MMP-7 and TIMP-1, New Targets in Predicting Poor Wound Healing in Apical Periodontitis

Introduction

Matrix metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinases (TIMPs) are strongly associated with tissue destruction because of inflammation. In this study, we investigated the expression of MMPs and TIMPs messenger RNA and protein levels in apical periodontitis lesions.

Methods

Tissue samples from patients presenting clinical signs of chronic apical abscess (CAA) or asymptomatic apical periodontitis (AAP) were collected postoperatively and used for gene expression analysis of MMP-2, -3, -7, -9, -14, -16, and -25; TIMP-1; and TIMP-2 in real-time polymerase chain reaction. Immunohistochemistry was also performed to detect the expression of MMP-7 and TIMP-1 proteins. Lastly, U-937 cells were induced to terminal differentiation into macrophages, infected with purified Escherichia coli lipopolysaccharide, and assessed for the expression of MMP-7 and TIMP-1 using immunocytochemistry and confocal microscopy.

Results

Significantly higher messenger RNA levels were found for all genes in AAP and CAA samples when compared with healthy control samples (P < .001). AAP cases exhibited significantly higher TIMP-1 when compared with CAA cases, whereas CAA cases showed higher MMP-2, MMP-7, and MMP-9 messenger RNA levels (P < .05). We also detected positive the expression of MMP-7 and TIMP-1 proteins in the tissue samples. The expression of both MMP-7 and TIMP-1 were increased in lipopolysaccharide-stimulated cells compared with nonstimulated cells and appear to colocalize in the Golgi apparatus.

Conclusions

MMPs appear to have an influential role in CAA cases in which ongoing tissue destruction is observed. TIMPs are preferentially associated with AAP, perhaps as a subsequent defense mechanism against excessive destruction. Taken together, our findings implicate MMP and TIMP molecules in the dynamics of inflammatory periapical lesion development.     

Immunohistochemical detection of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in ameloblastomas

BACKGROUND: To evaluate the roles of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in tumor progression, expression of MMP-1, -2 and -9 and TIMP-1 and -2 was analyzed in ameloblastomas as well as tooth germs. METHODS: Frozen tissue sections of seven tooth germs and 22 ameloblastomas were immunohistochemically examined using anti-MMP-1, -2 and -9 and anti-TIMP-1 and -2 antibodies. RESULTS: MMP-1, -2 and -9 and TIMP-1 and -2 were expressed strongly in mesenchymal components of tooth germs, and stromal cells of ameloblastomas. Immunoreactivity for MMP-9 in stromal cells of ameloblastomas was significantly stronger than in mesenchymal cells of dental follicles and dental papillae. Dental laminae showed weak MMP-2 expression in six tooth germs, MMP-9 expression in two tooth germs and TIMP-1 expression in six tooth germs. Some tumor cells showed weak MMP-2 expression in 19 ameloblastomas, MMP-9 expression in four ameloblastomas and TIMP-1 expression in all cases. TIMP-2 reactivity was prominently found in basement membrane zones of dental laminae in tooth germs, and tumor cell islands or nests in ameloblastomas. CONCLUSION: Expression of MMPs and TIMPs was considered to be associated with interactions between epithelial cells and mesenchymal components in normal and neoplastic odontogenic tissues; these molecules might play a role in regulation of tumor progression in ameloblastomas as well as regulation of developmental processes in tooth germs.     

Collagenolytic fragments and active gelatinase complexes in periodontitis

Background: Periodontal tissues remodel rapidly, which enables quick adaptation to mechanical changes. Matrix metalloproteinases (MMPs) are involved in these remodeling processes under control of tissue inhibitor of metalloproteinases (TIMPs). In periodontitis, overactivity of MMPs results in pathologic tissue degradation. The aim of this study was to analyze MMPs and TIMPs in healthy and diseased gingiva, periodontal ligament (PDL), and gingival crevicular fluid (GCF). Methods: Samples of gingiva, PDL, and GCF were obtained from healthy controls (gingiva: n = 18; PDL: n = 15; GCF: n = 8) and subjects with periodontitis (gingiva: n = 11; PDL: n = 18; GCF: n = 12). MMPs and TIMPs were analyzed by gelatin-, collagen-, and reverse zymography and by Western blotting. Total MMP activity was analyzed using a fluorogenic substrate. Results: TIMP-1 and -2, active and pro-MMP-2 and -9, and active MMP-1 and -8 were present in all samples. Large amounts of active MMP-2 complexes and collagenolytic fragments were also found. Their levels were higher in PDL and GCF from subjects with periodontitis. In general, TIMP levels were lower in diseased periodontal tissues. Especially diseased GCF contained more MMPs. Surprisingly, some MMPs were more abundant in healthy gingiva and PDL than in diseased tissue. Conclusions: Unexpected variations in MMP and TIMP levels in gingiva, PDL, and GCF may result from differences in subject characteristics and disease activity. The levels of active MMP-2 complexes and collagenolytic fragments are higher in the periodontium of subjects with periodontitis and might contribute significantly to periodontal destruction.     

Immunoexpression of matrix metalloproteinases and their inhibitors in different areas of oral squamous cell carcinoma

Background: Several studies have shown the participation of MMPs in oral squamous cell carcinoma, the most frequent malignant neoplasm of the oral cavity. The expression of some MMPs correlates with a more aggressive biological behaviour. The objective of this study was to determine which MMPs and TIMPs were expressed in both neoplastic and peritumoural stromal cells in different histopathology areas. Methods: Patients who underwent primary tumour neck dissection for oral squamous cell carcinoma were included. Immunoexpression of MMP-1, -2, -3, -7, -9, -11, -13, and TIMP-1 and -2 in different areas of pathologic specimens (in situ carcinoma, primary tumour, invasive front, distant invasion carcinoma, and lymph node metastasis) was evaluated. Enzyme expression on mucosa adjacent to tumour served as control. Results: Thirty cases were included. Only 6 MMPs and 1 TIMP were expressed in the studied areas. Statistically significant differences in the number of cases with positive MMPs or TIMP expression, in both neoplastic and peritumoural cells, between control and the rest of the areas were observed. MMP-2 expression was constant in the areas with a more aggressive biological behaviour. Conclusions: MMP-2 expression may represent a dynamic interaction between host and tumour that favours the establishment of neoplastic cells at distant sites.     

Matrix metalloproteinase-7, -8, -9, -25, and -26 and CD43, -45, and -68 cell-markers in HIV-infected patients'' saliva and gingival tissue

BACKGROUND: Matrix metalloproteinases (MMPs) process the extracellular matrix and act in tissue remodelling in many physiological and pathological conditions. Certain MMPs can also exert protective anti-inflammatory properties. The levels and expression of MMPs and tissue inhibitors of MMPs (TIMPs) in saliva and gingival tissues of human immunodeficiency virus-seropositive (HIV+) patients are unclear. METHODS: Enzyme-linked immunosorbent assay methods and Western blots were used to study levels and molecular forms of MMP-7, -8, -9, -25, and -26 and TIMP-1 from salivary samples of HIV+ patients (n = 55) and healthy controls (n = 10). The expression of MMPs was also studied by immunohistochemical means in gingival tissue specimens (n = 11, HIV+ patients; n = 10, healthy controls). RESULTS: The HIV+ patients' MMP-8 levels in saliva were statistically significantly higher only in the acquired immunodeficiency syndrome (AIDS)-phase. MMP-9 levels in ASX- and AIDS-phases showed increased expression. TIMP-1 levels were significantly decreased in lymphadenopathy syndrome (LAS)- and AIDS-related complex (ARC)-phases, while MMP-8/TIMP-1 and MMP-9/TIMP-1 molar ratios were increased in all phases in comparison with controls. The molecular forms of MMP-7, -25, and -26 were different between patients and controls as assessed by Western blot. Immunohistochemical studies showed slightly enhanced MMP-7, -8, -9, -25, and -26 staining in HIV+ gingival tissue samples in comparison with controls. CONCLUSIONS: This study confirmed and further demonstrated differences in salivary amounts and molecular forms of MMPs and TIMP-1 in HIV+ patients. The results may reflect alterations in host defence reactions associated with HIV infection.     

The matrix metalloproteinase system in oral squamous cell carcinoma

BACKGROUND: The matrix metalloproteinase (MMP) system is responsible for degradation of tissue in both normal and pathological processes, including tumour invasion and metastasis. AIM: To compare tissue concentrations of components of the MMP system between tumour tissue and normal tissue in patients with oral squamous cell carcinoma, and to correlate concentrations with pathological grade of tumour. METHODS: Thirty-eight paired tissue samples from tumours and normal tissue were analysed by three laboratory techniques: firstly, enzyme linked immunosorbent assays (ELISA) in ng/mg protein for MMP-1, MMP-3, and tissue inhibitors of metalloproteinases (TIMPs) -1 and -2. Secondly, gelatinase activity assays to measure concentrations of total and endogenous active gelatinases, MMP-2 and MMP-9 (ng/mg protein). And thirdly to use quenched fluorescent substrate hydrolysis to measure total MMP activity (pM/min). RESULTS: The concentration of all MMPs was significantly higher in tumour than in normal oral tissue (p < 0.05, Mann-Whitney U-test). Tissue concentrations of some of these factors correlated with clinical and pathological indices of aggressiveness of tumours, including T-stage, N-stage, tumour differentiation, and anatomical level of involved nodes. However, the study was not powered to show statistical significance. CONCLUSION: It is the balance between proteinases and their inhibitors that controls tissue degradation at each stage of tumour invasion and metastasis. Measurement of MMPs in oral mucosal biopsy samples may establish the invasive potential of tumours at their initial presentation.     

Extracellular matrix molecules in chronic obstructive sialadenitis: an immunocytochemical and Western blot investigation

The exact pathomechanism of inflammation progress and fibrosis in chronic sialadenitis is unknown. Connective tissue growth factor (CTGF), matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) have been implicated in the pathogenesis of various fibrotic conditions. These factors are thought to be essential in the regulation of extracellular matrix turnover and the development of tissue fibrosis. In the present study, the expression of CTGF, MMP-2, -3, -9, -13 and TIMP-3 was examined in chronic obstructive sialadenitis. Tissue samples of 13 patients with chronic sialadenitis of the submandibular gland associated with sialolithiasis and 4 normal tissue samples of the submandibular gland were analyzed immunohistochemically and by Western blot analysis. An intense CTGF immunoreactivity was observed in the ductal system of inflamed salivary glands, whereas in normal glands no reactivity or a very low CTGF immunoreactivity was present. Immunohistochemical studies revealed a low to strong reactivity of MMP-2, -3, -9, -13, and TIMP-3 in the ductal system, in acinar cells and in lymphomonocytic infiltrates in normal and inflamed tissues. The expression of MMP-2, -3, -9, -13, and TIMP-3 was confirmed by Western blotting in all cases. Over-expression of CTGF in chronic obstructive sialadenitis suggests that this factor may play a role in glandular fibrosis. However, the physiological role of MMP-2, -3, -9, -13, and TIMP-3 in normal glands, as well as their possible role in inflammation progress and fibrosis in chronic obstructive sialadenitis, remains to be elucidated.     

Expression profile of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs in mature human odontoblasts and pulp tissue

Previous studies have demonstrated that (at least) matrix metalloproteinase (MMP)-2, -8, -9, -14 and -20 are expressed by human odontoblasts. Here, we analysed the expression of 19 MMPs and their specific tissue inhibitors (TIMP)-1, -2 and -3) -1, -2 and -3 in mature human odontoblasts and pulp tissue. Since MMP-20 is almost exclusively expressed by the dentin-pulp complex cells, we further analysed the effect of transforming growth factor (TGF)-beta1 and bone morphogenetic protein (BMPs)-2 on its expression. Matrix metalloproteinase-9 served as a positive control for growth factor responsiveness. It was found that MMP-1, -2, -9, -10, -11, -13, -14, -15, -16, -17, -19, -20 and -23, in addition to TIMP-1, -2 and -3 were expressed by both odontoblasts and pulp tissue. Neither MMP-3 nor MMP-12 were expressed in odontoblasts or pulp tissue, and MMP-7, -8, -24 and -25 were expressed only in the odontoblasts; MMP-2, -10, -11, -14 and -20 were expressed more abundantly by odontoblasts, whereas pulp tissue expressed more MMP-13 and MMP-17. Transforming growth factor-beta1 (1 ng ml(-1)) and BMP-2 (100 ng ml(-1)) did not markedly affect MMP-20 mRNA expression. In contrast, TGF-beta1 alone and with BMP-2 significantly upregulated MMP-9 mRNA by 2.4-fold and by 2.6-fold, respectively, in odontoblasts, while in pulp tissue no effects could be detected. The wide-scale expression of MMPs and TIMPs by mature human odontoblasts and pulp tissue suggests that they may participate in dentin matrix organization prior to mineralization, and that growth factors may further control dentin matrix modeling by differentially regulating individual MMPs.     

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.     

Expression of MMP-7 and MT1-MMP in oral squamous cell carcinoma as predictive indicator for tumor invasion and prognosis

BACKGROUND: Squamous cell carcinoma of the oral cavity is a highly invasive neoplasm that spreads locally and metastasizes to regional lymph nodes. This process involves multiple proteolytic enzymes including matrilysin (MMP-7) and membrane type I-matrix metalloproteinase (MT1-MMP). This study was designed to explore the association between MMP-7 and MT1-MMP in the invasiveness and prognosis of oral squamous cell carcinoma (OSCC). METHODS: About 4-microM, formalin-fixed, paraffin-embedded tissue sections from 69 patients with OSCC were immunohistochemically studied using specific antibodies against MMP-7 and MT1-MMP proteins. Immunostaining was semiquantitatively scored, and results were correlated with histologic and clinical variables including clinical behavior and survival. RESULTS: MMP-7 was observed only in cancer cells, and MT1-MMP in both tumoral tissue and stroma. MMP-7 expression was significantly correlated with lymph node metastasis (P = 0.03; RR = 3.2). MT1-MMP showed a significant association with TIMP-2 (in N+ cases) and p53 expression (P = 0.01). MMP-7 and MT1-MMP displayed a survival relevance, and in multivariate analysis they were independent prognostic indicators, particularly in neck node-positive cases.     

Indices of extracellular matrix turnover in human masseter muscles as markers of craniofacial form--a preliminary study

Environmental remodelling of the craniofacial musculature is obligatory for successful outcomes following interventions such as functional appliance therapy or orthognathic surgery. Genetically driven remodelling of the craniofacial musculature is also seen in individuals with altered facial form. The processes that are involved in the remodelling of intramuscular connective tissue need to be activated in such situations. Such processes require activity of matrix metalloproteinases (MMPs) and the tissue inhibitors of matrix metalloproteinases (TIMPs), which are responsible for extracellular matrix (ECM) turnover. The aim of this study was, therefore, to establish the expression of MMP-2 and MMP-9 and their inhibitors, TIMP-1 and TIMP-2, in the masseter muscle of humans with both normal and increased vertical facial form and to assess whether this expression had any value as a predictor of facial form. Biopsies were taken from 20 subjects (10 with vertical facial deformity and 10 with normal vertical facial form to act as a control group). The sample group consisted of 15 females and 5 males and the average age of the donors +/- standard deviation (SD) was 26.04 +/- 6.16 years (range: 17.67-31.25 years). Biopsy samples were then subjected to zymography and reverse zymography to assess MMP and TIMP expression, respectively. Lateral skull cephalograms were analysed for each subject using Spearman's rho correlation coefficients and Mann-Whitney U-tests. TIMP-1 activity was consistently expressed in human masseter muscle. MMP-2, MMP-9, and TIMP-2 activity, when detected, was at a low level. These data indicate that in most individuals, an excess of TIMP-1, compared with MMP-2 and MMP-9, limits ECM turnover in human masseter muscle. There was a demonstrable variation in proteinase expression between different individuals. These preliminary findings, however, do not confirm that indices of ECM turnover are a reflection of an individual's vertical facial form.     

In situ hybridization study on tissue inhibitors of metalloproteinases (TIMPs) mRNA-expressing cells in human inflamed gingival tissue

This study presents the exact cell types and localization of tissue inhibitors of metalloproteinases (TIMPs) production sites in periodontal diseased gingiva by means of in situ hybridization. Gingival tissue specimens were fixed, embedded and hybridized in situ with specific digoxigenin-labeled cRNA probes (386 and 496 bp). TIMP-1 and -2 mRNAs were expressed on macrophages, mononuclear cells, capillary endothelial cells and some fibroblasts throughout the gingival tissue. In periodontitis, TIMP-1 and -2 mRNA-expressing cells showed significantly different localization. TIMP-1 mRNA was broadly observed in the gingival connective tissue while TIMP-2 mRNA was predominantly expressed in the connective tissue adjacent to the pocket epithelium (p < 0.01). Fewer TIMPs mRNA were observed in minimal gingivitis than in periodontitis, especially in the middle zone of gingival tissue. Thus, TIMP-1 and TIMP-2 mRNA was detected differentially and site-specifically in periodontal diseased gingival tissue.     

Hydrocortisone affects the expression of matrix metalloproteinases (MMP-1, -2, -3, -7, and -11) and tissue inhibitor of matrix metalloproteinases (TIMP-1) in human gingival fibroblasts

BACKGROUND: There is a positive correlation between the course of periodontal disease and psychosocial stress status. Stress leads to activation of the hypothalamic-pituitary-adrenal axis, resulting in increased cortisol release. The present study evaluates the effect of two different hydrocortisone concentrations on mRNA expression of matrix metalloproteinases (MMPs) and tissue inhibitor of matrix metalloproteinases (TIMPs) in cultured, human gingival fibroblasts. METHODS: Gingival fibroblasts were stimulated with 10(-7) or 10(-9) M hydrocortisone for 24 hours; untreated cells served as controls. Alterations in the expression of MMP-1, -2, -3, -7, -11 and TIMP-1 and -2 were evaluated using real-time polymerase chain reaction and Western blotting. beta-actin mRNA expression was used as a reference to normalize gene expression. RESULTS: Although the higher hydrocortisone concentration upregulated MMP-1, -2, -7, -11, and TIMP-1 (P <0.05) expression, the lower concentration induced downregulation or diminished upregulation. The lower hydrocortisone concentration induced a 23-fold increase in MMP-3 gene expression, whereas the higher concentration induced less upregulation; however, protein expression was regulated similarly by both hydrocortisone concentrations. The effect of hydrocortisone on TIMP-2 expression was not significant (P >0.05). CONCLUSIONS: Hydrocortisone produced a dose-dependent regulation of MMP and TIMP expression. The higher hydrocortisone concentration significantly upregulated expression of MMP-1, -2, -7, and -11 and TIMP-1 in human gingival fibroblasts, which may constitute a mechanism underlying the increased periodontal breakdown associated with psychosocial stress status.     

Expression of mRNAs encoding for alpha and beta integrin subunits, MMPs, and TIMPs in stretched human periodontal ligament and gingival fibroblasts

The biological mechanisms of tooth movement result from the cellular responses of connective tissues to exogenous mechanical forces. Among these responses, the degradation of the extracellular matrix takes place, but the identification of the molecular basis as well as the components implicated in this degradation are poorly understood. To contribute to this identification, we subjected human fibroblasts obtained from the periodontal ligament (PDLs) and from the gingiva (HGFs) to a continuous stretch to quantify the mRNAs encoding for various metalloproteinases (MMPs), their tissue inhibitors (TIMPs), and alpha and beta integrin subunits. Both cell lines reacted by inducing the expression of the mRNAs encoding for MMP-1, MMP-2, TIMP-1, and TIMP-2, while other mRNAs did not vary (MT1-MMP, TIMP-3) or were not expressed (MMP-9). PDLs expressed selectively the mRNAs encoding for alpha4 and alphav, with no difference measurable under stretching, while the mRNAs encoding for alpha6 and beta1 were increased and the one encoding for alpha5 was decreased. HGFs increased the mRNAs encoding for alpha2, alpha6, beta1, and beta3 and decreased the one encoding for alpha3. Analysis of our data indicated that stretched HGFs and PDLs induced the same pattern of mRNAs encoding for MMPs and TIMPs but differed for those encoding various integrin subunits, known to act as protein receptors in mechanotransduction.