• Journal of Periodontal and Implant Science
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• 제43권2호
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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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• 제55권5호
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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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• 제53권4호
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• pp.510-518
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• 2010

목 적: 가와사끼병은 급성 전신성 미세혈관염으로서, 소아의 후천성 심질환의 가장 흔한 원인이다. 가와사끼병의 관상동맥병변에 대한 조직병리학적 소견은 미만성 혈관염에 의한 관상동맥벽의 파괴를 시사하는데, matrix metalloproteinase (MMP)와 그 내인성 억제인자인 tissue inhibitor of metalloproteinases (TIMP)가 이러한 과정에 주요한 역할을 할 것으로 생각되고 있으며, 그 중에도 MMP-9이 최근 가장 주목을 받고 있다. 따라서 본 연구에서는, 한국의 가와사끼병 환아들에서 MMP-9과 그 억제인자들인 TIMP-1, TIMP-2가 갖는 임상적 중요성을 평가하고자 하였다. 방 법: 가와사끼병 환아 47명을 연구대상으로 하였고, 급성열성 세균감염 환아 14명을 대조군으로 하였다. 혈중 MMP-9, TIMP-1, TIMP-2의 농도를 kit를 통한 효소 면역법을 이용하여 측정하였고, 각 임상 단계 및 관상동맥병변의 유무에 따라 비교분석하였다. 결 과: 급성기에 MMP-9과 TIMP-1은 가와사끼군에서 대조군에 비해 유의하게 높았고, TIMP-2는 오히려 낮았다($P$<0.05). 급성기에 증가한 MMP-9은 아급성기와 회복기에 유의하게 감소하였다($P$<0.05). 관상동맥 확장군과 비확장군의 비교에서는, 확장군에서 급성기 MMP-9과 TIMP-1, MMP-9/TIMP-2가 낮았으며, 아급성기로 가면서 통계적으로 유의하게 증가하였다가 다시 회복기로 가면서 감소하는 양상을 보였다($P$<0.05). MMP-9은 급성기와 아급성기에 TIMP-1과 통계적으로 유의한 양의 상관관계를 보였고, TIMP-2와는 아급성기와 회복기에 음의 상관관계를 보였다($P$<0.05). 결 론: 이러한 결과들은 MMP-9과 TIMP-1의 증가, 그리고 MMP-9과 TIMP-2의 불균형이 가와사끼병, 특히 그 관상동맥병변의 병태생리에 핵심적인 역할을 한다는 사실을 시사한다 하겠다. 더 종합적이고 심도 깊은 연구를 위해서 좀 더 많은 대상자를 포함하는 큰 규모의 연구들이 향후 이루어져야 할 것이다.

• Asian-Australasian Journal of Animal Sciences
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• 제26권3호
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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.

• 약학회지
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• 제48권3호
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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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• 제13권12호
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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.

• 대한의생명과학회지
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• 제15권4호
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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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• 제35권1호
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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.

• 대한화장품학회:학술대회논문집
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• 대한화장품학회 2003년도 IFSCC Conference Proceeding Book I
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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.

• 한국임상수의학회지
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• 제26권2호
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• pp.144-149
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• 2009

퇴행성관절염(Osteoarthritis,OA)은 관절 부위의 퇴행성 변화가 특징이며, 이를 진단하기 위해서는 연골세포나 활액에서 유래된 표지인자가 일반적으로 사용된다. 이번 연구에서는 개를 이용하여 실험적으로 퇴행성관절염을 유도하고, 활액과 연골세포에서 단백질 분해 효소인 matrix metalloproteinase(MMPs)와 MMPs의 활성을 억제시키는 것으로 알려진 tissue inhibitors of metalloproteinases(TIMPs)의 발현 정도를 조사하였다. 20마리의 비글견이 퇴행성관절염 모델로 사용되었으며 MMP-2 와 -9은 Western blot 분석에 의해서, TIMP-2의 농도는 ELISA(enzyme-linked immunosorbent assays)에 의해 결정하였다. 퇴행성관절염 유도 4주 후에 연골에서 분리한 연골세포에서 MMP-2의 발현은 증가되었지만 MMP-9의 발현은 감소되었다. 그러나, 퇴행성관절염을 유도한 개의 활액에서는 MMP-2와 -9의 발현이 모두 증가하는 것을 보였다. TIMP-2의 농도는 퇴행성관절염을 유도한 연골에서 분리한 연골세포에서는 높았지만, 활액에서는 낮은 농도를 보였다. 이러한 결과는 MMP-9가 퇴행성관절염 시 연골 조직의 변성에 따른 연골세포의 손상에 의해 MMP-2보다 더 활액으로 방출된다는 것을 보여주며, 활액 내 TIMP-2의 감소에 따른 MMPs의 활성이 퇴행성관절염을 더욱 악화시키는 것을 제안해준다. 결국 MMPs의 활성은, 특히 MMP-9, 개의 퇴행성관절염의 조기 진단과 치료를 위한 표지인자로서 사용할 수 있을 것으로 사료된다.