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.     

MMPs及TIMPs在ACC-M、ACC-2裸鼠移植瘤中的表达

目的:探讨唾液腺腺样囊性癌高低转移细胞株(ACC-M、ACC-2)裸鼠移植瘤中基质金属蛋白酶(matrixmetalloproteinases,MMPs)及基质金属蛋白酶组织抑制剂(tissueinhibitorofmetalloproteinases,TIMPs)的表达。方法:采用ACC-M、ACC-2细胞株,建立ACC-M、ACC-2细胞株的裸鼠皮下移植瘤模型。在2组模型中,每组随机取3只裸鼠,用半定量RT-PCR分析移植瘤中MMP-2、MMP-7、MMP-9、TIMP-1、TIMP-2基因的表达水平,同时用免疫组化方法检测2个细胞株裸鼠移植瘤的MMP-2、MMP-7、MMP-9、TIMP-1、TIMP-2蛋白的表达情况,检测并记录其积分光密度值。所有检测数据用SPSS11.5软件进行均数t检验处理。结果:半定量RT-PCR和免疫组化结果均显示,MMP-2、MMP-7、MMP-9、TIMP-1的表达为ACC-M显著高于ACC-2(P<0.05);TIMP-2在2个细胞株裸鼠移植瘤的表达差异无统计学意义(P>0.05)。结论:MMP-2、MMP-7、MMP-9、TIMP-1的表达差异可能与ACC-M、ACC-2转移能力的大小相关。     

In vitro expression of matrix metalloproteinase-1, tissue inhibitor of metalloproteinase-1 and transforming growth factor-beta1 in human periodontal ligament fibroblasts

Extracellular matrix remodelling is mediated via matrix metalloproteinases (MMPs) and their regulatory factors such as tissue inhibitors of metalloproteinases (TIMPs) and transforming growth factor-beta (TGF-beta). The regulation of MMPs is thought to be associated with cytoskeletal changes. In this study, cytoskeletal changes in human periodontal ligament fibroblasts (PDFs) were induced using cytochalasin B (CB) which reorganizes actin microfilaments reversibly, and colchicine which disrupts microtubules irreversibly. The levels of MMP-1, TIMP-1 and TGF-beta secreted by the CB- or colchicine-treated PDFs were measured using an enzyme-linked immunosorbent assay. Differences between experimental and control groups were tested using analysis of variance (ANOVA) and Scheffé's ad hoc test. Although CB treatment did not significantly increase MMP-1 expression over the controls, colchicine treatment significantly increased the expression of MMP-1 (P < 0.01) in a time-dependent manner compared with the controls. Both CB and colchicine showed a time-dependent increase in TIMP-1 and TGF-beta1 expression and a dose-dependent increase in TIMP-1 and TGF-beta1 expression until threshold compared with the controls (P < 0.05, P < 0.01 and P < 0.001). In addition, CB treatment produced significantly increased TGF-beta1 expression over the controls (P < 0.05 and P < 0.001) from lower doses, with this effect occurring at earlier time points compared with colchicine treatment.     

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.     

Effect of avocado and soybean unsaponifiables on gelatinase A (MMP-2), stromelysin 1 (MMP-3), and tissue inhibitors of matrix metalloproteinase (TIMP- 1 and TIMP-2) secretion by human fibroblasts in culture.

BACKGROUND: In inflamed periodontal tissues, gingival fibroblasts are able to express matrix metalloproteinases (MMPs) and their natural inhibitors, tissue inhibitors of matrix metalloproteinases (TIMPs). They can also respond to growth factors and cytokines. In this study, the in vitro effects of avocado and soybean unsaponifiable residues (ASU), their fractions (avocado unsaponifiable [ASF] or soy unsaponifiable [SSF]) on MMP-2 and MMP-3, and the activity and secretion of their inhibitors TIMP-1 and TIMP-2 were investigated using cultured human gingival fibroblasts. METHODS: Gingival fibroblasts were cultured for 72 hours with ASU, ASF, and SSF at concentrations of 0. 1, 0.5, 2.5, 5, and 10 microgram/ml of culture medium, after pretreatment or no pretreatment for 1 hour with interleukin-1beta (IL-1beta). MMP-2 and MMP-3 were detected and quantified in the culture media after zymography and image analysis. TIMP-1, TIMP-2, MMP-2, and MMP-3 were also evidenced by dot blotting and quantified by image analysis. RESULTS: In the absence of IL-1beta, a slight decrease in the secretion of MMP-2 was observed with lower doses of ASU, ASF, and SSF. The decrease of MMP-3 secretion was clearly marked with all fractions especially at low concentrations (0.1 and 2.5 microgram/ml). A slight decrease in TIMP-2 secretion was seen for low doses of ASU, ASF, and SSF, while a small increase was seen at higher concentrations. Concerning TIMP-1, no significant variation was observed in culture medium for low concentrations, and a decrease was noted for 5 and 10 microgram/ml of ASU, ASF, and SSF. As anticipated, IL-1beta induced a marked release of MMP-2, MMP-3, and TIMP-1, but no variation for TIMP-2 was seen. ASU, ASF, and SSF reversed the IL-1beta effect on gingival fibroblasts for MMP-2 and MMP-3, particularly with doses varying from 0.1 to 2.5 microgram/ml and for TIMP-1, particularly with doses varying from 2.5 to 10 microgram/ml. CONCLUSIONS: These findings suggest a potential role for avocado and soy unsaponifiable extracts to prevent the deleterious effects of IL-1beta that occur during periodontal diseases.     

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.     

Nicotine increases the collagen-degrading ability of human gingival fibroblasts

BACKGROUND AND OBJECTIVE: The objective of this study was to determine the effects that nicotine and the combination of nicotine and Porphyromonas gingivalis supernatant have on human gingival fibroblast-mediated collagen degradation. MATERIAL AND METHODS: Human gingival fibroblasts were cultured with 25-500 microg/ml of nicotine in collagen-coated six-well plates. On days 1-5, the conditioned media was collected for zymography and western blot analyses of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). The cells were then removed and the collagen cleavage visualized by Coomassie blue staining. To examine the combined effect, 250 microg/ml of nicotine and 10% v/v culture supernatant of P. gingivalis ATCC 33277 were added to the human gingival fibroblasts. The mRNA levels of multiple MMPs and TIMPs were monitored. RESULTS: Nicotine increased the human gingival fibroblast-mediated collagen cleavage. The MMP-14 and MMP-2 produced by the nicotine-treated human gingival fibroblasts more readily underwent zymogen activation. Nicotine treatment resulted in TIMP-2 redistribution to the cell surface. The mRNAs of multiple MMPs and TIMPs were unaltered by nicotine. An additive collagen cleavage effect was observed when the human gingival fibroblasts were treated with both nicotine and P. gingivalis. CONCLUSION: Nicotine increased human gingival fibroblast-mediated collagen degradation, in part through the activation of membrane-associated MMPs. Nicotine and P. gingivalis had an additive effect on human gingival fibroblast-mediated collagen degradation.     

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.     

Cytokines, matrix metalloproteinases and tissue inhibitor of metalloproteinases-1 in gingival crevicular fluid from patients with Papillon-Lefèvre syndrome

The aim of the present study was to compare concentrations of cytokines, matrix metalloproteinases (MMPs) and a metalloproteinase inhibitor (TIMP-1) in gingival crevicular fluids (GCF) from sites with gingival inflammation in 28 young patients with Papillon-Lefèvre syndrome (PLS), and in age- and gender-matched controls. Each group consisted of 17 females and 11 males with a mean age of 11.0 years (range 4-22 years). In both groups, anterior upper sites with a clinical diagnosis of gingival inflammation and with pockets < or = 3 mm were selected for sampling of GCF, which was carried out with filter disks inserted into the gingival crevice until saturated. The concentrations of cytokines (IL-1alpha, IL-1beta, TNF-alpha, and IL-8), matrix metalloproteinases (MMP-1, MMP-3, MMP-8, and MMP-9), and their tissue inhibitor (TIMP-1) were analysed using commercial ELISA kits. Significantly higher levels of IL-1beta (P < 0.001) and MMP-8 (P < 0.05) were disclosed among the PLS patients compared with their controls, while the opposite was found for IL-8 (P < 0.05) and MMP-1 (P < 0.001). The individual variations were considerable in both groups. When comparing the expression of cytokines, MMPs, and TIMP-1 in PLS patients with clinically active and non-active periodontitis, the non-active PLS patients showed significantly higher values of IL-1beta than the patients with active periodontal disease (ANOVA, P < 0.01). In conclusion, this study was unable to demonstrate a clear-cut pathognomonic expression of cytokines or MMPs in patients with PLS, but further studies on cytokine and MMP output are warranted.     

Influence of phase I periodontal therapy on levels of matrix metalloproteinase 1 and tissue inhibitor of metalloproteinase 1

Background

Matrix metalloproteinase-1 (MMP-1) is a member of a family of enzymes that can degrade most extracellular matrix macromolecules. Extracellularly, MMPs are controlled by tissue inhibitors of metalloproteinases (TIMPs) and by mechanisms of pro-MMP activation. Levels of MMPs and TIMPs change during healing, inflammation, and normal tissue turnover. Herein we aimed to evaluate the levels of MMP-1 and TIMP-1 in gingival crevicular fluid (GCF) from periodontally healthy patients (control group) and chronic periodontitis patients before and after phase 1 therapy.

Methods

In this study we examined 30 patients who had chronic periodontitis with probing depth sites ⩾5 mm and a clinical attachment level (CAL) ⩾5 mm. We included 30 periodontally healthy patients as a control. Clinical measurements such as plaque (PI) and gingival (GI) indices, papillary bleeding index (PBI), probing depths (PD), and CAL were recorded both before treatment (BT) and after phase I periodontal treatment (AT). Assays for MMP-1 and TIMP-1 were performed with an enzyme-linked immunosorbent assay (ELISA) method.

Results

All clinical parameters were significantly reduced at the post-therapy visit. MMP-1 levels were significantly higher in patients BT than the controls; however, the patients AT were not statistically different than the controls. TIMP-1 levels in patients BT were significantly lower than in the controls and significantly lower than patients AT. We observed a significant positive correlation between GCF volume and MMP-1 levels. Furthermore, TIMP-1 levels were significantly negatively correlated with both GCF volume and all clinical parameters.

Conclusions

We observed that as the extent of periodontal destruction increases, MMP-1 concentration increases and TIMP-1 concentration decreases in GCF. When chronic periodontitis patients were treated by scaling and root planing (SRP), the average MMP-1 concentrations decreased and TIMP-1 concentrations increased in GCF.