Gene-expression analysis of matrix metalloproteinases 1 and 2 and their tissue inhibitors in chronic periapical inflammatory lesions.

BACKGROUND: Periapical inflammatory lesions have been investigated previously, but understanding of pathogenesis of these lesions (granulomas and radicular cysts) at the molecular level is still questionable. Matrix metalloproteinases (MMPs) are enzymes involved in the development of periapical pathology, specifically inflammation and tissue destruction. To elucidate pathogenesis of periapical granulomas and radicular cysts, we undertook a detailed analysis of gene expression of MMP-1, MMP-2 and their tissue inhibitors, TIMP-1 and TIMP-2. METHODS: A total of 149 samples were analyzed using real-time PCR (59 radicular cysts, 50 periapical granulomas and 40 healthy gingiva samples as controls) for expression of MMP-1, MMP-2, TIMP-1 and TIMP-2 genes. The determination of best reference gene for expression analysis of periapical lesions was done using a panel of 12 genes. RESULTS: We have shown that ß-actin and GAPDH are not the most stable reference controls for gene expression analysis of inflammatory periapical tissues and healthy gingiva. The most suitable reference gene was determined to be SDHA (a succinate dehydrogenase complex, subunit A, flavoprotein [Fp]). We found that granulomas (n = 50) and radicular cysts (n = 59) exhibited significantly higher expression of all four examined genes, MMP-1, MMP-2, TIMP-1, and TIMP-2, when compared to healthy gingiva (n = 40; P < 0.05). CONCLUSION: This study has confirmed that the expression of MMP-1, MMP-2, TIMP-1, and TIMP-2 genes is important for the pathogenesis of periapical inflammatory lesions. Since the abovementioned markers were not differentially expressed in periapical granulomas and radicular cysts, the challenge of finding the genetic differences between the two lesions still remains.

Immunoexpression of tryptase-positive mast cells in periapical granulomas and radicular cysts.

AIM: To evaluate and compare the immunoexpression of tryptase in samples of periapical granulomas (PGs) and radicular cysts (RCs) correlating it with the type of lesion, localization, intensity of the inflammatory infiltrate and thickness of the cystic epithelial lining, in order to gain insight into the phlogistic role of these cells in the lesions studied. METHODOLOGY: Twenty-five PGs and twenty-five RCs obtained from human teeth without endodontic treatment were submitted to morphological and immunohistochemical analysis using anti-tryptase antibody. Mast cells were identified and counted in three regions: intra-epithelial, central/superficial and deep portions. The data were analysed using the Mann-Whitney U-test (P < 0.05). RESULTS: In comparison with RCs, PGs exhibited higher immunoexpression of tryptase-positive mast cells located in both central/superficial and deep regions (P < 0.001 and P < 0.001, respectively). When considering the total number of mast cells and disregarding the location, the number of tryptase-positive mast cells increased gradually from RCs to PGs (P < 0.001). Lesions with inflammatory infiltrate grade III had greater number of tryptase-positive mast cells located in both central/superficial and deep regions than lesions with inflammatory infiltrates grade II (P = 0.045 and P = 0.025). When the location was ignored, the lesions with inflammatory infiltrate grade III also exhibited higher immunostaining of tryptase-positive mast cells (P = 0.01). CONCLUSIONS: Tryptase-positive mast cells were present in chronic periapical lesions in a larger number in periapical granulomas than in radicular cysts, in both central/superficial and deep regions.

Heparanase expression in periapical granulomas and radicular cysts.

Heparanase is an endo-ß-D-glucuronidase enzyme which degrades heparan sulfate glycosaminoglycan side chains of proteoglycans in the extracellular matrix and in basement membranes. The aim of this study was to evaluate the expression of heparanase in periapical granulomas (PGs) and radicular cysts (RCs). Immunohistochemistry was used to assess heparanase expression in PGs and RCs. Parameters including stain intensity, location and cell type were used to characterize heparanase expression in the periapical lesions. Ordered categories (from weak to strong) were used to compare the level of heparanase staining in the PG and RC groups. Both epithelial cells and inflammatory cells were positive for heparanase. The relative staining of the epithelial cells was strong, whereas the relative staining of the inflammatory cells was weak. Significant differences in immunohistochemical staining of epithelial cells were observed between RCs and PGs (p = 0.002). The relative expression of heparanase in epithelial cells in RCs was strong. In PGs, lesions with few or no epithelial cells, heparanase was predominantly expressed weakly by inflammatory cells. PGs and RCs have the same infectious origin. Therefore, the different cellular sources of heparanase in these periapical lesions may imply that this enzyme has specific pathogenetic functions in RCs and PGs.

Metalloproteinases 2 and 9 Immunoexpression in Periapical Lesions from Primary Endodontic Infection: Possible Relationship with the Histopathological Diagnosis and the Presence of Pain.

INTRODUCTION: The aim of this study was to evaluate the possible associations among the histopathological diagnosis, the inflammatory infiltrate profile, the presence of pain, and the immunoexpression of matrix metalloproteinases MMP-2 and MMP-9 in periapical lesions from primary endodontic infection. METHODS: Fifty-one primary periapical lesions obtained from extracted teeth were selected for this study. Patients were previously evaluated for the presence of pain and sinus tract related to the tooth to be extracted. Tissues were processed for microscopic examination and MMP-2 and MMP-9 immunoexpression. Microscopically, samples were classified as periapical granulomas or periapical cysts and the inflammatory infiltrate as chronic or mixed. The percentage of immunopositive cells for MMP-2 and MMP-9 of each case was performed based on 10 consecutive microscopic fields. The Student t or chi-square tests were used in the statistical analysis. RESULTS: Of the total, 28 cases were classified as periapical granulomas (54.90%) and 23 cases as periapical cysts (45.10%). Seventeen patients (33.33%) reported pain associated with the extracted tooth, with 12 cases of periapical granulomas (70.58%) and 5 cases of periapical cysts (29.42%). All cases showed immunopositivity for MMP-2 and MMP-9 in a high percentage of cells, mainly in the cytoplasm of the leukocytes. MMP-2 was expressed more in periapical granulomas than periapical cysts (P < .05) and in symptomatic cases (P < .05). CONCLUSIONS: According to the results, we may conclude that MMP-2 and MMP-9 are highly expressed in periapical lesions from a primary endodontic infection. Moreover, we may suggest MMP-2 is expressed more in periapical granuloma and in cases associated with pain.

Localization of active and inactive elastase, alpha-1-proteinase inhibitor, and alpha-2-macroglobulin in human gingiva.

Biochemically, there is usually much less elastase activity in gingival tissue than in crevicular fluid. The tissue distributions of active and inactive elastase and the endogenous inhibitors alpha-1-proteinase inhibitor (alpha 1PI) and alpha-2-macroglobulin (alpha 2M) were therefore compared. Inflamed tissue was obtained from chronic periodontitis patients, and cryostat sections were incubated with the histochemical elastase substrate MeOSuc-Ala-Ala-Pro-Val-MNA. Adjacent sections were examined immunocytochemically with antibodies to neutrophil elastase, alpha 1PI, alpha 2M, and leukocyte differentiation antigens. Antigenic elastase was widely distributed in CD15-positive granulocytes in both the epithelium and lamina propria as well as in granulomatous tissue from infrabony defects. However, there was very limited histochemical staining of these cells, and biochemical activity against the equivalent substrate MeOSuc-Ala-Ala-Pro-Val-AFC could be extracted only from sections with such staining. The pH optimum and effector response of the activity in the extracts were, nevertheless, consistent with those of leukocyte elastase. The large difference between the total elastase content of the tissue, as determined immunocytochemically, and the limited amount of active enzyme, as demonstrated histochemically, indicated that the majority was in an inactive form. The involvement of tissue inhibitors was suggested by the fact that extracts from sections with no histochemical staining reduced biochemical elastase activity in crevicular fluid. alpha 2M was found in many fibroblasts and also some CD68-positive macrophages, which additionally contained alpha 1PI.(ABSTRACT TRUNCATED AT 250 WORDS)

Histochemical and immunocytochemical localization of dipeptidyl peptidases II and IV in human gingiva.

Dipeptidyl peptidase (DPP) II and IV activities were demonstrated in unfixed cryostat sections of gingival tissue from chronic periodontitis patients using histochemistry with 2-methoxy-4-naphthylamine (MNA) substrates. In the case of DPP IV, enzyme localization was confirmed by immunocytochemistry with mouse monoclonal antihuman DPP IV (CD26) antibody. Inflammatory cells containing enzyme were identified in adjacent sections with mouse monoclonal antibodies directed against leukocyte differentiation antigens. Lys-Ala-MNA and Ala-Pro-MNA staining in acid buffer for DPP II was only found in a few fibroblasts in superficial tissue. Staining with Gly-Pro-MNA and Ala-Pro-MNA in alkaline buffer for DPP IV was localized in some CD4 and CD8 positive T lymphocytes, CD68 positive macrophages, and fibroblasts and these cells also reacted with the enzyme antibody. DPP IV-containing macrophages and T lymphocytes were seen in the epithelium. In deeper granulomatous tissue Gram positive and negative bacteria stained with the histochemical substrates, but not the DPP IV antibody. Fibroblast DPP II and IV might participate in cellular interactions with collagen, while T lymphocyte DPP IV may be involved in cell signalling.

Tissue levels of matrix metalloproteinases in pulps and periapical lesions.

The purpose of this study was to evaluate the tissue levels of matrix metalloproteinase (MMP)-1, -2, -3 and their distributions in inflamed human dental pulps and periapical lesions. Samples were subjected to enzyme-linked immunosorbent assay and/or immunohistochemistry by using specific antibodies to MMP-1, -2, and -3. Results from enzyme-linked immunosorbent assay were analyzed by using the Mann-Whitney U test and presented as p values. The concentrations of MMP-1 in all experimental groups were significantly higher than in the control (p < 0.05). The acute pulpitis and control groups were significant different in terms of their MMP-2 levels (p < 0.05). The concentration of MMP-3 in acute pulpitis was significantly higher than the control and chronic pulpitis groups (p < 0.05). Immunohistochemically, MMP-1 and MMP-3 were localized in the infiltrating neutrophils, macrophages, and extracellular matrix of the acute pulpitis group. These results suggest that MMPs play an important role in the pulp tissue destruction of acute, inflamed pulp.

Characteristics of collagenase in experimental periapical granulomas from the teeth of dogs.

Periapical granulomas were induced, with a success rate of about 60% (66 granulomas produced out of 109 roots treated) in mandibular premolars. The average wet weight of the granulomas, 46.1 +/- 34.5 mg (mean +/- SD, n = 22), was sufficient to allow individual specimens to be used for most of the biochemical analyses. High collagenase activity was extracted directly from the granulomas with 4 M urea solution. The enzyme was a typical animal collagenase (EC 3.4.24.7) which clove native collagen molecules into three-quarter (alpha A) and one-quarter (alpha B) length fragments. The collagenase was activated by 1 mM p-aminophenylmercuric acetate. This activated enzyme broke down collagen I rather than collagen III preferentially, which is similar to the activity of human polymorphonuclear leucocyte collagenase. The molecular weights of the latent and activated collagenases were 67 and 49 K, respectively.

Collagenase-3 expression in periapical lesions: an immunohistochemical study.

Collagenase-3 (matrix metalloproteinase-13) is a metalloproteinase (MMP) that is associated with bone lesions and exhibits variable expression patterns in odontogenic cysts; it may play a role in regulating focal proliferation and maturation of jaw cyst epithelium. We studied the localization, staining intensity and distribution of collagenase-3 in 13 periapical granulomas with epithelium, 16 periapical granulomas without epithelium and 10 radicular cysts using archived formalin fixed, paraffin embedded tissues. A monoclonal antibody against human collagenase-3 was used to evaluate its expression. Immunohistochemical staining intensities of collagenase-3 in all periapical lesions were (-), 4 (10%); (+), 1 (3%); (++), 22 (56%) and (+++), 12 (31%); differences were not statistically significant. Immunohistochemical distribution of collagenase-3 in epithelial cells was (-), 17 (44%); (+), 17 (44%); (++), 5 (13%); in fibroblasts it was (-), 8 (20%); (+), 23 (59%); (++), 8 (21%); in plasma cells it was (-), 7 (18%); (+), 22 (56%); (++), 10 (26%); in macrophages it was (-), 7 (18%); (+), and 15 (38%); and (++), 17 (44%). Statistically significant differences were found in epithelial cells (p = 0.00) and fibroblasts (p = 0.02), whereas differences were not statistically significant for plasma cells and macrophages. Collagenase-3 may play a role in the conversion of a periapical granuloma with epithelium to radicular cyst. MMP's influence not only epithelial rest cell migration, but also invasion of various stromal cells into granulomatous tissue.

Immunohistochemical expression of apoptotic factors, cytokeratins, and metalloproteinase-9 in periapical and epithelialized gingival lesions.

The aim of the study was to assess the involvement of apoptotic factors, cytokeratins and metalloproteinase-9 in the histogenesis of both Epithelialized Gingival Lesions (EGL) and Periapical Lesions (PAL). 55 consecutive patients, 30 with PAL and 25 with EGL, were selected for the study after clinical and radiological examinations. The PAL patients had severe periapical lesions and tooth decay with exposure of the pulp chamber.All PAL and EGL biopsies were surgically extracted, fixed in 10% buffered formalin, and processed for routine light microscopy. Ten biopsies of each category were processed for immunohistochemistry (IHC). Serial paraffin sections were stained by IHC with appropriate antibodies to detect cytokeratins (CKs) 1, 5, 8, 10 and 14, caspase-3 and -9, metalloproteinase-9, and for PCNA and TUNEL assays. Both PAL and EGL showed a high expression of the cytokeratin 1, 5 and 8 with higher expression in EGL. Moreover, CK10 was markedly less intense expressed in EGL compared to PAL, while CK14 was almost three times stronger expressed in EGL. The expression of caspase-3 and -9 was stronger in PAL compared to EGL, however, the difference was only significant for caspase-9. In PAL apoptosis detected by TUNNEL method and the expression of MMP-9 were higher than in EGL, whereas PCNA was significantly more expressed in EGL. The results clearly suggest that both lesions have exclusively an epithelial origin and that epithelial proliferation was correlated with the degree of apoptosis in both entities. PAL and EGL presented mostly similar cytokeratin expression except for CK10 and CK14, though with marked differences in the distribution and intensity of IHC reactions. Finally, the degradation of extracellular matrix in both lesions could be partially attributed to the strong presence of MMP-9.

High matrix metalloproteinase activity is a hallmark of periapical granulomas.

INTRODUCTION: The inability to distinguish periapical cysts from granulomas before performing root canal treatment leads to uncertainty in treatment outcomes because cysts have lower healing rates. Searching for differential expression of molecules within cysts or granulomas could provide information with regard to the identity of the lesion or suggest mechanistic differences that may form the basis for future therapeutic intervention. Thus, we investigated whether granulomas and cysts exhibit differential expression of extracellular matrix (ECM) molecules. METHODS: Human periapical granulomas, periapical cysts, and healthy periodontal ligament tissues were used to investigate the differential expression of ECM molecules by microarray analysis. Because matrix metalloproteinases (MMP) showed the highest differential expression in the microarray analysis, MMPs were further examined by in situ zymography and immunohistochemistry. Data were analyzed by using one-way analysis of variance followed by the Tukey test. RESULTS: We observed that cysts and granulomas differentially expressed several ECM molecules, especially those from the MMP family. Compared with cysts, granulomas exhibited higher MMP enzymatic activity in areas stained for MMP-9. These areas were composed of polymorphonuclear cells (PMNs) in contrast to cysts. Similarly, MMP-13 was expressed by a greater number of cells in granulomas compared with cysts. CONCLUSION: Our findings indicate that high enzymatic MMP activity in PMNs together with MMP-9 and MMP-13 stained cells could be a molecular signature of granulomas unlike periapical cysts.

Collagenase-3 (MMP-13) is expressed in periapical lesions: an immunohistochemical study.

AIM: To determine whether or not matrix metalloproteinase 13 (MMP-13) is present in periapical granulomas with and without epithelium. METHODOLOGY: Seventeen open periapical granulomas of pulpal origin (seven lesions without epithelium and 10 with proliferating epithelium) were fixed in formalin and then embedded in paraffin prior to being processed for immunohistochemical analysis. A monoclonal antibody against human MMP-13 was used to evaluate MMP-13 expression. Immunocomplexes were subsequently treated with the secondary antibody and then detected by means of streptavidin peroxidase. Immunoreactivity was visualized by development with 3,3'-diaminobenzidine. RESULTS: An immunopositive cytoplasmatic reaction for MMP-13 was observed in all the specimens, although the immunostaining by anti-MMP-13 antibody was heterogeneous and its levels varied according to histopathological findings. In periapical lesions without epithelium MMP-13 immunolabelling was detected in a few fibroblast-like cells, and in some plasma cells within the granulomatous tissue. A clear upregulation of MMP-13 expression was detected in periapical lesions with epithelium, especially in small island and thin strands of epithelium. CONCLUSIONS: The expression pattern of MMP-13 demonstrates that it is involved in the conversion of a periapical granuloma with epithelium into a radicular cyst. This property is related to the ability of MMP-13 to influence not only the migration of epithelial cell but also the invasion of granulomatous tissue.

[Periapical odontogenic processes of inflammatory origin. Enzyme activity]. / Procesos odontógenos periapicles de origen inflamatorio. Actividad ezimática.

A histoenzymological study was carried out on 40 tissues specimens removed at biopsy and for surgical operations of the following lesions: 5 normal oral mucosa, 5 periapical granulomas, and 30 periapical inflammatory cysts. The purpose of this study was to study some possibly significant variations in levels o activities of oxidative enzymes, and hydrolaxes enzymes. In inflammatory cysts, enzymatic activities were similar to normal epithelium. There was high levels of acid phosphatase LDH and G6PDH activity in the central cells of apical granulomas and in the exfoliating epithelial cells of periapical inflammatory cysts. There were differency in the glycosaminoglicans activity on the different epitelial pattern.

The role of reactive oxygen intermediates in the pathogenesis of chronic apical periodontitis.

The level of malondialdehyde, a stable end product of lipid peroxidation induced by reactive oxygen intermediates and the activity of two potent antioxidant enzymes, superoxide dismutase and glutathione peroxidase, was investigated in tissue homogenates of 22 surgical periapical granuloma specimens. Malondialdehyde levels were significantly higher and glutathione peroxidase activity was significantly lower in periapical granuloma samples than in healthy gingival tissue homogenates, which were used as controls. The activity of superoxide dismutase was similar in periapical granuloma and in control samples. Our results indicate an altered balance between the production and the elimination of toxic oxygen metabolites in chronic apical periodontitis. We hypothesize that reactive oxygen intermediates, which are being produced by activated phagocytic cells abundantly present in periapical granulomas, can contribute to periapical tissue injury and bone loss in this disease.