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Biomedical subjects

P Maisi

Publications and source records attributed to P Maisi.

At least 19 recordsLinked to original sources
PubMed2005

Dose responses to inhalation of endotoxin, hay dust suspension and Aspergillus fumigatus extract in horses as measured by levels and activation of matrix metalloproteinase-9.

REASONS FOR PERFORMING STUDY: Airway matrix metalloproteinases (MMPs) increase after endotoxin (LPS) exposure, but there are no reports describing dose-dependent increases or activation following exposure. OBJECTIVES: To study matrix metalloproteinase-9 (MMP-9) and -2 (MMP-2) responses in bronchoalveolar lavage fluid (BALF) from heaves-susceptible and control horses following inhalation of hay dust suspension (HDS), LPS and Aspergillus fumigatus extract (AFE). METHODS: Heaves-susceptible (n = 7) and control (n = 6) horses received inhalation challenges with 3 different doses of HDS and LPS. Heaves-susceptible horses (n = 6) also received 3 different doses of AFE and one dose of AFE depleted of endotoxin (AFE-LPS). BALF collected following inhalation challenges was analysed using gelatin zymography. Gelatinolytic bands were identified as complex, pro-MMP-9, active MMP-9, pro-MMP-2 and active MMP-2 based on molecular weights. RESULTS: Each challenge substance induced a dose-dependent elevation in gelatinolytic activity. The dose-dependency was most evident for pro-MMP-9 and total MMP-9 levels in heaves-susceptible horses following LPS challenges. CONCLUSIONS: There is a dose-dependent elevation in MMP-9 in BALF of heaves-susceptible and control horses following inhalation challenge with organic dust and some of its components, elevation being more marked in heaves-susceptible horses. Organic dust components vary in their pro-inflammatory potential. POTENTIAL RELEVANCE: This study supports the role of MMPs in the pathogenesis of heaves and highlights the potential value of protease inhibitors in attenuating the airway inflammatory response to inhaled organic dust.

Animals↗
PubMed2005

Effect of composition and different fractions of hay dust suspension on inflammation in lungs of heaves-affected horses: MMP-9 and MMP-2 as indicators of tissue destruction.

REASONS FOR PERFORMING STUDY: Airway matrix metalloproteinases (MMPs) increase following inhalation of organic dust. The relative contribution of dust components to this elevation is unknown. OBJECTIVE: To identify components of organic dust responsible for elevated MMP levels in equine airways. METHODS: Bronchoalveolar lavage (BALF) from 7 heaves-susceptible horses, collected 6 h following inhalation challenges with saline, 2 different hay dust suspensions (HDS-1 and -2) and soluble and particulate fractions of HDS-1, were analysed for MMP-2 and -9 using SDS-page gelatin zymography. RESULTS: HDS-1 challenge increased BALF proMMP-9 and total MMP-9. HDS-1 fractions, or the particulate fraction with added lipopolysaccharide, increased BALF proMMP-9 and total MMP-9 in combination, but not when inhaled separately. HDS-2 inhalation elevated BALF complex forms, proMMP-9, active MMP-9, total MMP-9 and total MMP-2. CONCLUSIONS: The results suggest synergistic action of soluble and particulate organic dust components. The fact that HDS-1 and HDS-2 had different glucan concentrations supports a role for moulds in the activation of MMP-9. POTENTIAL RELEVANCE: Activation and release of MMPs in response to inhaled moulds are involved in the aetiopathogenesis of heaves. Endotoxin contributes to the synergistic action of the dust components, but the overall MMP response to organic dust inhalation in heaves-susceptible horses largely reflects the mould content of the dust. In the future, inhibition of MMP production and release may offer therapeutic means for treatment and prevention of heaves and recommendations for acceptable dust levels can be given.

Animals↗
PubMed2002

Increase in milk metalloproteinase activity and vascular permeability in bovine endotoxin-induced and naturally occurring Escherichia coli mastitis.

An endotoxin-induced mastitis model was used to study permeability changes associated with increased milk matrix metalloproteinase (MMP) activity in early inflammation. One quarter of two cows was inoculated with endotoxin (Escherichia coli 055:B5). Blood, milk, and whey were collected before and repeatedly after inoculation for 48 h. The profile and amounts of gelatinolytic MMPs were determined by zymography; gelatinase A (72 kD MMP-2) and gelatinase B (92 kD MMP-9) were identified by Western immunoblotting. Bovine serum albumin (BSA) and trypsin inhibitor capacity (TIC) were used as markers of capillary permeability with parallel examination of neutrophil penetration from blood to milk. Five clinical E. coli mastitis milk samples and five milk samples from cows with healthy udders were analyzed to detect whether increased levels of gelatinolytic MMP-2 and MMP-9 have a role in naturally occurring mastitis with endotoxin involvement. Milk MMP levels increased 2h after the endotoxin challenge. Both MMP-2 and MMP-9 were involved in this early proteolytic event. These increased MMP levels are associated with increased capillary permeability, evidenced first by the penetration of small molecular weight proteins, such as BSA and TIC. Later, 6-12h post endotoxin inoculation, neutrophilic leucocytes also entered the site, as they require larger tissue damage in basal membrane and interstitial tissue structures than BSA and TIC to extravasate. In naturally occurring disease, increased MMP-2 and MMP-9 levels were detected in milk. Thus, gelatinases, especially MMP-2, are involved in the early degradation of the blood-milk barrier, which precedes the penetration of blood-derived cellular components into milk in endotoxin-induced mastitis. In the future, measuring MMP in milk/whey might be a useful tool for diagnosing early mastitis.

Animals↗
PubMed2002

Collagenase-2 (MMP-8) and collagenase-3 (MMP-13) in adult periodontitis: molecular forms and levels in gingival crevicular fluid and immunolocalisation in gingival tissue.

AIM: To determine the cellular and molecular forms of MMP-8 (collagenase-2) and MMP-13 (collagenase-3) associated with chronic adult periodontitis by examining the species present in gingival crevicular fluid (GCF) and enzyme distribution in gingival tissue. METHODS: 30-s GCF samples were collected directly from the periodontal pockets of 12 untreated patients using filter paper strips. After elution into buffer, the samples were examined by Western immunoblotting with polyclonal antibodies for MMP-8 and MMP-13 and quantification by scanning image analysis. Individual band intensities were expressed as a percentage of total sample absorbance and mean patient values were calculated. Gingival tissue from 6 patients was fixed in formalin and embedded in paraffin wax. MMP-8 and MMP-13 were localised using the same antibodies and an avidin-biotin-peroxidase detecting system. Double staining was performed with a contrasting substrate reaction. RESULTS: The majority of MMP-8 staining in pre-treatment GCF was present in 80, 75 and 60 kD bands corresponding to prepro-, pro- and active forms of PMN-type enzyme. 43 and 38 kD bands evidently represented active, fibroblast-type MMP-8. Immunoreactivities at >100 kD and < or =30 kD were probably enzyme-inhibitor complex and degraded fragments, respectively. MMP-13 was seen mainly as 60 kD proenzyme with some 40 kD active enzyme and a small proportion of >100 kD complex. The percentages of MMP-8 PMN-type enzyme and MMP-13 proenzyme bands correlated significantly with gingival and bleeding indices (p<0.05). Immunohistochemistry demonstrated MMP-8 in PMNs, sulcular epithelial and also plasma cells in inflamed gingival connective tissue. MMP-13 immunoreactivity was detected in the sulcular epithelium and in macrophage-like cells. CONCLUSION: Multiple species and elevated levels of both MMP-8 and MMP-13 from many rather than single cellular sources in the diseased periodontium are identified in untreated periodontitis GCF and active forms contribute to GCF collagenase activity.

Adult↗
PubMed2002

Matrix metalloproteinases and tissue inhibitor of metalloproteinase-1 in sarcoidosis and IPF.

The purpose of this study was to examine the role of interstitial collagenases, members of the family of matrix metalloproteinases, in the development of pulmonary fibrosis. The activity, levels and molecular forms of collagenases (matrix metalloproteinases (MMP)-1, -8 and -13), gelatinase B (MM P-9) and its main endogenous inhibitor, tissue inhibitor of metalloproteinase-1 (TIMP-1) were assessed in bronchoalveolar lavage fluid (BALF) from patients with idiopathic pulmonary fibrosis (IPF) and sarcoidosis patients with varying degrees of pulmonary parenchymal involvement. Collagenase activity was elevated in IPF and group 3 sarcoidosis patients. A positive correlation between BALF collagenase activity and MMP-8 levels was also observed. Western immunoblotting revealed the presence of two isoforms of MMP-8 in patient samples; an 80 kD form representing latent enzyme from polymorphonuclear neutrophils and a 55 kD form representing the fibroblast-type proform. MMP-9 levels were also elevated in both IPF and group 3 sarcoidosis patients, while TIMP-1 levels remained normal, indicating a shift in the balance between the enzyme and inhibitor, favouring MMP-9. Matrix metalloproteinase-8 is the major contributor to the bronchoalveolar lavage fluid collagenase activity in the airways of patients with idiopathic pulmonary fibrosis and sarcoidosis and may initiate collagen destruction and remodelling leading to the development of pulmonary fibrosis.

Aged↗
PubMed2002

Inhalation of organic dusts and lipopolysaccharide increases gelatinolytic matrix metalloproteinases (MMPs) in the lungs of heaves horses.

We report the effects of mouldy hay/straw exposure, inhaled hay dust suspension (HDS) and inhaled lipopolysaccharide (LPS) on bronchoalveolar lavage fluid (BALF) gelatinolytic matrix metalloproteinase (MMP) levels and degree of activation in healthy (n = 6) and heaves- (previously termed chronic obstructive pulmonary disease) affected (n = 6 or 7) horses. Gelatinolytic MMPs in BALF were quantified by zymography, and gelatinases were shown by Western immunoblotting to be MMP-2 and MMP-9. Hay/straw and HDS challenges increased BALF total gelatinolytic activity only in heaves horses, with the majority of gelatinolytic activity comprising pro- and active MMP-9. The 5 h duration hay/straw challenge increased BALF gelatinolytic MMP activity in heaves horses at 5 and 24 h after the start of this challenge, with activity returning to baseline by Day 4. In contrast to hay/straw and HDS challenges, LPS inhalation increased BALF gelatinolytic MMP activity in both groups. For all challenges, absolute BALF neutrophil counts were highly significantly correlated (P<0.0001) with levels of proMMP-9 and active MMP-9, but not with levels of MMP-2 (P>0.05). As gelatinolytic MMPs are pro-inflammatory agents, they may contribute to lung dysfunction and tissue destruction in heaves horses exposed to airborne organic stable dusts.

Animals↗
PubMed2001

In vivo localization of gelatinases (MMP-2 and -9) by in situ zymography with a selective gelatinase inhibitor.

In situ zymography provides a tool to localize proteolytic activity in tissues in vivo. However, it has been difficult to discriminate between the proteases responsible for the detected activity. We used a selective tissue-permeable gelatinase inhibitor, the CTTHWGFTLC-peptide (CTT) in inflamed human gingiva. The CTT-peptide was evidenced to home, target to, and selectively inhibit the areas of gelatinolytic activity in inflamed human gingiva expressing MMP-2 and -9. Gelatinolytic activity, MMP-9 immunoreactivity, and mRNA expression as well as CD-45-positive inflammatory cells colocalized well in the inflamed human gingival connective tissue. Gelatinolytic activity corresponding to MMP-2 colocalized with laminin-5 gamma2-chain immunoreactivity and was detected in the close vicinity of the sulcular basement membrane region. Furthermore, the CTT-peptide inhibited beta-caseinolysis by human MMP-2 and MMP-9 as well as laminin-5 gamma2-chain degradation by MMP-2 in vitro. Thus, the CTT-peptide may prove to be a useful tool (i) to discriminate between gelatinolytic proteases detected by in situ zymography and (ii) to preventMMP-2-dependent induction of epithelial cell migration and gelatinase-dependent tissue destruction in inflammatory and malignant diseases.

Cell Adhesion Molecules↗
PubMed2001

In vivo collagenase-2 (MMP-8) expression by human bronchial epithelial cells and monocytes/macrophages in bronchiectasis.

The purpose of this study was to determine whether other cellular sources than neutrophils can express matrix metalloproteinase (MMP)-8 protein and mRNA in bronchiectatic (BE) lung. The molecular forms of MMP-8 in the BE bronchoalveolar lavage fluid (BALF) and healthy control BALF were analysed by western immunoblotting. MMP-8 expression was demonstrated by immunohistochemistry and in situ hybridization in BE lung tissue and by immunohistochemistry in control lung tissue. In the BE BALF, different MMP-8 species were detected: 70-80 kD MMP-8 apparently of polymorphonuclear leukocyte (PMN) origin and also 40-60 kD MMP-8 from non-PMN cellular sources, such as bronchial epithelial cells, glandular cells or monocytes/macrophages. Both of these MMP-8 species were elevated and converted to a significant extent to activated forms in BE BALF compared with healthy control BALF. The levels of high molecular weight (>80 kD) MMP-8 complexes, evidently representing MMP-8 trapped by endogenous MMP inhibitors and/or MMP-8 dimers, were significantly elevated in BE BALF compared with healthy control BALF. In BE lung tissue, the MMP-8 protein and mRNA expression was found in bronchial ciliated epithelial cells, glandular cells, neutrophils, and monocytes/macrophages infiltrating the bronchial epithelial area. Minimal MMP-8 expression was observed in neutrophils, monocytes/macrophages, and epithelial cells in control lung tissues. In this study, new potential cellular sources have been demonstrated for MMP-8 in the inflamed lung. MMP-8 from multiple cellular sources, including inflamed lung epithelium, was activated to a significant extent in the BE BALF, indicating a major role for MMP-8 in the destruction of lung and bronchial tissues.

Blotting, Western↗
PubMed2001

Expression and induction of collagenases (MMP-8 and -13) in plasma cells associated with bone-destructive lesions.

Matrix metalloproteinases (MMPs) collectively degrade extracellular matrix and basement membrane proteins in chronic inflammation and bone-destructive lesions. This study examined the ability of immunoglobulin-producing plasma cells, typically present in sites of chronic inflammation, to express collagenases (MMP-8 and -13) in vivo and in vitro. Phorbol-12-myristate-13-acetate, interleukin-6, and tumour necrosis factor-alpha and heparin with the tumour promoter or cytokines potently enhanced (up to nine-fold) MMP-8 and -13 expression by the RPMI 8226 myeloma cell line, as evidenced by western blotting and semi-quantitative reverse transcriptase-polymerase chain reaction. Immunohistochemical analysis and in situ hybridization revealed that plasma cells expressed MMP-8 and -13 focally in periapical granulomas, odontogenic cysts, and malignant plasmacytomas. MMP-8 and MMP-13 from plasma cells can participate in bone organic matrix destruction at sites of chronic inflammation and neoplastic growth. Since MMP-13 was more frequently expressed than MMP-8 in plasma cells of strongly recurring keratocysts and malignant plasmacytomas, it is concluded that plasma cell MMP-13 has a particularly important role in benign and malignant bone-destructive lesions.

Bone Diseases↗
PubMed2001

MMP-9 as a marker of inflammation in tracheal epithelial lining fluid (TELF) and in bronchoalveolar fluid (BALF) of COPD horses.

Gelatinolytic activity was analysed to study whether elevated activity previously found at the tracheal level of the respiratory tract of horses with chronic obstructive pulmonary disease (COPD) could also be found at the lower part of the respiratory tract. Furthermore, presence and significance of the gelatinolytic matrix metalloproteinases (MMPs) MMP-2 and MMP-9 in respiratory secretions of healthy and COPD horses were determined. Elevated gelatinolytic matrix metalloproteinases were detected in bronchoalveolar and tracheobronchial secretions from COPD horses. The main pathologically elevated MMP was characterised to be MMP-9. Significantly increased MMP-9 activities as measured by gelatin zymography and Western blotting were found in all the respiratory samples from COPD horses compared to healthy horses. Elevation of active MMP-9 paralleled with increased gelatinase-associated lipocalin levels. Bronchoalveolar lavage fluid (BALF) epithelial cells, macrophages, neutrophils and lymphocytes expressed MMP-9 immunoreactivity demonstrated by immunocytochemistry and MMP-9 mRNA was expressed by bronchial epithelial cells of lung tissue section shown by in situ hybridisation. MMP-2 seems not to play a major role in chronic lung inflammation. No clear differences in MMP-2 or MMP-14 (a potent MMP-2 activator) levels were found when comparing the samples from COPD or healthy horses. These results suggests that MMP-9 could serve as a potential diagnostic marker for the active ongoing tissue remodelling in the acute phase of equine COPD. Increased gelatinolytic activity could be found at both tested respiratory tract levels. Therefore, tracheal epithelial lining fluid (TELF) samples can usefully serve as diagnostic material for detection of increased levels of the main gelatinolytic MMP, MMP-9, representing the entire diseased lung.

Animals↗
PubMed2001

Matrix metalloproteinases-2, -8, and -9 and TIMP-2 in tracheal aspirates from preterm infants with respiratory distress.

OBJECTIVES: Matrix metalloproteinases (MMPs) are a family endoproteinases that act in degradation of extracellular matrix and basement membranes. The development of bronchopulmonary dysplasia (BPD) is characterized by early pulmonary inflammation, increased microvascular permeability, and subsequently by disordered repair. The aims of our study were to characterize the presence and molecular weight forms of MMP-2, -8, and -9 and their specific inhibitor, tissue inhibitor of metalloproteinases (TIMP)-2, in lungs of preterm infants during the early postnatal period and to determine whether levels of these MMPs and TIMP-2 in tracheal aspirate fluid (TAF) are associated with acute or chronic lung morbidity of the preterm infant. METHODS: TAF samples were collected from 16 intubated preterm infants (gestational age 27.0 +/- 2.0 weeks; birth weight 875 +/- 246 g) during their first 5 postnatal days. The presence and molecular weight forms of MMPs and TIMP-2 were identified by Western immunoblotting, and their levels were evaluated by densitometric scanning. RESULTS: MMP-8 in TAF was higher in infants who needed treatment with surfactant (25.4 +/- 6.3 vs 10.6 +/- 1.5 arbitrary unit/secretory component of immunoglobulin A [AU/SC]) and in whom BPD developed (N = 6; 27.6 +/- 5.2 vs 15.1 +/- 5.0 AU/SC). TIMP-2 levels were lower in infants with initial arterial to alveolar oxygen tension ratios <0.22 (2.7 +/- 1.1 vs 16.8 +/- 7.4 AU/SC) and in infants needing mechanical ventilation for >1 week (5.2 +/- 2.1 vs 22.8 +/- 11.7 AU/SC). CONCLUSIONS: In preterm infants, an imbalance between pulmonary MMP-8 and TIMP-2 participates in the acute inflammatory process in respiratory distress syndrome and may contribute to the development of chronic lung injury.

Bronchopulmonary Dysplasia↗
PubMed2001

Wound healing in ovariectomized rats: effects of chemically modified tetracycline (CMT-8) and estrogen on matrix metalloproteinases -8, -13 and type I collagen expression.

Cutaneous wound healing is a complex process involving interactions of various cell types. Skin, in addition to certain other organs, is dependent on estrogen; and estrogen-deficiency is associated with impaired wound healing. Wound healing involves the action of collagenolytic matrix metalloproteinases (MMPs). We investigated the expression and localization of collagenolytic MMPs -8 and -13 by collagenase activity assay, Western immunoblot analysis, in situ hybridization and immunohistochemical staining as well as type I collagen by hydroxyproline content analysis and immunohistochemical staining in cutaneous wounds from aged Sham and ovarioectomized (OVX) rats. After wounding, OVX rats were treated with either placebo, chemically modified tetracycline-8 (CMT-8) or estrogen. We found that MMP-8 and MMP-13 mRNA were expressed in wound epithelium of all samples examined as evidenced by in situ hybridization. Type I collagen, which was abundant in all groups examined, was decreased in OVX rats, but was increased by both CMT-8 and estrogen treatments to the level of Sham group. Hydroxyproline analysis revealed similar results. Western blot data showed that all forms of MMP-8 and MMP-13 were clearly reduced in the CMT-8 treated group compared to OVX. Analysis of collagenolytic activity confirmed the decreased collagenolysis in skin wound extracts from CMT-treated rats when compared with skin wound extracts from OVX rats. Our results show for the first time that MMP-8 mRNA and protein are expressed in rat wound epithelium. We further show that CMT-8 and estrogen have a beneficial effect on skin wound healing in OVX rats by increasing the collagen content and reducing the MMP-mediated collagenolysis.

Animals↗
PubMed2001

Association of trypsin-2 with activation of gelatinase B and collagenase-2 in human bronchoalveolar lavage fluid in vivo.

BACKGROUND: Tissue injury mediated by matrix metalloproteinases (MMPs) is a hallmark of inflammatory lung diseases. Latent secreted proMMPs must be activated to be catalytically competent. AIM: Our aim was to analyse an involvement of the trypsin-2, trypsin-2-alpha1-proteinase inhibitor (PI) complex and tumour-associated trypsin inhibitor (TATI) in the in vivo activation of proMMP-8, -9 and -2. METHODS: Concentrations of trypsin-2, trypsin-2-alpha1-PI complex and TATI in bronchoalveolar lavage fluid (BALF) were analysed by immunofluorometry. Molecular forms and expression of trypsin-2 and trypsin-2-alpha1-PI complex were identified by Western immunoblot and immunocytochemistry. Gelatinolytic and collagenolytic activities were measured by substrate-based activity assays. RESULTS: BALFs from 16 of 43 patients and BALFs from five of 15 healthy controls contained trypsin-2-alpha1-PI complex. TATI was found in all healthy control BALFs (median 0.12 microg/L, range 0.02-0.66 microg/L) whereas 8 of 43 BALFs from patients (median 0, range 0-0.64 microg/L, P = 0.0001) contained TATI. Patient BALFs showed significantly increased activation of MMP-9 and MMP-8 compared with healthy controls. The concentrations of trypsin-2-alpha1-PI complex correlated with the in vivo activation of MMP-9 and -8 (r = 0.68, P = 0.002 and r = 0.61, P = 0.008) but not with the activation of MMP-2 in BALFs. CONCLUSION: Results show a key role of trypsin-2 in the in vivo activation of proMMP-8 and -9 in inflammatory lung diseases.

Adult↗
PubMed2000

Differences in the concentration of various synovial fluid constituents between the distal interphalangeal joint, the metacarpophalangeal joint and the navicular bursa in normal horses.

As a prerequisite for the identification of navicular disease markers, the concentrations of cartilage oligomeric matrix protein (COMP), total glycosaminoglycans (GAG), hyaluronan, metalloproteinases (MMP) 2 and 9 and total protein were measured in synovial fluid samples obtained from the distal interphalangeal joint (DIP), the metacarpophalangeal joint (MCP) and the navicular bursa of 24 horses. Mean GAG, COMP and total protein levels were significantly higher in the DIP joint and in the navicular bursa compared to the MCP joint. Hyaluronan content was lower. MMP -2 activity was present in all fluids measured and had similar levels in different joints. MMP -9 was present in 42 per cent of MCP joint samples and 58 per cent of DIP joint samples and of navicular bursal samples. In relation to the constituents measured, the composition of navicular bursal fluid was similar to the articular synovial fluids, in particular that obtained from the DIP joint. Correlation between the constituents of DIP joint fluid and navicular bursal fluid obtained from the same legs was statistically significant for all the parameters measured.

Animals↗
PubMed2000

The in vivo expression of the collagenolytic matrix metalloproteinases (MMP-2, -8, -13, and -14) and matrilysin (MMP-7) in adult and localized juvenile periodontitis.

Periodontal inflammation is characterized by irreversible degradation of periodontal ligament collagen fibers leading to loss of tooth attachment. Cultured gingival keratinocytes and fibroblasts express, in vitro, various matrix metalloproteinases (MMPs) which can degrade fibrillar collagens. We hypothesized that several MMPs are also synthesized in vivo by sulcular epithelium, and analyzed the collagenolytic MMPs (MMP-2, -8, -13, and -14) and matrilysin (MMP-7) in gingival tissue specimens and gingival crevicular fluid from adult and localized juvenile periodontitis patients by in situ hybridization, immunohistochemistry, and Western immunoblotting. MMP-2, -7, -8, and -13 were expressed in gingival sulcular epithelium. MMP-7 and -13 were also located in fibroblasts and macrophages, and MMP-8 in neutrophils. MMP-8- and -13-positive cells/mm2 were higher in periodontitis gingiva when compared with healthy control tissue (p < 0.01). In periodontal diseases, gingival sulcular epithelium expresses several, rather than a single, collagenolytic MMPs, and this proteolytic cascade is evidently responsible for the tissue destruction characteristic of adult and juvenile periodontitis.

Adolescent↗
PubMed1999

Matrix metalloproteinase 2 (gelatinase A) is related to migration of keratinocytes.

The role of matrix metalloproteinases (MMPs) in cell migration was studied by measuring cell growth, migration, and production of MMP-2 and -9 in oral mucosal and skin keratinocytes cultured in the presence of synthetic MMP inhibitors. MMP-2 was the major gelatinolytic MMP produced by these cells while MMP-9 was produced at a low basal level. Inhibitor effects on MMP-9 production were therefore studied in keratinocytes stimulated by tumor necrosis factor alpha (TNFalpha). Tetracycline analogues at concentrations that inhibited the production of MMP-2 but not MMP-9 were able to drastically inhibit migration of both mucosal and skin keratinocytes. Tetracycline analogues also inhibited keratinocyte growth, an effect not found for the other inhibitors tested. Heterocyclic carbonate-derived compounds (LWs) that inhibited MMP-9 but not MMP-2 production had no effect on cell migration. Batimastat, a potent MMP inhibitor, did not have any effect on MMP production or cell growth but did inhibit keratinocyte migration. Tumor growth factor beta (TGFbeta) increased keratinocyte migration as well as both cell-associated and secreted MMP-2 production in wounded cell cultures. The secreted enzyme was partially converted into an active form. In this model batimastat totally blocked TGFbeta-promoted keratinocyte migration. Immunostaining of keratinocytes advancing into the wound revealed that MMP-2 was localized in extracellular matrix contactlike structures against the endogenously produced laminin-5-rich matrix. MMP-9 was localized diffusely along the cell membranes. Using in situ hybridization we observed that in chronically inflamed human gingiva MMP-2 is expressed in epithelium extending into subepithelial connective tissue. These results suggest that MMP-2 plays a specific role in epithelial migration, possibly by detaching the advancing cells from the pericellular matrix or by activating other MMPs.

Cell Adhesion Molecules↗
PubMed1999

MMP inhibition and downregulation by bisphosphonates.

Bisphosphonates are a group of drugs capable of inhibiting bone resorption, and are thus used for the treatment of bone diseases, such as Paget's disease, osteoporosis, and for bone metastases of malignant tumors. Their primary cellular target is considered to be the osteoclast. The molecular mechanisms responsible for the downregulation of bone resorption by bisphosphonates have remain unclear. We have discovered that various matrix metalloproteinases (MMPs) are inhibited in vitro by several bisphosphonates. This novel finding may, in part, explain the efficacy of bisphosphonates in their current indications in humans. In enzyme activity tests using purified and recombinant enzymes, we have observed the inhibition of MMP-1, -2, -3, -7, -8, -9, -12, -13, and -14 by clondronate, alendronate, pamidronate, zolendronate, nedrinate, and clodrinate. The IC50s range from 50 to 150 microM. We have also shown that clodronate can downregulate the expression of MT1-MMP protein and mRNA in several cell lines. Additionally, several bisphosphonates decrease the degree of invasion of malignant melanoma (C8161) and fibrosarcoma (HT1080) cells through artificial basement membrane (Matrigel) in cell cultures at IC50s of 50-150 microM and below. Having low toxicity and proven to be well tolerated after several years in human use, bisphosphonates have the potential to become one of the main MMP-inhibitors for MMP-related human soft and hard tissue-destructive diseases in the near future.

Alendronate↗