is likely to be the primary mediator in invasion of the trophoblast into the decidual endometrium, as well as in vascular remodeling and angiogenesis in the first trimester of pregnancy. The high expression of TIMP-1 and TIMP-2 in EVTs and glandular epithelium suggests that a restricted and balanced expression of these molecules is important for matrix remodeling and controlled trophoblast invasion during placentation. We conclude that (1) MMP-2 and MMP-9 and their inhibitors TIMP-1, and TIMP-2 determine the invasive behavior of trophoblast into the endometrium, and in particular, (2) MMP-2 may be the key regulator of trophoblast invasion in early human pregnancy.     

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.     

Immunolocalisation studies of matrix metalloproteinases-1, -2 and -3 in human melanoma

The matrix metalloproteinases (MMPs) are considered to have an important role in connective tissue degradation and have been implicated in the mechanisms of tumour invasion and metastatic spread. We have used immunohistochemistry to examine and compare the tissue distributions of collagenase-1 (MMP-1), gelatinase A (MMP-2) and stromelysin-1 (MMP-3) in 18 specimens of malignant melanoma, viz. 10 superficial spreading and 8 nodular melanomas. MMPs-1, -2 and -3 were demonstrated within melanoma and host tissue cells, especially at the periphery of some tumours, but were usually restricted to less than 10% of total melanoma cells. The MMPs were absent from ’normal’ skin tissue distant from the tumour. MMP-2 was localised to discrete groups of cells and was especially evident at the epidermal:tumour interface, whereas MMP-3 was mainly confined to the deeper margins of melanoma. No regular pattern of MMP expression was observed for either the superficial spreading or the nodular melanomas. The variable distributions of the MMPs suggested that enzyme expression was subject to local microenvironmental regulation, possibly in response to matrix components and the cellular heterogeneity observed at the tumour margins. These in situ observations add weight to the concept that specific MMPs contribute to the mechanisms of tumour invasion. Received: 10 March 1999 / Accepted: 6 July 1999     

The role of sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) in regulation of osteoclastic and osteoblastic cells

The bioactive lipid molecules, sphingosine-1-phosphate (S1P) and lysophosphatidic acid LPA) have recently emerged as potentially highly significant physiological and pathophysiological regulators of bone cell biology. Since compromised signaling by these bioactive lipids has been implicated in the etiology of disorders such as inflammatory and autoimmune diseases, their role in bone biology can be a key influence in the coordination of the events underlying osteoimmunology. Both S1P and LPA have been shown to have receptor-mediated effects on osteoblastic cell proliferation and differentiation critical to bone formation and on osteoclastic cell action and regulation of bone resorption. This review of the recent studies on these processes provides insight into the potential role of S1P and LPA as autocrine and paracrine mediators of bone remodeling and their potential interaction with immune cells that have emerged as important players in skeletal biology.     

Down-regulation of tissue inhibitor of matrix metalloprotease-1 (TIMP-1) in aged human skin contributes to matrix degradation and impaired cell growth and survival

Up regulation of matrix metalloproteinases (MMPs), particularly collagenase-1 (MMP-1), stromelysin-1 (MMP-3) and gelatinase A (MMP-2) is responsible for the lysis of dermal collagen and elastin fibers during chronological skin aging. Tissue inhibitor of metalloproteinase-1 (TIMP-1) is one representative of the natural MMP inhibitor family, encompassing four members. Its expression is decreased with fibroblast senescence, both ex vivo and in vivo, thus contributing to increased catabolic activity within dermis. TIMP-1 displays multiple biological functions. It inhibits most MMPs, except membrane-type MMP subfamily, with Ki in the subnanomolar range, but also interacts with the hemopexin-like (PEX) domain of pro MMP-9. Besides, it exhibits keratinocyte and fibroblast growth factor-like activity and has been described as a cell survival factor.     

骨骼生长发育中生长激素释放肽的重要作用

文题释义：生长激素释放肽(Ghrelin)：也被称为饥饿素及内源性脑肠肽,20世纪80年代人们通过从大鼠胃组织中分离提取出一种多肽类激素,发现其具有促进生长激素分泌而被命名为生长激素释放肽。骨代谢：骨的功能是为肌肉收缩提供附着处及保护内脏等重要的生命器官。一般认为骨在细胞水平上是不活跃的,事实上骨的细胞在不停地进行着细胞代谢,不仅骨的细胞之间会相互作用,还存在骨髓中的红细胞生成细胞、基质细胞相互作用,以进行骨的改建和重建。背景：生长激素释放肽具有促进生长激素分泌、调节机体能量代谢平衡及一些尚在探索的药理学特性,这些都与机体代谢状态密切相关。目的：就生长激素释放肽对于骨骼细胞功能的调节及其在骨代谢和能量代谢中的整合作用做一综述,以期阐明生长激素释放肽在骨骼发育过程中发挥的重要作用。方法：检索 PubMed数据库1993年1月至2017年1月相关文献,检索词为“ghrelin,Bone metabolism,leptin,osteoblast,osteoclast,castric resection,cartilage cells”。排除重复研究及与综述内容关系不密切的文献,对最终纳入的74篇文献进行分析。结果与结论：机体骨骼系统的重塑是一个高度耗能的过程,与之相应的是,骨骼的生长发育涉及到外周和中枢的能量代谢,包括交感神经系统和瘦素等激素的作用。国外研究表明,生长激素释放肽能够调节成骨细胞的分化和功能,诱导骨髓干细胞的增殖及分化,通过不同的方式调节生长激素-胰岛素轴的状态,并能够通过与瘦素相作用来调配骨骼代谢与机体能量代谢从而影响骨骼的生长状态。ORCID: 0000-0003-1053-8093(叶楠)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     

Expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in human chondrosarcomas

The aim of the present study was to characterise the ability of malignant chondrosarcomas to invade normal bone by analysing their production of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). For this purpose 12 chondrosarcomas were investigated for the expression of mRNAs for several MMPs and all 4 TIMPs by Northern hybridisation, and for immunohistochemical localisation of the proteins. A characteristic finding of these analyses was increased expression of MMP-13, MMP-14 and TIMP-2 mRNAs in chondrosarcomas when compared with nonmalignant control samples. Individual chondrosarcomas also exhibited elevated levels of MMP-1, MMP-7 and MMP-9 mRNAs. The results of Northern hybridisations were supported by immunohistochemical stainings of the corresponding tumour areas for MMP-2, MMP-14 and TIMP-2, further suggesting that these may have prognostic value for determining whether individual chondrosarcomas are locally aggressive or have a probability of recurrence. Another finding of the present study was a marked heterogeneity in histologic appearance and gene expression of the chondrosarcomas, emphasising the importance of analysing several areas of these tumours to get representative results. These findings suggest that analysis of MMPs could be a useful diagnostic indicator in patients with cartilaginous tumours and could help in differentiating between a low-grade malignant chondrosarcoma and a benign growing enchondroma.     

基质金属蛋白酶及其组织抑制因子在皮肤增生性瘢痕中的作用

目的探讨基质金属蛋白酶（MMPs）MMP2、MMP9及其抑制物（TIMPs）TIMP-1在皮肤增生性瘢痕中的作用。方法取3-6个月、6-12个月皮肤增生性瘢痕，以正常皮肤组织为对照，ELISA方法测定其MMP2、MMP9以及TIMP-1的含量，采用SPSS统计软件，以成组设计的单因素方差分析，分析它们在不同时段瘢痕组织中变化的意义。结果（1）3-6月瘢痕组织和6-12月瘢痕组织的MMP2，均较正常皮肤显著增高（110.70±5.23ng／ml VS 54.59±3.01，P〈0.01；77.23±7.10ng／ml VS 54.59±3.01，P〈0.01），而3-6月瘢痕组织的MMP2较6-12月瘢痕组织的MMP2亦有显著增高（110.70±5.23 VS 77．23±7．10，P〈0．01）；3-6月瘢痕组织和6-12月瘢痕组织的MMP9之间（3.85±0.88 VS 3.61±0.43，P〉0.05）以及它们与正常皮肤组织的MMP9相比（3.85±0.88 VS 4.13±0.33，P〉0.05；3.61±0.43 VS 4.13±0.33，P〉0.05）均无显著性差异；（2）3-6月瘢痕组织和6-12月瘢痕组织的TIMP-1与正常皮肤组织相比有显著增高（4．74±0．35 VS 3．01±0．11，P〈0．01；5．12±0．34 VS 3.01±0．11，P〈0．01），6-12月瘢痕组织较3-6月瘢痕组织的TIMP-1有显著增高（5.12±0.34 VS 4.74±0．35，P〈0．05）；（3）3-6月瘢痕组织和6-12月瘢痕组织的MMP2与TIMP-1的比值与正常皮肤组织相比均有显著增高（23．38±1．01 VS 18．15±0．58，P〈0.01；15.10±1.45 VS 18．15±0．58，P〈0．01），3-6月瘢痕组织较6-12月瘢痕组织的MMP2与TIMP-1的比值有显著降低（23.38±1.01 VS 15.10±1.45，P〈0.01）。结论MMPs以及TIMPs参与了皮肤瘢痕的增生过程，MMP2、TIMP-1含量及其比值可作为瘢痕生长和预后的指标。