Tissue Inhibitor of Metalloproteinase 3: Unravelling Its Biological Function and Significance in Oncology.

Tissue inhibitor of metalloproteinases-3 (TIMP3) is vital in regulating several biological processes. TIMP3 exerts antitumour effects via matrix metalloproteinase (MMP)-dependent and MMP-independent pathways. Due to promoter methylation and miRNA binding, TIMP3 expression has been observed to decrease in various cancers. Consequently, the migration and invasion of cancer cells increases. Conflicting results have reported that expression levels of TIMP3 in primary and advanced cancers are higher than those in healthy tissues. Therefore, the role of TIMP3 in cancer biology and progression needs to be elucidated. This review provides an overview of TIMP3, from its biological function to its effects on various cancers. Moreover, gynaecological cancers are discussed in detail. TIMP3 has been associated with cervical adenocarcinoma as well as cancer development in serous ovarian cancer and breast cancer metastasis. However, the relationship between TIMP3 and endometrial cancers remains unclear. TIMP3 may be a useful biomarker for gynaecological cancers and is a potential target for future cancer therapy.

Impact of tissue inhibitor of metalloproteinases-3 genetic variants on clinicopathological characteristics of urothelial cell carcinoma.

To investigate the distribution of single nucleotide polymorphism (SNP) of tissue inhibitor of metalloproteinases-3 (TIMP-3) in patients with/without urothelial cell carcinoma (UCC), three loci of TIMP-3 SNPs (rs9862 C/T, rs9619311 T/C, rs11547635 C/T) were genotyped via TaqMan allelic discrimination for 424 UCC patients and 848 non-UCC participants. Furthermore, the TIMP-3 mRNA expression and its correlation with clinical characters of urothelial bladder carcinoma was analyzed using The Cancer Genome Atlas database (TCGA). The distribution of all 3 studied SNPs of TIMP-3 was insignificantly different between the UCC and non-UCC groups. However, significantly lower tumor T status was found in TIMP-3 SNP rs9862 CT + TT variant than the wild type (OR: 0.515, 95% CI: 0.289-0.917, P = 0.023). Moreover, the muscle invasive tumor type was significantly correlated to the TIMP-3 SNP rs9619311 TC + CC variant in the non-smoker subgroup (OR: 2.149, 95% CI: 1.143-4.039, P = 0.016). With the TIMP-3 expression data provided in TCGA, significantly higher TIMP-3 mRNA expression was observed in UCC with high tumor stage (P < 0.0001), high tumor T status (P < 0.0001) and high lymph node status (P = 0.0005). In conclusions, TIMP-3 SNP rs9862 variant is associated with lower tumor T status of UCC while TIMP-3 SNP rs9619311 variant is correlated to muscle invasive UCC development in non-smoker.

Effect of tissue inhibitor of metalloproteinases-3 genetics polymorphism on clinicopathological characteristics of uterine cervical cancer patients in Taiwan.

Single nucleotide polymorphisms (SNPs) of tissue inhibitor of metalloproteinases-3 (TIMP-3) have been revealed to be related to various cancers. To date, no study explores the relationships between TIMP-3 polymorphisms and uterine cervical cancer. The purposes of this research were to investigate the associations among genetic variants of TIMP-3 and development and clinicopathological factors of uterine cervical cancer, and patient 5 years survival in Taiwanese women. The study included 123 patients with invasive cancer and 97 with precancerous lesions of uterine cervix, and 300 control women. TIMP-3 polymorphisms rs9619311, rs9862 and rs11547635 were checked and their genotypic distributions were determined by real-time polymerase chain reaction. It showed that women with genotypes CT/TT in rs9862 were found to display a higher risk of developing cervical cancer with moderate and poor cell differentiation. Moreover, it revealed that cervical cancer patients carrying genotypes CC in rs9619311 exhibited a poorer 5 years survival, as compared to those with TT/TC in Taiwanese women, using univariate analysis. In addition, pelvic lymph node metastasis was determined to independently predict 5 years survival in cervical cancer patients using multivariate analysis. Conclusively, TIMP-3 SNPs polymorphisms rs9619311 are related to cervical patient survival in Taiwanese women.

TIMP3 attenuates cerebral ischemia/reperfusion-induced apoptosis and oxidative stress in neurocytes by regulating the AKT pathway.

Ischemic stroke seriously threatens human health and creates a large social burden. The present study investigated whether tissue inhibitor of metalloproteinases-3 (TIMP3) prevented cerebral ischemia/reperfusion (I/R), with the aim to explore the underlying mechanism. A transient middle cerebral artery occlusion model was conducted in mice, and oxygen glucose deprivation and reoxygenation (OGD/R) was investigated in PC12 cells to mimic cerebral ischemia-reperfusion injury (CIRI). Western blotting was used to determine the expression of TIMP3, Bax, Bcl-2 and AKT. TUNEL was used to detect apoptosis in cerebral tissues or cultured PC12 cells. Expression levels of reactive oxygen species (ROS), superoxide dismutase (SOD) and malondialdehyde (MDA) were detected to reveal oxidative stress. The results demonstrated that TIMP3 expression was significantly decreased after I/R in vivo or OGD/R in vitro, and the number of TUNEL-positive cells was reduced by the overexpression of TIMP3. The attenuation of Bax/Bcl-2 ratio in OGD/R-induced PC12 cells suppressed the expression levels of ROS and MDA; while also elevating SOD activity in the OGD/R-induced neurocytes in vitro. In addition, TIMP3-overexpression reversed the downregulation of phosphorylated-AKT (Thr308 and Ser473) in OGD/R-treated PC12 cells. However, the anti-apoptotic and anti-oxidative stress roles of TIMP3 in OGD/R-induced PC12 cells were partially abolished after treatment with the AKT inhibitor, AZD5363. Overall, TIMP3 exerted an anti-apoptotic and anti-oxidative stress role in CIRI through the AKT pathway, which may be a potential therapeutic target for the treatment of CIRI.

Long non­coding RNA GAS5 protects against Mycoplasma pneumoniae pneumonia by regulating the microRNA­222­3p/TIMP3 axis.

Mycoplasma pneumoniae pneumonia (MPP) is a type of pneumonia induced by M. pneumoniae (MP) infection. The present study investigated the effect of long non­coding RNA growth arrest­specific 5 (GAS5) in MPP and the underlying molecular mechanism of this. The expression of GAS5, microRNA­222­3p, (miR­222­3p) and tissue inhibitor of metalloproteinases­3 (TIMP3) in MPP was investigated using reverse transcription­quantitative PCR. Lipid­associated membrane protein (LAMP)­induced THP­1 cells were used to model MPP. The viability of LAMP­induced THP­1 cells was analyzed using an MTT assay. Expression levels of interleukin (IL)­1ß, IL­6 and tumor necrosis factor­α (TNF­α) pro­inflammatory cytokines, and the anti­inflammatory cytokine heme oxygenase­1 (HO­1) in LAMP­induced THP­1 cells were measured by ELISA. A dual­luciferase reporter assay assessed the associations among GAS5, miR­222­3p and TIMP3. The expression of GAS5 and TIMP3 was downregulated in MPP. Expression of miR­222­3p was upregulated. GAS5­overexpression increased the viability of LAMP­induced THP­1 cells. GAS5 upregulation decreased the levels of IL­1ß, IL­6, TNF­α and HO­1 levels in LAMP­induced THP­1 cells. GAS5 directly interacted with miR­222­3p. TIMP3 was a target of miR­222­3p. miR­222­3p upregulation or TIMP3­knockdown reversed the promotion effect on cell viability as well as the inhibitory effect on inflammation caused by GAS5­overexpression in LAMP­induced THP­1 cells. GAS5­overexpression increased the viability and decreased the inflammation of LAMP­induced THP­1 cells by regulating the miR­222­3p/TIMP3 axis. These results demonstrated a potential therapeutic target for MPP treatment.

MicroRNA-21 Contributes to Cutaneous Squamous Cell Carcinoma Progression via Mediating TIMP3/PI3K/AKT Signaling Axis.

BACKGROUND: Though the therapeutic potentials of microRNAs (miRNAs) are extensively explored in cutaneous squamous cell carcinoma (CSCC), the concrete function of miR-21 in this disorder has not been thoroughly comprehended. Therein, this work is launched to clarify the miR-21-pivoted mechanism in CSCC from the perspective of tissue inhibitor of metalloproteinases-3 (TIMP3) and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway. METHODS: Microarray-based analysis was utilized to screen out miR-21 with the most up-regulated expression in CSCC tissues. The relation between miR-21 and TIMP3 expression in tissues, and the overall survival of CSCC patients was evaluated. Loss-of-function assays were performed in cells to explore the independent and combined functions of miR-21 and TIMP3 in CSCC cell progression. Mice were injected with miR-21 inhibitor or TIMP3 si for identifying their roles in tumor formation and liver metastasis. The mechanism among miR-21, TIMP3 and PI3K/AKT pathway was interpreted. RESULTS: MiR-21 was up-regulated while TIMP3 was down-regulated in CSCC tissues, which were connected with unsatisfactory survival of patients. Down-regulating miR-21 inhibited CSCC cell progression and retarded CSCC tumor formation and metastasis in mice. Silencing of TIMP3 reversed the effects of miR-21 down-regulation on CSCC progression. Besides, down-regulating miR-21 inhibited PI3K/AKT pathway activation in CSCC cells via mediating TIMP3. CONCLUSION: It is elucidated that miR-21 depletion impedes CSCC cell invasion and metastasis via enhancing TIMP3 and suppressing PI3K/AKT pathway activation.

Associations of TIMP-3 Genetic Polymorphisms with EGFR Statuses and Cancer Clinicopathologic Development in Lung Adenocarcinoma Patients.

Lung adenocarcinoma (LADC) is a major subtype of lung cancer, particularly among populations of East Asia. The epidermal growth factor receptor (EGFR) is the most frequently mutated oncogene promoting LADC progression and can serve as a therapeutic target in LADC. The tissue inhibitor of metalloproteinases (TIMP)-3 is a major regulator of extracellular matrix turnover via targeting of matrix metalloproteinases (MMPs), and thus, plays a critical role in tumor development and progression. The purpose of this study was to investigate potential associations among TIMP-3 genetic polymorphisms, EGFR statuses, and cancer clinicopathologic development in patients with LADC. In this study, 277 LADC patients with different EGFR statuses were recruited to dissect the allelic discrimination of TIMP-3 -1296 T>C (rs9619311), TIMP3 249T>C (rs9862), and TIMP3 261C>T (rs11547635) polymorphisms using a TaqMan allelic discrimination assay. Our data showed that compared to those LADC patients with wild-type CC homozygotes of TIMP-3 rs9862, patients harboring TT homozygotes of rs9862 were at a higher risk of developing mutant EGFR (adjusted odds ratio (AOR) = 2.530; 95% confidence interval (CI): 1.230-5.205; p = 0.012), particularly the EGFR L858R point mutation (AOR = 2.975; 95% CI: 1.182-7.488; p = 0.021). Moreover, we observed that TIMP-3 TT homozygotes of rs9862 were correlated with the incidence of EGFR mutations in patients with a smoking habit (p = 0.045). Within male patients harboring a mutant EGFR, TIMP-3 rs9862 T (CT+TT) allele carriers were at higher risk of developing an advanced stage (p = 0.025) and lymph node metastasis (p = 0.043). Further analyses of clinical datasets revealed correlations of TIMP-3 expression with a favorable prognosis in patients with LADC. In conclusion, the data suggest that TIMP-3 rs9862 polymorphisms may contribute to identify subgroups of lung cancer patients at high risk for tumor progression, among carriers of LADC-bearing mutant EGFR.

MicroRNA-221 promotes proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs) by targeting tissue inhibitor of metalloproteinases-3 (TIMP3).

BACKGROUND: Aberrant vascular smooth muscle cell (VSMC) proliferation and migration play an important role in the development of cardiovascular diseases including pulmonary arterial hypertension (PAH). MicroRNAs (miRNAs, miRs) have been considered to be implicated in the progression of PAH pathogenesis. In this study, we aim to clarify the role of miR-221 on proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs) and identify the target genes involved in this biological process. METHODS: PASMCs were isolated from the pulmonary arteries of male Sprague-Dawley (SD) rats. Cell proliferation of PASMCs was detected by 5-ethynyl-2'-deoxyuridine (EdU) assay. Cell migration was determined by a scratch wound assay. Quantitative real-time PCR was used to determine the expression of miR-221 while western blot analysis was used to determine the expression of TIMP3. Luciferase assay was used to confirm that TIMP3 was a direct target gene of miR-221. Monocrotaline (MCT) induced-PAH rat model was established and miR-221 and TIMP3 levels were checked in lung tissue and PASMCs from PAH rats. RESULTS: miR-221 was able to promote the proliferation and migration PASMCs. TIMP3 were negatively regulated by miR-221 at the protein level in PASMCs. In addition, TIMP3 was identified to be a direct target gene of miR-221 in PASMCs based on luciferase assays. TIMP3 knockdown abolished the inhibitory effect of miR-221 inhibitor on PASMCs proliferation and migration, suggesting TIMP3 mediated the effects of miR-221 in PASMCs. Finally, we found that miR-221 was increased while TIMP3 was down-regulated in PASMCs in MCT-treated rats. CONCLUSIONS: In conclusion, miR-221 promotes PASMCs proliferation and migration by targeting TIMP3. MiR-221 and TIMP3 could be potential therapeutic targets for the treatment of PAH.

Disturbed balance in the expression of MMP9 and TIMP3 in cerebral amyloid angiopathy-related intracerebral haemorrhage.

Cerebral amyloid angiopathy (CAA) is characterized by the deposition of the amyloid ß (Aß) protein in the cerebral vasculature and poses a major risk factor for the development of intracerebral haemorrhages (ICH). However, only a minority of patients with CAA develops ICH (CAA-ICH), and to date it is unclear which mechanisms determine why some patients with CAA are more susceptible to haemorrhage than others. We hypothesized that an imbalance between matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) contributes to vessel wall weakening. MMP9 plays a role in the degradation of various components of the extracellular matrix as well as of Aß and increased MMP9 expression has been previously associated with CAA. TIMP3 is an inhibitor of MMP9 and increased TIMP3 expression in cerebral vessels has also been associated with CAA. In this study, we investigated the expression of MMP9 and TIMP3 in occipital brain tissue of CAA-ICH cases (n = 11) by immunohistochemistry and compared this to the expression in brain tissue of CAA cases without ICH (CAA-non-haemorrhagic, CAA-NH, n = 18). We showed that MMP9 expression is increased in CAA-ICH cases compared to CAA-NH cases. Furthermore, we showed that TIMP3 expression is increased in CAA cases compared to controls without CAA, and that TIMP3 expression is reduced in a subset of CAA-ICH cases compared to CAA-NH cases. In conclusion, in patients with CAA, a disbalance in cerebrovascular MMP9 and TIMP3 expression is associated with CAA-related ICH.

MiR-21 antagomir improves insulin resistance and lipid metabolism disorder in streptozotocin-induced type 2 diabetes mellitus rats.

BACKGROUND: Type 2 diabetes mellitus (T2DM) is a major cause of death with an increasing incidence at an epidemic rate. The existing treatments for T2DM lack long-term effective blood glucose control. In this study, the effects of miR-21 antagomir on T2DM and the related mechanism were investigated using streptozotocin (STZ)-induced T2DM rats. METHODS: 30 T2DM rats were randomly divided into 3 groups (n=10): T2DM group, T2DM rats with miR-21 antagomir group, T2DM rats with NC antagomir group. The expression of miR-21 in rats was detected by qRT-PCR. blood glucose, triacylglycerol (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-Cho), insulin, adiponectin, ITT and GTT were detected. The expression of TIMP3 in si-TIMP3 rats and the expression of TIMP3 in T2DM rats with miR-21 antagomir and si-TIMP3 was detected by Western blotting. RESULTS: We found that miR-21 antagomir reduced blood glucose concentration in T2DM rats. MiR21 antagomir improved lipid metabolic disorder by decreasing the levels of triacylglycerol (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-Cho) and increasing the level of high-density lipoprotein cholesterol (HDL-Cho). Also, miR-21 antagomir reduced the value of homeostasis model assessment of insulin resistance (HOMA-IR), hemoglobin A1c (HbAc1), plasma insulin, and up-regulated the plasma adiponectin. These results, combined with insulin tolerance tests (ITT) and glucose tolerance tests (GTT) results, showed that miR-21 improved insulin resistance in STZ-induced T2DM rats. Then the target relationship between miR-21 and tissue inhibitor of metalloproteinases 3 (TIMP3) was proved by luciferase reporter assay. More impressively, miR-21 significantly increased the expression level of TIMP3 in STZ-induced T2DM rats. CONCLUSIONS: Our study taken together has shown that miR-21 antagomir improved insulin resistance and lipid metabolism disorder in STZ-induced T2DM rats by up-regulating the expression level of TIMP3. This study suggested that miR-21 antagomir could be used as an effective therapeutic strategy and the underlying mechanism was revealed.

TIMP-3 suppression induces choroidal neovascularization by moderating the polarization of macrophages in age-related macular degeneration.

PURPOSE: To investigate the role of tissue inhibitor of metalloproteinases-3 (TIMP-3) as a key moderator of macrophage polarization in choroidal neovascularization (CNV) lesions of model mice and in bone marrow-derived macrophage (BMDM). METHOD: We used siR-TIMP-3 to transfect BMDM and gave an intravitreal injection of siR-TIMP-3 to laser-induced CNV mice model, real time-PCR and western blot were applied for detecting the expressions of TIMP-3 and macrophages' biomarker. Besides, CNV lesions in different treatment groups of animal model were examined by the optical coherence tomography angiography (OCTA). RESULTS: Our experimental data showed that lack of TIMP-3 stimulated M2 polarization proved by real time-PCR and western blot in BMDMs and CNV mice model. Moreover, intravitreal injection of siR-TIMP-3 accelerated CNV formation using OCTA, which indicated that TIMP-3 suppression is related to pro-angiogenesis of M2 macrophage. CONCLUSION: We showed that the absence of TIMP-3 leads to a more pro-angiogenic microenvironment, playing a key role in CNV formation by positively modulating M2 polarization. The role of TIMP-3 in the regulating inflammation and novel therapeutic target of nAMD needs to be further studied.

miR-222-3p promotes osteosarcoma cell migration and invasion through targeting TIMP3.

BACKGROUND: Abnormal expression of miRNAs has been reported in osteosarcoma (OS), and miR-222-3p levels have been found to be increased in the serum of OS patients. However, the exact role of miR-222-3p in OS remains unclear. In the present study, we aimed to identify the molecular mechanism underlying the role of miR-222-3p in the development of OS. METHODS: We examined the expression level of miR-222-3p in OS tissues and OS cells using reverse-transcription quantitative PCR (RT-qPCR) analysis. MTT, colony formation, and transwell invasion assays were used to analyze the effects of miR-222-3p on the proliferation and invasion ability of OS cells. Luciferase reporter gene assays were used to confirm the target gene of miR-222-3p in OS cells. Tumor xenografts were then used to investigate the role of miR-222-3p in OS growth in vivo. RESULTS: The data of the present study demonstrated that miR-222-3p levels were increased in OS tissues and OS cells. Downregulation of miR-222-3p significantly inhibited the proliferation, migration, and invasion of OS cells in vitro. Further analysis revealed that tissue inhibitors of metalloproteinases 3 (TIMP3) is one of the functional target genes of miR-222-3p, and inhibition of TIMP3 efficiently rescues the blocking of cell proliferation and invasion mediated by miR-222-3p inhibitor in OS cells. CONCLUSION: Our findings constitute evidence that miR-222-3p promotes OS cell proliferation and invasion through targeting TIMP3 mRNA and provide novel insight into the mechanism underlying the development of OS.

Tissue inhibitor of the metalloproteinases-3 gene polymorphisms and carotid plaque susceptibility in the Han Chinese population.

Tissue inhibitor of metalloproteinases (TIMPs) are endogenous inhibitors of matrix metalloproteinases that are involved in normal cellular processes and in the development and progression of atherosclerosis. Our purpose was to evaluate the polymorphisms of the TIMP-3 genes for their associations with carotid plaques or with serum protein levels in the Han Chinese population. Two promoter variants, -915A/G (rs2234921) and -1296T/C (rs9619311), were genotyped in 548 subjects with no plaques, 462 subjects with echogenic plaques, and 427 subjects with mixture plaques. The serum TIMP-3 levels were measured using an enzyme-linked immunosorbent assay (ELISA). There was a strong linkage disequilibrium between -1296T/C and -915A/G (D' = 1.0, r2 = 0.991). The individuals with the genotype (TC+CC) were 1.8 times more likely to have mixture plaques than the individuals with the TT genotype (P = 0.001, OR: 1.836, 95%CI: 1.269-2.665). The frequency of the C allele in the mixture plaque group was significantly higher than in the no plaque group (P = 0.009, CI: 1.119-2.187). We observed a significant elevation of the TIMP-3 levels in the serum of patients affected with mixture plaques compared to those with no plaques (P = 0.013). The current data suggest that genetic variation in the TIMP-3 genes may contribute to individual differences in mixture plaque susceptibility in the Han Chinese population.

Effect of magnesium-calcium alloy extract on matrix metalloproteinase-9 and matrix metalloproteinase inhibitor-3 in human colonic epithelial cells / 新乡医学院学报

Objective To investigate the effect of different concentrations of magnesium-calcium alloy extract on the expression of matrix metalloproteinase-9 (MMP9) and tissue inhibitor of metalloproteinase-3 (TIMP3) in human colonic epithelial NCM460 cells.Methods The different concentrations of extracts (the volume fraction was 10％,50％ and 100％ respectively) were made with magnesium-calcium alloy.The 5 × 106 L-1 NCM460 suspension was randomly divided into control group,experimental group 1,experimental group 2 and experimental group 3.The cells in the control group were cultured by 2 000 μL high glucose Dulbecco's modified Eagle's medium (containing 10％ volume fraction of fetal bovine serum).The cells in the experimental group 1,2 and 3 were cultured by 2 000 μL magnesium-calcium alloy extract with volume fraction of 10％,50％ and 100％ respectively.The expressions of MMP9 and TIMP3 mRNA in NCM460 cells was detected by real-time fluorescence quantitative polymerase chain reaction,and the expression of MMP9 and TIMP3 protein in NCM460 cells was detected by Western blot at after one,three and five days of cultivation respectively.Results The expression of MMP9 mRNA and TIMP3 mRNA in NCM460 cells of the experimental group 1,2 and 3 was significantly lower than that in the control group after one day of cultivation (P ＜ 0.05).After three and five days of cultivation,the expression of MMP9 mRNA in NCM460 cells of the experimental group 1 was significantly lower than that in the control group (P ＜ 0.05),but the expression of MMP9 mRNA in the NCM460 cells of the experimental group 2 and 3 was significantly higher than that in the control group and the experimental group 1 (P ＜ 0.05).The expression of MMP9 mRNA in NCM460 cells of the experimental group 3 was significantly higher than that in the experimental group 2 after five days of cultivation (P ＜ 0.05).The expression of MMP9 mRNA in NCM460 cells of the experimental group 1,2 and 3 after three and five days of cultivation was significantly higher than that after one day of cultivation(P ＜ 0.05).There was no significant difference in the expression of MMP9 mRNA in NCM460 cells of the experimental group 1 between three and five days of cultivation (P ＞ 0.05).The expression of MMP9 mRNA in NCM460 cells of the experimental group 2 and 3 after five days of cultivation was significantly higher than that after three days of cultivation(P ＜ 0.05).The expression of TIMP3 mRNA in NCM460 cells of the experimental group 2 and 3 was significantly higher than that in the experimental group 1 after one day of euhivation (P ＜ 0.05).After three days of cultivation,the expression of TIMP3 mRNA in NCM460 cells of the experimental group 1,2 and 3 was significantly lower than that in the control group (P ＜ 0.05);the expression of TIMP3 mRNA in NCM460 cells of the experimental group 2 was significantly lower than that in the experimental group 1 and 3 (P ＜ 0.05).After five days of cultivation,the expression of TIMP3 mRNA in NCM460 cells of the experimental group 1,2 and 3 was significantly higher than that in the control group (P ＜ 0.05).The expression of TIMP3 mRNA in NCM460 cells after three and five days of cultivation was significantly higher than that after one day of cultivation (P ＜ 0.05),and the expression of TIMP3 after five days of cultivation was significantly higher than that after three days of cultivation in the experimental group 1 (P ＜ 0.05).The expression of TIMP3 mRNA in NCM460 cells after three days of cultivation was significantly lower than that after one day of cultivation (P ＜ 0.05),and the expression of TIMP3 after five days of cultivation was significantly higher than that after one and three days of cultivation in the experimental group 2 (P ＜ 0.05).The expression of TIMP3 mRNA in NCM460 cells after five days of cultivation was significantly higher than that after one and three days of cultivation in the experimental group 3 (P ＜ 0.05).After five days of cultivation,there was no significant difference in the expression of MMP9 protein in NCM460 cells between the experimental group 1 and control group (P ＞ 0.05),the expression of MMP9 protein in NCM460 cells of the experimental group 2 and 3 was significantly higher than that in the control group and the experimental group 1 (P ＜ 0.05),but there was no significant difference in the expression of MMP9 protein in NCM460 cells between the experimental group 2 and 3 (P ＞ 0.05).After five days of cultivation,the expression of TIMP3 protein in NCM460 cells of the experimental group 1,2 and 3 was significantly higher than that in the control group (P ＜0.05);but there was no significant difference in the expression of TIMP3 protein in NCM460 cells among the experimental group 1,2and 3 (P ＞ 0.05).Conclusions The high concentration of magnesium-calcium alloy extract has certain influence on the expression of MMP9 and TIMP3 gene in NCM460 cells,which may lead to the early inflammatory reaction,and the mechanism may be related to the calcium ion concentration in the extract.

Amelioration of Dalton's lymphoma-induced angiogenesis by melatonin.

For tumor to grow beyond 1-2 mm3 size, tumor recruits new blood vessels referred as angiogenesis; therefore, targeting angiogenesis can be a promising strategy to suppress cancer progression. In this study, in order to develop a good angiogenesis model, we investigated effect of Dalton's lymphoma on angiogenesis and further monitored the role of melatonin on regulation of angiogenesis. To evaluate angiogenesis, endothelial cells were isolated from main thoracic aorta and cultured in vitro in the presence or absence of Dalton's lymphoma supplemented with or without melatonin to monitor their role on its proliferation and migration, a hallmark of angiogenesis. Chick chorioallantoic membrane as well as mice mesentery which allows in vivo studies of tumor angiogenesis and testing of anti-angiogenic molecules was used to validate the in vitro analysis. To further extend our understanding about the regulation of the angiogenesis, we evaluated expression of tissue inhibitor of metalloproteinases 3, vascular endothelial growth factor, vascular endothelial growth factor receptor, and fibroblast growth factor in Dalton's lymphoma cells and mesentery by semiquantitative and quantitative reverse transcription polymerase chain reaction analysis. Dalton's lymphoma ascites induced significant increase in endothelial cell proliferation, migration, and sprouting of the tertiary branching in chorioallantoic membrane and mesentery of Dalton's lymphoma-bearing mice, whereas melatonin treatment led to their inhibition in a dose-dependent manner. Semiquantitative and quantitative reverse transcription polymerase chain reaction analysis of melatonin-treated Dalton's lymphoma cells and mesentery tissue clearly demonstrated restoration of angiogenesis-related genes tissue inhibitor of metalloproteinases 3 and reduction of vascular endothelial growth factor, vascular endothelial growth factor receptor, and fibroblast growth factor messenger RNA expression. Taken together, our results strongly demonstrate that Dalton's lymphoma provides pro-angiogenic environment leading to significant increase in angiogenesis, and further melatonin treatment reduced the Dalton's lymphoma ascites-induced angiogenesis implying that Dalton's lymphoma can serve as a very good model to study angiogenesis as well as for screening of drugs that can target angiogenesis.

Myocardial overexpression of TIMP3 after myocardial infarction exerts beneficial effects by promoting angiogenesis and suppressing early proteolysis.

Myocardial infarction (MI) results in loss of cardiomyocytes, adverse extracellular matrix (ECM) and structural remodeling, and left ventricular (LV) dilation and dysfunction. Tissue inhibitors of metalloproteinase (TIMPs) inhibit matrix metalloproteinases (MMPs), the main regulators of ECM turnover. TIMPs also have MMP-independent functions. TIMP3 levels are reduced in the heart within 24 h of MI in mice. We investigated if overexpression of TIMP3 post-MI limits adverse remodeling and LV dilation and dysfunction. MI was induced by left anterior descending coronary artery ligation in 10- to 12-wk-old male C57BL/6J mice, and adenoviral constructs expressing human (h)TIMP3 (Ad-hTIMP3) or no TIMP (Ad-Null) were injected in the peri-infarct zone (5.4 × 107 plaque-forming units/heart, 5 injections/heart). Cardiac function assessed by echocardiography showed improved LV physiology and reduced LV dilation after TIMP3 overexpression compared with the Ad-Null-MI group. Post-MI adverse remodeling was attenuated in the Ad-hTIMP3-MI group, as assessed by greater cardiomyocyte density, less infarct expansion, and ECM disruption. TIMP3 overexpression blunted the early rise in proteolytic activities post-MI. A higher density of coronary arteries and a greater number of proliferating endothelial cells were detected in the infarct and peri-infarct regions in the Ad-hTIMP3-MI group compared with the Ad-Null-MI group. In vitro three-dimensional angiogenesis assay confirmed that recombinant TIMP3 promotes angiogenesis in human endothelial cells, although biphasically and in a dose-dependent manner. Intriguingly, overexpression of Ad-hTIMP3 at 10-fold higher concentration had no beneficial effects, consistent with antiangiogenic effects of TIMP3 at higher doses. In conclusion, optimal overexpression of TIMP3 can be a promising therapeutic approach to limit adverse post-MI remodeling by dually inhibiting early proteolysis and promoting angiogenesis.NEW & NOTEWORTHY Here, we report that tissue inhibitor of metalloproteinase 3 overexpression after myocardial infarction improves myocardial structural remodeling and function by promoting angiogenesis and inhibiting early proteolysis. This demonstrates the therapeutic potential of preserving the local balance of tissue inhibitor of metalloproteinase 3 in the heart given its diverse functions in modulating different processes involved in the adverse postmyocardial infarction remodeling.

Proximal inhibition of p38 MAPK stress signaling prevents distal axonopathy.

The p38 mitogen-activated protein kinase (MAPK) isoforms are phosphorylated by a variety of stress stimuli in neurodegenerative disease and act as upstream activators of myriad pathogenic processes. Thus, p38 MAPK inhibitors are of growing interest as possible therapeutic interventions. Axonal dysfunction is an early component of most neurodegenerative disorders, including the most prevalent optic neuropathy, glaucoma. Sensitivity to intraocular pressure at an early stage disrupts anterograde transport along retinal ganglion cell (RGC) axons to projection targets in the brain with subsequent degeneration of the axons themselves; RGC body loss is much later. Here we show that elevated ocular pressure in rats increases p38 MAPK activation in retina, especially in RGC bodies. Topical eye-drop application of a potent and selective inhibitor of the p38 MAPK catalytic domain (Ro3206145) prevented both the degradation of anterograde transport to the brain and degeneration of axons in the optic nerve. Ro3206145 reduced in the retina phosphorylation of tau and heat-shock protein 27, both down-stream targets of p38 MAPK activation implicated in glaucoma, as well as expression of two inflammatory responses. We also observed increased p38 MAPK activation in mouse models. Thus, inhibition of p38 MAPK signaling in the retina may represent a therapeutic target for preventing early pathogenesis in optic neuropathies.

Effect of dehydroepiandrosterone on the balance of ADAMTS/tissue inhibitor of metalloproteinases-3 system in rabbit osteoarthritis models / 中华风湿病学杂志

Objective To study the effect of intra-articular injection of dehydroepiandrosterone (DHEA) on the balance of ADAMTS/TIMP-3 system in a rabbit osteoarthritis models.Methods Sixty rabbits were underwent bilateral anterior cruciate ligament transection (ACLT).Rabbits were randomizedn to the following treatment:one knee of each rabbit was treated with 100 μmol/L DHEA dissolved in dimethylsulphoxide (the experimental group) and the other knee was treated under the same schedule using dimethylsulphoxide (the control group) 4 weeks after transection,once a week for eight weeks.Twelve weeks after ACLT,all rabbits were killed after X-ray assessment and the knee joints were evaluated by gross morphology and histology.The concentration of hydroxyproline and glycosaminoglycan in the cartilage were analyzed.The mRNA expression of ADAMTS-4,ADAMTS-5,tissue inhibitor of metalloproteinases-3 (TIMP-3),transforming growth factor (TGF)-β1.Aggrecan and Collagen Ⅱ in the cartilage were analyzed using reverse transcription polymerase chain reaction (RT-PCR).The protein expression of aggrecan ARGxx and Collagen Ⅱ in the cartilage were analyzed by Western blot.Results By Mann-Whitney test,Gross morphologic scores on femoral condyle and tibial plateau in the control group were significantly higher than the experi-mental group (Z=-3.517,P＜0.01 ; Z=-2.518,P＜0.05).By unpaired Student's t test,histological evaluation showed that the grade of cartilage damage in the experimental group [(5.3±1.2) μg/ml] were less severe than that in the control group (10.1 ± 1.3,P＜0.01).The concentration of hydroxyproline [(5.7±0.3,23.6± 1.7) μg/ml] and glycosaminoglycan (30±4) in the experimental group increased significantly when compared with the control group [(4.6±0.5),(18.5±1.4),(24±4) μg/ml,P＜0.01].The mRNA expression of ADAMTS-4 (0.15±0.03)and ADAMTS-5 (0.10±0.04) in the experimental group decreased significantly compared with the control group (0.29±0.08,0.15±0.05; all P＜0.05).The mRNA expression of TIMP-3 (0.85±0.10),TGF-β1(1.2±0.4),Aggrecan (0.87±0.31) and Collagen Ⅱ (2.74±0.59) in the experimental group increased significantly when compared with the control group (0.70±0.13,0.8±0.4,0.49±0.16,2.2±0.5; all P＜0.05).The protein expression of Aggrecan ARGxx (0.53±0.10) in the experimental group decreased significantly when compared with the control group (0.81±0.12,P＜0.01).The protein expression of Collagen Ⅱ (2.3±0.7) in the experimental group increased significantly when compared with the control group (1.7±0.5,P＜0.05).Conclusion DHEA protects against cartilage degradation and inhibits the progression of OA,TGF-β1,Aggrecan and Collagen Ⅱ in cartilage may be the mechanism of the protective effect of DHEA on OA.

Recombinant adenovirus carrying tissue inhibitor of metalloproteinase-3 gene regulates the matrix of rabbit intervertebral disc in vivo / 中国矫形外科杂志

To investigate the influence of recombinant adenovirus carrying tissue inhibitor of metalloproteinase-3 (RAdTIMP-3) on the main compositions of rabbits intervertebral discs and to assess its potential in treatment for intervertebral disc degeneration.[Method]RadTIMP-3 and empty adenovims vector with Lac-Z gene (Rad66) was propagated in 293 Cells and was purified, identified and tittered. Thirty Japanese white rabbits were randomly divided into 5 groups. And 25 μl of various reagents were injected to the L4、5 and L5、6 intervertebral discs of the rabbits as follows:normal saline in group 1, 1.0×1010 OPU/ml of RAd66 in Group 2, and 1.0×1010 OPU/ml of RAdTIMP-3 in group 3, 4 and 5. The intervertebral discs of each group were collected after 2, 2, 1, 2 and 4 weeks after injection respectively.Then X-gal staining, And Group 1, RT-PCR for TIMP-3 and aggrecan core protein,TUNEL staining, immunohistochemical staining for TIMP-3 and type I! Collagen and Safranin O-Fast green staining was carried out to assess the effects of RadTIMP-3 transfection.[Result](1)concentration of RAdTIMP-3 reached 1.9×1012 OPU/ml after propagation and purification. (2)RT-PCR shows that the expression of TIMP-3 was significantly raised in group 3, 4, 5, as compared with group 1 or 2. And the expression of core protein gene in group 3, 4, 5 increased slightly than in group 1 and 2. (3) TUNEL staining revealed that there was not significant difference between the positive-staining rates of any two of the groups. (4)TIMP-3 staining exhibited an obvious increase of positive-staining rates in group 3, 4 and 5 as compared with groupi or 2. The staining density of Safranin O-Fast Green staining and immunohistochemical staining for type II collagen of group 5 was obviously higher than that of group 1 or 2.[Conclusion]RAdTIMP-3 can express widely and safely in rabbit intervertebral discs, and improve the quantity and quality of matrix. It has the potential to be used in treatment for intervertabral disc degeneration.

Expression of matrix metalloproteinase-2,9 and it′s tissue inhibitor-1,2 in endometrial carcinoma / 中华妇产科杂志

Objective To study the expression of matrix metalloproteinase(MMP)-2,9 and tissue inhibitor of metalloproteinase(TIMP)-1,2 protein in human endometrial carcinoma tissue and its relation to the invasion and metastasis of endometrial carcinoma. Methods Immunocytochemistry and zymography techniques were used to measure the MMP-2,MMP-9,TIMP-1,TIMP-2 protein levels and activities in endometrial carcinoma tissue of 37 patients and control group composed of 7 normal postmenstrual endometrial samples. Results The MMP-2,MMP-9,TIMP-1 and TIMP-2 proteins mainly expressed in endometrial carcinoma cells, glandular cells and endothelial cells. The strongly positive expression proportions of MMP-2,9 and TIMP-1 proteins in grade Ⅲ carcinoma cells were respectively 73%, 20% and 67%, which were higher than those in gradeⅡ (13%, 0, 27%) and gradeⅠ (0) ones ( P