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.     

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.     

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.     

Identification of proteases from periodontopathogenic bacteria as activators of latent human neutrophil and fibroblast-type interstitial collagenases.

Activation of latent human fibroblast-type and neutrophil interstitial procollagenases as well as degradation of native type I collagen by supra- and subgingival dental plaque extracts, an 80-kDa trypsinlike protease from Porphyromas gingivalis (ATCC 33277), a 95-kDa chymotrypsinlike protease from Treponema denticola (ATCC 29522), and selected bacterial species commonly isolated in periodontitis was studied. The bacteria included were Prevotella intermedia (ATCC 25261), Prevotella buccae (ES 57), Prevotella oris (ATCC 33573), Porphyromonas endodontalis (ES 54b), Actinobacillus actinomycetemcomitans (ATCC 295222), Fusobacterium nucleatum (ATCC 10953), Mitsuokella dentalis (DSM 3688), and Streptococcus mitis (ATCC 15909). None of the bacteria activated latent procollagenases; however, both sub- and supragingival dental plaque extracts (neutral salt extraction) and proteases isolated from cell extracts from potentially periodontopathogenic bacteria P. gingivalis and T. denticola were found to activate latent human fibroblast-type and neutrophil interstitial procollagenases. The fibroblast-type interstitial collagenase was more efficiently activated by bacterial proteases than the neutrophil counterpart, which instead preferred nonproteolytic activation by the oxidative agent hypochlorous acid. The proteases were not able to convert collagenase tissue inhibitor of metalloproteinase (TIMP-1) complexes into active form or to change the ability of TIMP-1 to inhibit interstitial collagenase. None of the studied bacteria, proteases from P. gingivalis and T. denticola, or extracts of supra- and subgingival dental plaque showed any significant collagenolytic activity. However, the proteases degraded native and denatured collagen fragments after cleavage by interstitial collagenase and gelatinase. Our results indicate that proteases from periodontopathogenic bacteria can act as direct proteolytic activators of human procollagenases and degrade collagen fragments. Thus, in concert with host enzymes the bacterial proteases may participate in periodontal tissue destruction.     

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.     

Evidence of gelatinase secretion by the submandibular gland in prepubescent rats

The gelatin cleaving activities in secretions of cultured fragments of male rat submandibular glands were studied using zymography. Gelatinolytic activities of 88-, 64-, and 57-kDa proteins detected in the tissues from 22-28-day old animals were undetectable in 31-70-day old rats. The traces of gelatinolytic activity associated with 28-kDa protein were detectable from 22-day old rats in serum-free media, and this activity of the enzyme markedly increased with aging from 38-days old. At 52-days and the subsequent stages, in addition to 28-kDa, activities associated with 60-, 32-, and 29-kDa proteins were strong. When the conditioned media were treated with 1,10-phenanthroline and diisopropyl fluorophosphate (DFP), both products inhibited activity of 88-kDa enzyme, indicating that this enzyme is Cls-like enzyme. The 64- and 57-kDa activities were inhibited by 1,10-phemanthroline, but not by DFP; when the conditioned medium of the tissue from 24-day old rats was treated with p-aminophenylmercuric acetate, gelatinolytic activity associated with 64-kDa converted to 57-kDa. Therefore, 64- and 57-kDa activities were concluded to be progelatinase A and gelatinase A, respectively. On the other hand, the gelatinolytic activities associated with 60-, 32-, 29- and 28-kDa proteins were inhibited by DFP but not by 1,10-phenanthroline, indicating that these enzymes belong to the family of serine proteinase, most probably kallikrein-related enzymes. From these findings, it was suggested that gelatinase A, along with Cls-like enzyme, participates in the maturation of the submandibular gland before it becomes active as an exocrine organ.     

MT1-MMP-dependent remodeling of cardiac extracellular matrix structure and function following myocardial infarction

The myocardial extracellular matrix (ECM), an interwoven meshwork of proteins, glycoproteins, proteoglycans, and glycosaminoglycans that is dominated by polymeric fibrils of type I collagen, serves as the mechanical scaffold on which myocytes are arrayed for coordinated and synergistic force transduction. Following ischemic injury, cardiac ECM remodeling is initiated via localized proteolysis, the bulk of which has been assigned to matrix metalloproteinase (MMP) family members. Nevertheless, the key effector(s) of myocardial type I collagenolysis both in vitro and in vivo have remained unidentified. In this study, using cardiac explants from mice deficient in each of the major type I collagenolytic MMPs, including MMP-13, MMP-8, MMP-2, MMP-9, or MT1-MMP, we identify the membrane-anchored MMP, MT1-MMP, as the dominant collagenase that is operative within myocardial tissues in vitro. Extending these observations to an in vivo setting, mice heterozygous for an MT1-MMP-null allele display a distinct survival advantage and retain myocardial function relative to wild-type littermates in an experimental model of myocardial infarction, effects associated with preservation of the myocardial type I collagen network as a consequence of the decreased collagenolytic potential of cardiac fibroblasts. This study identifies MT1-MMP as a key MMP responsible for effecting postinfarction cardiac ECM remodeling and cardiac dysfunction.     

Differences in proliferation and invasion by normal,transformed and NF1 Schwann cell cultures are influenced by matrix metalloproteinase expression

Loss of negative growth regulation and high invasive potential are neoplastic traits often associated with abnormal expression of matrix metalloproteinases (MMPs). We previously found MMP-3 (stromelysin/transin) was secreted by quiescent rat Schwann cell cultures and expressed potent antiproliferative activity. In the present study we observed that human Schwann cells and cutaneous neurofibroma Schwann cell cultures secreted abundant MMP-3 and their proliferation was inhibited by autologous and rat Schwann cell conditioned media. Antiproliferative activities were depleted by immunoadsorption with anti-stromelysin antibodies. In contrast, plexiform neurofibroma cultures did not secrete MMP-3 and failed to respond to Schwann cell antiproliferative activities associated with MMP-3. Quiescent Schwann cells constitutively secreted low levels of MMP-2 (gelatinase A) and showed a low invasion potential in filter-based assays of basement membrane invasion. Cyclic AMP elevation, which profoundly influences cell differentiation, increased the invasion potential of rat Schwann cells and caused a corresponding increase in secretion of MMP-2. Schwann cells immortalized by protracted elevation of CAMP, as well as a schwannoma cell line (D6P2T), also rapidly invaded a reconstituted basement membrane and over-expressed MMP-2. Similarly, neurofibroma Schwann cells were highly invasive and secreted up to 10-fold more MMP-2 than normal human Schwann cells. Additionally, only cutaneous neurofibroma Schwann cell cultures secreted MMP-9 (gelatinase B) and MMP-1 (interstitial collagenase) and also invaded native type I collagen barriers. Cultures of normal Schwann cells and plexiform neurofibroma tumor expressed little or no MMP-1 and did not invade type I collagen barriers. These results suggest a role for MMPs in the control of proliferation and invasion by Schwann cells and in the formation of peripheral nerve sheath tumors.     

Androgens influence expression of matrix proteins and proteolytic factors during cutaneous wound healing

Excessive proteolytic activity is a feature of chronic wounds such as venous ulcers, in which resolution of the inflammatory response fails and restorative matrix accumulation is delayed as a consequence. The inflammatory actions of native androgens during the healing of acute skin wounds have lately been characterized. We have now investigated the hypothesis that such activities may impact upon the balance between anabolic and catabolic processes during wound healing. We report that wound deposition of both type I collagen and fibronectin is increased in castrated rats compared with control animals. This response is accompanied by early increases and later decreases in overall wound levels of the key collagenolytic enzymes, matrix metalloproteinase (MMP)-1 and MMP-13. Moreover, the activities of MMP-2 and MMP-9, two further enzymes that contribute to collagen digestion during venous ulceration, were significantly decreased in the wounds of castrated rats. Additional analyses provide evidence that androgens directly stimulate dermal fibroblast collagen production, which supports the suggestion that increased wound collagen deposition in androgen-deprived rats results from reduced matrix degradation (as opposed to enhanced matrix protein biosynthesis). Androgen-mediated dysregulation of the parallel processes of collagen deposition and turnover may underscore the delayed healing of cutaneous wounds in elderly male patients and further contribute to the increased incidence of non-healing wounds in this population.     

The Effects of Collagen Fragments on the Extracellular Matrix Metabolism of Bovine and Human Chondrocytes

Cartilage matrix degradation generates collagen type II fragments. The objective of this study is to explore the possibility that these collagen fragments may be part of an endogenous metabolic feedback. Initially, collagen fragments were extracted from normal or osteoarthritic cartilage, as part of a matrix fragment preparation. Later, collagen fragments were generated by digestion of bovine collagen type II with bacterial collagenase (col2f). These fragments were added to cultures of isolated chondrocytes (bovine and human) and cartilage explants (human). In a dose-dependent manner, col2f caused inhibition of cell attachment to collagen, inhibition of collagen synthesis, and induction of matrix degradation. In addition, when col2f were added to human cartilage explants, an induction of gelatinase activity was detected in the media. These data sets present first evidence that degradation products of collagen may be directly involved in the regulation of cartilage homeostasis.     

低氧诱导肌成纤维细胞生成并通过ERK1/2途径促进Ⅰ型胶原的表达

目的：探讨低氧能否激活成纤维细胞转化为肌成纤维细胞,以及低氧环境对肾皮质肌成纤维细胞表达Ⅰ型胶原（Col-Ⅰ）的影响及其可能的信号通路。方法：（1）采用正常大鼠肾成纤维细胞系（NRK-49F），应用Western blotting方法检测低氧诱导因子-1α(HIF-1α)的表达;比较低氧和正常氧条件下α-平滑肌肌动蛋白(α-SMA)的蛋白水平。（2）原代培养正常大鼠肾皮质肌成纤维细胞，Western blotting方法检测低氧和正常氧条件下，HIF-1α和Ⅰ型胶原蛋白水平以及ERK1/2的活化及其特异阻断剂 PD98059的阻断效应；RT-PCR方法检测Ⅰ型胶原mRNA表达水平；细胞免疫化学法检测HIF-1α胞内表达部位的变化；明胶酶谱法检测细胞上清中基质金属蛋白酶-2（MMP-2）和基质金属蛋白酶-9（MMP -9）的活性。结果：（1）低氧刺激6 h，NRK-49F细胞和肌成纤维细胞胞内和核内均有HIF-1α蛋白表达，核内更明显。（2）低氧培养12 h,NRK-49F细胞表达α-SMA蛋白明显增高，是正常氧组的187%±32%（P<0.05），验证了低氧可引起成纤维细胞表型转化。（3）低氧刺激原代培养的正常大鼠肾皮质肌成纤维细胞6 h、12 h上清中Ⅰ型胶原蛋白水平增加，分别为正常氧组的171%±27%（P<0.05）和256%±61% （P<0.05）；低氧刺激4 h、6 h，Ⅰ型胶原mRNA表达增加，为正常氧组的189%±28%（P<0.05）和221%±44%（P<0.05）。（4）低氧刺激肌成纤维细胞6 h、12 h、24 h，培养上清液中MMP-9、MMP-2活性无明显变化。（5）低氧刺激肌成纤维细胞15 min 即可使ERK1/2活化，阻断实验显示，PD98059可以使低氧12 h引起Ⅰ型胶原增加（低氧刺激组为正常氧对照组的273%±51%，P<0.05)显著减少（PD98059 +低氧组为正常氧组的108%±19%，P>0.05)。结论： 低氧促肾纤维化可能与其诱导肾成纤维细胞转化为肌成纤维细胞并经ERK1/2途径增加Ⅰ型胶原蛋白的表达有关。     

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.     

Hepatocyte production of modulators of extracellular liver matrix in normal and cirrhotic rat liver

In the present study, we found collagenolytic and gelatinolytic activity in the supernatants of hepatocyte cultures from rats with experimental CCl(4)-induced liver cirrhosis, in levels significantly higher than in comparable supernatants of hepatocyte cultures from normal rats. In addition, we clearly detected the messenger ribonucleic acids (mRNA) of four matrix metalloproteinases (MMP-2, MMP-3, MMP-10, and MMP-13) and of two tissue inhibitors of matrix metalloproteinases (TIMP-1 and TIMP-2) in hepatocytes from both normal and cirrhotic rats by RT-PCR and by in situ hybridization. Finally, we demonstrated MMP-2, MMP-3, and MMP-13 and TIMP-1 and TIMP-2 proteins in the same hepatocyte preparations by immunostaining. We conclude that rat hepatocytes produce the major enzymes and inhibitors involved in liver ECM modulation and therefore suggests that they might participate actively in the pathophysiology of liver cirrhosis in rats.     

Differential expression of matrix metalloproteinases 2 and 9 by glial Müller cells: response to soluble and extracellular matrix-bound tumor necrosis factor-alpha

Glial Müller cells are known to undergo functional and morphological changes during retinal proliferative disorders, but very little is known of the contribution of these cells to extracellular matrix deposition during retinal wound healing and gliosis. This study constitutes the first demonstration that retinal Müller cells express two major matrix metalloproteinases (MMPs), gelatinase A (MMP-2) and gelatinase B (MMP-9), implicated in cell migration and matrix degradation. Although mRNA and gelatinolytic activity of MMP-2 remained unchanged in cultured Müller cells, basal levels of MMP-9 mRNA observed after subculture at 24 hours, markedly declined after 48 or 72 hours. This correlated with the expression of MMP-9 gelatinolytic activity that peaked at 24 hours, but gradually decreased at 48 and 72 hours. Tumor necrosis factor-alpha, in both a soluble form or bound to collagen and fibronectin, increased MMP-9 mRNA and gelatinolytic activity, but not MMP-2 expression, and its effect could be blocked by anti-tumor necrosis factor-alpha antibodies. The results suggest that Müller cells may aid in the local control of extracellular matrix deposition during retinal proliferative disorders, and that interaction of these cells with matrix-bound cytokine may influence their pathological behavior. Control of Müller cell production of MMP-9 may constitute an important target for the design of new therapeutic approaches to treat and prevent retinal proliferative disease.     

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.     

Matrix metalloproteinase 9 (gelatinase B) is expressed in multinucleated giant cells of human giant cell tumor of bone and is associated with vascular invasion.

Human giant cell tumor (GCT) consists of multinucleated giant cells and mononuclear stromal cells, and is characterized by frequent vascular invasion without distant metastases. To study the role of matrix metalloproteinases (MMPs) in the vascular invasion, we examined production of MMP-1 (tissue collagenase), -2 (gelatinase A), -3 (stromelysin-1), -9 (gelatinase B), and tissue inhibitors of metalloproteinases (TIMP-1 and -2) in GCT. MMP-9 was highly and predominantly expressed in giant cells by both immunohistochemistry and in situ hybridization. Expression of other MMPs was also observed in some cases but was inconstant. Sandwich enzyme immunoassays demonstrated that MMP-9 is the predominant MMP secreted by GCT. There was a definite imbalance between the amounts of MMP-9 and those of TIMPs in the culture media of GCT, leading to detectable gelatinolytic activity in an assay using 14C-gelatin. Gelatin zymography demonstrated the main activity at about 90 kd, which was identified as the zymogen of MMP-9 by immunoblotting. Immunohistochemistry for type IV collagen and laminin, major basement membrane components, showed that disappearance of the proteins is closely associated with MMP-9-positive giant cells. These results indicate the production of MMP-9 by multinucleated giant cells and suggest that the metalloproteinase may contribute to proteolysis associated with vascular invasion and local bone resorption in human GCT.     

Gelatinase is the main matrix metalloproteinase involved in granuloma-induced cartilage degradation.

This study was performed to characterize the matrix metalloproteinases (MMPs) produced during the degradation of cotton-wrapped cartilage, implanted into the murine air pouch. One, two or three weeks following cartilage implantation, proteins were extracted from the granulation tissue and MMP activities were measured. Although collagenase-, gelatinase- and stromelysin-like activities were detected at each time point, gelatinase activity was by far the most prominent. These enzymes were inhibited by EDTA, but not by NEM or PMSF, indicating that these proteinases were metalloproteinases. Gelatin zymography revealed several lysis zones amongst which a major 92-kDa band shifted to 83- and 68-kDa species during the course of implantation. The emergence of these species coincided with enhanced gelatinolytic activity and collagen loss from the implanted cartilage.     

Therapeutic failure in American cutaneous leishmaniasis is associated with gelatinase activity and cytokine expression

Cutaneous lesions caused by Leishmania braziliensis infection occasionally heal spontaneously, but with antimonials therapy heal rapidly in approximately 3 weeks. However, about 15% of the cases require several courses of therapy. Matrix metalloproteinase-2 (MMP-2) and MMP-9 are gelatinases that have been implicated in other chronic cutaneous diseases and skin re-epithelialization. These enzymes are controlled by their natural inhibitors [tissue inhibitors of metalloproteinase (TIMPs)] and by some cytokines. Uncontrolled gelatinase activity may result in intense tissue degradation and, consequently, poorly healing wounds. The present study correlates gelatinase activity to therapeutic failure of cutaneous leishmaniasis (CL) lesions. Our results demonstrate an association between gelatinase activity and increased numbers of cells making interferon (IFN)-γ, interleukin (IL)-10 and transforming growth factor (TGF)-β in lesions from poor responders. Conversely, high levels of MMP-2 mRNA and enhanced MMP-2 : TIMP-2 ratios were associated with a satisfactory response to antimonials treatment. Additionally, high gelatinolytic activity was found in the wound beds, necrotic areas in the dermis and within some granulomatous infiltrates. These results indicate the importance of gelatinase activity in the skin lesions caused by CL. Thus, we hypothesize that the immune response profile may be responsible for the gelatinase activity pattern and may ultimately influence the persistence or cure of CL lesions.