Involvement of MT1‐MMP and TIMP‐2 in human periodontal disease

Oral Diseases (2010) 16 , 388–395 Objectives: Periodontal disease is characterized by an increased collagen metabolism. Although membrane type‐1 matrix metalloproteinase (MT1‐MMP) plays a critical role in collagen degradation, its involvement in human periodontitis remains to be determined. Methods: MT1‐MMP and TIMP‐2 expression and distribution were evaluated in gingival tissue samples derived from 10 healthy and 12 periodontitis‐affected human subjects. MT1‐MMP and TIMP‐2 expression were assessed through Western‐blot of tissue homogenates. The main cell types involved in MT1‐MMP and TIMP‐2 production were evaluated by means of immunohistochemistry. Results: Both MT1‐MMP and TIMP‐2 were significantly increased in periodontitis‐affected gingival tissues when compared to healthy gingiva. Moreover, the balance between MT1‐MMP and its inhibitor TIMP‐2 was altered in periodontitis‐affected tissues, suggesting an imbalance in this proteolytic axis. Immunohistochemistry demonstrated the expression of MT1‐MMP in fibroblasts and macrophages in gingival tissues. MT1‐MMP was detected in cells in close association with the gingival collagen matrix. TIMP‐2 expression was identified in fibroblasts, macrophages and epithelial cells. Conclusions: Our observations show an increased expression of MT1‐MMP and TIMP‐2 in periodontitis‐affected gingival tissues. The altered balance between these two molecular mediators of collagen remodeling suggests their involvement in human periodontal disease.     

Crevicular fluid matrix metalloproteinase‐8, ‐13, and TIMP‐1 levels in type 2 diabetics

Oral Diseases (2010) 16 , 476–481 Objectives: To evaluate whether type 2 diabetes mellitus (DM) enlarged and if so the quantum of such increase in the gingival crevicular fluid (GCF) levels of matrix metalloproteinase‐8 (MMP‐8), MMP‐13 and tissue inhibitor of metalloproteinases‐1 (TIMP‐1). Methods: Subjects (n = 73) were divided into five groups as follows: 12 DM patients with gingivitis (DM‐G), 12 DM patients with periodontitis (DM‐P), 12 systemically healthy patients with gingivitis (H‐G), 13 systemically healthy patients with periodontitis (H‐P) and 24 periodontally, systemically healthy volunteer subjects (H‐C). Full‐mouth clinical periodontal measurements were performed at six sites per tooth. Gingival crevicular fluid samples were obtained from two sites representing the clinical periodontal diagnosis in single‐rooted teeth. Gingival crevicular fluid levels of MMP‐8, MMP‐13 and TIMP‐1 were analysed by immunofluorometric MMP assay (IFMA), enzyme‐linked immunosorbent assay (ELISA). Data were tested statistically by parametric tests. Results: All clinical periodontal measurements were similar in both diabetic and systemically healthy patients with periodontal disease (all P > 0.05). Total amounts of MMP‐8 in GCF samples were significantly lower in H‐C group than DM‐G, DM‐P, H‐P groups (all P < 0.05). Matrix metalloproteinase‐13, TIMP‐1 total amounts were similar in study groups (P > 0.05). Diabetes mellitus patients exhibited similar levels of MMP‐8, MMP‐13, TIMP‐1 with systemically healthy gingivitis/periodontitis patients (P > 0.05). Conclusions: Within the limits of this study, DM does not seem to significantly affect GCF levels of MMP‐8, MMP‐13, TIMP‐1 or clinical periodontal status.     

Proteolytic roles of matrix metalloproteinase (MMP)‐13 during progression of chronic periodontitis: initial evidence for MMP‐13/MMP‐9 activation cascade

Aim: Matrix metalloproteinases (MMP)‐13 can initiate bone resorption and activate proMMP‐9 in vitro, and both these MMPs have been widely implicated in tissue destruction associated with chronic periodontitis. We studied whether MMP‐13 activity and TIMP‐1 levels in gingival crevicular fluid (GCF) associated with progression of chronic periodontitis assessed clinically and by measuring carboxy‐terminal telopeptide of collagen I (ICTP) levels. We additionally addressed whether MMP‐13 could potentiate gelatinase activation in diseased gingival tissue. Materials and Methods: In this prospective study, GCF samples from subjects undergoing clinical progression of chronic periodontitis and healthy controls were screened for ICTP levels, MMP‐13 activity and TIMP‐1. Diseased gingival explants were cultured, treated or not with MMP‐13 with or without adding CL‐82198, a synthetic MMP‐13 selective inhibitor, and assayed by gelatin zymography and densitometric analysis. Results: Active sites demonstrated increased ICTP levels and MMP‐13 activity (p<0.05) in progression subjects. The MMP‐9 activation rate was elevated in MMP‐13‐treated explants (p<0.05) and MMP‐13 inhibitor prevented MMP‐9 activation. Conclusions: MMP‐13 could be implicated in the degradation of soft and hard supporting tissues and proMMP‐9 activation during progression of chronic periodontitis. MMP‐13 and ‐9 can potentially form an activation cascade overcoming the protective TIMP‐1 shield, which may become useful for diagnostic aims and a target for drug development.     

Matrix Metalloproteinase (MMP)‐8 and Tissue Inhibitor of MMP‐1 (TIMP‐1) Gene Polymorphisms in Generalized Aggressive Periodontitis: Gingival Crevicular Fluid MMP‐8 and TIMP‐1 Levels and Outcome of Periodontal Therapy

Background: The aim of the present study is to investigate matrix metalloproteinase (MMP)‐8 and tissue inhibitor of MMP‐1 (TIMP‐1) gene polymorphisms in generalized aggressive periodontitis (GAgP) and to assess the effects of MMP‐8 and TIMP‐1 genotypes on the outcomes of non‐surgical periodontal therapy. Methods: Genomic DNA was obtained from peripheral blood of 100 patients with GAgP and 167 periodontally healthy controls. MMP‐8 +17 C/G, ?799 C/T, ?381 A/G and TIMP‐1 372 T/C, *429 T/G polymorphisms were determined by polymerase chain reaction‐restriction fragment length polymorphism. Patients with GAgP received non‐surgical periodontal therapy and were followed for 6 months. Clinical periodontal parameters and gingival crevicular fluid (GCF) samples were collected at baseline and at follow‐up visits. GCF biomarkers were analyzed by immunofluorescence assay and enzyme‐linked immunosorbent assay. Results: Distribution of the MMP‐8 ?799 C/T genotypes was significantly different between the GAgP and control groups (P <0.005). TIMP‐1 372 T/C and *429 T/G genotypes in males were also significantly different between study groups (P <0.004). GCF MMP‐8 levels decreased until 3 months after non‐surgical therapy compared with baseline in T and G alleles, as well as G and C allele carriers (P <0.0125), whereas no significant decreased was observed in non‐carriers (P >0.0125). Conclusion: On the basis of the present findings, it can be suggested that MMP‐8 ?799 C/T and TIMP‐1 372 T/C, *429 T/G gene polymorphisms in males may be associated with the susceptibility to GAgP in the Turkish population.     

Smoking and matrix metalloproteinases,neutrophil elastase and myeloperoxidase in chronic periodontitis

Oral Diseases (2010) 17 , 68–76 Objectives: To investigate possible relationship between smoking and serum concentrations of matrix metalloproteinase‐8,‐9 (MMP‐8, MMP‐9), tissue inhibitor of matrix metalloproteinases‐1 (TIMP‐1), neutrophil elastase (NE), myeloperoxidase (MPO) in chronic periodontitis (CP) patients relative to periodontally healthy subjects. Methods: Serum samples were obtained from 111 subjects before initiation of any periodontal intervention. Fifty‐five CP patients (39 non‐smokers, 16 smokers) and 56 periodontally healthy subjects (39 non‐smokers, 17 smokers) were recruited. Serum concentrations of MMP‐8 were determined by IFMA and MPO, MMP‐9, TIMP‐1, NE concentrations by ELISA. ANCOVA and Pearson correlation analysis was utilized for statistical analysis. Results: Serum MPO, NE concentrations were higher in smoker CP than non‐smoker CP patients (P = 0.002 and P < 0.001, respectively), whereas these were similar in smoker, non‐smoker periodontally healthy groups (P > 0.05). TIMP‐1 concentration was higher in non‐smoker CP than smoker CP group (P < 0.05). MMP‐9/TIMP‐1 ratios were higher in smoker CP than non‐smoker CP group (P = 0.01). MMP‐8 concentrations, MMP‐8/TIMP‐1 and MMP‐9/TIMP‐1 ratios in CP group were not significantly different from those in periodontally healthy group (P > 0.05). Conclusions: Our findings of significantly elevated serum MMP‐9, MPO, NE together with decreased TIMP‐1 in smoker CP patients than non‐smokers support that smoking together with periodontal destruction may expose/predispose to cardiovascular diseases.     

Oral rinse MMP‐8 point‐of‐care immuno test identifies patients with strong periodontal inflammatory burden

Oral Diseases (2010) 17 , 115–122 Objective: To determine whether oral rinse matrix metalloproteinase (MMP)‐8 levels, measured by three different methods, tissue inhibitor of matrix metalloprotease‐1 (TIMP‐1) levels and elastase activity differentiate subjects with different periodontal condition; and second, to find out if MMP‐8 levels were comparable among the methods used. Methods: MMP‐8 levels were analysed with an immunofluorometric method (IFMA), dentoELISA and commercial ELISA. Also TIMP‐1 levels and elastase activity were measured. For statistical analysis 214 study subjects were categorized into four groups, specified by the presence and number of moderate (4–5 mm) and deep (≥6 mm) periodontal pockets, and bleeding on probing percentage. Results: MMP‐8 levels especially measured by dentoELISA and adjusted to the number of teeth per subject differentiated the study group with strong periodontal inflammatory burden from groups with lower levels. This was also verified with receiver operating characteristic ( ROC) analysis. Elastase activity associated with higher IFMA and dentoELISA MMP‐8 levels. IFMA MMP‐8/TIMP and dentoELISA MMP‐8/TIMP‐1 tended to be higher with the increasing level of periodontal inflammatory burden. TIMP‐1 levels decreased with increasing age. Conclusions: Oral rinse MMP‐8 together with TIMP‐1 analysis may have potential in complementary periodontal diagnostics. dentoELISA can be applied in quantitative oral rinse chair side biomarker diagnostics.     

Induction of MMP‐2 at the interface between epithelial cells and fibroblasts from human periodontal ligament

Shimonishi M, Takahashi I, Terao F, Komatsu M, Kikuchi M. Induction of MMP‐2 at the interface between epithelial cells and fibroblasts from human periodontal ligament. J Periodont Res 2010; 45: 309–316. © 2009 John Wiley & Sons A/S Background and Objective: MMP‐2 can degrade type IV collagen and MMP‐14 can activate pro MMP‐2. The present study was undertaken to examine the expression of MMP‐2 and MMP‐14 with respect to interaction between the cells of the epithelial rests of Malassez and fibroblasts from human periodontal ligament. Material and Methods: Explants of human periodontal ligament tissues produced outgrowths containing both putative epithelial rests of Malassez cells and human periodontal ligament fibroblasts after incubation in a modified serum‐free medium. The distribution and expression of MMP‐2 and MMP‐14 were analysed using immunohistochemistry, in situ hybridization and RT‐PCR analysis. The conditioned media and cell extracts were collected for western blot analysis for MMP‐2. Results: Putative epithelial rests of Malassez cells at the interface between the cells of the epithelial rests of Malassez and fibroblasts expressed MMP‐2 and MMP‐14 strongly. However, in situ hybridization analysis revealed that human periodontal ligament fibroblasts expressed MMP‐2 mRNA while putative epithelial rests of Malassez cells expressed MMP‐14 mRNA at the interface. The RT‐PCR analysis showed that the expression of MMP‐2 mRNA was significantly higher when putative epithelial rests of Malassez cells and human periodontal ligament fibroblasts were cultured together than when cultured alone. Western blot analysis showed that the active form of MMP‐2 was detected at higher levels in the conditioned medium of the co‐cultured cells. Conclusion: These findings indicate that putative epithelial rests of Malassez cells stimulate the production of MMP‐2 in human periodontal ligament fibroblasts. Up‐regulated proMMP‐2 bound by MMP‐14 expressed in epithelial rests of Malassez cells can degrade matrix molecules, such as type IV collagen, in the basal membrane between putative epithelial rests of Malassez cells and human periodontal ligament fibroblasts.     

Nicotine modulates gelatinase B (MMP‐9) and epilysin (MMP‐28) expression in reconstituted human oral epithelium

J Oral Pathol Med (2011) 40 : 33–36 Oral epithelial keratinocytes express nicotinic cholinergic receptors which activation modulates keratinocytes differentiation and migration through different metabolic pathways. Matrix metalloproteinases (MMPs) are Zn‐dependent enzyme involved in cell migration. Among them, gelatinase B (MMP‐9) and epilysin (MMP‐28) are two MMPs expressed by human keratinocytes during both wound healing and proliferation. Their expression has been investigated in a reconstituted human oral epithelium (HOE) exposed to nicotine (Nic, 1–50 μM) for 72 h both in the absence and presence of the nicotinic antagonist mecamylamine (Mec), H7, a PKC inhibitor and PD98059, a MAPK inhibitor (PD). At the end of treatment, MMP‐28 expression has been analyzed in epithelium sections using an anti‐MMP‐28 antibody, whereas MMP‐9 presence and activity has been measured in cell‐conditioned medium analyzed by gelatine zymography. The expression of MMP‐9 was reduced by Nic in a dose‐dependent fashion and this effect was antagonized by Mec, H7 and PD. On the other hand, Nic increased the expression of MMP‐28, and this effect was blocked both by H7 and PD, whereas Mec even enforced it. Nic effects on MMP‐9 and MMP‐28 expression by oral keratinocytes were not previously reported and these data suggest MMPs expression mediated by PKC and MAPK as a possible target for Nic toxicity in oral epithelium.     

第三磨牙近中阻生对邻近第二磨牙龈沟液中MMP8和TIMP1的影响

目的:探讨第三磨牙近中阻生对邻近磨牙龈沟液中MMP8和TIMP1的影响。方法:选择64例口腔科就诊的战士,分为4组,A组下颌第二磨牙伴第三磨牙近中阻生,第三磨牙无冠周炎病史,龈瓣颜色正常;B组下颌第二磨牙伴第三磨牙近中阻生,6个月内第三磨牙有冠周炎病史,龈瓣颜色正常;C组下颌第二磨牙伴第三磨牙近中阻生,有冠周炎;D组无下颌第三磨牙,第二磨牙作为对照。收集下颌第二磨牙龈沟液,检测MMP8和TIMP1水平,同时测定第二磨牙龈沟出血指数、探诊深度和菌斑指数等指标。结果:MMP8和TIMP1各组间均有统计学差异(C组>B组>A组>D组)。MMP8/TIMP1 B组和A组之间无明显差别,其余各组间差异有统计学意义。临床指标中,C组的菌斑指数明显高于D组,A、B、C组的探诊深度大于D组。结论:第三磨牙近中阻生在无炎症状态时可引起第二磨牙龈沟液MMP8、TIMP1、MMP8/TIMP1和探诊深度等牙周炎潜在致病因素的变化,出现冠周炎症时变化更加显著。     

Salivary Interleukin‐6 and ‐8 in Patients With Oral Cancer and Patients With Chronic Oral Inflammatory Diseases

Background: Previous research has indicated that salivary interleukin (IL)‐6 and IL‐8 are potential biomarkers for oral squamous cell carcinoma (OSCC). However, their levels have been found to be significantly elevated in patients with chronic periodontitis (CP) or oral lichen planus (OLP). The data also showed wide variations in levels among the different studies, and no standardization procedure was ever performed. Therefore, the objective of this study is to determine whether CP or OLP confounds the use of IL‐6 or IL‐8 for OSCC detection. Methods: Saliva samples were collected from five groups: OSCC before treatment (n = 18); CP (n = 21); disease‐active OLP (n = 21); disease‐inactive OLP (n = 20); and healthy controls (n = 21). IL‐6 and IL‐8 concentrations (determined by enzyme‐linked immunosorbent assays) were compared, using total salivary protein–standardized levels to validate the data. The Kruskal–Wallis test (α = 0.05) followed by pairwise Mann–Whitney U (post hoc) tests with Bonferroni adjustments (α = 0.00625) were used for statistical analysis. Results: Salivary IL‐6 levels were significantly higher in patients with OSCC than in patients with CP (P <0.001), disease‐active OLP (P = 0.001), disease‐inactive OLP (P <0.001), and healthy controls (P <0.001). Salivary IL‐8 levels were significantly higher in patients with OSCC than in patients with CP (P <0.001), but only marginally significantly higher than in healthy controls (P = 0.014). Statistical results of standardized IL‐6 and IL‐8 levels were consistent with the non‐standardized levels in all pairs except one. Conclusion: Salivary IL‐6 may be a useful biomarker in the detection of OSCC, unconfounded by CP or OLP.