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Results for “Matrix Metalloproteinase 8”

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PubMed2002

Release and activation of matrix metalloproteinase 8 from human neutrophils triggered by the leukotoxin of Actinobacillus actinomycetemcomitans.

Matrix metalloproteinase 8 (MMP 8) degrades type I collagen and may be involved in the pathogenesis of periodontitis. Latent MMP 8 is stored in neutrophil granules and can be activated when released extracellularly. The periodontitis-associated bacterium Actinobacillus actinomycetemcomitans produces an RTX-toxin, leukotoxin, that degranulates and lyses human neutrophils. This study deals with the ability of leukotoxic A. actinomycetemcomitans to trigger the release and activation of MMP 8. Whole bacteria of three A. actinomycetemcomitans strains or leukotoxin purified from the highly toxic strain HK 1519 were incubated with human neutrophils. The extracellularly released latent and active forms of MMP 8 were detected by an immunoblot technique using specific antibodies against the protease. The activity of MMP 8 was determined by a collagen degradation assay. All strains induced release and activation of MMP 8. The effect was more pronounced under aerobic than anaerobic conditions and correlated with the leukotoxicity of the strains. Pure leukotoxin also induced MMP 8 release and activation in a concentration-dependent manner. Under aerobic conditions, oxidising substances formed by the neutrophils contributed to the rapid activation of the latent enzyme. Upon anaerobic incubation, the activation was slow and mainly caused by other proteases released during neutrophil degranulation. The activation was totally abolished in the presence of serum, probably due to the serum-protease inhibitors. Compared to the calcium ionophore A 23187, a well-known stimulus of neutrophil degranulation, leukotoxin was a more powerful inducer of MMP 8 release, since it triggered the process at a 1000-fold lower concentration. The present findings reveal a specific mechanism that can be induced by A. actinomycetemcomitans leukotoxin and which may contribute to the degradation of periodontal tissues under certain conditions.

Aerobiosis↗

PubMed2005

Matrix metalloproteinase 2 is associated with stable and matrix metalloproteinases 8 and 9 with vulnerable carotid atherosclerotic lesions: a study in human endarterectomy specimen pointing to a role for different extracellular matrix metalloproteinase inducer glycosylation forms.

BACKGROUND AND PURPOSE: We studied matrix metalloproteinases (MMP) 2, 8, and 9 and extracellular matrix metalloproteinase inducer (EMMPRIN) levels in relation to carotid atherosclerotic plaque characteristics. METHODS: Carotid atherosclerotic plaques (n=150) were stained and analyzed for the presence of collagen, smooth muscle cell (SMC), and macrophages. Adjacent segments were used to isolate total protein to assess MMP-2 and MMP-9 activities and gelatin breakdown, MMP-8 activity, and EMMPRIN levels. RESULTS: Macrophage-rich lesions revealed higher MMP-8 and MMP-9 activities, whereas SMC-rich lesions showed higher MMP-2 activity. The levels of less glycosylated EMMPRIN-45kD were higher in SMC-rich lesions and lower in macrophage-rich plaques. EMMPRIN-45kD was associated with MMP-2 levels, whereas EMMPRIN-58kD was related to MMP-9 levels. CONCLUSIONS: MMP-2, MMP-8, and MMP-9 activities differed among carotid plaque phenotypes. Different EMMPRIN glycosylation forms are associated with either MMP-2 or MMP-9 activity, which suggests that EMMPRIN glycosylation may play a role in MMP regulation and plaque destabilization.

Analysis of Variance↗

PubMed2001

The 1.8-A crystal structure of a matrix metalloproteinase 8-barbiturate inhibitor complex reveals a previously unobserved mechanism for collagenase substrate recognition.

The individual zinc endoproteinases of the tissue degrading matrix metalloproteinase (MMP) family share a common catalytic architecture but are differentiated with respect to substrate specificity, localization, and activation. Variation in domain structure and more subtle structural differences control their characteristic specificity profiles for substrates from among four distinct classes (Nagase, H., and Woessner, J. F. J. (1999) J. Biol. Chem. 274, 21491-21494). Exploitation of these differences may be decisive for the design of anticancer or other drugs, which should be highly selective for their particular MMP targets. Based on the 1.8-A crystal structure of human neutrophil collagenase (MMP-8) in complex with an active site-directed inhibitor (RO200-1770), we identify and describe new structural determinants for substrate and inhibitor recognition in addition to the primary substrate recognition sites. RO200-1770 induces a major rearrangement at a position relevant to substrate recognition near the MMP-8 active site (Ala206-Asn218). In stromelysin (MMP-3), competing stabilizing interactions at the analogous segment hinder a similar rearrangement, consistent with kinetic profiling of several MMPs. Despite the apparent dissimilarity of the inhibitors, the central 2-hydroxypyrimidine-4,6-dione (barbiturate) ring of the inhibitor RO200-1770 mimics the interactions of the hydroxamate-derived inhibitor batimastat (Grams, F., Reinemer, P., Powers, J. C., Kleine, T., Pieper, M., Tschesche, H., Huber, R., and Bode, W. (1995) Eur. J. Biochem. 228, 830-841) for binding to MMP-8. The two additional phenyl and piperidyl ring substituents of the inhibitor bind into the S1' and S2' pockets of MMP-8, respectively. The crystal lattice contains a hydrogen bond between the O(gamma) group of Ser209 and N(delta)1 of His207 of a symmetry related molecule; this interaction suggests a model for recognition of hydroxyprolines present in physiological substrates. We also identify a collagenase-characteristic cis-peptide bond, Asn188-Tyr189, on a loop essential for collagenolytic activity. The sequence conservation pattern at this position marks this cis-peptide bond as a determinant for triple-helical collagen recognition and processing.

Amino Acid Sequence↗

PubMed2001

Systematic separation and purification of elastase, gelatinase (matrix metalloproteinase 9), and collagenase (matrix metalloproteinase 8) from polymorphonuclear leukocytes in dialyzers previously used by patients with renal failure.

We developed a simple and effective method for the systematic separation and purification of human polymorphonuclear leukocyte (PMN) proteinases, elastase, gelatinase (matrix metalloproteinase 9, type IV collagenase), and collagenase (matrix metalloproteinase 8), derived from the extracts of hollow fiber dialyzers that had been utilized in the treatment of patients with renal failure. The fraction containing elastase was grossly separated from that containing gelatinase and collagenase by heparin-Sepharose chromatography and purified in an aprotinin column. The remaining two enzymes were then separated using the gelatin-Sepharose column after gel chromatography following ammonium sulfate precipitation. Gelatinase and collagenase were further purified by gelatin-Sepharose chromatography as a latent form and by collagen-Sepharose chromatography as an activated form. This novel method offers procedural advantages over existing methods that separate PMNs from the whole blood of volunteers for experimental research purposes.

Animals↗

PubMed2000

Human neutrophil collagenase (matrix metalloproteinase 8) in parturition, premature rupture of the membranes, and intrauterine infection.

OBJECTIVES: The mechanisms by which microbial invasion of the amniotic cavity leads to membrane weakening and rupture are poorly understood. Recently, endogenous host enzymes have been implicated in this process. Matrix metalloproteinases are a family of potent enzymes that degrade components of the extracellular matrix. Collagen type I provides the main tensile strength of the fetal membranes. Matrix metalloproteinase 8 (MMP-8), or neutrophil collagenase, degrades interstitial collagens, acting preferentially on collagen type I. This study was undertaken (1) to determine whether MMP-8 is present in amniotic fluid and whether its concentrations are changed in preterm and term labor and membrane rupture with and without intra-amniotic infection and (2) to determine whether the amniotic fluid concentrations of MMP-8 in labor at term are different in the lower and upper uterine compartments. STUDY DESIGN: A cross-sectional study was conducted and transabdominal amniocentesis was performed in women in the following categories: (1) midtrimester (n = 25), (2) preterm labor in the presence and absence of microbial invasion of the amniotic cavity (n = 86), (3) preterm premature rupture of the membranes in the presence and absence of microbial invasion of the amniotic cavity (n = 51), (4) term patients in labor and not in labor (n = 51), and (5) term premature rupture of membranes (n = 20). Additional paired samples of amniotic fluid were retrieved by transabdominal amniocentesis (upper compartment) and transvaginal amniocentesis (lower or forebag compartment) from 14 term patients (28 samples) in spontaneous labor with intact membranes. Amniotic fluid MMP-8 concentrations were determined with a sensitive and specific immunoassay. RESULTS: MMP-8 was detected in 95.4% (249/261) of all samples. (1) Spontaneous human parturition was associated with a significant increase in amniotic fluid concentrations of MMP-8 in both term and preterm gestation. Term (no labor median, 3.3 ng/mL; range, <0.06-38.6 ng/mL; vs labor median, 16.6 ng/mL; range, 0. 33-1650 ng/mL; P <.05). Patients with preterm labor who delivered preterm (in the absence of microbial invasion of the amniotic cavity) had a significantly higher median amniotic fluid MMP-8 concentration than those with preterm labor who delivered at term (preterm labor, term delivery median, 3.1 ng/mL; range, <0.06-415.1 ng/mL; vs preterm labor, preterm delivery median, 32.5 ng/mL; range, <0.06-6006.6 ng/mL;P <.003). (2) Spontaneous rupture of membranes in preterm gestation but not in term gestation was associated with elevated amniotic fluid concentrations of MMP-8. Preterm gestation (preterm labor, intact membranes median, 3.1 ng/mL; range, <0.06-415. 1 ng/mL; vs preterm premature rupture of membranes median, 35.1 ng/mL; range, 0.71-1184.1 ng/mL; P <.05). Term gestation (intact membranes median, 3.3 ng/mL; range, 0.24-38.6 ng/mL; vs rupture of membranes median, 5.6 ng/mL; range, 0.22-19.8 ng/mL; P =.9). (3) Microbial invasion of the amniotic cavity was associated with a significant increase in amniotic fluid MMP-8 concentration in patients with preterm labor and intact membranes, as well as in patients with preterm premature rupture of membranes. Preterm labor (no microbial invasion of the amniotic cavity, preterm delivery median, 32.5 ng/mL; range, <0.06-6006.6 ng/mL; vs microbial invasion of the amniotic cavity median, 208.1 ng/mL; range, 4.2-14,600 ng/mL; P <.001). Preterm premature rupture of membranes (no microbial invasion of the amniotic cavity median, 35.1 ng/mL; range, 0.71-1184. 1 ng/mL; vs microbial invasion of the amniotic cavity median, 317.9 ng/mL; range, 2.16-14,500 ng/mL; P <.01). (4) The median amniotic fluid MMP-8 concentrations were significantly higher in fluid obtained from the forebag compartment than in that obtained from the upper compartment (median, 66.2 ng/mL; range, 7.4-170 ng/mL; vs median, 13.3 ng/mL; range, 2-170 ng/mL; respectively; P <.01). (ABSTRACT TRUNCATED)

Amniocentesis↗

PubMed1996

A one-step sandwich enzyme immunoassay for human matrix metalloproteinase 8 (neutrophil collagenase) using monoclonal antibodies.

A one-step sandwich enzyme immunoassay (EIA) system for human matrix metalloproteinase 8 (MMP-8, neutrophil collagenase, EC 3.4.24.7) has been established with a pair of monoclonal antibodies prepared against the zymogen of MMP-8 purified from human neutrophils. MMP-8 in samples simultaneously reacted with both solid-phase and peroxidase-labeled antibodies. Sensitivity of this EIA system was 0.34 micrograms/l (5.7 pg/assay) and linearity was obtained between 0.5 and 500 micrograms/l (8.3-8300 pg/assay). The EIA system recognized both precursor and active forms of MMP-8 but not MMP-8 complexed with tissue inhibitors of metalloproteinases. There was no difference in the MMP-8 levels between the plasma samples from patients with rheumatoid arthritis or osteoarthritis and those from healthy subjects (median 6.2 micrograms/l, range 1.5-28 micrograms/l). However, the level in synovial fluids from patients with rheumatoid arthritis (median 345 micrograms/l, range 84-2860 micrograms/l) was shown to be higher than that from osteoarthritic patients. MMP-8 levels in human whole saliva from patients with periodontal diseases (median 282 micrograms/l, range 0-1420 micrograms/l) were also significantly higher than those from clinically healthy subjects (median 25 micrograms/l, range 0-100 micrograms/l). Immunoreactivity analyses showed that MMP-8 species in normal human plasma exists as a precursor but not as a complex form with tissue inhibitor of metalloproteinases (TIMP)-1 or TIMP-2.

Animals↗

PubMed2005

The expression of matrix metalloproteinases 8 and 9 by neutrophils of Wistar albino rats with severe qualitative and quantitative protein malnutrition.

Neutrophils are the major cellular immune components in response to bacterial infections. Neutrophil enzymes are important in invasion, inflammation, and infection processes. In order to understand the basic effects of protein malnutrition on neutrophils we studied matrix metalloproteinases 8 and 9 (MMP-8 and MMP-9) production in severe quantitative and qualitative protein malnutrition in rats. Wistar rats (2 months old) were divided into four groups each with three subgroups and fed various protein-containing diets (24% protein, 20% gelatin-containing and N-free) for 7, 14, 21, and/or 28 days. Neutrophil enzyme expression was determined by Western blotting. Leukocytes decreased significantly due to malnutrition (p = 0.001 ) whilst the percentage of neutrophils increased (p = 0.02) in protein-deprived groups. Neutrophils of malnourished rats produced lower levels of MMP-8 at early stages of protein deprivation with an increase in the following weeks. MMP-9 production by neutrophils from N-free diet fed animals was highest after one week. Serum MMP-9 levels decreased in the qualitative but not in the quantitative protein malnutrition groups. Results suggest that neutrophils might be important in reuse of body cell proteins during fasting or malnutrition conditions and dietary manipulation might have profound effects on MMP-8 and -9 production in rats.

Animals↗

PubMed1994

Reduction of matrix metalloproteinase 8-neutrophil collagenase levels during long-term doxycycline treatment of reactive arthritis.

The aim of this work was to determine whether human polymorphonuclear neutrophilic interstitial collagenase (matrix metalloproteinase 8 [MMP-8]) levels are reduced during long-term doxycycline treatment in humans with reactive arthritis. Serum MMP-8 levels were reduced (mean +/- standard error of the mean, 678.9 +/- 185.6 versus 491.2 +/- 144.8 ng of MMP-8 per ml), but not statistically significantly. However, the reduction of salivary MMP-8 levels was statistically significant (3,729 +/- 1,905.3 versus 1,866 +/- 780.0 ng of MMP-8 per ml, P < 0.05). This study demonstrated that a 2-month regimen of doxycycline can reduce MMP-8 levels in serum and especially in body fluids (i.e., saliva) containing inflammatory exudates and thus may contribute to reduced tissue destruction.

Adult↗

PubMed2005

Repair after cholestatic liver injury correlates with neutrophil infiltration and matrix metalloproteinase 8 activity.

BACKGROUND: Although timely surgical treatment of liver disease can interrupt inflammation and reduce fibrosis, the mechanisms of repair are unknown. We questioned whether these mechanisms of repair include changes in the inflammatory infiltrate and associated biological activity of matrix metalloproteinases (MMPs) 8 and 2. METHODS: Rats (n >or= 3) underwent biliary ductal suspension for 7 days followed by decompression. Livers were collected after 7 days of obstruction (d0) and after 2, 5, and 7 days of repair (d2, d5, d7, respectively), and assessed morphometrically for collagen, polymorphonuclear cells (PMNs), Kupffer cells (KCs), and inflammatory mononuclear phagocytes (MNPs). In situ zymography was performed by using fluorogenic substrates for MMP-8 and MMP-2 to spatially localize enzymatic activity. RESULTS: Cholestatic injury resulted in significantly elevated (P <or= .001) collagen deposition (3-fold), and elevated numbers of MNPs (10-fold), KCs (5-fold), and PMNs (4-fold), compared with shams. PMNs remained elevated through d7, while collagen deposition, KCs, and MNPs returned to sham levels by d2. In situ zymography showed no significant changes in MMP-2 activity after cholestatic injury and repair. MMP-8 activity was significantly (P <or= .05) elevated only during repair. Activity was localized to fibrotic portal triads containing PMNs. CONCLUSIONS: Cholestatic injury results in increased fibrosis, MNPs, KCs, and PMNs but no MMP-2 or MMP-8 activity. Biliary decompression results in increased MMP-8 activity co-localized to areas of portal fibrosis and PMN accumulation. We conclude that secretion of MMP-8 by neutrophils may play a critical role in resolving the fibrotic scar generated during cholestasis.

Animals↗

PubMed2004

Pivotal molecular determinants of peptidic and collagen triple helicase activities reside in the S3' subsite of matrix metalloproteinase 8 (MMP-8): the role of hydrogen bonding potential of ASN188 and TYR189 and the connecting cis bond.

The mechanism of triple helical collagen unwinding and cleavage by collagenases in the matrix metalloproteinase (MMP) family is complex and remains enigmatic. Recent reports show that triple helicase activity is initiated by the hemopexin C domain of membrane type 1-MMP, whereas catalytically inactive full-length interstitial collagenase (MMP-1) exhibits full triple helicase functionality pointing to active site determinants that are needed to complete the triple helicase mechanism. In MMP-8, the neutrophil collagenase, a conserved Gly at the S(3)' substrate specificity subsite is replaced by Asn(188) that forms a highly unusual cis bond with Tyr(189), a conserved active site residue in the collagenases. Only in MMP-1 is the S(3)' Gly also replaced, and there too a cis configured Glu-Tyr occurs. Thus, this high energy peptide bond coupled to the canonical Tyr may be important in the collagenolytic process. In a systematic mutagenesis investigation of the MMP-8 S(3)' subsite we found that introducing an S(3)' Gly(188) into MMP-8 reduced collagenolytic efficiency by approximately 30% with a corresponding reduction in cleavage of a synthetic peptide fluorescence resonance energy transfer substrate analogue of the alpha2(I) collagen chain cleavage site. The substitution of Asn(188) to Leu, a hydrophobic residue of similar size to the highly polar Asn and designed to retain the cis bond, revealed the importance of hydrogen bonding to bound substrate with both collagenolytic and peptidic activities reduced approximately 3-fold. In contrast, the specificity for type I collagen of the mutant Y189F dropped 3-fold without any significant alteration in general peptidase activity. Therefore, S(3)' and in particular the hydrogen bonding potential of Tyr(189) is a specific molecular determinant for MMP-8 triple helicase activity. The cis bond connection to Asn(188) juxtaposes these two side chains for closely spaced hydrogen bonding with substrate that improves collagenolytic and general catalytic efficiency that could be exploited for new collagenase-specific inhibitor drugs.

Amino Acid Sequence↗

PubMed2005

Matrix metalloproteinase 8 (neutrophil collagenase) in the pathogenesis of abdominal aortic aneurysm.

BACKGROUND: Loss of elastin is the initiating event in abdominal aortic aneurysm (AAA) formation, whereas loss of collagen is required for continued expansion. The elastolytic matrix metalloproteinases (MMPs) 2 and 9 are well described, but the source of excessive collagenolysis remains undefined. The aim of this study was to determine the expression of MMP-8, a potent type I collagenase, in normal aorta and AAA. METHODS: Infrarenal aortic biopsies were taken from 40 AAA and ten age-matched normal aortas. The concentrations of MMP-8 protein and its inhibitors, tissue inhibitor of metalloproteinase (TIMP) 1 and TIMP-2, were quantified by enzyme-linked immunosorbent assay. Immunohistochemistry was used to localize MMP-8 expression. RESULTS: MMP-8 concentrations were significantly raised in AAA compared with normal aorta (active MMP-8: 4.5 versus 0.5 ng per mg protein, P < 0.001; total MMP-8: 16.6 versus 2.8 ng per mg protein, P < 0.001). Levels of TIMP-1 and TIMP-2 were significantly lower in AAA than in normal aortic samples (TIMP-1: 142.2 versus 302.8 ng per mg protein; P = 0.010; TIMP-2: 9.2 versus 33.1 ng per mg protein, P < 0.001). Immunohistochemistry localized MMP-8 to mesenchymal cells within the adventitia of the aortic wall. CONCLUSION: The high concentration of MMP-8 in aortic aneurysms represents a potent pathway for collagen degradation, and hence aneurysm formation and expansion.

Aged↗

PubMed2004

Altered metastatic behavior of human breast cancer cells after experimental manipulation of matrix metalloproteinase 8 gene expression.

Previous work in our laboratory led to the cloning, from the same parent tumor cell line (MDA-MB-435), of two human breast cancer cell lines (M-4A4 and NM-2C5) with opposite metastatic phenotypes. Additional investigations revealed that the nonmetastatic cell line NM-2C5 overexpressed the neutrophil collagenase, matrix metalloproteinase (MMP)-8, relative to its partner. Because other studies have implicated the MMP family in promoting tumor metastasis, we investigated the apparently paradoxical expression of MMP-8 in these cell lines. By genetic engineering, we inverted its relative levels of expression in the two partners and studied the effects on the behavior of the tumors that they generated in athymic mice. Knock-down of expression in NM-2C5 cells by transduction with a sequence encoding a specific ribozyme and overexpression of MMP-8 in M-4A4 cells by retroviral transduction both strikingly changed metastatic performance in opposite directions, indicating that this gene plays a role in the regulation of tumor metastasis.

Animals↗

PubMed2001

Correlation among macrophage inflammatory protein 1alpha levels, matrix metalloproteinase 8 levels, and systemic inflammation in rheumatoid arthritis: comment on the articles by Yamanaka et al and Mattey et al.

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Arthritis, Rheumatoid↗

PubMed2003

Expression of matrix metalloproteinase 8 (MMP-8) and tyrosinase-related protein-1 (TYRP-1) correlates with the absence of metastasis in an isogenic human breast cancer model.

The multi-step nature of metastasis poses difficulties in both design and interpretation of experiments to unveil the mechanisms causing the process. In order to facilitate such studies, we have previously derived a pair of breast tumor cell lines that originate from the same breast tumor but which have diametrically opposite metastatic capabilities. In this system, the monoclonal cell line M-4A4 is metastatic to the lungs of athymic mice, whereas clone NM-2C5 is equally tumorigenic but non-metastatic. Here, we report that representational difference analysis (RDA) of cDNA obtained from the two clonal populations revealed an increased expression of tyrosinase-related protein-1 (TYRP-1) and the matrix metalloproteinase-8 (MMP-8) genes in the non-metastatic cell line. RNA and protein analyses in cultured cells and in primary xenograft tissues confirmed that the non-metastatic cell line expresses TYRP-1 and MMP-8 at levels that are at least 20-fold higher than the metastatic counterpart. Other members of the MMP family (MMP-9 and MMP-2) and the tissue inhibitor of metalloproteinase-2 (TIMP-2) were found to be expressed at similar levels in both populations. The effects of MMP-8 and TYRP-1 on in vitro invasion and migration were assessed in cells whose expression of these genes was altered by stable transduction with sense and antisense constructs. Specific down-regulation of MMP-8 in non-metastatic NM-2C5 cells resulted in a 2.5-fold increased capacity to invade through Matrigel. Unlike other members of the matrix metalloproteinase family, MMP-8 has not previously been implicated in the processes of tumorigenesis or metastasis. The successful identification of two proteins that are differentially expressed in these matched clonal cell lines and the tumors that they produce demonstrates the feasibility of using this approach to search for genes that are associated with aberrant differentiation toward metastatic behavior.

Animals↗

PubMed1999

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.

Chlorhexidine↗

PubMed1994

Degradation of kinins, angiotensins and substance P by polymorphonuclear matrix metalloproteinases MMP 8 and MMP 9.

1. The kinetics of the degradation of the kinins bradykinin and Met-Lys-bradykinin, angiotensins I and II and the tachykinin substance P by PMNL-collagenase (MMP 8), PMNL-gelatinase (MMP 9) and by the recombinant catalytic domain of MMP 8 (rcd-PMNL-c) was examined by RP-HPLC. The resulting fragments were identified by automated Edman degradation or by amino acid analysis. 2. The initial degradation rates of substance P at a substrate concentration of 25 microM were 5 min-1 for MMP 9 and 150 min-1 for MMP 8. The kinetic constants KM and kcat were determined by concentration-dependent measurements. For MMP 8/substance P the constants were KM = 78 +/- 14 microM and kcat = 412 +/- 67 min-1. For MMP 9/substance P the constants were KM = 91 +/- 15 microM and kcat = 25 +/- 4 min-1. Both enzymes cleaved substance P between Gln6 and Phe7 and between Gly9 and Leu10. 3. Under the same conditions, MMP 8 degraded angiotensin I at an initial rate of 20 h-1, resulting mainly in the vasoactive fragments angiotensin II and angiotensin(1-7). At a substrate concentration of 25 microM and an enzyme/substrate ratio of 1:100, angiotensin II was degraded very slowly (19% in 24 h) by MMP 8. Under these conditions, MMP 9 degraded angiotensin I to a lesser extent than MMP 8 (25% in 24 h) and was unable to cleave angiotensin II. 4. Under the same conditions, bradykinin and Met-Lys-bradykinin were cleaved by PMNL-collagenase at a rate of 20% in 24 h, producing BK(1-7) and BK(1-8). PMNL-gelatinase was unable to cleave the kinins under these conditions. 5. In all cases, rcd-PMNL-c produced the same fragments as wild type PMNL-collagenase, but at a significantly lower rate.

Amino Acid Sequence↗

PubMed2002

Inhibition of type I procollagen production in photodamage: correlation between presence of high molecular weight collagen fragments and reduced procollagen synthesis.

Three-dimensional lattices of reconstituted, polymerized type I collagen were subjected to partial hydrolysis by organ culture fluid from human skin or by various matrix metalloproteinases, including matrix metalloproteinase-1 (interstitial collagenase), -2 (72 kDa gelatinase A), -8 (neutrophil collagenase), -9 (92 kDa gelatinase B), or -13 (collagenase 3). Following partial digestion, human dermal fibroblasts were incubated on the enzyme-treated or control lattices and examined for ability to contract the collagen lattice and synthesize type I procollagen. Collagen lattices partially degraded by organ culture fluid were contracted by fibroblasts under conditions in which control collagen lattices were not. On the partially degraded collagen, fibroblasts synthesized reduced amounts of type I procollagen (approximately 70% reduction). Purified matrix metalloproteinases with collagenolytic activity duplicated the effects of the human skin organ culture fluid, although matrix metalloproteinases 8 and 13 were less efficient than matrix metalloproteinase-1 (65% vs 40% and 18% reduction in type I procollagen production for matrix metalloproteinases 1, 8, and 13, respectively). Matrix metalloproteinases 2 and 9 were without effect on intact collagen; however, when collagen lattices were subjected to digestion by a combination of matrix metalloproteinases 1 and 9, fragments produced by matrix metalloproteinase-1 were further degraded by the gelatinase. Collagen contraction and inhibition of procollagen synthesis were both reduced. Matrix metalloproteinase-2 was less effective than matrix metalloproteinase-9 in clearing matrix metalloproteinase-1-generated fragments. Matrix metalloproteinase-2 was also less effective in preventing contraction and inhibiting the downregulation of type I procollagen synthesis. These observations suggest that in the presence of high molecular weight fragments of type I collagen, type I procollagen synthesis is inhibited. As these fragments are processed further, there is less inhibition of type I procollagen production.

Cell Size↗

PubMed2000

Analysis of in situ protease activity in chronic adult periodontitis patients: expression of activated MMP-2 and a 40 kDa serine protease.

BACKGROUND: Periodontitis is characterized by extensive destruction of the gingival tissues and associated supporting structures of the teeth. Although the pathogenesis of the various forms of this disease is not completely understood, host-derived proteases are believed to have an important role. In this study, we analyzed human tissue samples from chronic adult periodontitis patients to assess the levels of specific proteases and determine the effect of pH and tetracyclines on their activity. METHODS: Gingival tissue samples were obtained from patients with chronic adult periodontitis (probing depths ranged from 5 to 9 mm) and periodontally healthy controls. Tissue extracts were prepared and analyzed for protease activity by zymography and Western blotting. RESULTS: Maximal protease activity from clinically normal and diseased tissues was observed at pH 8. Latent matrix metalloproteinase (MMP)-9 and MMP-2 were expressed in all samples examined, while active MMP-2 was detected only in tissues obtained from patients with clinical disease. The MMP activities were differentially inhibited by derivatives of tetracycline. At pH 6, a protease with a mass of approximately 40 kDa was observed in diseased samples. The enzymatic activity was inhibited by phenylmethylsulfonyl fluoride, suggesting it is a serine protease. CONCLUSIONS: The results of the current study substantiate the proposed role of host-derived proteases in the pathogenesis of chronic adult periodontitis. Specifically, they indicate that activated MMP-2 and a 40 kDa serine protease are involved in tissue destruction associated with this form of periodontal disease and also suggest that tissue pH influences protease activity in situ.

Adult↗