• Journal of Periodontal and Implant Science
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• v.43 no.2
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• pp.87-95
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• 2013

Purpose: The purpose of this study was to analyze the expression of inducible nitric oxide synthases (iNOS), tissue inhibitors of metalloproteinase $(TIMP)_{-3}$, and $TIMP_{-4}$ in the gingival tissues of periodontal patients with or without type 2 diabetes mellitus (DM). Methods: Depending on the patient's systemic condition and clinical criteria of the gingiva, each gingival sample was classified into one of three groups. Sixteen clinically, systemically healthy patients (group 1), 16 periodontal patients (group 2), and 16 periodontal patients with DM (group 3) were included. Tissue samples in each group were collected, prepared, and analyzed by western blotting. Quantification of the relative amount of $TIMP_{-3}$, $TIMP_{-4}$, and iNOS was performed. Results: The expression levels of iNOS and $TIMP_{-3}$ both increased in group 1, group 2, and group 3 in increasing order, and were significantly higher in both group 2 and group 3 as compared to group 1 (P<0.05). The expression levels of $TIMP_{-4}$ increased in the same order, but significantly increased in group 2 as compared to group 1, in group 3 as compared to group 1, and group 3 as compared to group 2 (P<0.05). Conclusions: This study demonstrated that iNOS, $TIMP_{-3}$, and $TIMP_{-4}$ might be involved in the progression of periodontal inflammation associated with type 2 DM. It is thought that further study of these factors can be applied practically for the diagnosis and control of periodontitis in diabetics.

• Journal of Korean Neurosurgical Society
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• v.55 no.5
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• pp.237-243
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• 2014

Objective : Symptomatic disc degeneration develops from inflammatory reactions in the annulus fibrosus (AF). Although inflammatory mediators during annular inflammation have been studied, the roles of matrix metalloproteinases (MMPs) and their inhibitors have not been fully elucidated. In this study, we evaluated the production of MMPs and tissue inhibitors of metalloproteinase (TIMPs) during annular inflammation using an in vitro co-culture system. We also examined the effect of notochordal cells on annular inflammation. Methods : Human AF (hAF) pellet was co-cultured for 48 hours with phorbol myristate acetate-stimulated macrophage-like THP-1 cells. hAF pellet and conditioned media (CM) from co-cultured cells were assayed for MMPs, TIMPs, and insulin-like growth factor (IGF)-1 levels using real-time reverse-transcriptase polymerase chain reaction and enzyem-linked immunosorbent assay. To evaluate whether notochordal cells affected MMPs or TIMPs production on annular inflammation, hAF co-cultured with notochordal cells from adult New Zealand White rabbits, were assayed. Results : MMP-1, -3, -9; and TIMP-1 levels were significantly increased in CM of hAF co-cultured with macrophage-like cells compared with hAF alone, whereas TIMP-2 and IGF-1 levels were significantly decreased (p<0.05). After macrophage exposure, hAF produced significantly more MMP-1 and -3 and less TIMP-1 and -2. Interleukin-$1{\beta}$ stimulation enhanced MMP-1 and -3 levels, and significantly diminished TIMP-2 levels. Co-culturing with rabbit notochordal cells did not significantly influence MMPs and TIMPs production or COL1A2 gene expression. Conclusion : Our results indicate that macrophage-like cells evoke annular degeneration through the regulation of major degradative enzymes and their inhibitors, produced by hAF, suggesting that the selective regulation of these enzymes provides future targets for symptomatic disc degeneration therapy.

• Clinical and Experimental Pediatrics
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• v.53 no.4
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• pp.510-518
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• 2010

Purpose : Kawasaki disease (KD) is a systemic vasculitis, a leading cause of pediatric acquired heart disease. Histopathological findings of coronary artery lesion (CAL) in KD indicate destruction of the coronary artery wall with diffuse vasculitis. Matrix metalloproteinases (MMPs) and their endogenous tissue inhibitors (TIMPs) might play central roles in this process. Special attention to MMP-9 has recently been emerging. This study was performed to investigate the clinical significance of MMP-9 and its inhibitors, TIMP-1 and TIMP-2, in KD. Methods : We compared 47 KD patients with 14 febrile controls. Serum MMP-9 and TIMP-1, TIMP-2 were measured by ELISA and compared according to clinical stages and coronary involvement. Results : In acute stage, MMP-9 and TIMP-1 were significantly higher, whereas TIMP-2 was lower, in KD than those in febrile controls ($P$<0.05). The elevated MMP-9 levels in acute phase significantly decreased during the subacute and convalescent phases ($P$<0.05). During acute phase, the MMP-9, TIMP-1, and MMP-9/TIMP-2 levels in the CAL group were lower than those in the non-CAL group, but they increased significantly in the subacute phase ($P$<0.05). MMP-9 has a positive correlation with TIMP-1 in the acute and subacute phases, and negative correlation with TIMP-2 in the subacute and convalescent phases ($P$<0.05). Conclusion : These results suggest that MMP-9, TIMP-1, and the imbalance in MMP-9 and TIMP-2 might play important roles on the pathophysiology of KD and especially on the development of CAL. However, further larger studies are needed.

• Asian-Australasian Journal of Animal Sciences
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• v.26 no.3
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• pp.334-342
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• 2013

Proteases and protease inhibitors play key roles in most physiological processes, including cell migration, cell signaling, and cell surface and tissue remodeling. Among these, the matrix metalloproteinase (MMPs) pathway is one of the most efficient biosynthetic pathways for controlling the activation of enzymes responsible for protein degradation. This also indicates the association of MMPs with the maturation of spermatozoa. In an attempt to investigate the effect of MMP activation and inhibitors in cultures with various hormones during sperm capacitation, we examined and monitored the localization and expression of MMPs (MMP-2 and MMP-9), tissue inhibitors of metalloproteinases (TIMP-2 and TIMP-3), as well as their expression profiles. Matured spermatozoa were collected from cultures with follicle-stimulating hormone (FSH), luteinizing hormone (LH), and Lutalyse at 1 h, 6 h, 18 h, and 24 h. ELISA detected the expression of MMP-2, MMP-9, TIMP-2, and TIMP-3 in all culture media, regardless of medium type (FSH-supplemented fertilization Brackett-Oliphant medium (FFBO), LH-supplemented FBO (LFBO), or Lutalyse-supplemented FBO (LuFBO)). TIMP-2 and TIMP-3 expression patterns decreased in LFBO and LuFBO. MMP-2 and MMP-9 activity in FBO and FFBO progressively increased from 1 h to 24 h but was not detected in LFBO and LuFBO. The localization and expression of TIMP-2 and TIMP-3 in sperm heads was also measured by immunofluorescence analysis. However, MMPs were not detected in the sperm heads. MMP and TIMP expression patterns differed according to the effect of various hormones. These findings suggest that MMPs have a role in sperm viability during capacitation. In conjunction with hormones, MMPs play a role in maintaining capacitation and fertilization by controlling extracellular matrix inhibitors of sperm.

• YAKHAK HOEJI
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• v.48 no.3
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• pp.189-196
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• 2004

Matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) playa key role in tumor invasion and metastasis. As an inhibitor of MMP-2, TIMP-2 is known to block both the invasive and metastatic behavior of cancer cells, and decrease tumor growth activity. We performed this study to investigate the effects of TIMP-2 over-expression induced by retroviral mediated gene transfer in vitro and in vivo. The human colon cancer cell line SW480 was transfected with the retroviral vector encoding TIMP-2. The effects of TIMP-2 over-expression were analyzed by invasion assay and gelatinase activity test in colon cancer cells and tumorigencity in nude mice. In evaluation of the transfection efficiency of the retroviral vector encoding TIMP-2 in colon cancer cells, we confirmed up-regulation of TIMP-2 expression dependent on the time of cell culture. In addition, inhibition of MMP-2 expression in SW480/TIMP-2 was shown by gelatin zymography. In the in vitro invasion assay SW480/TIMP-2 inhibited the invasiveness on matrigel coated with collagen. To determine whether TIMP-2 can modulate in vivo tumorigenicity and metastasis, SW480/TIMP-2 cells were injected subcutaneously in nude mice. The tumor mass formation of SW480/TIMP-2 cells in nude mice was markedly decreased compared to nontransfected cancer cells. These results showed that colon cancer cells transfected with the retroviral vector encoding TIMP-2 inhibits the invasiveness in vitro and tumorigenicity in vivo.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.12
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• pp.6363-6368
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• 2012

Objective: The study aim was to prepare poly-DL-lactide-poly (PELA) microspheres encapsulating recombinant tissue inhibitors of metalloproteinase-1 (TIMP-1) in an adenovirus to investigate its inhibition on the proliferation of hepatocellular carcinoma cells HepG2. Methods: Microspheres were prepared by encapsulating the recombinant TIMP-1 adenovirus into biodegradable PELA. The particle size, viral load, encapsulation efficiency and in-vitro release were measured. Microspheres were used to infect HepG2 cells, then infection efficiency was examined under a fluorescent microscope and ultrastructural changes assessed by TEM. Expression of TIMP-1 mRNA in HepG2 cells was examined by semi-quantitative RT-PCR and proliferation by MTT and cell growth curve assays. Results: We successfully prepared microspheres encapsulating recombinant TIMP-1 adenovirus with a diameter of $1.965{\mu}m$, an encapsulation efficiency of 60.0%, a viral load of $10.5{\times}10^8/mg$ and approximate 60% of virus release within 120 h, the total releasing time of which was longer than 240 h. The microspheres were confirmed to be non-toxic with blank microspheres. Infected HepG2 cells could stably maintain in-vitro expression of TIMP-1, with significantly effects on biological behaviour Conclusion: PELA microspheres encapsulating a recombinant TIMP-1 adenovirus can markedly inhibit the proliferation of HepG2 cells, which provides an experimental basis for polymer/chemistry-based gene therapy of hepatocellular carcinomas.

• Biomedical Science Letters
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• v.15 no.4
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• pp.309-317
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• 2009

Occupational exposure to inorganic dusts such as coal and silica has been identified as a chronic obstructive pulmonary disease (COPD) risk factor. This risk factor causes lung inflammation and protease-antiprotease imbalance. This abnormal inflammatory response of the lung induces parenchymal tissue destruction and leads to progressive airflow limitation that is characteristics of COPD. The aim of this study was to determine the relationship of proteases such as neutrophil elastase (NE) and matrix metalloproteinase (MMP)-9 and antiproteases such as alpha-1 antitrypsin (AAT) and tissue inhibitors of metalloproteinase (TIMP)-1 with lung function. The study population contained 223 retired workers exposed to inorganic dusts. We performed lung function test, including percent of forced expiratory volume in one second ($%FEV_1$) predicted and $%FEV_1$/forced vital capacity (FVC). We analyzed serum MMP-9, AAT, TIMP-1 and plasma NE concentrations by sandwich enzyme immunoassay. NE, AAT, and TIMP-1 concentrations in workers, who had $%FEV_1$<80% predicted, were higher than those of workers who had $%FEV_1{\geq}80%$ (P<0.05). Both AAT and TIMP-1 concentrations in workers with airflow limitation were higher than those of workers with normal airflow (P<0.05). $%FEV_1$ predicted showed significant negative correlation with AAT (r=-0.255, P<0.0l), TIMP-1 (r=-0.232, P<0.01), and NE (r=-0.196, P<0.01). $%FEV_1$/FVC predicted showed significant negative correlation with NE (r=-0.172, P<0.05). From the results of stepwise multiple regression analysis about $%FEV_1$ and $%FEV_1$/FVC, significant independents were NE (r=-0.135, P=0.001) and AAT (r=-0.100, P=0.013) in $%FEV_1$, and NE (r=-0.160, P=0.014) in $%FEV_1$/FVC. In the present study, there were significant correlations between airflow limitation and protease concentration and between airflow limitation and antiprotease concentration. Serum protease and antiprotease concentrations, however, may be affected by the biological and inflammatory responses. It is necessary to evaluate specimens more reflected the effects of proteases and antiproteases in the lung such as lung tissue, bronchoalveolar lavage fluid, and exhaled breath condensate (EBC).

• Journal of Periodontal and Implant Science
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• v.35 no.1
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• pp.21-30
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• 2005

Matrix metalloproteinases (MMPs) are a family of host-derived proteolytic enzymes and implicated in the remodeling and degradation of extracellular matrix under both physiological and pathological conditions. Connective tissue degradation in periodontal diseases is thought to be due to excessive MMP activities over their specific inhibitors. The effects of lipopolysaccharide (LPS) from Prevotella intermedia, one of the major putative pathogens of periodontitis, on the expression of mRNA for MMPs and tissue inhibitors of metalloproteinases (TIMPs) in human gingival and periodontal ligament fibroblasts were examined by reverse transcriptase-polymerase chain reaction (RT-PCR). The expression of mRNAs encoding MMP-1, -2, -3, -10, and -14 was increased in human gingival fibroblasts treated with p. intermedia LPS, whereas MMP-11 and TIMP-2 mRNA expression was decreased in these cells stimulated with LPS. P. intermedia LPS increased the MMP-1, -2, -10, -11, and -14 mRNA expression and decreased TIMP-1 and -2 mRNA expression in human periodontal ligament fibroblasts. These findings imply that P. intermedia LPS may play an important role in the connective tissue degradation in periodontitis.

• Proceedings of the SCSK Conference
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• 2003.09a
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• pp.590-600
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• 2003

The matrix metalloproteinases (MMPs) are a family of enzymes responsible for degrading connective tissue. MMPs catalyze the breakdown of collagen from the extracellular matrix, leading to wrinkle formation and accelerated skin aging. Furthermore, ultraviolet irradiation causes increased expression of certain MMPs. In the extracellular matrix turnover, MMPs are interacting with endogenous regulators named tissue inhibitors of metalloproteinases (TIMPs). Using peptide substrate assays, it has been demonstrated that TIMP-MMP complexes interact highly specifically with $K_{i}$ values of 10$^{-9}$ -10$^{-16}$ M. Therefore applications for TIMP as inhibitor of collagen degradation are suggested for cosmetic anti-aging products to prevent wrinkle formation and loss of elasticity. To date four TIMP proteins (TIMP-1, TIMP-2, TIMP-3 and TIMP-4) have been identified which show a high degree in sequence similarity. The production of human TIMP-2, a 194-residue nonglycosylated protein, was performed by fed-batch culture of Escherichia coli. TIMP-2 accumulated in the bacterial cells in an insoluble form as inclusion bodies. The inclusion bodies were solubilized and the protein refolded to yield the native TIMP-2 in the active form. The integrity of the protein was confirmed by mass analysis, Edman sequencing and gel shift experiments with authentic samples. The inhibitory activity of the refolded and purified TIMP-2 was demonstrated with MMP-1 and MMP-2 assays using synthetic fluorogenic peptide substrates.s.

• Journal of Veterinary Clinics
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• v.26 no.2
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• pp.144-149
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• 2009

Chondrocytes and synovial fluid derived markers are used to monitor for osteoarthritis(OA). Specific inhibitors, known as tissue inhibitors of metalloproteinases(TIMP), regulate the proteolytic activity of matrix metalloproteinases(MMP). This study investigated whether MMP and TIMP levels were altered in synovial fluid and cartilage following the experimental induction of OA in canines. Twenty mature beagle dogs underwent a unilateral surgical transection of the cranial cruciate ligament and the medial collateral ligament as well as a medial meniscectomy. Matrix metalloproteinase-2 and MMP-9 levels were assayed using Western blot and TIMP-2 levels were measured with enzyme-linked immunosorbent assays four weeks after OA induction. Increased MMP-2 expression was observed in chondrocytes isolated from cartilage following OA induction, but MMP-9 expression decreased. Matrix metalloproteinase-2 and MMP-9 levels in synovial fluid from the OA induced joint significantly increased compared to those of the sham group. Tissue inhibitors of metalloproteinase-2 concentrations were higher in chondrocytes from the OA cartilage, yet TIMP-2 remained lower in the synovial fluid of OA. This suggests the elevated release of MMP-9 over MMP-2 into the synovial fluid following the cartilage degradation-related death of chondrocytes after OA. Osteoarthritis can be further deteriorated by increased MMP activity in the synovial fluid because TIMP-2 exist low concentration into the extracellular matrix. As a result, MMP activity, particularly MMP-9 activity, can be useful as a biomarker in diagnosing and monitoring the early stages of canine OA.