Matrix metalloproteinase-9 and tissue inhibitor of matrix metalloproteinase-1 in blood as markers for early atherosclerosis in subjects with chronic periodontitis

Background and Objectives: An association has been found between periodontal disease and the development of atherosclerosis. We investigated the hypothesis that periodontal disease triggers the expression of matrix metalloproteinase‐9 (MMP‐9) and tissue inhibitor of matrix metalloproteinase‐1 (TIMP‐1) in blood. Increased levels of these parameters might then indicate early atherosclerosis. Material and Methods: In this cross‐sectional study, the material comprised 80 subjects with chronic periodontitis and 31 subjects with no periodontal disease. Sixteen years after diagnosis of periodontal disease ultrasonography revealed a statistically significant difference (p < 0.001) of carotid intima–media thickness between the subjects with chronic periodontitis and the periodontally healthy subjects. Matrix metalloproteinase‐9 and TIMP‐1 were analyzed from blood as periodontal and systemic inflammatory markers. The relationship between MMP‐9, TIMP‐1 and MMP‐9/TIMP‐1 as dependent variables and several independent variables (age, sex, smoking, education, body mass index, hypertension, periodontal disease and cholesterol) were analyzed in multiple logistic regression models to assess the value of the inflammatory markers in predicting carotid atherosclerosis. Results: Matrix metalloproteinase‐9 and TIMP‐1 were significantly higher in plasma from subjects with periodontal disease and atherosclerosis. Periodontal disease was identified as the principal independent predictor both for atherosclerosis (odds ratio 3.89 for increase in bilateral carotid intima–media thickness) and for increased MMP‐9, TIMP‐1 and MMP‐9/TIMP‐1 (odds ratio 2.58, 5.53 and 3.41, respectively). Classical atherosclerosis risk factors, such as increased total cholesterol, age and sex (women), were significant predictors in the model. Conclusion: Matrix metalloproteinase‐9, TIMP‐1 and MMP‐9/TIMP‐1 in blood from subjects with periodontal disease could be useful laboratory markers for increased carotid artery intima–media thickness.     

Substance P regulates the expression of matrix metalloproteinases and tissue inhibitors of metalloproteinase in cultured human gingival fibroblasts

Background and Objective: Substance P may play a role in the pathogenesis of periodontal disease; however, its mechanisms of modulation are not clear. This study evaluated the effect of two concentrations of Substance P on the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in cultured human gingival fibroblasts. Material and Methods: Fibroblasts were stimulated for 48 h with 10^?4 or 10^?9 m Substance P; untreated fibroblasts served as controls. The expression of MMP‐1, ‐2, ‐3, ‐7 and ‐11 and of TIMP‐1 and ‐2 was evaluated using real‐time polymerase chain reaction and western blotting. Results: There was a significant, concentration‐dependent stimulatory effect of Substance P on MMP‐1, ‐2, ‐3 and ‐7 and TIMP‐2 gene expression (p < 0.05), and a probable effect on MMP‐11 (p = 0.06). At the higher concentration (10^?4 m Substance P), MMP‐1, ‐2, ‐3, ‐7 and ‐11 and TIMP‐2 showed the greatest up‐regulation; at the lower concentration (10^?9 m Substance P), MMP‐1, ‐3 and ‐7 and TIMP‐2 exhibited diminished up‐regulation, with MMP‐2 and ‐11 showing down‐regulation (p < 0.05). Expression of TIMP‐1 was not affected by Substance P (p > 0.05). Western blotting confirmed that Substance P up‐regulated MMP‐1, ‐2, ‐3 and ‐11 and TIMP‐2. MMP‐1, ‐3 and ‐11 and TIMP‐2 showed greater up‐regulation at the higher Substance P concentration and diminished up‐regulation at the lower concentration. MMP‐2 was up‐regulated to a similar degree at both Substance P concentrations. Conclusion: In gingival fibroblast cells, Substance P at the higher concentration (10^?4 m ) induced greater up‐regulation of MMP‐1, ‐3 and ‐11 and TIMP‐2 expression, but at the lower concentration (10^?9 m ) induced diminished up‐regulation, which may represent a mechanism for modulating periodontal breakdown.     

The associations between gingival crevice fluid matrix metalloproteinase-9, tissue inhibitor of metalloproteinase-1 and periodontitis in human immunodeficiency virus-positive patients

BACKGROUND AND OBJECTIVE: The study aimed to determine whether matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in gingival crevice fluid could serve as prognostic factors for the progression of periodontitis in human immunodeficiency virus (HIV) -positive patients. Activated inflammatory cells produce inflammatory mediators, which stimulate the production of MMPs and their inhibitors. It is likely that the compromised immune system contributes to the pathogenesis of periodontitis in HIV-positive patients. METHODS: Clinical measurements including gingival index, plaque index, bleeding index, probing depth, attachment loss, and gingival crevice fluid samples were taken from two healthy sites (including sites with gingival recession, gingival index = 0; probing depth < or = 3 mm; attachment loss < or = 2 mm), three gingivitis sites (gingival index > 0; probing depth < or = 3 mm; attachment loss = 0) and three periodontitis sites (gingival index > 0; probing depth > or = 5 mm; attachment loss > or = 3 mm) of each of the 35 patients at baseline visits and 6-month visits by means of paper strips. Gingival crevice fluid levels of MMP-9 and TIMP-1 were determined by sandwich enzyme-linked immunosorbent assays. RESULTS: The mean amounts of MMP-9 and TIMP-1 in the gingivitis and periodontitis sites sites were significantly higher than in the healthy sites (P < 0.0001). The progressing site was defined as a site that had 2 mm or more attachment loss during the 6-month study period. Gingival crevice fluid levels of MMP-9 were significantly correlated with probing depth, attachment loss, TIMP-1, age, smoking pack years, and viral load values at baseline and 6-month visits (0.0001 < P < 0.001). TIMP-1 levels were only correlated with CD4, viral load, attachment loss, and MMP-9 (0.001 < P < 0.01). Repeated measures analysis of 11 active sites vs. 269 inactive sites indicated that MMP-9 and TIMP-1 levels were significantly higher in active sites than in inactive sites (P < 0.0001). These data indicate that sites with high ginigval crevice fluid levels of MMP-9 and TIMP-1 in HIV-positive patients are at significantly greater risk for progression of periodontitis.     

Enamel matrix derivative induces the expression of tissue inhibitor of matrix metalloproteinase‐3 in human gingival fibroblasts via extracellular signal‐regulated kinase

Zeldich E, Koren R, Dard M, Weinberg E, Weinreb M, Nemcovsky CE. Enamel matrix derivative induces the expression of tissue inhibitor of matrix metalloproteinase‐3 in human gingival fibroblasts via extracellular signal‐regulated kinase. J Periodont Res 2010; doi: 10.1111/j.1600‐0765.2009.01218.x © 2009 John Wiley & Sons A/S Background and Objective: Periodontal disease is characterized by increased expression and activity of matrix metalloproteinases (MMPs) and insufficient expression/activity of their inhibitors, tissue inhibitors of matrix metalloproteinases (TIMPs). This altered MMP–TIMP balance results in progressive destruction of gingival and periodontal extracellular matrix. Enamel matrix derivative (EMD), clinically used for periodontal regeneration in a device called Emdogain^®, has been suggested to enhance gingival healing following periodontal procedures in humans. We previously showed that EMD increases the proliferation of human and rat gingival fibroblasts and protects them from tumor necrosis factor‐induced apoptosis. In the present study, the modulation of MMP and TIMP expression by EMD was investigated. Material and Methods: Primary human gingival fibroblasts were treated in vitro with tumor necrosis factor, EMD or both in serum‐free conditions, and RNA was analyzed with an extracellular matrix‐focused microarray and quantitative real‐time polymerase chain reaction. Results: Microarray analysis showed detectable expression of MMP‐1, MMP‐2, MMP‐3, MMP‐7 and MMP‐13, as well as TIMP‐1 and TIMP‐3 in untreated cells. There was no apparent regulation of the expression of MMP‐2, MMP‐7, MMP‐13 and TIMP‐1 by either tumor necrosis factor or EMD. In contrast, tumor necrosis factor significantly increased MMP‐1 expression, and EMD reduced it when both agents were present. Also, EMD significantly induced TIMP‐3 expression, an effect which was dependent on activation of extracellular signal‐regulated kinase 1/2, since it was totally abolished by a selective extracellular signal‐regulated kinase pathway inhibitor. Conclusion: These data suggest that EMD may affect gingival health by ways other than cell proliferation/survival, i.e. by stimulation of TIMP‐3 production, which could improve the MMP–TIMP balance in gingival tissue and curb extracellular matrix destruction.     

Effects of phase I periodontal treatment on gingival crevicular fluid levels of matrix metalloproteinase-3 and tissue inhibitor of metalloproteinase-1

OBJECTIVES: The aim of this study was to evaluate the effects of phase I periodontal treatment on gingival crevicular fluid (GCF) levels of matrix metalloproteinase (MMP)-3 and tissue inhibitors of metalloproteinase (TIMP)-1. METHODS: Plaque index, gingival index, pocket depth and clinical attachment loss were recorded and GCF samples were collected from 20 chronic periodontitis (CP) patients and 20 periodontally healthy controls (C) before treatment. CP patients received phase I periodontal treatment and all clinical parameters were recorded and GCF samples were collected once more after treatment. Assays were performed by an enzyme-linked immunosorbent assay. RESULTS: All of the clinical parameters improved significantly after the therapy (p<0.05). Baseline GCF levels of MMP-3 were significantly higher than C and that level was reduced significantly by treatment compared with baseline levels (p<0.05). Baseline GCF levels of TIMP-1 were lower than post-treatment levels and C (p<0.05). GCF levels of TIMP-1 increased significantly by treatment compared with baseline levels (p<0.05). CONCLUSION: This study shows that the clinical improvements after phase I periodontal therapy are accompanied by reduction in MMP-3 and increasing in TIMP-1 GCF levels.     

Signal pathways involved in the production of MMP-1 and MMP-3 in human gingival fibroblasts

Periodontitis is associated with enhanced production of cytokines, prostaglandins and matrix metalloproteinases (MMPs). The aim of this study was to investigate the production and regulation of MMP-1 and MMP-3 in human gingival fibroblasts challenged with the cytokines interleukin-lbeta (IL-1beta), tumor necrosis factor alpha (TNFalpha) or epidermal growth factor (EGF). The results showed that gingival fibroblasts constitutively produce MMP-1 and MMP-3, and that the cytokines IL-1beta, TNFalpha and EGF increase both MMP-1 and MMP-3 production in gingival fibroblasts. The upregulation by the cytokines was apparent at 8 h of incubation and increased thereafter continuously during 48 h of incubation. The upregulation of MMPs, induced by IL-1beta or TNFalpha, was reduced by the cyxlooxygenase-2 (COX-2) inhibitor NS-398, the p38 MAP-kinase inhibitor SB 203580, and the tyrosine kinase inhibitor herbimycin A. In addition, MMP-1 and MMP-3 production, induced by IL-1beta, TNFalpha or EGF, was strongly reduced by the presence of the glucocorticoid dexamethasone. Our findings demonstrate that the cytokines IL-1beta, TNFalpha and EGF, respectively, enhance both MMP-1 and MMP-3 production in human gingival fibroblasts, and that the signal pathways COX-2, MAP-kinases and tyrosine kinases are partly involved in the production of MMPs.     

实验性牙根吸收组织中基质金属蛋白酶-1及其抑制剂-1的定位表达

目的检测小型猪实验性牙根吸收组织中基质金属蛋白酶(MMP)-1及基质金属蛋白酶抑制剂(TIMP)-1的表达,探讨二者在牙根吸收过程中的作用。方法选用6头小型猪的12颗下颌乳侧切牙,随机分为4组,每组3颗下颌乳侧切牙,分别加力0、0.98、1.96、2.94 N,每2周加力1次。第1次加力后45 d切取标本,应用SABC免疫组化方法检测MMP-1及TIMP-1在根吸收区牙周组织中的定位表达。结果加力0.98、1.96、2.94 N时牙根吸收区牙周组织中MMP-1阳性染色均比不加力强,加力0.98、1.96 N时牙根吸收区牙周组织中TIMP-1阳性染色均比不加力强。结论MMP-1与TIMP-1参与细胞外基质的代谢活动;MMP-1与TIMP-1在根吸收活动中起着重要的作用。     

Gingival fibroblasts grown from cyclosporin-treated patients show a reduced production of matrix metalloproteinase-1 (MMP-1) compared with normal gingival fibroblasts, and cyclosporin down-regulates the production of MMP-1 stimulated by pro-inflammatory cytokines

BACKGROUND AND OBJECTIVE: Cyclosporin-induced gingival overgrowth arises from an alteration in collagen homeostasis and is enhanced by inflammatory changes in the gingival tissues. The aim of this study was to investigate the interaction among interleukin-1, oncostatin M, cyclosporin and nifedipine in promoting the up-regulation of matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinase by gingival fibroblasts. MATERIAL AND METHODS: Fibroblast cultures (n = 5) were obtained from healthy controls and from patients with cyclosporin-induced gingival overgrowth, and cells were harvested between the fourth and ninth passages. Cells were stimulated with interleukin-1 and oncostatin M, alone or in combination, and with different concentrations of cyclosporin (0-2000 ng/mL) and nifedipine (0-200 ng/mL). MMP-1 and tissue inhibitor of metalloproteinase-1 production was determined using an enzyme-linked immunosorbent assay technique. A CyQuant cell proliferation assay was used to determine the DNA concentration in the sample. RESULTS: Fibroblasts obtained from patients with cyclosporin-induced gingival overgrowth produced significantly lower levels of MMP-1 than control fibroblasts (p < 0.001); tissue inhibitor of metalloproteinase-1 levels were significantly lower (p < 0.05), and the ratio of MMP-1 to tissue inhibitor of metalloproteinase-1 was reduced, in the conditioned medium of patients with cyclosporin-induced gingival overgrowth compared with controls. Interleukin-1 and oncostatin M produced a significant increase in the up-regulation of MMP-1, which was reversed when cyclosporin and nifedipine were added to the cell cultures (p < 0.05). CONCLUSION: Pro-inflammatory cytokines significantly up-regulate MMP-1 in cultured gingival fibroblasts. Up-regulation is attenuated by both cyclosporin and nifedipine. The interaction may account for the synergism between inflammation and cyclosporin-induced gingival overgrowth.     

Transforming growth factor-beta stimulates interleukin-11 production by human periodontal ligament and gingival fibroblasts

BACKGROUND: Transforming growth factor (TGF)-beta is a potent multifunctional polypeptide, abundant in the bone matrix. Interleukin (IL)-11 is a pleiotropic cytokine with effects on multiple cell types. The present study was performed to evaluate the regulatory effects of TGF-beta on IL-11 production by human periodontal ligament cells (PDL) and human gingival fibroblasts (HGF). MATERIAL AND METHODS: The expression of TGF-beta receptor in PDL and HGF were observed using flow cytometry. PDL and HGF were stimulated with TGF-beta with or without protein kinase C (PKC) inhibitors and activator. IL-11, bone morphogenetic protein-2 (BMP-2) and TGF-beta mRNA expression was quantified by real-time polymerase chain reaction (PCR). IL-11 production was measured using enzyme-linked immunosorbent assay. RESULTS: PDL and HGF expressed both TGF-beta receptor I and TGF-beta receptor II on the cell surfaces. IL-11 mRNA expression and IL-11 production were augmented by TGF-beta in both PDL and HGF, with higher values in PDL. PKC inhibitors partially suppressed TGF-beta-induced IL-11 production in PDL and HGF, whereas activator enhanced it. TGF-beta mRNA and BMP-2 mRNA expression were up-regulated by TGF-beta in PDL. CONCLUSION: These results suggest that PDL produce IL-11 in response to TGF-beta.     

Interleukin-1beta induces matrix metalloproteinase-1 expression in cultured human gingival fibroblasts: role of cyclooxygenase-2 and prostaglandin E2

OBJECTIVE: Matrix metalloproteinases (MMPs) degrade extracellular matrices and are responsible for excessive connective tissue breakdown in inflammatory disorders. We investigated the mechanism of MMP-1 expression in human gingival fibroblasts in response to the stimulation with interleukin-1beta (IL-1beta), and the role of inducible-type cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) in the regulation of MMP-1 expression. MATERIALS AND METHODS: We stimulated cultured human gingival fibroblasts with r(h)IL-1beta, and examined the expression of MMP-1 mRNA and protein by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. The effect of indomethacin, dexamethasone, or cycloheximide (CHX) on the IL-1beta-induced expression of MMP-1 was examined. The expression of MMP-1 in gingival fibroblasts stimulated with PGE2 was also examined. RESULTS: IL-1beta stimulated the expressions of mRNA and protein for MMP-1, in cultured fibroblasts, in time- and concentration-dependent manners. Pretreatment of the cells with indomethacin or dexamethasone inhibited the IL-1beta-induced MMP-1 expression. CHX, a protein synthesis inhibitor, also suppressed the MMP-1 expression. IL-1beta also induced COX-2 expression in gingival fibroblasts, and PGE2, a major COX-2 product, was found to enhance MMP-1 expression. CONCLUSION: The IL-1beta-induced MMP-1 expression in gingival fibroblasts may be mediated, at least in part, by COX-2 and its product PGE2.     

Levels of tissue inhibitor of metalloproteinases-1 and matrix metalloproteinases-1 and -8 in gingival crevicular fluid following treatment with enamel matrix derivative (EMDOGAIN)

The mechanism of enamel matrix derivative (EM D) action on the periodontal wound healing process is not well understood. However, earlier in vitro studies from our laboratory demonstrated that EMD stimulated the proliferation of both periodontal ligament and gingival fibroblast cells. Therefore, the purpose of this study was to further evaluate the effect of EMD on the early wound healing process by assessing the protein levels of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in gingival crevicular fluid (GCF). Sixteen patients, each of whom had one or two pairs of infrabony defects located contralaterally in the same arch, were included in this clinical trial. Thirty-six infrabony defects were randomly assigned treatment with flap surgery plus EMD or flap surgery plus placebo. At baseline and at 2, 4 and 12 week follow-up evaluation visits, GCF was sampled with paper strips. After determination of GCF volume, TIMP-1, MMP-1 and MMP-8 GCF levels were measured by an enzyme-linked immunosorbent assay. Intragroup analysis: At week 2 following surgery, when compared to baseline all parameters in each study group, except MMP-1, significantly increased (p<0.05). There were no significant differences between 4 or 12 weeks and baseline in either study group. Intergroup analysis: At 4 weeks after surgery, GCF volume and TIMP-1 levels showed a significant decrease (p<0.05) in the EMD group, when compared to the placebo group. MMP-1 levels at weeks 2, 4 and 12, and MMP-8 levels at weeks 4 and 12 were significantly lower (p < 0.05) in the EMD group compared to the placebo group. EMD compared to placebo treated sites demonstrated a more rapid return to baseline levels of TIMP-1, MMP-1 and MMP-8. These findings suggest that treatment with flap surgery and EMD, compared to flap surgery with placebo, accelerated healing at an earlier stage of wound healing following surgery.     

NH4C1 and protein metabolism in human gingival fibroblasts

Abstract – The biologic effect of ammonia was studied in cultures of fibroblasts isolated from human gingiva. NH_CI in the range 2–20 mu was found to exhibit a concentration dependent growth inhibitory effect, with a delayed action wllich was most pronounced at low concentrations. Concomitant with the growth inhibitor)'effect a significant cellular accumulation of protein was evident. No effect on protein synthesis in general, as measured by G-protine incorporation, was found, whereas some inhibitory effect on collagen biosynthesis was indicated. Secretion of C-collagen and other labeled proteins was not affected. The only pronounced effect of ammonia on metabolism of the l4C-labeled proteins was an inhibition of the intracellular degradation of newly synthesized collagen, the lysosomes being suggested as the site for this degradation.