Inhibition of the activities of matrix metalloproteinases 2, 8, and 9 by chlorhexidine

Matrix metalloproteinases (MMPs) are a host cell-derived proteolytic enzyme family which plays a major role in tissue-destructive inflammatory diseases such as periodontitis. The aim of the present study was to evaluate the inhibitory effect of chlorhexidine (CHX) on MMP-2 (gelatinase A), MMP-9 (gelatinase B), and MMP-8 (collagenase 2) activity. Heat-denatured type I collagen (gelatin) was incubated with pure human MMP-2 or -9 activated with p-aminophenylmercuric acetate (APMA), and the proteolytic degradation of gelatin was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie blue staining. The effect of CHX on MMP-8 activity was also studied with a cellular model addressing the ability of phorbol myristate acetate (PMA)-triggered human peripheral blood neutrophils (polymorphonuclear leukocytes [PMNs]) to degrade native type I collagen. CHX inhibited the activities of both gelatinases (A and B), but MMP-2 appeared to be more sensitive than MMP-9. Adding calcium chloride to the assay mixtures almost completely prevented the inhibition of MMP-9 activity by CHX, while the inhibition of MMP-2 activity could be reversed only when CHX was used at a low concentration. This observation suggests that CHX may act via a cation-chelating mechanism. CHX dose-dependently inhibited collagenolytic activity of MMP-8 released by PMA-triggered PMNs. MMP-8 without APMA activation was inhibited clearly more efficiently than APMA-activated MMP-8. Our study suggests that the direct inhibition of the MMPs' activities by CHX may represent a new valuable effect of this antimicrobial agent and explains, at least in part, the beneficial effects of CHX in the treatment of periodontitis.     

11-羰基-β-乙酰乳香酸（AKBA）调节基质金属蛋白酶-1,-2,-9的活性(英文)

目的：降低创面的高MMPs活性是治疗慢性皮肤溃疡的新途径。本研究主要观察11-羰基-β-乙酰乳香酸（AKBA,中药乳香的一种活性成分）对MMP-1、MMP-2和MMP-9活性的调节作用。方法： 人间质胶原酶（MMP-1）或者明胶酶A（MMP-2）被醋酸氨基苯汞（p-aminophenylmercuric acetate, APMA)激活后,与不同浓度的AKBA共同孵育1h,通过底物裂解法观察其活性的改变。MMP-9在中性粒细胞（polymorphonuclear neutrophils,PMNs)中含量丰富,因此以大鼠腹腔PMN作为MMP-9的来源。PMN裂解产物与不同浓度的AKBA共同孵育1 h,通过明胶酶谱法观察其中MMP-9活性的改变。我们建立了3个细胞模型：由TNF-α活化的人皮肤成纤维细胞模型;PMA活化的THP-1细胞模型和成纤维细胞-THP-1共培养细胞模型。AKBA与这3个细胞模型共同孵育24 h后,用ELISA法检测细胞上清中MMP-1、MMP-2和MMP-9的含量,用明胶酶谱法检测细胞上清中 MMP-2、MMP-9的活性。结果： AKBA在0.1-0.8 mmol/L浓度范围内对MMP-1、MMP-2的活性有抑制作用,IC50分别为0.18 mmol/L和0.27 mmol/L;在0.05-0.85mmol/L浓度范围内对MMP-9活性表现出不同程度的抑制作用（P<0.01）,其抑制作用呈浓度依赖性。AKBA促进成纤维细胞分泌MMP-2,但是,对THP-1细胞分泌MMP-9表现出抑制作用。在共培养细胞模型中,AKBA对MMP-1、MMP-2和MMP-9的分泌均表现出抑制作用。结论： AKBA作为乳香的一种活性成分,它对MMPs活性的直接抑制作用和对MMPs分泌的抑制作用可能是中药乳香治疗慢性皮肤溃疡的机制之一。     

Molecular mechanism for connective tissue destruction by dipeptidyl aminopeptidase IV produced by the periodontal pathogen Porphyromonas gingivalis

Porphyromonas gingivalis is a pathogen associated with adult periodontitis. It produces dipeptidyl aminopeptidase IV (DPPIV), which may act as a virulence factor by contributing to the degradation of connective tissue. We investigated the molecular mechanism by which DPPIV contributes to the destruction of connective tissue. DPPIV itself did not show gelatinase or collagenase activity toward human type I collagen, but it promoted the activity of the host-derived matrix metalloproteinase 2 (MMP-2) (gelatinase) and MMP-1 (collagenase). DPPIV bound to fibronectin and mediated the adhesion of P. gingivalis to fibronectin. Mutant DPPIV with catalytic Ser mutagenized to Ala (DPPSA) did not accelerate the degradation of collagen and gelatin by MMPs but retained fibronectin-binding activity. The adhesion of human gingival fibroblasts and NIH 3T3 cells to fibronectin was inhibited by DPPIV. Strain 4351ADPPSA exhibited an intermediate level of virulence in mice, between that of the strain expressing wild-type DPPIV (4351ADPP) and that of the strain harboring only the plasmid vector (4351AVEC). It is suggested that both activity promoting the degradation of collagen and gelatin and binding to fibronectin are required for full virulence. These results reveal novel biological functions of DPPIV and suggest a pathological role in the progression of periodontitis.     

92-kd type IV collagenase (matrix metalloproteinase-9) activity in human amniochorion increases with labor.

To determine whether specific collagenolytic enzymes are expressed in human fetal membranes with labor, we examined gelatinase activity in extracts of amniochorion by zymography. The 92-kd gelatinase (MMP-9) was barely detectable in extracts of fetal membranes before the onset of labor but was readily demonstrable in extracts prepared from membranes isolated from laboring women or membranes collected immediately after delivery. In contrast, the 72-kd gelatinase (MMP-2) was detectable in extracts from pre- and post-labor membranes. Ethylenediaminetetracetic acid and the tissue inhibitor of metalloproteinases, TIMP-1, inhibited the gelatinase activities detected by zymography, confirming that the enzymes are metalloproteinase. Assay of amniochorion gelatinase activity using a radiolabeled denatured collagen substrate revealed a more than twofold increase in activity comparing pre-labor with post-labor fetal membrane extracts. A function-blocking anti-MMP-9 monoclonal antibody inhibited pre-labor membrane gelatinase activity by approximately 11.5%, which was only slightly greater inhibition than observed with irrelevant monoclonal antibodies. However, post-labor membrane gelatinase activity was reduced by 53% by the function-blocking antibody, indicating that MMP-9 is a major contributor to the increased gelatinase activity extractable from post-labor membranes. Western blot analyses demonstrated increased MMP-9 protein in amniochorion extracts after onset of labor. MMP-9 protein and mRNA were co-localized in amnion epithelium, underlying macrophages and chorion laeve trophoblast and decidual cells after labor. We conclude that 1) MMP-9 activity and protein in human amniochorion increases with labor and 2) MMP-9 is expressed by amnion epithelium, macrophages and chorion laeve trophoblast and decidual cells. The increased expression of MMP-9 may result in degradation of the extracellular matrix of the fetal membranes and facilitate their rupture under both physiological and pathological conditions.     

MRL/lpr狼疮小鼠肾脏明胶酶表达随增龄变化及意义

目的 观察不同周龄MBL/lpr狼疮小鼠肾脏明胶酶的表达及其随增龄而发生的活性变化及意义。方法 取8、16与24周龄RMRL/lpr狼疮小鼠的肾组织进行常规病理检测。利用含有放射自显影的成像乳胶对冷冻肾组织切片进行原位明胶酶活性的检测;利用免疫组织化学检测肾脏明胶酶A与明胶酶B的酶B的蛋白表达变化,十二氨基磺酸钠-聚丙烯酰胺凝胶电泳（SDS-PACE）明胶酶谱法检测肾脏明胶酶A、B的活性变化。结果 8周龄狼疮小鼠肾组织吵仅在血管处检测到明胶酶的活性;16与24周龄狼疮小鼠肾小球内明胶酶的净活性明显增加。在肾小球以及肾小管间质上也可检测到明胶酶的活性,乙二胺四乙酸（EDTA）能够抑制肾脏明胶酶的活性,免疫组织化学与SDS-PAGE明胶酶谱法结果显示其肾组织中明胶酶A与明胶酶B的蛋白质表达及活性均明显增加。结论 明胶酶A、B在自发性狼疮小鼠肾炎中的表达及活化随增龄均明显增加,活化态的明胶酶可能在狼疮性肾炎细胞外基质重塑中发挥重要的作用。     

Sequential degradation of interstitial collagen by metalloproteinases extracted from tumors of murine ascites hepatomas

Gelatinolytic and collagenolytic proteinases were separately isolated by different extraction methods from the mouse ascites hepatoma MH134, and from rat ascites hepatoma AH109A. The activities of two proteinases in each extract showed no significant differences, but after trypsin activation the activities of proteinases from the highly metastic MH134 were significantly increased compared to the emzyme activities in AH109A, which has low metastatic potential. The total activities of collagenase and gelatinase were increased 7.2- and 5.1-fold; their specific activities were increased 5.2-and 4.8-fold, respectively. Gelatinase and collagenase from MH134 were characterized on gelatin zymography. The gelatinase had a molecular weight of 99 and activation by 4-aminophenylmercuric acetate (APMA) or trypsin resulted in its conversion to 79 or 79–95 kD, respectively. The collagenase revealed a major gelatinolytic band at 89 kD, which was converted to 85 and 70 kD by APMA-activation, and a minor gelatinolytic band at 60 kD. These proteinases could degrade native type I collagen to small fragments in a cooperative manner. Trypsin inhibitor, which affects the trypsin activation of latent gelatinase, was extracted together with gelatinase. The inhibitory activity of the enzyme from AH109A showed a 4.1-fold higher specific activity and 3.7-fold greater total activity than that from MH134. The proteinase(s) capable of activating the gelatinase was also extracted from MH134.     

Focal expression and final activity of matrix metalloproteinases may explain irregular dysfunctional endometrial bleeding

Irregular dysfunctional bleeding of the endometrium (ie, metrorrhagia without organic lesion) is common in women, whether treated or not with ovarian hormones. Several matrix metalloproteinases (MMPs) become normally expressed and/or activated at menstruation and cause extracellular matrix breakdown. We therefore explored whether episodes of irregular dysfunctional bleeding could be associated with untimely MMP activity. By histology, foci of stromal breakdown were exclusively found in the endometrium of metrorrhagic women at bleeding. In these foci, 1) expression of estrogen receptor-alpha and progesterone receptor was altered; 2) collagenase-1 (MMP-1), stromelysin-1 (MMP-3), and gelatinase B (MMP-9) became detected in stromal cells, together with MMP-9 in neutrophils; and 3) gelatinase A (MMP-2) was more expressed and immunolocalized at the membrane of stromal cells. By biochemistry, endometrial lysates from nonbleeding metrorrhagic patients contained more latent and active MMP-2 and -9 than age-matched controls; at bleeding, collagenase activity, MMP-9, and active MMP-2 were strikingly increased whereas tissue inhibitor of metalloproteinases-1 (TIMP-1) was considerably decreased. As a functional assay, in situ gelatin zymography revealed large areas of gelatinolytic activity only in endometrium of bleeding patients. Altogether, these results strongly suggest that inappropriate focal expression and activation of several MMPs, combined with decreased inhibition, trigger irregular dysfunctional endometrial bleeding.     

Enhanced activation of matrix metalloproteinase-9 correlates with the degree of papillary thyroid carcinoma infiltration

Aim

To determine whether matrix metalloproteinase-9 (MMP-9) may be a useful adjunctive tool for predicting unfavorable biological behavior of papillary thyroid carcinoma (PTC) by evaluating the expression profile and proteolytic activity of MMP-9 in PTC by different techniques and correlating the findings with clinicopathological prognostic factors.

Methods

Immunohistochemical localization of MMP-9 was analyzed with antibodies specific for either total or active MMP-9. Activation ratios of MMP-9 were calculated by quantifying gel zymography bands. Enzymatic activity of MMP-9 was localized by in situ zymography after inhibiting MMP-2 activity.

Results

Immunostaining of total and active MMP-9 was observed in tumor tissue and occasionally in non-neoplastic epithelium. Only active MMP-9 was significantly associated with extrathyroid invasion, lymph-node metastasis, and the degree of tumor infiltration (P < 0.001, P = 0.004, and P < 0.001, respectively). Gelatin zymography revealed a correlation between the MMP-9 activation ratio and nodal involvement, extrathyroid invasion, and the degree of tumor infiltration. In situ zymography showed that gelatinases exerted their activity in tumor parenchymal and stromal cells. Moreover, after application of MMP-2 inhibitor, the remaining gelatinase activity, corresponding to MMP-9, was highest in cancers with the most advanced degree of tumor infiltration.

Conclusions

This is the first report suggesting that the evaluation of active MMP-9 by immunohistochemistry and determination of its activation ratio by gelatin zymography may be a useful adjunct to the known clinicopathological factors in predicting tumor behavior. Most important, in situ zimography with an MMP-2 inhibitor for the first time demonstrated a strong impact of MMP-9 activity on the degree of tumor infiltration during PTC progression.Papillary thyroid carcinoma (PTC) is the most common malignancy of the thyroid, with a rapid global rise in incidence in the recent decades. Despite its generally indolent behavior, a small proportion of PTC patients develop more aggressive forms of the disease. Several clinical and histopathological parameters, such as sex, age, histologic grade, and tumor stage have been reported as useful in improving prognostic stratification (1). However, the prognosis of tumor behavior for individual patients with thyroid cancer can vary greatly, partly due to the marked clinical heterogeneity among such patients, even within a particular histologic group. Therefore, it is important to understand the molecular mechanisms responsible for cancer development, progression, and metastasis, and to establish novel strategies for predicting biological behavior of thyroid cancer and for clinical management of patients.In cancer research, much interest has been devoted to alterations in expression grade and activity of various matrix metalloproteinases (MMPs) and their corresponding inhibitors (2). MMPs are a family of zinc-dependent endopeptidases with the capacity to degrade extracellular matrix proteins and basement membranes (3), and are therefore strongly implicated in multiple stages of cancer progression. Among the MMPs, a subset called gelatinases, consisting of MMP-2 (gelatinase A) and MMP-9 (gelatinase B), has gained the most attention in studies on the acquisition of invasive and metastatic tumor properties, as they degrade collagen IV, the major component of the basement membrane (4,5). Their proteolytic activity is regulated at various levels, including expression, balance between amounts of enzymes and their inhibitors, pericellular localization, and most importantly, latent form activation. Namely, gelatinases are secreted as inactive proenzymes and are activated after cleavage of the pro-peptide domain of the molecule (6). MMP-9 is of special interest because its basal expression is normally low, whereas it is highly expressed in most human cancers in response to various growth factors and cytokines (7,8). It has been shown that MMP-9-deficient mice exhibit impaired metastasis formation and tumor growth (9). In this respect, up-regulation of MMP-9 expression in various types of human cancers contributes to tumor progression, invasion, and metastasis (10-13).There is much evidence demonstrating that MMP-9 is overexpressed in various tumor types when compared to normal tissue (14-16). On the other hand, this protein has not been widely investigated in thyroid tumors and there are still some controversies regarding its usefulness as a marker for diagnosis or prognosis of these tumors (17-21). Moreover, the ratio of active-to-total MMP-9 and the precise localization of enzymatic activity in thyroid tissue have not yet been investigated. To localize MMP-9 expression in PTC and corresponding non-tumor tissue, we used two commercial antibodies that recognize either both pro-active and active or only active form of MMP-9 for immunohistochemistry. To our knowledge, active form of MMP-9 has not been immunohistochemically evaluated on thyroid tissue sections previously. We also used gelatin zymography, which is a sensitive, quantifiable assay to analyze pro-active and active form of MMP-9, and sensitive dye-quenched gelatin (DQ-gelatin) in situ zymography with a selective MMP-2 inhibitor to localize the gelatinase activity corresponding to MMP-9 in tissue. Thus, we explored the expression profiles, activation ratio, and localization of MMP-9 activity in tissue sections of papillary thyroid carcinomas and correlated the findings with clinicopathological prognostic factors with the aim of determining whether MMP-9 may be a useful adjunctive tool for predicting unfavorable biological behavior of PTC.     

Association of matrix metalloproteinase-9 in eosinophilic meningitis of BALB/c mice caused by Angiostrongylus cantonensis

Induction of gelatinase in eosinophilic meningitis of BALB/c-strain mice was caused by Angiostrongylus cantonensis. Time-course studies showed that the molecular weight of 94-kDa gelatinase was detected at day 10 post-inoculation (PI), and reached a high intensity from days 15 to 25 PI. The 94-kDa gelatinase activity was clearly inhibited by EDTA and 1,10-phenanthroline, but not by leupeptin and phenylmethanesulphonyl fluoride. When immunoblots were performed using specific antiserums against the 94-kDa gelatinase B (matrix metalloproteinase-9; MMP-9) with cerebrospinal fluid (CSF), the 94-kDa immunopositive band was MMP-9. Immunohistochemistry studies demonstrated MMP-9 localisation within eosinophils and macrophages. The increased MMP-9 activity was closely associated with the rapid rise of CSF eosinophils, and the inflammatory reaction of the subarachnoid space. In contrast to changes in MMP-9, MMP-2 activity was constitutive and unaffected in this parasitic meningitis. These results show that MMP-9 was associated with eosinophilic meningitis, and that the enzyme may be a useful marker for angiostrongyliasis meningitis.     

Inhibition of human gelatinases by metals released from dental amalgam.

The interaction between metal ions and the oral environment is a major subject matter in dental research. Matrix metalloproteinases (MMPs) have been implicated in pathologic oral processes such as periodontal tissue destruction, root caries, tumor invasion and temporomandibular joint disorders. The aim of this study was to test the effect of metal ions released from dental amalgam on the major gingival gelatinolytic MMPs. Gingival human explants were cultured overnight in DMEM and the activity of secreted enzymes was analyzed by gelatin zymography in buffers conditioned with dispersed phase or concentional phase dental amalgams. The major enzymes present in conditioned media were characterized as MMP-2 and MMP-9 by immunoprecipitation. The proteolytic activities of MMP-2 and MMP-9 were strongly inhibited by dispersed phase amalgams conditioned buffers. Inhibition of MMP-2 and MMP-9 activities was partly prevented by the addition of 1,10 phenanthroline, a divalent metal chelator, to the amalgam conditioned buffers. Dental amalgam conditioned buffer also inhibited the degradation of denatured type I collagen by purified MMP-2 on liquid phase assays. These findings suggest that the activity of oral tissue MMPs may be modulated by metal ions released from dental amalgam.     

Comparison of techniques for measurement of gelatinases/type IV collagenases: enzyme-linked immunoassays versus substrate degradation assays

Radiolabeled substrate degradation assays and gelatin zymography are routinely employed to assay 72 kDa gelatinase A (MMP-2) and 92 kDa gelatinase B (MMP-9) in biological fluids. Enzyme-linked immunosorbent assays (ELISA) have recently been developed for the quantitation of these matrix metalloproteinases (MMP). In this study, we have compared ELISA to standard substrate degradation assays for measurement of MMP-2 and MMP-9 in human plasma and tumor-conditioned media. Gelatin Sepharose chromatography and gel filtration chromatography were employed as partial purification procedures for MMP-2 and MMP-9. The ELISA data for MMP-2 and MMP-9 are linear on a log:log regression curve over a wide range of MMP concentrations and are specific for the designated gelatinase, with no overlap detected with related metalloproteinases. The minimum detectable concentrations of MMP-2 and MMP-9 were approximately 0.5 ng/ml and 0.2 ng/ml, respectively, in the ELISA as compared to 4 ng/ml and 3 ng/ml, respectively, in gelatin zymography. The [³H]gelatin degradation assay required a combination of >50 ng/ml of MMP-2 and MMP-9 for detection. Although gelatin zymography was less sensitive than ELISA (primarily due to the smaller sample volume employed) and was more difficult to quantitate, this procedure offers the important advantage of being able to distinguish between latent and activated gelatinases.     

Macrophages contain 92-kd gelatinase (MMP-9) at the site of degenerated internal elastic lamina in temporal arteritis.

Inflammation precedes erosion and rupture of atherosclerotic atheromas and aneurysms. Inflammatory infiltrates of macrophages have been shown to secrete proteolytic enzymes, including matrix metalloproteinases (MMPs), that weaken the arterial wall. The effect of inflammation on arterial structure and remodeling can be studied in primary vascular inflammatory diseases such as in temporal arteritis. We examined the 72-kd gelatinase (MMP-2) and the 92-kd gelatinase (MMP-9) in inflamed and uninvolved temporal arteries from 10 patients with temporal arteritis and 5 controls by immunohistochemistry. The substrates of these enzymes, type IV collagen and elastin, were detected by immunohistochemistry and histochemical staining, respectively. Both diseased and normal artery specimens had moderate staining for immunoreactive MMP-2. Temporal arteritis specimens had clearly enhanced immunostaining for MMP-9 compared with normal arteries. MMP-9 was specifically localized to macrophages in regions of internal elastic lamina disruption, which may thus be of pathological significance.     

Salivary histatin 5 is an inhibitor of both host and bacterial enzymes implicated in periodontal disease

One of the salient features of periodontitis and gingivitis is the increase in the levels of bacterial and host-derived proteolytic enzymes in oral inflammatory exudates. This study evaluated the potential of histatin 5, a 24-residue histidine-rich salivary antimicrobial protein, to inhibit these enzymes. Using biotinylated gelatin as a substrate, histatin 5 was found to inhibit the activity of the host matrix metalloproteinases MMP-2 and MMP-9 with 50% inhibitory concentrations (IC50s) of 0.57 and 0.25 microM, respectively. To localize the domain responsible for this inhibition, three peptides containing different regions of histatin 5 were synthesized and tested as inhibitors of MMP-9. Peptides comprising residues 1 to 14 and residues 4 to 15 of histatin 5 showed much lower inhibitory activities (IC50, 21.4 and 20.5 microM, respectively), while a peptide comprising residues 9 to 22 showed identical activity to histatin 5 against MMP-9. These results point to a functional domain localized in the C-terminal part of histatin 5. To evaluate the effect of histatin 5 on bacterial proteases, a detailed characterization of histatin 5 inhibition of gingipains from Porphyromonas gingivalis was carried out using purified Arg- and Lys-specific enzymes. Kinetic analysis of the inhibition of the Arg-gingipain revealed that histatin 5 is a competitive inhibitor, affecting only the Km with a K(i) of 15 microM. In contrast, inhibition of Lys-gingipain affected both the Km and Vmax, suggesting that both competitive and noncompetitive competitive processes underlie this inhibition. The inhibitory activity of histatin 5 against host and bacterial proteases at physiological concentrations points to a new potential biological function of histatin in the oral cavity.     

Secretion of matrix metalloproteinase-2, matrix metalloproteinase-9 and tissue inhibitor of metalloproteinases into the intrauterine compartments during early pregnancy.

Matrix metalloproteinases (MMPs) are important enzymes in tissue remodelling, a key event for the development of the fetal membranes and placenta and establishing the feto-maternal interface during early pregnancy. This study has examined the secretion of the gelatinases, MMP-2 (72 kDa) and MMP-9 (92 kDa), and the endogenous tissue inhibitors of metalloproteinases (TIMPs) into extra-embryonic coelomic and amniotic fluids, the two principal intra-uterine compartments of the first trimester, and compared them to amniotic fluid collected later in gestation. In extra-embryonic coelomic fluid, gelatin zymography demonstrated that MMP-2 (72 kDa) was the predominant gelatinase, with some MMP-9 present. A broad range of TIMPs corresponding to TIMP-1 and TIMP-2, glycosylated and unglycosylated TIMP-3 and TIMP-4 was detected in this compartment by reverse zymography and immunoblot analyses. There was little gelatinase or TIMP activity in amniotic fluid in the first trimester. In amniotic fluid from the second trimester after fusion of the membranes obliterating the extra-embryonic coelom, and at term elective caesarean section, MMP-2 is the predominant gelatinase present, with a broad spectrum of TIMPs. These findings demonstrate that predominantly MMP-2 and also MMP-9, regulated by a range of TIMPs, are involved in intra-uterine tissue remodelling during the establishment of pregnancy.     

Activation of a 66-kilodalton human endothelial cell matrix metalloprotease by Streptococcus pyogenes extracellular cysteine protease.

Human umbilical vein endothelial cells (HUVECs) were used to gain insight into the molecular mechanism whereby the major extracellular protease from group A streptococci damages host tissue. HUVECs exposed to streptococcal cysteine protease (SCP) for various times exhibited cytopathic effect and cell detachment from the culture vessel. Gelatin substrate zymography showed that a time- and concentration-dependent increase in the level of activity of an approximately 66-kDa gelatinase occurred in culture medium taken from cells exposed to enzymatically active SCP. This gelatinase comigrated in gelatin zymograms with the activated form of purified recombinant matrix metalloprotease 2 (MMP-2) and had type IV collagenase activity. In contrast, medium taken from cells exposed to inactivated (boiled) SCP and cells exposed to SCP inhibited by treatment with N-benzyloxycarbonyl-leucyl-valyl-glycine diazomethyl ketone lacked the 66-kDa gelatinase. Appearance of the 66-kDa gelatinase activity was also prevented by 1,10-phenanthroline, a zinc chelator and MMP inhibitor. Inasmuch as proteolytically active SCP is required for the emergence of this gelatinase and MMP activation occurs by proteolytic processing, the 66-kDa gelatinase may be a proteolytic cleavage product of a latent MMP expressed extracellularly by HUVECs. Direct SCP treatment of culture supernatant taken from HUVECs not exposed to SCP also produced the 66-kDa gelatinase. The data show that SCP activates an MMP produced by human endothelial cells, a process that may contribute to endothelial cell damage, tissue destruction, and hemodynamic derangement observed in some patients with severe, invasive group A streptococcal infection.     

Amorphous Calcium Phosphate-Mediated Binding of Matrix Metalloproteinase-9 to Fibrin is Inhibited by Pyrophosphate and Bisphosphonate

Coordinate regulation of fibrinolytic and collagenolytic systems is essential for normal tissue remodeling and wound healing. To define the molecular mechanisms which link these two proteolytic systems, we have investigated the role of fibrin in matrix metalloproteinase (MMP) function. Both active and latent forms of MMP-9 (gelatinase B) bind to fibrin in a selective, dose-dependent manner; latent enzyme is activated by plasmin during fibrinolysis. Fibrin binding of MMP-9 is mediated by amorphous calcium phosphate (ACP), and proceeds in a step-wise fashion with formation of ACP as the first and rate-limiting step. MMP-9 rapidly binds preformed ACP to yield a transient ACP: MMP-9 complex that avidly binds fibrin. Here we report the effect(s) on fibrin: ACP: MMP-9 formation/dissociation of pyrophosphate (POP), an endogenous calcification inhibitor, and its bisphosphonate analog, alendronate (PCP). MMP-9 was obtained from neutrophil lysate and ACP formation was monitored turbidimetrically. Free MMP-9, ACP: MMP-9 and fibrin: ACP: MMP-9 complexes were analyzed by gelatin zymography. POP at physiologic concentrations (0.5-2.5 microM) inhibited both ACP formation and subsequent fibrin binding of MMP-9 at orthophosphate concentrations of 250 microM. PCP exhibited a similar inhibitory effect. With both substances, inhibition was slightly overcome (>2.5 microM) by higher phosphate (500 microM). In contrast, supraphysiologic concentrations of either POP or PCP (>50 microM) were required to inhibit MMP-9 binding to preformed ACP or to induce dissociation of preformed ACP: MMP-9 complexes (50-100 microM). Neither POP nor PCP had any effect on preformed fibrin: ACP: MMP-9 at concentrations up 1 mM. POP is an endogenous by-product of numerous metabolic pathways and may regulate bone turnover, soft tissue calcification, and contribute to the pathogenesis of calcium pyrophosphate crystal disease (CPPD). These studies support another role for POP and fibrin: ACP: MMP-9 complexes in physiologic and pathologic processes, including tumorigenesis and cancer metastasis.     

Matrix metalloproteinase-9 in myeloid cells: implications for allergic inflammation

Matrix metalloproteinase-9 (MMP-9; 92 kDa gelatinase) is utilized by myeloid and lymphoid cells for migration across basement membranes. Although eosinophils are commonly seen infiltrating asthmatic airways, the role of basophils in allergic inflammation is debated. This study was undertaken to evaluate the content of MMP-9 in purified basophils compared with eosinophils and neutrophils. Peripheral blood basophils were isolated to greater than 95% purity using negative selection with antibody-coated magnetic beads using a 12-antibody cocktail. Eosinophils of greater than 98% purity were obtained by negative selection and neutrophils by positive selection using anti-CD16 magnetic beads. MMP-9 activity was assessed by gelatin zymography of cell lysates. Under parallel conditions, neutrophils contained 1,000-fold more MMP-9 than eosinophils. No activity was detected from 2x10(5) basophils. Immunocytochemistry with an anti-MMP-9 antibody showed bright staining of all neutrophils, lesser staining of eosinophils and no detectable staining of basophils. The failure to find MMP-9 in basophils may explain their paucity in asthmatic airway inflammation or suggest they secrete other enzymes capable of degrading type IV collagen.     

Fluorescence Polarization Assay and SDS-PAGE Confirms Matrilysin Degrades Fibronectin and Collagen IV whereas Gelatinase: A Degrades Collagen IV but not Fibronectin

Matrilysin and gelatinase A are hypothesized to have significant roles in uterine and ovarian function. However, proteolytic activity assays for these enzymes are limited. We describe the development of simple and rapid assays for the proteolysis of fluorescein-labeled full-length substrates, collagen IV (Col-IV) and fibronectin (FN), and demonstrate the selectivity of matrilysin (MMP-7) compared to gelatinase A (MMP-2) for fibronectin. Changes in fluorescence intensity (FIU) and fluorescence polarization (mP) resulting from the protease activity of matrilysin and gelatinase A were measured. These studies show that the fluorescently labeled substrates, Col-IV and FN, are as reliable and amenable to rapid in vitro assay as peptide substrates. In addition, they are easier to use than previously described, non-fluorescent methods. The results demonstrate that assays using full-length, biological matrix proteins are more sensitive indicators of MMP-specific substrate activity than peptide based assays.     

Fluorescence polarization assay and SDS-PAGE confirms matrilysin degrades fibronectin and collagen IV whereas gelatinase A degrades collagen IV but not fibronectin.

Matrilysin and gelatinase A are hypothesized to have significant roles in uterine and ovarian function. However, proteolytic activity assays for these enzymes are limited. We describe the development of simple and rapid assays for the proteolysis of fluorescein-labeled full-length substrates, collagen IV (Col-IV) and fibronectin (FN), and demonstrate the selectivity of matrilysin (MMP-7) compared to gelatinase A (MMP-2) for fibronectin. Changes in fluorescence intensity (FIU) and fluorescence polarization (mP) resulting from the protease activity of matrilysin and gelatinase A were measured. These studies show that the fluorescently labeled substrates, Col-IV and FN, are as reliable and amenable to rapid in vitro assay as peptide substrates. In addition, they are easier to use than previously described, non-fluorescent methods. The results demonstrate that assays using full-length, biological matrix proteins are more sensitive indicators of MMP-specific substrate activity than peptide based assays.     

人巨细胞病毒感染对体外培养肺成纤维细胞中明胶酶的影响

目的探讨人巨细胞病毒（HCMV）感染对体外培养肺成纤维细胞（HEL）中明胶酶活性的影响。方法体外培养HEL细胞感染HCMV，分为低感染复数（MOI）组及高MOI组，每组重复6例。明胶酶谱法检测HEL细胞中MMP-2及MMP-9的明胶酶活性，用半定量RT-PCR检测各组HEL细胞中MMP-9及TIMP-1的转录水平。结果在低MOI组及高MOI组HEL细胞中MMP-9及MMP-2活性均增强（P〈0．05），高MOI组MMP-9及MMP-2活性较低MOI组显著增加（P〈0．05）。进一步检查MMP-9及TIMP-1的mRNA水平发现，正常对照组HEL细胞中MMP-9及TIMP-1的mRNA处于一个较低的水平，HCMV感染使HEL细胞中MMP-9及TIMP-1的mRNA水平均明显升高（P〈0．05），低MOI组和高MOI组差异无统计学意义（P〉0．05）。在低MOI组及高MOI组，HEL细胞MMP-9／TIMP-1的比值和正常对照组相比明显升高（P〈0．05），表明MMP-9升高更为显著。高MOI组和低MOI组中MMP-9／TIMP-1的比值差异无统计学意义（P〉0．05）。结论HCMV感染可以造成MMP-9和TIMP-1转录和MMP-9／TIMP-1的失衡，同时造成MMP-9及MMP-2明胶酶活性增强，导致肺泡结构的破坏和肺纤维化的发生，这在CMV肺炎的发病机制中起着重要的作用。