Tissue Inhibitor Of Metalloproteinase-1 Gene Research Articles

The GC + CC genotype at position -418 in TIMP-2 promoter and the -1575GA/-1306CC genotype in MMP-2 is genetic predisposing factors for prevalence of moyamoya disease.

BackgroundTo investigate the association of single-nucleotide polymorphisms (SNPs) in matrix metalloproteinases (MMPs)-2, -3, and -9 and tissue inhibitor of metalloproteinase (TIMP)-2 with moyamoya disease (MMD). We conducted a case-control study of MMD patients by assessing the prevalence of six SNPs of MMP-2 -1575G > A [rs243866], MMP-2 -1306C > T [rs243865], MMP-3 -1171 5a/6a [rs3025058], MMP-9 -1562C > T [rs3918242], MMP-9 Q279R [rs17576], and TIMP-2 -418G > C [rs8179090].MethodsKorean patients with MMD (n = 107, mean age, 20.9 ± 15.9 years; 66.4% female) and 243 healthy control subjects (mean age, 23.0 ± 16.1 years; 56.8% female) were included. The subjects were divided into pediatric and adult groups. The genotyping of six well-known SNPs (MMP-2 -1575G > A, MMP-2 -1306C > T, MMP-3 -1171 5a/6a, MMP-9 -1562C > T, MMP-9 Q279R, and TIMP-2 -418G > C) in MMP and TIMP genes was performed by polymerase chain reaction-restriction fragment length polymorphism assays.ResultsA significantly higher frequency of the GC genotype for TIMP-2 -418 G > C was found in MMD patients. The MMP-9 Q279R GA + AA genotype showed a protective effect for MMD. The GA/CC MMP-2 -1575/-1306 genotype was significantly more prevalent in MMD patients.ConclusionsOur findings demonstrate that TIMP-2 -418 GC + CC and MMP-2 -1575GA/-1306CC genotypes could be genetic predisposing factors for MMD development.Electronic supplementary materialThe online version of this article (doi:10.1186/s12883-014-0180-5) contains supplementary material, which is available to authorized users.

Effects of captlpril on tissue inhibitorof metalloproteinase-1 in rat hepatic stellate cells

Objective To investigate the effects of angiotensin Ⅱ (Ang Ⅱ) and captlpril on tissue inhibitorof metalloproteinase-1 (TIMP-1) mRNA expression in culured hepatic stellate cells (HSCs) in vivo.Methods HSC-T6 rat hepatic stellate cell line was chosen as the study model of the activated HSCs.The cultured HSCs were randomized into control group,Ang Ⅱ group (1 × 10-5,1 × 10-7 mol/L) and AngⅡ + captlpril group (1 × 10-5,1 × 10-7 mol/L).HSCs were harvested to measure the TIMP-1 mRNA expression by reverse transcription-polymerase chain reaction (RT-PCR).Results TIMP-1 gene expression levels in different groups of Ang Ⅱ (1 × 10-5 mol/L,1 × 10-7 mol/L) were 1.145 ±0.219 and 0.860±0.115 respectively,and those in control group was 0.523 ± 0.056.TIMP-1 expression levels in different groups of Ang Ⅱ ± captlpril (1 × 10-5 mol/L,1 × 10-7 mol/L) were 0.740 ± 0.089 and 0.590 ± 0.073 respectively,and those in Ang Ⅱ group were 0.850 ± 0.107 and 0.620 ± 0.103 respectively.Conclusion Angiotensin Ⅱ can increase the type Ⅰ collage mRNA expression of HSCs,which can be inhibited by captlpril. Key words: Angiotensin Ⅱ ; Captlpril; Hepatic stellate cells; Tissue inhibitorof metalloproteinase-1

Association of tissue inhibitor of metalloproteinase gene polymorphisms and unexplained recurrent spontaneous abortions in Han Chinese couples

ObjectiveTo investigate the association of tissue inhibitor of metalloproteinase (TIMP) gene polymorphisms with unexplained recurrent spontaneous abortions (URSA) in a well-defined group of Han Chinese couples. Study designThis is a case-control association study. Genomic DNA was extracted from peripheral blood samples from 84 couples with histories of three or more pregnancy losses and 69 age-matched healthy couples with at least one live birth and no histories of pregnancy loss. Polymerase chain reactions (PCRs) and sequencing with the fluorescent dye dideoxy-termination method were used to detect the rs4898 in TIMP-1, rs2277698 in TIMP-2, rs2234921 and rs5749511 in TIMP-3 and rs17035945 in TIMP-4 genotypes and allele frequencies. ResultsNeither the allele frequencies nor any of the genetic model of the five TIMP gene SNPs (i.e., TIMP-1-rs4898, TIMP-2-rs2277698, TIMP-3-rs5749511 and rs2234921, and TIMP-4-rs7035945) were significantly differences between the URSA couples and the control group. ConclusionsNo evidence was found for any associations between the TIMP-1,-2,-3, or -4 genes SNPs with URSA in this Han Chinese Han.

Tissue inhibitor of metalloproteinase gene from pearl oyster Pinctada martensii participates in nacre formation

Tissue inhibitors of metalloproteinases (TIMPs) are nature inhibitors of matrix metalloproteinases and play a vital role in the regulation of extracellular matrix turnover, tissue remodeling and bone formation. In this study, the molecular characterization of TIMP and its potential function in nacre formation was described in pearl oyster Pinctada martensii. The cDNA of TIMP gene in P. martensii (Pm-TIMP) was 901bp long, containing a 5′ untranslated region (UTR) of 51bp, a 3′ UTR of 169bp, and an open reading fragment (ORF) of 681bp encoding 226 amino acids with an estimated molecular mass of 23.37kDa and a theoretical isoelectric point of 5.42; The predicted amino acid sequence had a signal peptide, 13 cysteine residues, a N-terminal domain and a C-terminal domain, similar to that from other species. Amino acid multiple alignment showed Pm-TIMP had the highest (41%) identity to that from Crassostrea gigas. Tissue expression analysis indicated Pm-TIMP was highly expressed in nacre formation related-tissues, including mantle and pearl sac. After decreasing Pm-TIMP gene expression by RNA interference (RNAi) technology in the mantle pallium, the inner nacreous layer of the shells showed a disordered growth. These results indicated that the obtained Pm-TIMP in this study participated in nacre formation.

Gene polymorphism -418 G/C of tissue inhibitor of metalloproteinases 2 is associated with abdominal aortic aneurysm

The pathogenesis of abdominal aortic aneurysm (AAA) is connected with abnormal extracellular matrix remodeling, with the assistance of extracellular matrix metalloproteinases and tissue inhibitors of metalloproteinases (TIMPs). A decrease in tissue inhibitor of metalloproteinases 2 (TIMP2) gene expression was detected in AAA patients. Recently, a -418 G/C (rs8179090) polymorphism of the TIMP2 gene promoter, influencing the transcription rate of the gene, has been described. This study investigated whether the -418 G/C gene polymorphism is associated with AAA in the Polish population. The TIMP2 gene promoter polymorphism was evaluated by polymerase chain reaction, followed by restriction enzyme analysis and pyrosequencing in 128 patients affected by AAA and in 180 individuals included as references. The control group was directly matched to patients according to known risk factors for vascular diseases. The frequency of the C allele was significantly higher in AAA patients than in the control group (odds ratio [OR], 2.516; P = .0005). The distribution of genotypes also differed significantly between the groups (CC + CG vs GG: OR, 2.906; P = .0037) or was close to being significantly different (CC vs GG + GC: OR, 2.144; P = .0501). A similar trend was observed in men but not in women. The multivariate logistic regression analysis indicated the TIMP2 gene promoter polymorphism is risk factor of AAA in the Polish population, with an adjusted OR of 4.99, when applied to a dominant inheritance model. Our study supports the hypothesis that TIMP2 and the -418G/C polymorphism located in the promoter of the TIMP2 gene are important in AAA pathophysiology.

Abstract T MP109: Tocotrienol Induces TIMP1 Expression and Pro-arteriogenic Remodeling of Cerebrovascular Collaterals

Introduction: Tocotrienols (TCT), lesser-known vitamin E family members, improve perfusion to brain tissue and attenuate stroke injury. Mechanisms underlying TCT improvement of collateral perfusion during ischemic stroke, however, remain unclear. Arteriogenesis is defined by the growth of functional collaterals in brain tissue where tissue inhibitor of metalloproteinase-1 (TIMP1) is believed to play a key role in extracellular matrix remodeling. Objective/Hypothesis: We tested the effect of prophylactic TCT on arteriogenesis by measuring collateral diameter and quantity in stroke-affected brain. Gene and protein expression of TIMP1 was queried in isolated collaterals as a key regulator of arteriogenesis. We hypothesize that TCT increases collateral size and number, and induces TIMP1 expression in collaterals of stroke-affected brain. Methods: C57/BL6 mice (male, 5 wks) were orally gavaged daily with 50mg/kg body weight of TCT or volume matched placebo (n=12) for 10 wks prior to middle cerebral artery occlusion (MCAO). During MCAO, cerebral perfusion was monitored using laser speckle flowmetry. While ischemia persisted, mice were perfused with FITC-conjugated lectin to identify patent collaterals in the MCA territory of the stroke-affected hemisphere. Collaterals size (diameter) and number were quantified as CD31+/FITC-lectin+ arterioles in stroke-affected S1 cortex and collected by laser capture microdissection. TIMP1 gene and protein expression in laser-captured collaterals was determined by RT-PCR and Western blot. Results: Compared to placebo, TCT treatment significantly increased perfusion (38.7%, p<0.05), collateral size (21.2%, p<0.05), and collateral number (5.7-fold, p<0.05) during MCAO. Prophylactic TCT supplementation significantly induced TIMP1 expression 9.6-fold (p<0.05) in laser-captured collaterals as compared to placebo controls. Conclusions: TCT induced expression of TIMP1 in brain collaterals and enabled arteriogenic remodeling for functional collateral growth and protection against ischemic stroke. As TCT is a safe, natural nutrient, proposal outcomes may quickly translate toward clinical applications for high-risk stroke patients, such as those who suffer a transient ischemic attack (TIA).

Molecular cloning and characterisation of the tissue inhibitor of metalloproteinase-3 gene from canine mammary tumour

Inhibition of matrix metalloproteinases (MMPs) by tissue inhibitors of metalloproteinases (TIMPs) has been reported to decrease metastasis and tumour-associated angiogenesis. The present study is aimed at characterising the gene encoding TIMP-3 from canine mammary tumour. We identified and cloned the full-length cDNA encoding TIMP-3 from canine mammary tumour. The entire open reading frame consisted of 636 nucleotides and 213 residues (accession number JF508171). Nucleotide and translated protein sequence were close to Sus scrofa but clustered away from the rodent group. Synonymous substitution (dS) was higher than non-synonymous substitution (dN), suggesting that the TIMP-3 gene was not under positive selection and that no advantageous mutations had any significant role in its evolutionary adaptation. The predicted SWISS-MODEL of canine TIMP-3 protein is composed of 7 α-helices and 12 β-turns. The G-factor score for the TIMP-3 protein model based on Ramachandran plot was found to be −0.47 for dihedral bonds, −0.08 for covalent bonds and −0.31 overall, which suggests that the model obtained for TIMP-3 is a reliable one.

Polymorphisms of the Tissue Inhibitor of Metalloproteinase 3 Gene Are Associated with Resistance to High-Altitude Pulmonary Edema (HAPE) in a Japanese Population: A Case Control Study Using Polymorphic Microsatellite Markers

IntroductionHigh-altitude pulmonary edema (HAPE) is a hypoxia-induced, life-threatening, high permeability type of edema attributable to pulmonary capillary stress failure. Genome-wide association analysis is necessary to better understand how genetics influence the outcome of HAPE.Materials and MethodsDNA samples were collected from 53 subjects susceptible to HAPE (HAPE-s) and 67 elite Alpinists resistant to HAPE (HAPE-r). The genome scan was carried out using 400 polymorphic microsatellite markers throughout the whole genome in all subjects. In addition, six single nucleotide polymorphisms (SNPs) of the gene encoding the tissue inhibitor of metalloproteinase 3 (TIMP3) were genotyped by Taqman® SNP Genotyping Assays.ResultsThe results were analyzed using case-control comparisons. Whole genome scanning revealed that allele frequencies in nine markers were statistically different between HAPE-s and HAPE-r subjects. The SNP genotyping of the TIMP3 gene revealed that the derived allele C of rs130293 was associated with resistance to HAPE [odds ratio (OR) = 0.21, P = 0.0012) and recessive inheritance of the phenotype of HAPE-s (P = 0.0012). A haplotype CAC carrying allele C of rs130293 was associated with resistance to HAPE.DiscussionThis genome-wide association study revealed several novel candidate genes associated with susceptibility or resistance to HAPE in a Japanese population. Among those, the minor allele C of rs130293 (C/T) in the TIMP3 gene was linked to resistance to HAPE; while, the ancestral allele T was associated with susceptibility to HAPE.

Identification of miR-1293 potential target gene: TIMP-1

Tissue inhibitor of metalloproteinases 1 (TIMP-1) is a glycosylated protein with multiple activities in the regulation of biological processes, such as cell growth and apoptosis as well as tumor invasion and metastasis. Bioinformatics analysis using TargetScan and miRanda suggested tissue inhibitors of TIMP-1 are among the targets of miR-1293. To confirm this, we cloned both wild-type and mutant TIMP-1 3'UTR fragments by overlap extension PCR, constructed the recombinant plasmids pGL3-TIMP-1-wt, -mut, and pcDNA 3.1(+)/TIMP-1-CDS and, respectively, co-transfected them into 293T cells with the miR-1293 inhibitor, mimics or the miR inhibitor-NC using a BTX ECM 2001 square-wave electroporator. We used a luciferase assay to investigate binding of miR-1293 to the 3'UTR of TIMP-1. Effects on the levels of the TIMP-1 protein were analyzed by Western blot experiments. The luciferase reporter assay showed a statistically significant (P<0.05) upregulation of activity. Western blot analysis showed a significant increase of expression of the TIMP-1 gene co-transfected with the miR-1293 inhibitor, and demonstrated direct binding of miR-1293 to the 3'UTR of TIMP-1. In this study, we identified TIMP-1 as a novel direct target for miR-1293, which provides the basis for further study of the multifunctional mechanisms of miR-1293 and TIMP-1 in the regulation of a variety of diseases.

Promoter methylation and expression of TIMP3 gene in gastric cancer

BackgroundGastric carcinoma development is a multi-stage process that involves more than one gene. Aberrant changes in DNA methylation are considered as the third mechanism that leads to anti-oncogene inactivation, which plays an essential role in tumor development. In this study, we assessed the relationship among the aberrant methylation of the promoter CpG islands of tissue inhibitor of metalloproteinase 3 (TIMP3) gene, its protein expression, and the clinicopathological features of gastric adenocarcinoma.MethodsThe methylation status of the promoter CpG islands and the protein expression of TIMP3 gene in tumors and adjacent normal mucosal tissues of 78 patients with gastric adenocarcinoma were detected by methylation-specific PCR (MSP) and immunohistochemistry.ResultsThe CpG island methylation of TIMP3 was detected in tumor tissues, cancer-adjacent tissues, and lymph nodes with metastasis. In increasing order, the hypermethylation frequency of these tissues were 35.9% (28 of 78 non-neoplastic tissues), 85% (17 of 20 early-stage cases), 89.7% (52 of 58 progressive-stage cases), and 100% (78 of 78 metastatic lymph node). A marked difference was found between tumors and non-neoplastic tissues (P < 0.05), but no difference existed among the subgroups of tumors (P > 0.05). Immunohistochemistry analysis confirmed TIMP3 down-regulation in tumor tissues. The rate of TIMP3 gene expression was 100% in non-neoplastic tissues but apparently decreased to various extents at different stages, i.e., decreased to 30% (6/20) at the early stage, to 3.4% (2/58) at the progressive stage, and to 0% (0/78) in metastatic lymph nodes. Among the 70 tumor tissues with negative TIMP3 expression, 64 (91.4%) were hypermethylated and 6 were unmethylated (8.6%), indicating a significant association between hypermethylation and reduced or negative TIMP3 expression (P < 0.01).ConclusionThe hypermethylation of the promoter region in CpG islands is the main mechanism of TIMP3 gene expression and may provide evidence for the molecular diagnosis and stage evaluation of gastric cancer.Virtual slidesThe virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1756134016954958

THU0054 Role of Fra-2 in TLR8-Mediated TIMP-1 Production in Systemic Sclerosis

BackgroundSystemic sclerosis (SSc) is an autoimmune connective tissue disease characterised by fibrosis, vascular dysfunction and abnormal activation of immune cells. We and others have shown that monocytes along with fibroblasts...

Antifibrotic Effects of a Recombinant Adeno-Associated Virus Carrying Small Interfering RNA Targeting TIMP-1 in Rat Liver Fibrosis

Elevated tissue inhibitor of metalloproteinase 1 (TIMP-1) expression contributes to excess production of extracellular matrix in liver fibrosis. Herein, we constructed a recombinant adeno-associated virus (rAAV) carrying siRNA of the TIMP-1 gene (rAAV/siRNA-TIMP-1) and investigated its effects on liver fibrosis in rats. Two models of rat liver fibrosis, the carbon tetrachloride and bile duct ligation models, were treated with rAAV/siRNA-TIMP-1. In the carbon tetrachloride model, rAAV/siRNA-TIMP-1 administration attenuated fibrosis severity, as determined by histologic analysis of hepatic collagen accumulation, hydroxyproline content, and concentrations of types I and III collagen in livers and sera. Levels of mRNA and active matrix metalloproteinase (MMP) 13 were elevated, whereas levels of mRNA and active MMP-2 were decreased. Moreover, a marked decrease was noted in the expression of α-smooth muscle actin, a biomarker of activated hepatic stellate cells (HSCs), and transforming growth factor-β1, critical for the development of liver fibrosis. Similarly, rAAV/siRNA-TIMP-1 treatment significantly alleviated bile duct ligation-induced liver fibrosis. Furthermore, this treatment dramatically suppressed TIMP-1 expression in HSCs from both model rats. These data indicate that the administration of rAAV/siRNA-TIMP-1 attenuated liver fibrosis by directly elevating the function of MMP-13 and diminishing activated HSCs. It also resulted in indirect decreased expression of type I collagen, MMP-2, and transforming growth factor-β1. In conclusion, rAAV/siRNA-TIMP-1 may be an effective antifibrotic gene therapy agent.

Tumor progression locus 2/Cot is required for activation of extracellular regulated kinase in liver injury and toll-like receptor–induced TIMP-1 gene transcription in hepatic stellate cells in mice

Toll-like receptors (TLRs) function as key regulators of liver fibrosis and are able to modulate the fibrogenic actions of nonparenchymal liver cells. The fibrogenic signaling events downstream of TLRs on Kupffer cells (KCs) and hepatic stellate cells (HSCs) are poorly defined. Here, we describe the MAP3K tumor progression locus 2 (Tpl2) as being important for the activation of extracellular regulated kinase (ERK) signaling in KCs and HSCs responding to stimulation of TLR4 and TLR9. KCs lacking Tpl2 display defects with TLR induction of cytokines interleukin (IL)-1β, IL-10, and IL-23. tpl2(-/-) HSCs were unable to increase expression of fibrogenic genes IL-1β and tissue inhibitor of metalloproteinase 1 (TIMP-1), with the latter being the result of defective stimulation of TIMP-1 promoter activity by TLRs. To determine the in vivo relevance of Tpl2 signaling in liver fibrosis, we compared the fibrogenic responses of wild-type (WT) and tpl2(-/-) mice in three distinct models of chronic liver injury. In the carbon tetrachloride and methionine-choline-deficient diet models, we observed a significant reduction in fibrosis in mice lacking Tpl2, compared to WT controls. However, in the bile duct ligation model, there was no effect of tpl2 deletion, which may reflect a lesser role for HSCs in wounding response to biliary injury. We conclude that Tpl2 is an important signal transducer for TLR activation of gene expression in KCs and HSCs by the ERK pathway and that suppression of its catalytic activity may be a route toward suppressing fibrosis caused by hepatocellular injuries. (HEPATOLOGY 2013).

A NOVEL MUTATION AT THE N-TERMINAL DOMAIN OF THE TIMP3 GENE IN SORSBY FUNDUS DYSTROPHY

To report a novel mutation occurring in the N-terminal domain of the tissue inhibitor of metalloproteinase 3 (TIMP3) gene in Sorsby fundus dystrophy. Retrospective review of medical records of two patients who had clinical features consistent with Sorsby fundus dystrophy. Genetic testing confirmed a mutation in the TIMP3 gene in both patients. Both patients had findings of drusenlike deposits, retinal pigment epithelial and photoreceptor atrophy, and bilateral, recurrent choroidal neovascularization. A strong family history of early onset macular degeneration was present in both. The patients developed choroidal neovascularization at the age of 45 and 48 years, and both had multiple recurrences in both eyes. Genetic testing in both patients confirmed a heterozygous nucleotide change of C113G, causing a Ser38Cys change in Exon 1 of the N-terminal domain of the TIMP3 gene. All previously reported mutations in Sorsby fundus dystrophy occur at Exon 5 in the C-terminal domain. We report 2 patients with novel mutations in Exon 1 of the N-terminal domain. Although the mutation occurs at a different location on the TIMP3 gene, the clinical features are similar to other reported patients with Sorsby fundus dystrophy. This finding assists in understanding the pathogenesis of this disorder.

Release of tissue inhibitor of metalloproteinase-2 from alginate microcapsule encapsulating genetically engineered cells

BackgroundIn this study, 293T cells were genetically engineered to secrete tissue inhibitor of metalloproteinase-2 (TIMP2) and encapsulated into alginate microcapsules to continuously release TIMP2 protein.MethodsThe anti-invasive potential of the microcapsules was studied in vitro using brain tumor cells. The TIMP2 gene was transfected to 293T cells, and genetically engineered 293TIMP2 cells were encapsulated into alginate microcapsules. Release of TIMP2 protein was detected with Western blot analysis and the anti-invasive potential against U87MG cells was tested using gelatin zymography and a Matrigel assay.ResultsCell viability within the alginate microcapsules was maintained at a cell density of 5 × 106. Because polycationic polymers are helpful for maintaining the mechanical strength of microcapsules with good cell viability, the alginate microcapsules were reinforced with chitosan (0.1% w/v). Expression of TIMP2 protein in cell lysates and secretion of TIMP2 into the conditioned medium was confirmed by Western blot analysis. Alginate microcapsules encapsulating 293TIMP2 cells released TIMP2 protein into the medium efficiently, where the TIMP2 protein participated in degradation of the matrix metalloproteinase-2 enzyme and inhibited invasion of U87MG cells.ConclusionAlginate microcapsules encapsulating 293TIMP2 cells are promising candidates for anti-invasive treatment of glioma.

S-adenosylmethionine, a methyl donor, up regulates tissue inhibitor of metalloproteinase-2 in colorectal cancer

DNA methylation is a fundamental epigenetic mechanism in regulating the expression of genes controlling crucial cell functions in cancer development. Gene silencing via CpG island methylation/demethylation in the promoter region is one of the mechanisms by which different genes are inactivated/activated in human cancers. Tissue inhibitor of metalloproteinase-2 (TIMP-2) is known to antagonize matrix metalloproteinase (MMP) activity and to suppress tumor growth, angiogenesis, invasion, and metastasis. TIMP-2 expression has been found to be both upregulated and downregulated in various cancers. The inconsistent TIMP-2 expression and unclear epigenetic regulation lead us to investigate its role in colorectal cancer in the presence of a methylating agent. Highly invasive human colorectal cells SW-620 were treated with the methyl donor S-adenosylmethionine (SAM) and its effect was evaluated by cell proliferation, cell cycle, invasion and migration assay. The ability of SAM to down regulate a panel of activated prometastatic, angiogenesis and growth- and cell cycle-regulatory genes was evaluated using end-point and real-time PCR. Treatment of SW-620 with SAM diminished cell proliferation and altered cell cycle kinetic G2/M phase cell cycle arrest. An in vitro matrigel invasion assay of SAM-treated cells showed a significant reduction in the invasive potential compared to untreated SW-620 cells. Treatment of SW-620 cells with SAM resulted in activation of TIMP-2 and inhibition of the expression of genes such as MMP (MMP-2, MT1-MMP), urokinase plasminogen activator, and vascular endothelial growth factors. The level of expression of tumor suppressor and apoptotic genes was not significantly higher compared to the untreated control. No changes in the levels of expression of genes (growth and cell cycle regulator), such as TGF-β, Smad2, Smad4, and p21 were observed. Our data support the hypothesis that TIMP-2, along with other prometastatic genes, is hypomethylated and expressed differently in colorectal cancer. Further in-depth analysis is warranted to confirm the promoter region CpG methylation pattern of the TIMP-2 gene.

Regulation of carthamus tinctorius on gene expression of stromelysin-1 and tissue inhibitor of metalloproteinase-1 in HSC-T6 cells

Objective To investigate the effects of carthamus tinctorius on gene expression of stromelysin-1 (MMP3) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in HSC-T6 cells.Methods HSC-T6 cells were cultured in the medium containing different concentrations of carthamus tinctorius (1.0,and 0.5 g/L) and collected after incubation for 48 h.Total RNA of HSC-T6 cells was isolated and gene expression levels of MMP3 and TIMP-1 were measured by using reverse transcription polymerase chain reaction (RT-PCR).Results MMP3 gene expression levels in 1.0 and 0.5 g/L carthamus tinctorius groups were 2.80 ±0.36 and 2.52 ±0.32 respectively,and that in control group was 2.13 ±0.30.TIMP-1 gene expression level in 1.0 and 0.5 g/L carthamus tinctorius groups were 1.66 ± 0.23 and 2.04 ± 0.30 respectively,and that in control group was 4.03 ± 0.56.Conclusion Carthamus tinctorius may enhance the expression of MMP3,but significantly inhibit TIMP-1 gene expression of HSC-T6 cells in a concentration-dependent manner. Key words: Hepatic stellate cell; Carthamus tinctorius; Stromelysin-1; Tissue inhibitor of metalloproteinase-1 ; Gene expression

Loss of TIMP3 selectively exacerbates diabetic nephropathy

Diabetic nephropathy is the most common cause of end-stage renal disease. Polymorphism in the tissue inhibitor of metalloproteinase-3 (TIMP3) gene, and the ECM-bound inhibitor of matrix metalloproteinases (MMPs), has been linked to diabetic nephropathy in humans. To elucidate the mechanism, we generated double mutant mice in which the TIMP3 gene was deleted in the genetic diabetic Akita mouse background. The aggravation of diabetic injury occurred in the absence of worsening of hypertension or hyperglycemia. In fact, myocardial TIMP3 levels were not affected in Akita hearts, and cardiac diastolic and systolic function remained unchanged in the double mutant mice. However, TIMP3 levels increased in Akita kidneys and deletion of TIMP3 exacerbated the diabetic renal injury in the Akita mouse, characterized by increased albuminuria, mesangial matrix expansion, and kidney hypertrophy. The progression of diabetic renal injury was accompanied by the upregulation of fibrotic and inflammatory markers, increased production of reactive oxygen species and NADPH oxidase activity, and elevated activity of TNF-α-converting enzyme (TACE) in the TIMP3(-/-)/Akita kidneys. Moreover, while the elevated phospho-Akt (S473 and T308) and phospho-ERK1/2 in the Akita mice was not detected in the TIMP3(-/-)/Akita kidneys, PKCβ1 (but not PKCα) was markedly elevated in the double mutant kidneys. Our data provide definitive evidence for a critical and selective role of TIMP3 in diabetic renal injury consistent with gene expression findings from human diabetic kidneys.

Experimental research on the prevention of autologous vein graft restenosis by fibrin glue combined with tissue inhibitor of metalloproteinase-1

Objective To investigate the mechanism of attenuation of restenosis by applying fibrin glue combined with tissue inhibitor of metalloproteinase-1 (TIMP-1) locally on vein graft.Methods Twenty-four New Zealand white rabbits were randomly divided into 3 groups with 8 rabbits in each group:non-support group (NS group),fibrin glue group (FS group) and fibrin glue + TIMP-1 group (TS/FS group).All animals underwent unilateral jugutar vein into common carotid artery interposition grafting.After operation,vein grafts were harvested at 28th day for histopathologic measurement of intimal thickness,media thickness and intimal areas,media areas,reverse transcription-polymerase chain reaction (RTPCR) and Western blotting analyses of TIMP-1 gene expression.Results At 28th day after the operation,the mean intimal thickness and intimal areas of the TS/FS group were (37.98 ±4.19) μm and (0.557 ±0.049) mm2 respectively,which were reduced obviously as compared with NS group and FS group (P ＜0.05).The mean media thickness and media areas in the TS/FS group were (28.45 ± 3.01 ) μm and (0.458 ±0.053) mm2 respectively,which were decreased statistically as compared with NS group and FS group (P ＜ 0.05).RT-PCR and Western blotting analysis revealed that the gene expression of TIMP-1 was increased in TS/FS group as eompared with NS group and FS group (P ＜ 0.05).Conclusion The expression of TIMP-1 in vein grafts can be up-regulated by adventitial delivery of TIMP-1.The fibrin glue combined with TIMP-1 has synergistic effect on reducing neointimal and media thickness of vein grafts. Key words: Vein graft; Restenosis; Fibrin glue; Tissue inhibitor of metalloproteinase-1

Curcumin Effect on MMPs and TIMPs Genes in a Breast Cancer Cell Line

Curcumin (CM) possesses anti-cancer activity against a variety of tumors. Matrix metalloproteinases (MMPs) play an important role in remodeling the extracellular matrix and their activities are regulated by tissue inhibitor of metalloproteinases (TIMPs) family. Control of MMP and TIMP activity are now of great significance. In this study, the effect of CM is investigated on metastatic MMPs and anti-metastatic TIMPs genes on MDA breast cancer cells cultured in a mixture of DMEM and Ham's F12 medium and treated with different concentrations of CM (10, 20 and 40 μM for various lengths of time. Reverse transcription followed by quantitative real time PCR was used to detect the gene expression levels of MMPs and TIMPs in CM-treated versus untreated cases and the data were analyzed by one-way ANOVA. At high concentrations of curcumin, TIMP-1, -2, -3 and -4 genes were up-regulated after 48 hours of treatment, their over-expression being accompanied by down-regulation of MMP-2 and MMP-9 gene expression levels in a concentration- and time-dependent manner. These results suggest that curcumin plays a role in regulating cell metastasis by inhibiting MMP-2 and MMP-9 and up-regulating TIMP1 and TIMP4 gene expression in breast cancer cells.