Increased Levels Of MMP-9 Research Articles

Host CD147 blockade by small interfering RNAs suppresses growth of human colon cancer xenografts

Tumor cells can stimulate matrix metalloproteinase (MMP) production by stromal cells through cell-cell interactions mediated by cell adhesion molecules such as extracellular matrix metalloproteinase inducer (human CD147/EMMPRIN, mouse CD147/Basigin). This study sought to characterize whether specific tumor-stromal cell interactions mediated by CD147 promote colon cancer growth by utilizing small interfering (si)RNAs directed against human CD147/EMMPRIN or mouse CD147/Basigin in co-cultures of cancer cells with macrophages and fibroblasts and established human SW620 colon cancer xenograft models in immune deficient mice. We show that blockade of host (mouse) CD147/Basigin expression, but not cancer cell-derived CD147/EMMPRIN, suppresses tumor growth in human colon cancer xenografts. Experiments in vitro indicated that colon cancer cell-stromal cell interactions mediated by CD147 lead to increased MMP-2 expression in fibroblasts but not macrophages. Furthermore, expression of host VEGF-A in both fibroblasts and macrophages is independent of CD147 in vitro and in vivo. Interestingly, inhibition of cancer cell-derived EMMPRIN leads to increased MMP-9 levels in vivo. Our findings provide new insights into CD147-mediated tumor-host interactions mediating colon cancer growth.

Upregulation of EMMPRIN after permanent focal cerebral ischemia

Elevated activities of matrix metalloproteinases (MMPs) following ischemic stroke have been shown to mediate ischemic injury as well as neurovascular remodeling. The extracellular MMP inducer (EMMPRIN) is a 58-kDa cell surface glycoprotein, which has been known to play a key regulatory role for MMP activities. The roles of EMMPRIN in stroke injury are not clearly understood. In this study, we investigated changes of EMMPRIN in a mouse model of permanent focal cerebral ischemia, and examined potential association between EMMPRIN and MMP-9 expression. Adult male CD-1 mice were subjected to permanent focal ischemia by intraluminal occlusion of the left middle cerebral artery (MCAO) under anesthesia. EMMPRIN expression was markedly upregulated in the peri-infarct area at 2–7 days after ischemia compared to the contralateral non-ischemic hemisphere by Western blot analysis. Immunofluorescent double staining demonstrated that EMMPRIN signals co-localized with vwF-positive endothelial cells and GFAP-positive peri-vascular astrocytes. In contrast, EMMPRIN signal did not co-localize with NeuN-positive neurons, or MPO-positive neutrophils. Dual fluorescent staining revealed that EMMPRIN co-localized with MMP-9. Our data also demonstrated that increased EMMPRIN expression correlated with increased MMP-9 levels in a temporal manner. In summary, we report for the first time that EMMPRIN expression was significantly increased in a mouse model of permanent focal cerebral ischemia. The spatial and temporal association between increased EMMPRIN expression and elevated MMP-9 levels suggest that EMMPRIN may modulate MMP-9 activity, and participate in neurovascular remodeling after ischemic stroke.

Effects of Topical Glutathione Treatment in Rat Ischemic Wound Model

Oxidative stress secondary to ischemia can cause physiopathologic changes that adversely affect wound healing. In this experimental study, we hypothesized that the topical use of esterified glutathione, a well-known antioxidant, can minimize the effects of oxidative stress by an increase in intracellular glutathione and accelerate wound healing by increasing the contraction capacity of fibroblasts and preventing keratinocytes from apoptosis in a rat ischemic wound model. Experimental models were divided into 3 groups as treatment, control, and healthy. Bipedicled flaps were elevated from the dorsum of the rats, and 6-mm punch wounds were created at the end of the first day when the ischemia is most apparent. Wounds were followed histopathologically and immunohistochemically, and matrix metalloproteinase (MMP)-1 and tissue inhibitors of metalloproteinase (TIMP-1) levels were measured by ELISA. Samples were collected at 0, 5, 8, 10, and 12 days. Histopathologic evaluation revealed significant extracellular matrix deposition and reepithelization every fifth day in treatment and healthy groups when compared with control group. Immunohistochemical evaluation revealed increased apoptosis in basal keratinocytes in the control group when compared with the other groups. The evaluation of the samples collected at 5 and 8 days revealed increased MMP-1 levels in treatment and control groups, but the increase in TIMP-1 levels was more significant than MMP-1 levels in treatment group. MMP-1/TIMP-1 ratio was significantly low in the treatment group.Our results showed that topical GSH treatment can reduce oxidative stress, and the reestablishment of the MMP-1/TIMP-1 ratio gives way to adequate and regular extracellular matrix production and reepithelization. It is concluded that esterified GSH, which is experimentally shown to be effective in ischemic wound healing, can be used clinically in ischemic wounds.

Concluding remarks

Concluding remarks

Regulation of type I procollagen and MMP-1 expression after single or repeated exposure to infrared radiation in human skin

Human skin is daily exposed to infrared (IR) radiation from natural sunlight. However, the effects of IR irradiation on collagen metabolism have not been investigated in human skin in vivo. Here, we examined whether single or repeated (three times a week for 4 weeks) exposure to IR irradiation changes the expressions of type I procollagen and interstitial collagenase (MMP-1). By using immunostaining, Western blotting, and semi-quantitative RT-PCR, we analyzed the protein and mRNA levels of type I procollagen and MMP-1 in young buttock skin. A single dose of IR to human skin increased the expression of type I procollagen within 24 h, but did not change the expression of MMP-1. On the other hand, multiple IR doses reduced the expression of type I procollagen and increased the expression of MMP-1. We also found that TGF-βs may mediate type I procollagen synthesis in IR-irradiated human skin. Our results demonstrate that the regulations of the expressions of type I procollagen and MMP-1 differ in acute and chronically IR-irradiated skin. In particular, decreased collagen levels and increased MMP-1 levels in chronic IR-irradiated skin may be associated with connective tissue damage. Thus, we suggest that repeated exposure to IR irradiation might induce premature skin aging (photoaging) in human skin in vivo.

EFFECT OF NOS INHIBITION ON RAT GASTRIC MATRIX METALLOPROTEINASE PRODUCTION DURING ENDOTOXEMIA

Matrix metalloproteinases (MMPs) degrade the extracellular matrix and contribute to LPS-induced gastric injury. MMPs are closely modulated by their activators, membrane type-MMP (MT-MMPs) and their endogenous inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). As LPS-induced gastric injury is mediated in part by iNOS, and NO modulates MMP production in vitro, we hypothesized that NOS inhibition would similarly modulate LPS-induced gastric MMP production. Therefore, the purpose of these studies was to compare the effects of selective and nonselective NOS inhibition on LPS-induced gastric MMP production. Sprague-Dawley rats were given either the nonselective NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME; 5 mg/kg, s.c.), a selective iNOS inhibitor, aminoguanidine (45 mg/kg, i.p.) or L-N-iminoethyl-lysine (L-NIL; 10 mg/kg, i.p.), or vehicle 15 min before saline or LPS (20 mg/kg, i.p.) and killed 24 h after LPS administration. Stomachs were assessed for macroscopic injury (computed planimetry), and gastric mucosal MMP production was assessed by gelatin zymography, in situ zymography, and Western analysis for MMP-2, MT1-MMP, and TIMP-2. (n > or = 4/group; ANOVA). Aminoguanidine treatment decreased LPS-induced macroscopic gastric injury as well as MMP-2 and MT1-MMP protein production while having no effect on TIMP-2 protein levels. L-NIL similarly attenuated the induction of MMP-2 and MT1-MMP by LPS. L-NAME failed to attenuate LPS induced gastric injury or MT1-MMP protein induction and increased MMP-2 levels. L-NAME similarly had no effect on gastric TIMP-2 production. Selective iNOS inhibition decreases gastric MMP-2 activity after LPS administration, whereas nonselective inhibition increases MMP-2 levels. The ability of selective iNOS inhibition to ameliorate LPS-induced gastric injury may be due in part to its inhibition of active MMP-2 production, whereas nonselective NOS inhibitors increase MMP-2 levels and maintain gastric injury after LPS administration.

Protein Kinase Cδ Mediates Platelet-Induced Breast Cancer Cell Invasion

Platelets play an important role in carcinogenesis, but the underlying molecular mechanisms remain poorly understood. To investigate the effects of platelets on in vitro invasion of MCF7 human breast cancer cells, human MCF7 cells were used to study their interactions with platelets using aggregometry and cell invasion chambers. Zymography and quantitative polymerase chain reaction (PCR) were used to study matrix metalloproteinases (MMPs), whereas Western blot was used to study protein kinase C (PKC) delta in MCF7 cells. We observed that platelets promoted invasion of MCF7 cells (3-fold increase, p<0.05, n=3) and that this process correlated with a dramatic increase in MMP-9 (8 fold-increase, p<0.001, n=3), which is known to facilitate cancer cell invasion. Because both platelets and MCF7 cells have been shown to release MMP-9, we investigated the cellular source that accounted for this increase. The time course and the use of specific protein synthesis inhibitors demonstrated that most of the increase in MMP-9 levels derived from de novo synthesis of this protease by cancer cells. Furthermore, platelets activated PKCdelta in MCF7 cells after 1 h of incubation (18.45+/-4.75% increase, p<0.05, n=4-7), which, in turn, led to an up-regulation of MMP-9 mRNA (from 60+/-20 to 1040+/-100 pg, p<0.001, n=3) and protein levels (18-fold increase, p<0.001, n=3), with the subsequent cell invasion-promoting effects. PKCdelta plays a crucial role in transducing the invasion-promoting effects of platelets in breast cancer cells, and the specific inhibition of PKCdelta may be a strategy to decrease platelet-mediated cancer cell invasion.

Influence of Therapeutic Hypothermia on Matrix Metalloproteinase Activity after Traumatic Brain Injury in Rats

Recent evidence suggests that matrix metalloproteinases (MMPs) contribute to acute edema and lesion formation following ischemic and traumatic brain injuries (TBI). Experimental and clinical studies have also reported the beneficial effects of posttraumatic hypothermia on histopathological and behavioral outcome. The purpose of this study was to determine whether therapeutic hypothermia would affect the activity of MMPs after TBI. Male Sprague-Dawley rats were traumatized by moderate parasagittal fluid-percussion (F-P) brain injury. Seven groups (n=5/group) of animals were investigated: sham-operated, TBI with normothermia (37 degrees C), and TBI with hypothermia (33 degrees C). Normothermia animals were killed at 4, 24, 72 h and 5 days, and hypothermia animals at 24 or 72 h. Brain temperature was reduced to target temperature 30 mins after trauma and maintained for 4 h. Ipsilateral and contralateral cortical, hippocampal, and thalamic regions were analyzed by gelatin and in situ zymography. In traumatized normothermic animals, TBI significantly (P<0.005) increased MMP-9 levels in ipsilateral (right) cortical and hippocampal regions, compared with contralateral or sham animals, beginning at 4 h and persisting to 5 days. At 1, 3, and 5 days after TBI, significant increases in MMP-2 levels were observed. In contrast to these findings observed with normothermia, posttraumatic hypothermia significantly reduced MMP-9 levels. Hypothermic treatment, however, did not affect the delayed activation of MMP-2. Clarifying the mechanisms underlying the beneficial effects of posttraumatic hypothermia is an active area of research. Posttraumatic hypothermia may attenuate the deleterious consequences of brain trauma by reducing MMP activation acutely.

Thrombin-dependent regulation of matrix metalloproteinase (MMP)-9 levels in human fetal membranes

Objective. Amniochorion matrix metalloproteinase (MMP)-9 levels increase during labor, reaching a maximum in patients with preterm premature rupture of membranes (PPROM). Bleeding is a major risk factor for PPROM. Since such hemorrhage into the tissue factor-enriched decidua induces intense thrombin formation, we determined whether thrombin stimulates MMP levels in amniochorionic membranes.Study design. Fetal membrane (amniochorion) cultures were maintained in media with and without thrombin, lipopolysaccharide (LPS), thrombin receptor agonist peptide (TRAP)-14, and the anti-inflammatory steroid, dexamethasone (DEX). Concentrations of MMP-9, MMP-1, and tissue inhibitor of metalloproteinase (TIMP)-1 in culture media were measured by ELISA and normalized to total cell protein.Results. The presence of thrombin induced MMP-9 levels. TRAP-14, a thrombin receptor agonist, also significantly increased MMP-9 levels, suggesting that thrombin-induced changes in MMP-9 expression were mediated through the thrombin receptor. Conversely, levels of MMP-1 and TIMP-1 were not affected by thrombin treatment, indicative of specificity of its action. The presence of LPS increased the concentration of MMP-9 and MMP-1. In contrast, DEX treatment significantly reduced MMP-9 levels.Conclusion. Our findings clearly demonstrated that thrombin treatment selectively increased the concentration of MMP-9 in culture media of amniochorionic membranes. Our results provide a potential mechanism through which alterations in hemostasis promote PPROM through thrombin-dependent stimulation of MMP-9.

Decreased MMP-9 production in primary progressive multiple sclerosis patients.

An increase in MMP-9 levels has been found in relapsing-remitting (RR) multiple sclerosis (MS) showing correlation with magnetic resonance (MR) parameters mainly during relapses. However, data regarding primary progressive (PP) MS is scarce. To determine both the pro and active forms of MMP-9 in PPMS and transitional progressive (TP) MS, RRMS and healthy controls (HC), and to assess the relationship between MMP-9 levels and clinical and radiological variables in PP/TPMS. 73 patients with PP/TPMS, 50 RRMS and 43 HC were studied. Levels of pro and active forms of MMP-9 in serum were measured with ELISA. EDSS and MSFC scores were recorded and T2- and T1-weighted MR scans were obtained at the time of blood sampling and one and two years later for PP/TP MS cases. MMP-9 levels were 202.27 ng/ml for PP/TPMS, 242.20 ng/ml for RRMS and 274.49 ng/ml for HC. MMP-9 levels were significantly lower in PP/TPMS compared to RRMS (P= 0.026) and HC (P= 0.001). No significant correlations were found between MMP-9 levels and clinical scores or radiological parameters. These results point to different regulatory mechanisms of MMP-9 production and/or activity between PP/TPMS and RRMS.

Effects of Leukocyte Depletion Filters on Matrix Metalloproteinase Activation in an Extracorporeal Circulation Circuit

Matrix metalloproteinases (MMPs) are a family of endopeptidases that function to remodel tissue during both normal physiology and pathology. Research performed by the Medical University of South Carolina found an increased release of several MMP species during cardiopulmonary bypass (CPB), including the subtype MMP-9, but whether and to what degree the extracorporeal circulation circuit (ECC) induces the release of MMPs has yet to be determined. Human bank whole blood scheduled for discard was obtained and exposed to an ECC. The first set of studies (N = 8) was performed with a loop circuit using a standard arterial line filter. A leukoreduction filter was incorporated during the first 30 min of the pump run for the second set of trials; the leukoreduction filter was then bypassed and a standard arterial filter used for the remaining 60 minutes on pump (N = 8). Blood samples were drawn at four time points for analysis (baseline, 30, 60, and 90 min). Data were analyzed using repeated measures analysis of variance with betweensubjects factors, and a p value of less than .1 was considered statistically significant. The MMP-9 level increased by 130.44% at 90 min on pump in the standard arterial filter group and decreased by 34.62% at 90 min on pump in the leukoreduction group. There was a significant difference between the baseline MMP-9 level and the MMP-9 concentrations at 30, 60, and 90 min for both groups (p = .0348); there was a significant difference in MMP-9 levels between the two filter groups (p = .0611). The present study found a significant increase in MMP-9 levels when blood was exposed to an ECC with a standard arterial filter. The use of a leukoreduction filter significantly reduced MMP-9 concentrations as compared to baseline levels in this study. Leukocyte depletion filtration may serve to benefit CPB patients by attenuating the inflammatory response and disrupting pathways that govern such mediators as the MMPs.

Plasma concentrations and genetic variation of matrix metalloproteinase 9 and prognosis of patients with cardiovascular disease.

Matrix metalloproteinase (MMP)-9 secretion by macrophages and other inflammatory cells accelerates atherosclerotic progression and destabilizes vulnerable plaque in animal models. However, epidemiological data evaluating the prognostic impact of circulating concentrations and functional genetic variations of MMP-9 are lacking. In a prospective study of 1127 patients with documented coronary artery disease, we measured baseline plasma MMP-9 levels and determined the MMP-9/C-1562T and MMP-9/R279Q genotypes. During the follow-up period (mean of 4.1 years), 97 patients died from cardiovascular (CV) causes. Median concentrations of MMP-9 were significantly higher among patients who experienced a fatal CV event than among those who did not (62.2 versus 47.8 ng/mL; P<0.0001). The crude hazard risk ratio of CV death associated with increasing quartiles of MMP-9 was 1.4 (95% CI, 1.2 to 1.8; P<0.0001), and after adjustment for clinical and therapeutic confounders, it was 1.3 (95% CI, 1.1 to 1.6; P=0.005). Additional adjustment for highly sensitive CRP, interleukin-6, fibrinogen, and interleukin-18 revealed a hazard risk ratio to 1.2 (95% CI, 0.9 to 1.6; P=0.15). The T allele of the C-1562T polymorphism was associated with increased MMP-9 levels in a fairly codominant fashion (P=0.004). Although none of the polymorphisms was significantly related with future CV death, there was a significant association (P=0.02) between the R279Q polymorphism and CV events in patients with stable angina. Plasma MMP-9 concentration was identified as a novel predictor of CV mortality in patients with coronary artery disease. Whether it provides independent prognostic information compared with other inflammatory markers will have to be additionally assessed.

Activity and cellular origin of gelatinases in patients with colon and rectal carcinoma differential activity of matrix metalloproteinase-9.

Expression and enzymatic activity of gelatinases were examined in biopsy specimens from patients with colon and rectal neoplasms. The objective of this study was to determine whether the activity of these enzymes is altered between tumor areas compared with areas of noninvolved, normal mucosa and between colon and rectal carcinoma. Matrix metalloproteinase (MMP) production was analyzed by Western immunoblot analysis and gelatin zymography. mRNA was determined by quantitative, real-time polymerase chain reaction analysis. Patients with colon carcinoma (n = 20 patients) showed a significant increase in levels of MMP-9 (92 kDa and 88 kDa) and MMP-2 (72 kDa and 62 kDa) in tumor areas compared with noninvolved regions. In contrast, patients with rectal carcinoma (n = 10 patients) had revealed the same high activity of MMP-9 in tumor regions and corresponding healthy tissue. Confirming activity measurements, in colon tumors, but not in rectal tumors, there was significant up-regulation of MMP-9 transcription compared with healthy tissue in the same patients. There were no significant changes in the tissue inhibitor of metalloproteinase-1 protein when colon and rectal tumor tissues were compared with the corresponding noninvolved regions. Cell culture experiments revealed fibroblasts as the cellular origin of MMPs. The findings showed that the secretion and activation of gelatinases depend on soluble factors secreted by tumor cells and are influenced by extracellular matrix components. This is the first report showing differences in MMP-9 activity between rectal carcinoma and colon carcinoma. Previous results indicating an active involvement of stromal cells in the generation of MMPs during tumor invasion are extended. Because the abundance of gelatinases increases in colorectal carcinoma, inhibitors of these proteases may be of therapeutic value.

Effects of Matrix Metalloproteinase-9 Gene Knock-Out on Morphological and Motor Outcomes after Traumatic Brain Injury

Matrix metalloproteinases (MMPs) belong to a class of extracellular proteinases responsible for maintaining and remodeling the extracellular matrix. In addition to multiple functions in normal physiology, abnormal MMP expression and activity may also participate in the pathophysiology of cerebral disease. Here, we show that MMP-9 (gelatinase B; EC.3.4.24.35) contributes to the pathophysiology of traumatic brain injury. After controlled cortical impact in mice, MMP-9 was increased in traumatized brain. Total MMP-9 levels at 24 hr were significantly increased as measured by a substrate cleavage assay. Zymograms showed that MMP-9 was elevated as early as 3 hr after traumatic brain injury, reaching a maximum at approximately 24 hr. Increased MMP-9 levels persisted for up to 1 week. Western blot analysis indicated increased profiles of MMP-9 expression that corresponded with the zymographic data. Knock-out mice deficient in MMP-9 gene expression were compared with wild-type littermates in terms of morphological and motor outcomes after trauma. Motor outcomes were measured at 1, 2, and 7 d after traumatic brain injury by the use of a rotarod device. MMP-9 knock-out mice had less motor deficits than wild-type mice. At 7 d, traumatic brain lesion volumes on Nissl-stained histological sections were significantly smaller in MMP-9 knock-out mice. These data demonstrate that MMP-9 contributes to the pathophysiology of traumatic brain injury and suggest that interruption of the MMP proteolytic cascade may be a possible therapeutic approach for preventing the secondary progression of damage after brain trauma.

The tripeptide-copper complex glycyl-L-histidyl-L- lysine-Cu 2+ stimulates matrix metalloproteinase-2 expression by fibroblast cultures

Glycyl-histidyl-lysine-Cu 2+ (GHK-Cu) is a tripeptide-copper complex known to be a potent wound healing agent. We previously showed its ability to stimulate in vitro and in vivo the synthesis of extracellular matrix components. The aim of this study was to determine the effects of GHK-Cu on MMP-2 synthesis by dermal fibroblasts in culture. We showed that GHK-Cu increased MMP-2 levels in conditioned media of cultured fibroblasts. This effect was reproduced by copper ions but not by the tripeptide GHK alone. This stimulation was accompanied by an increase of MMP-2 mRNA level. We also showed that GHK-Cu increased the secretion of the tissue inhibitors of metalloproteinases, TIMP-1 and TIMP-2. Taken together, our results underline that GHK-Cu is not only an activator of connective tissue production but also of the remodeling of the extracellular matrix. It is able to modulate MMP expression by acting directly on wound fibroblasts.

MMP/TIMP imbalance in amniotic fluid during PROM: an indirect support for endogenous pathway to membrane rupture.

We theorize that excessive degradation of the fetal membrane extracellular matrix (ECM) by specific matrix metalloproteinases (MMPs) results in preterm premature rupture of the membranes (PROM). Active, inhibitor free MMP2 and 9 (gelatinase A and B respectively) can degrade the amniochorion basement membrane Type IV collagen to initiate rupture. This study examines the levels of the gelatinases and their natural inhibitors (tissue inhibitor of matrix metalloproteinases-TIMPs) in the amniotic fluid during PROM, preterm labor (PTL) and at term. A total of 51 AF samples were collected from the following groups of patients. Group 1: Women with PTL and no ROM (n = 16) Group 2: Women with PROM (n = 16) irrespective of labor status Group 3: Women at term with intact membranes undergoing cesarean delivery irrespective of labor status (n = 19). ELISA was used to assay MMP2, MMP9, TIMP1 and TIMP2 levels in the amniotic fluid. The active, TIMP free levels of MMP2 were quantitated by zymography followed by computerized densitometry. Active MMP9 was measured using a bioassay that specifically detects MMP9 activity. Statistical analysis was performed by Tukey-Kramer multiple comparison method. PROM is associated with increased MMP2 levels (mean 2125 ng/ml;) when compared with term (mean 1455 ng/ml; p < 0.01) or PTL where a non significant increase was seen (mean 1862 ng/ml; p = ns). MMP9 levels were higher in PROM (mean 15.03 ng/ml) than at term (mean 1.14 ng/ml; p < 0.001) or PTL (mean 3.75 ng/ml; p < 0.01). TIMP1 levels were slightly increased during PROM (mean 3143 ng/ml) compared to term (mean 1892 ng/ml; p < 0.05) pr PTL where a non significant change was seen (mean 2406 ng/ml; p = ns). TIMP2 levels were decreased in PROM (mean 98 ng/ml) compared with term (mean 176 ng/ml; p < 0.05) and PTL (mean 236 ng/ml; p < 0.001). Active, TIMP free MMP2 levels were increased during PROM (mean 233 pg/ml) compared to those at term (mean 132 pg/ml; p < 0.05) or PTL (mean 132 pg/ml; p < 0.05). Active forms of MMP9 were seen only during PROM (mean 632 pg/ml). Active, TIMP free forms of MMP2 and 9 are increased in the amniotic fluid of women with PROM. These MMPs can degrade the amniochorion basement membranes and other ECM components resulting in PROM.

Modulation of host matrix metalloproteinases by bacterial virulence factors relevant in human periodontal diseases

Bacterial pathogens involved in periodontal diseases exert a part of their destructive effect by triggering and inducing host cells to elevate their secretion of matrix metalloproteinases (MMPs). Pathogen-secreted phospholipase (PLC) is one bacterial product that may trigger this host response. The roles of exogenous PLC leading to the release, secretion and expression of MMPs by peripheral blood neutrophils (PMNs), cultured epithelial cells of human gingiva and porcine periodontal ligament were investigated. Also the activities of PLC in the diseased and healthy gingival sulcular fluid (crevicular fluid, GCF) and molecular forms of gelatinases present in dental plaque were investigated. GCF, salivary and dental plaque samples were analyzed for PLC and proteinase activities. The abilities of PLC to induce PMNs and oral epithelial cells to release and express their MMPs were examined by specific functional, immunological and molecular biology means. PMN-derived MMPs were found to predominate in periodontitis GCF and plaque, and PLC activities were higher in GCF of adult periodontitis patients than in healthy controls. Purified bacterial PLC (1 mU ml(-1)) efficiently induced PMN degranulation. PLC also induced MMP expression in the cultured epithelial cells. The strongest response was seen in MMP-9 and less in MMP-2. The induction was dose-dependent in the range of 0.0-1.0 U ml(-1) PI-PLC, and quiescent cultures were more responsive than proliferating ones. PLC induction of MMPs was polar, with increased levels of MMP-9 in the apical region and increased MMP-2 levels secreted in the basal direction. Northern analysis showed a strong increase in mRNA levels of MMP-9 and a smaller increase for MMP-2 and MMP-1. In the second part of the study we investigated the molecular forms of the released MMPs during periodontitis. In bacterial plaque of periodontitis patients the MMP-9 were found to be converted into lower molecular weight forms. Isolated proteinase from Porphyromonas gingivalis (ATCC 33277) was able to convert human proMMPs to their active forms. Bacterial PLC may induce degranulation of PMN MMPs and increase MMP expression in oral epithelial cells. The released proteases can be converted into active form by the proteases of plaque bacteria. Thereby, the pathogenic oral bacteria may indirectly participate in the destruction of periodontal tissues.

Metalloproteinase Activation Cascade After Burn Injury: A Longitudinal Analysis of the Human Wound Environment

In this paper, we present a longitudinal study on metalloproteinases in wound-fluid samples collected from three patients with partial- to full-thickness burn wounds. Gelatin zymography showed that 92-kDa gelatinase (MMP-9) and its 225-kDa complex could be detected in burn fluid beginning as early as 4-8 h after injury. Marked increases in MMP-9 levels as well as activation of the proenzyme occurred between day 0 and day 2. The 72-kDa gelatinase (MMP-2) proenzyme was not detected until day 2 and activated enzyme did not appear until day 4. Stromelysin (MMP-3), both proenzyme and activated-enzyme forms, was first observed on day 4. Fluid-phase proteinase activity detected by azocoll degradation roughly corresponded with the level of stromelysin rather than the gelatinases. Our results provide evidence for a regulated metalloproteinase activation cascade following acute traumatic injury and demonstrate in vivo expression of metalloproteinase activity.

Immunoassay of type IV collagenase / gelatinase (MMP-2) in human plasma

We have developed a sensitive and specific sandwich type enzyme-linked immunosorbent assay (ELISA) to detect type IV collagenase (MMP-2) in human plasma which employs the combination of affinity purified rabbit polyclonal antibodies and mouse monoclonal antibodies to human MMP-2. The MMP-2 ELISA detects latent and activated MMP-2, MMP-2 complexed with TIMP and MMP-2 complexed with α 2 macroglobulin (65% efficiency). To determine whether physiologic conditions associated with increased connective tissue turnover are accompanied by increased MMP-2 levels in plasma, we compared enzyme levels in pregnant and nonpregnant women. Plasma MMP-2 (mean±standard deviation) was significantly increased ( p<0.05) in the second half of pregnancy (650±312) was compared to early pregnancy (356±130) or the nonpregnanat state (387±193). As a result of the linkage between type IV collagenase production by cancer cells and the metastatic phenotype, the assay of MMP-2 in plasma is of potential clinical value in cancer.