Cathepsin C, matrix metalloproteinases, and their tissue inhibitors in gingiva and gingival crevicular fluid from periodontitis-affected patients

Successive active phases observed in periodontal diseases may be explained either by a sudden activation of the pro-forms of tissue-stored degradative enzymes such as metalloproteinases (MMPs) or by an imbalance between metalloproteinases and their tissue inhibitors (TIMPs). To discriminate between these two hypotheses, we quantified the levels, the percentage of active form, and the activities of four metalloproteinases (MMPs -1, -2, -3, and -9), as well as the levels of two tissue inhibitors of metalloproteinases (TIMP-1 and -2) and the activity of cathepsin C in tissue extract supernatants and their corresponding gingival crevicular fluid samples collected from periodontitis-affected and healthy patients. Our results supported evidence that tissue destruction results from an imbalance of metalloproteinases over their tissue inhibitors rather than from a sudden activation of the pro-forms of these enzymes. A significant reduction in the activity of cathepsin C also contributed to the degradative process.     

Longitudinal analysis of metalloproteinases, tissue inhibitors of metalloproteinases and clinical parameters in gingival crevicular fluid from periodontitis-affected patients

BACKGROUND: The aim of this work was to improve the assessment of the periodontal disease status through measurements of extracellular matrix metalloproteinases (MMPs) and their tissular inhibitors (TIMPs) in the gingival crevicular fluid from patients diagnosed with chronic periodontitis. METHODS: Gingival crevicular fluid samples from patients (n = 13) were taken from 60 sites initially, and from 51 and 41 sites, respectively, 3 and 6 months after scaling and root planing. Gingival crevicular fluid samples were also taken from healthy subjects (n = 11, 24 sites). The presence of MMP-9 and MMP-8 was assessed by zymography and immunowestern blotting, respectively. The actual MMP activity (gelatinase and collagenase) was measured using the fluorogenic substrate assay. TIMP-1 and -2 levels were measured by immunodot blot. RESULTS: The fluorogenic substrate assay determinations showed higher MMP activity in sites with probing depth > or = 4 mm, with significant reduction post-treatment. Gelatinase activity followed by zymography consisted mainly of MMP-9. A different pattern of MMP-8 in control and patient sites was found. Controls only showed species of a partially active form (69 kDa), whereas patient sites showed a high frequency of the active form (56 kDa), and in some cases the latent form (85 kDa) was also observed. The active form reduced its frequency in sites with probing depth > or = 4 mm. TIMP-1 and -2 levels in patients were significantly lower than in controls, and after treatment the recovery of TIMP-1 level similar to control was observed. CONCLUSION: Significant correlations between the severity of the periodontal disease and the actual MMP activity, the active form of MMP-8 and the low level of both TIMP-1 and TIMP-2 were found.     

Altered gene expression in leukocyte transendothelial migration and cell communication pathways in periodontitis-affected gingival tissues

Abe D, Kubota T, Morozumi T, Shimizu T, Nakasone N, Itagaki M, Yoshie H. Altered gene expression in leukocyte transendothelial migration and cell communication pathways in periodontitis‐affected gingival tissues. J Periodont Res 2011; 46: 345–353. © 2011 John Wiley & Sons A/S Background and Objective: Gene expression is related to the pathogenesis of periodontitis and plays a crucial role in local tissue destruction and disease susceptibility. The aims of the present study were to identify the expression of specific genes and biological pathways in periodontitis‐affected gingival tissue using microarray and quantitative real‐time RT‐PCR analyses. Material and Methods: Healthy and periodontitis‐affected gingival tissues were taken from three patients with severe chronic periodontitis. Total RNAs from six gingival tissue samples were used for microarray analyses. Data‐mining analyses, such as comparisons, gene ontology and pathway analyses, were performed and biological pathways with a significant role in periodontitis were identified. In addition, quantitative real‐time RT‐PCR analysis was performed on samples obtained from 14 patients with chronic periodontitis and from 14 healthy individuals in order to confirm the results of the pathway analysis. Results: Comparison analyses found 15 up‐regulated and 13 down‐regulated genes (all of which showed a change of more than twofold in expression levels) in periodontitis‐affected gingival tissues. Pathway analysis identified 15 up‐regulated biological pathways, including leukocyte transendothelial migration, and five down‐regulated pathways, including cell communication. Quantitative real‐time RT‐PCR verified that five genes in the leukocyte transendothelial migration pathway were significantly up‐regulated, and four genes in the cell communication pathway were significantly down‐regulated, which was consistent with pathway analysis. Conclusion: We identified up‐regulated genes (ITGB‐2, MMP‐2, CXCL‐12, CXCR‐4 and Rac‐2) and down‐regulated genes (connexin, DSG‐1, DSC‐1 and nestin) in periodontitis‐affected gingival tissues; these genes may be related to the stimulation of leukocyte transendothelial migration and to the the impairment of cell‐to‐cell communication in periodontitis.     

基质金属蛋白酶及其组织抑制剂在涎腺多形性腺瘤生物学行为中的意义

目的 研究涎腺多形性腺瘤中基质金属蛋白酶(MMP)-2、9及其组织抑制剂(TIMP)-1、2的表达变化与其生物学行为的关系。方法 用免疫组化SP法和明胶酶谱法分别检测9例普通型和14例生长活跃型多形性腺瘤中MMP-2、9及TIMP-1、2的阳性表达及细胞定位，分析其中酶原与活性酶的含量比例。结果 MMP-2及MMP-2／TIMP-1、MMP-2／TIMP-2的比值在生长活跃型多形性腺瘤中的表达高于普通型，活性MMP-2、MMP-9酶原和活性酶在生长活跃型多形性腺瘤中的表达明显高于普通型，涎腺良性肿瘤与普通型多形性腺瘤间、涎腺恶性肿瘤与生长活跃型多形性腺瘤间差异无统计学意义。结论 涎腺生长活跃型多形性腺瘤与涎腺恶性肿瘤有相似的MMP-2、9和TIMP-1、2表达特征，而普通型多形性腺瘤中MMP-2、9和TIMP-1、2的表达与涎腺良性肿瘤相近。检测MMPs、TIMPs有助于判断涎腺多形性腺瘤的生物学行为。     

The effect of epidermal growth factor on matrix metalloproteinases and tissue inhibitors of metalloproteinase gene expression in cultured human gingival fibroblasts

OBJECTIVE: Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) play a role in the breakdown of the extracellular matrix during normal physiological processes, and in pathological processes, including periodontitis. The aim of this study was to evaluate the effect of epidermal growth factor (EGF) on the expression of MMPs and TIMPs in cultured human gingival fibroblasts. METHODS: Fibroblasts were stimulated with 10(-3), 10(-6) or 10(-12)M EGF for 24h; untreated fibroblasts served as controls. Alterations in the expression of MMP-1, 2, 3, 7, 11, TIMP-1 and 2 were evaluated using real-time PCR and Western blotting. beta-Actin expression was used as a reference to normalize gene expression. RESULTS: Increased MMP-1, 3, 7 and 11 expressions were observed at all EGF concentrations (p<0.05). At the lowest EGF concentration, MMP-1, 3 and 7 presented the lowest expression and MMP-11 presented the greatest expression; at higher EGF concentrations, MMP-1, 3 and 7 presented greater up-regulation, and MMP-11 lower up-regulation (p<0.05). Protein expression was similarly regulated by EGF: increased up-regulation of MMP-1, 3 and 7 was observed with increasing EGF concentrations, except for MMP-11 that exhibited greater up-regulation at the lower EGF concentration. The gene expression of MMP-2, TIMP-1 and 2 was not affected by EGF (p<0.05). CONCLUSIONS: We conclude that EGF regulates expression for MMP-1, 3, 7 and 11 in a dose-dependent manner, suggesting that EGF may play a role in periodontal destruction and wound repair.     

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.     

Regional odontodysplasia: expression of matrix metalloproteinases and their natural inhibitors

Regional odontodysplasia is a localized disorder of tissues of dental origin that results in a ghost-like appearance of the affected teeth. We present a case with a study of gingival tissue around the follicle. The results show evidence of the role of the matrix metalloproteinases and their natural inhibitors by resident cells in this pathosis. An imbalance in the amounts of matrix metalloproteinases and their natural inhibitors is associated with the pathologic breakdown of the collagen.     

龈沟液中基质金属蛋白酶8及基质金属蛋白酶组织抑制物1水平与牙周病的关系

目的 探讨龈沟液中基质金属蛋白酶8(matrix metalloproteinases-8,MMP-8)及金属蛋白酶组织抑制物1(tissue inhibitors of metalloproteinases,TIMP-1)的水平变化与牙周病的关系,以期为临床提供参考.方法 试验人群共48例,分为牙周健康组16例、慢性牙龈炎组18例及慢性牙周炎组14例,采用酶联免疫吸附测定法检测各组人群龈沟液中MMP-8和TIMP-1水平,并计算MMP-8与TIMP-1的比值.结果 牙周健康组、慢性牙龈炎组及慢性牙周炎组中MMP-8的平均质量浓度分别为:(0.62±0.09)、(0.68 ±0.02)及(0.78±0.05) mg/L,3组之间差异有统计学意义(P＜0.01),并且两两比较差异均有统计学意义(P＜0.05);牙周健康组、慢性牙龈炎组及慢性牙周炎组龈沟液中MMP-8与TIMP-1的比值分别为:0.16 ±0.06、0.23±0.09及0.32 ±0.15,3组相比差异有统计学意义(P＜0.05).结论 龈沟液中MMP-8的质量浓度及MMP-8/TIMP-1比值的变化均可以作为判断牙周炎症水平有意义的临床指标.     

Langerhans cells express matrix metalloproteinases 9 and 2 and tissue inhibitors of metalloproteinases 1 and 2 in healthy human gingival tissue and in periodontitis

BACKGROUND: As antigen-presenting cells, Langerhans cells may play an important role in the initiation and maintenance of periodontal disease. This study is the first report that extends our knowledge of the expression of matrix metalloproteinases and their endogenous tissue inhibitors by Langerhans cells in healthy and diseased gingival tissues. METHODS: Single and double immunolabeling procedures were carried out using monoclonal antibodies against CD1a, matrix metalloproteinases 2 and 9, and tissue inhibitors of matrix metalloproteinases 1 and 2, and analyzed by conventional and confocal microscopes. RESULTS: Langerhans cells expressed matrix metalloproteinases 2 and 9, and tissue inhibitors of matrix metalloproteinases 1 and 2 in healthy and diseased gingival tissues. The tissue inhibitors of matrix metalloproteinase-positive Langerhans cells were mainly observed in the upper epithelial layers. Matrix metalloproteinase 9-positive Langerhans cells were observed especially during periodontitis and in the basal epithelial layer or crossing the basement membrane. CONCLUSION: During periodontal disease, changes in the expression of matrix metalloproteinases and their tissue inhibitors by gingival Langerhans cells could be implicated in the migration of the cells towards the connective tissue.     

口腔扁平苔藓中基质金属蛋白酶及其组织抑制剂的表达

目的 分析基质金属蛋白酶（MMP）2，膜型基质金属蛋白酶1（MT1-MMP），基质金属蛋白酶抑制剂（TIMP）2在口腔扁平苔藓（OLP）中的表达及意义。方法通过免疫组化检测其在OLP中的表达并与口腔鳞状细胞癌（OSCC）及正常口腔黏膜进行比较。结果从正常口腔黏膜（NOM）、非萎缩型OLP，到萎缩型OLP和OSCC3种酶表达依次增加。萎缩型OLP中MMP-2和MTl．MMP的表达明显高于NOM和非萎缩型OLP，与OSCC表达相似。TIMP-2的表达亦随着MMP的增高而增加，但与MMP-2和MT1-MMP相比，其增加量相对较低。结论MMP表达的高低可能成为判断OLP恶变潜能的检测指标之一。     

Differential gene and protein expression of tissue inhibitors of metalloproteinases (TIMP)-3 and TIMP-4 in gingival tissues from drug induced gingival overgrowth

Objectives

The purpose of this study was to analyse mRNA expression and protein localization of tissue inhibitors of metalloproteinases (TIMP)-3 and TIMP-4 in gingival tissues removed from drug (calcium-channel blocker) induced gingival overgrowth and periodontitis patients.

Design

Employing RT-PCR, we evaluated TIMP-3 and TIMP-4 mRNA levels of 20 human gingival tissue samples taken from patients suffering gingival overgrowth (GO) and periodontitis (P). Then, using immunohistochemistry we investigated the TIMP-3 and TIMP-4 protein localization of five sample tissues from each group.

Results

TIMP-4 mRNA levels in GO-gingiva tended to be lower than in P-gingiva but the results differed little (p = 0.22). Varying degrees of inflammation in the protein localization of TIMP-3 and TIMP-4 were found. TIMP-4 immunoreactivity (IR) was weak in the endothelial cells, fibroblasts, epithelial basal and parabasal cells while the degree of inflammation differed as well. TIMP-3 and TIMP-4 IR in inflammatory cells, including lymphocytes, plasma cells, and macrophages, were faint and intense respectively. For P-gingiva, both TIMP-3 and TIMP-4 IR expression was weak in the endothelial cells, fibroblasts, basal and parabasal epithelial layers. Expression of TIMP-3 was faint in the inflammatory cells, whereas TIMP-4 IR was strong.

Conclusion

Our findings suggest that TIMP-3 and TIMP-4 expression differs in GO and P-gingival tissues, both of which are potentially involved in pathogenesis.     

Healing following implantation of periodontitis-affected roots into gingival connective tissue

Abstract The aim of the present investigation was to examine if a new connective tissue attachment can be established on a previously periodontitis involved root surface, located in contact with gingival connective tissue during healing. A total of 28 teeth in one dog (beagle) and two monkeys (Macaca cynomolgus) were subjected to experimental periodontal tissue breakdown by placing cotton floss ligatures or orthodontic elastics around the teeth. The ligatures were left in situ until about 50% of the supporting tissues had been lost. Following resection of the crowns, the teeth were root filled and the exposed parts of the roots thoroughly scaled and planed. Each root was extracted and implanted into grooves prepared in edentulous areas of the jaws in such a way that the root was embedded to half its circumference in bone, leaving the remaining part to be covered by the gingival connective tissue of the repositioned flap of the recipient site. Root implantation and sacrifice of the animals were scheduled to allow for observation periods of 2 and 3 months of healing. An analysis of histologic specimens, obtained from biopsies of the recipient site tissues, disclosed that a new fibrous attachment failed to form on a previously “exposed” root surface located in contact with gingival connective tissue. In areas of the roots where the periodontal ligament tissue was preserved prior to transplantation, a fibrous reattachment occurred between the root and the adjacent gingival tissue. The results indicate that gingival connective tissue does not possess the ability to establish conditions which enable the formation of a new connective tissue attachment.     

Matrix metalloproteinases and their inhibitors in gingival crevicular fluid and saliva of periodontitis patients

Abstract Matrix metalloproteinases (MMPs) and serine proteinases seem to be related to tissue destruction in periodontitis. The presence of MMPs in gingival crevicular fluid (GCF) and saliva, however, has not been studied comprehensively with the enzyme-linked immunosorbent assay (ELISA)-technique, We therefore examined the levels of MMP-1,-3.-8 and -9, and their endogenous inhibitor, tissue inhibitor of matrix metalloproteinases (TIMP-1). in GCF and saliva of patients with adult periodontitiss (AP) and localized juvenile periodontitis (LJP). Elevated levels of MMP-1 were detected in LJP GCF compared to AP and control GCF. Elevated levels of TIMP-1 were also detected in LJP GCF in comparison to AP and control GCF Higher MMP-8 levels were detected in AP GCF compared to LJP and control GCF. The relative low levels of MMP-3 were present in all studied GCF samples. Elevated levels of MMP-8 were further detected in saliva of AP compared to LJP and the controls. Both MMP-1 and TIMP-1 were detected in all studied saliva samples, but not significant differences were detected between the studied groups. Our ELISA-results confirm that (i) PMN MMP-8 and MMP-9 are the main collagenase and gelatinase in AP GCF, whereas GCF collagenase in LJP seems to be of the MMP-1-type; (ii) only low levels of TIMP-1. endogenous MMP-inhibitor. are present in AP GCF. which emphasises the importance of doxycycline as a possible adjunctive drug in the treatment of AP patients; (iii) tests based on specific antibodies against PMN MMPs. especially MMP-8, might serve as a reliable method of measuring and monitoring enzyme levels in GCF from different periodontitis patients.     

Immunolocalization of matrix metalloproteinases and TIMP-1 (tissue inhibitor of metalloproteinases) in human gingival tissues from periodontitis patients

The matrix metalloproteinases (MMPs) collagenase, gelatinase A (72 kDa gela-tinase), stromelysin, and their specific inhibitor TIMP-1 (tissue inhibitor of metalloproteinases), were immunolocalized using specific polyclonal antisera in gingival tissues from 21 patients with chronic inflammatory periodontal disease. Monoclonal antibodies against macrophages (Leu-M5), B cells (Leu-14), helper T cells (OKT4), suppressor T cells (OKT8) and the HLA-DR epitope were also used to identify leukocyte subsets. MMPs were observed in connective tissues at sites that histologically showed signs of remodelling. The number and distribution of positive cells varied widely, however, not only between individual biopsy specimens, but also within the same specimen. The same was true for the composition and distribution of the inflammatory cell infiltrate. Moreover, although there was a positive correlation between the number of MMP-producing cells and the severity of inflammation in some specimens, for others with comparable leukocyte subset scoring the number was reduced and sometimes absent altogether. Cells secreting MMPs were fibroblasts, macrophages and epithelial cells. It was not possible to determine unequivocally whether a MMP-positive cell within the connective tissue was a fibroblast or a macrophage, since the antisera recognise both fibroblast and macrophage MMPs and the different fixation requirements for MMPs (4% paraformaldehyde) and Leu-M5 (acetone) precluded co-localization on the same section. TIMP-1 was immunolocalized within connective tissue cells at sites of tissue remodelling. Our results support the hypothesis that tissue-derived MMPs may be involved in tissue remodelling in periodontal disease and conclusively demonstrate that epithelial cells may be involved as well as connective tissue cells.