Tissue Inhibitor Of Metalloproteinase-1 mRNA Research Articles

Contrary Regulation of TIMP-1 and MMP-9 by Hepatocyte Growth Factor Antibody after Lung Injury

To study the influence of hepatocyte growth factor (HGF) antibody on the lung expression level of matrix metalloproteinases-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1). Thirty male Wistar rats were randomly divided into 3 groups: control group, model group, and intervention group. Endotoxin was intratracheally infused in the model and intervention groups. HGF antibody was injected in the rats of the intervention group from day 1 to day 14, while the same volume of saline was injected in the control group. The rats were sacrificed on day 28 after endotoxin treatment. The amounts of MMP-9 mRNA and TIMP-1 mRNA were measured by reverse transcription-polymerase chain reaction, and protein expression levels of MMP-9 and TIMP-1 were measured by immunohistochemistry. In the model group, both mRNA and protein expression levels of TIMP-1 were significantly increased, the same as MMP-9. In the intervention group, the increase of TIMP-1 was remarkably reduced compared with the model group, while the mRNA and protein expression levels of MMP-9 were still increased. HGF activity may accelerate the repair of lung injury through contrary regulating the expression levels of TIMP-1 and MMP-9.

Salivary tissue inhibitor of metalloproteinases-1 localization and glycosylation profile analysis

Salivary tissue inhibitor of metalloproteinases-1 (TIMP-1) has been associated with pathological conditions in the oral cavity, but the origin of TIMP-1 in saliva remains unknown. Hence, we studied the localization of TIMP-1 in salivary gland tissue and also investigated if TIMP-1 found in blood and saliva is identical. Human salivary gland tissue samples (four parotid gland and four submandibular gland biopsies) were analysed for the presence of TIMP-1 mRNA and protein expression. To assess TIMP-1 glycosylation profiles in blood and saliva, the protein was isolated from plasma and unstimulated and stimulated whole saliva as well as stimulated parotid and submandibular saliva and analysed by MALDI-TOF mass spectrometry. TIMP-1 protein was demonstrated in mucous acinar cells of the submandibular gland and in ductal cells of both the parotid and submandibular gland. However, no TIMP-1 mRNA was detected in any of these cells. The glycosylation profiles of TIMP-1 isolated from whole saliva and saliva from the major glands were highly similar. In contrast, a significant difference was found between the glycoprofiles of salivary TIMP-1 and plasma TIMP-1. Although no clear evidence of TIMP-1 transcription in major salivary glands was demonstrated our results suggest that TIMP-1 in saliva does not originate from plasma.

Effects of butyrate on matrix metalloproteinase-3 expression in intestinal epithelial cell line No.6

Objective To investigate the effects of butyrate on the expression of matrix metalloproteinase-3 (MMP-3) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in intestinal epithelial cell line No. 6 (IEC-6) cells. Methods IEC-6 cells of logarithmic growth phase were divided into three groups according to different concentrations of sodium butyrate being used. Low-concentration group, the final concentration of sodium butyrate in medium was 2 mmol/L; high-concentration group, the final concentration of sodium butyrate in medium was 14 mmol/L and control group with no sodium butyrate in medium. After cultured for 24 hours, expression of MMP-3 and TIMP-1 mRNA in IEC-6 cells was measured by reverse transcription-polymerase chain reaction. After cultured for 48 hours, Western blot was employed to examine the expression of MMP-3 and TIMP-1 protein in IEC-6 cells. The differences between groups were compared by ANOVA. Results Expression level of MMP-3 mRNA in group treated with butyrate in concentration of 14 mmol/L was 0. 566±0. 140,inactive form protein of MMP-3 in the group was 0. 756±0. 128, and expression level of active form protein was 0. 255±0. 057, which were higher than those of control group (0. 166±0. 041, 0. 312± 0. 057, 0. 042 ± 0. 038) (P＜0.01, respectively). While, there were no significant differences of those indexes between the control group and low-concentration group. In the high and low-concentration and control group, the levels of TIMP-1 mRNA were 0. 427±0. 042, 0. 383±0. 043 and 0. 355±0. 048,the expression of TIMP-1 protein were 0. 576±0. 140, 0. 524±0. 123 and 0. 355±0. 062, there were no singnificant difference among the three groups (F= 1.97 and 3.10, P＞ 0.05). Conclusions High concentration sodium butyrate could induce the expression of MMP-3 in IEC-6 cells, while not at low concentration. Key words: Enterocolitis, necrotizing; Cell line; Matrix metalloproteinase 3; Butyrates

Effects of a New PPARgamma Agonist on Tongue Carcinoma Cells

Objective: To investigate the effects of GW1929, a new specific PPARgamma agonist, on head and neck squamous carcinoma cell proliferation, apoptosis, and invasion. Method: Human well-differentiated tongue SCC15 cells were treated with 5, 10, 20 micromolar GW1929, and 1% DMSO in 2% FBS Media (control). Tetrazolilum salt WST-1 assay was used to assess proliferation. Cell cycle was analyzed with FACS. Caspase3 was measured with Caspase-Glo3/7 luminescent. RT-PCR measured metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 mRNA to assess anti-invasive effects. Results: GW1929 inhibited tumor cell proliferation. At 24, 48, and 72 hours, treatment with 5, 10, and 20 micromolar GW1929 inhibited SCC15 proliferation by 12% to 23%, 17% to 31%, 27% to 45% in a dose-dependent manner. FACS analysis showed that GW1929 prevented cells from entering S-phase of cell cycle. Confirming significant cellular apoptosis, levels of caspase 3 were 154%, 163%, 185% of control after a 24-hour treatment with 5, 10, 20 micromolar GW1929. GW1929 decreased expression of markers of extracellular matrix invasion in a dose-dependent manner. At 24 hours, levels of MMP-9 mRNA were decreased up to 2 fold, and levels of TIMP-1 mRNA were increased up to 2 fold. Conclusion: GW1929 is a potential antiproliferative agent, as evidenced by its pro-apoptotic, anti-proliferative, and anti-invasive effect on squamous carcinoma of the tongue.

Serum TIMP-1 Predicts Survival Outcomes of Invasive Breast Carcinoma Patients: A Meta-analysis

Tissue inhibitor of metalloproteinase-1 (TIMP-1) is a small secretory glycoprotein with multifunctional activity including anti-apoptosis and the inhibition of matrix metalloproteinase in invasive breast carcinomas. There have been contradictory results as to whether TIMP-1 is a poor or good prognostic factor in breast cancer patients. To address this controversy, we conducted a meta-analysis for the relationship between TIMP-1 levels and prognostic parameters in the breast cancer. The relevant published studies were pooled according to the defined selection criteria. The effect sizes of overall survival and prognostic parameters were calculated by a hazard ratio (HR) or an odds ratio (OR). HRs or ORs were combined using a random-effects model. Survival outcomes between high or elevated and low or normal serum TIMP-1 levels were compared by uni- and multivariate analyses involving 886 and 844 breast cancer patients, respectively. Patients with high or elevated serum TIMP-1 levels had unfavorable survival outcomes compared to patients with low or normal serum TIMP-1 levels in the uni- and multivariate analyses (HR, 1.7 and 2.4; p <0.001 and p= 0.033, respectively). However, no survival difference was evident in the data from tissue TIMP-1 levels by enzyme-linked immunosorbent assay and the expression of tissue TIMP-1 mRNA. The high or positive immunohistochemical expression of tissue TIMP-1 protein was not related to adjusted and unadjusted HRs, lymph node metastasis, and clinical stages. This meta-analysis indicates that serum TIMP-1 levels may be useful for predicting survival outcomes of invasive breast cancer patients.

Different expression of tissue inhibitor of metalloproteinase family members in rat dorsal root ganglia and their changes after peripheral nerve injury

Matrix metalloproteinase 9 (MMP9) and MMP2 are important in the development and maintenance of neuropathic pain behavior induced by peripheral nerve injury. The enzymatic activity of MMP9 and MMP2 is balanced specifically by tissue inhibitor of metalloproteinase 1 (TIMP1) and TIMP2, respectively. In present study, we measured the effect of peripheral nerve injury on the expression of TIMP1 and TIMP2 in adult dorsal root ganglia (DRG). A dramatic increase of TIMP1 mRNA and a decrease of TIMP2 in DRG after sciatic nerve transection (SNT) were displayed through a real-time PCR method. Furthermore, data showed by in situ hybridization that TIMP1 mRNA was only localized in DRG satellite cells under normal conditions. TIMP1 mRNA was increased in satellite cells, and induced within sensory neurons after SNT. Analysis of neuronal profiles showed that induced TIMP1 mRNA was mainly contained in small and medium DRG neurons. Further study displayed that induced TIMP1 mRNA was predominantly present in activating transcription factor 3 (ATF3)-positive injured DRG neurons. Comparatively, TIMP2 mRNA was mostly contained within sensory neurons and the overall amount decreased at the late stage after nerve injury. These data showed different change of TIMPs in DRG after peripheral nerve injury.

Tissue inhibitor of metalloproteinase 1 (TIMP-1) deficiency exacerbates carbon tetrachloride-induced liver injury and fibrosis in mice: involvement of hepatocyte STAT3 in TIMP-1 production

BackgroundTissue inhibitor of metalloproteinase 1 (TIMP-1), which is thought to be produced mainly by activated hepatic stellate cells and Kupffer cells in the liver, plays a pivotal role in matrix remodeling during liver injury and repair; while the effect of TIMP-1 on hepatocellular damage remains obscure.ResultsHepatic expression of TIMP-1 mRNA and protein was up-regulated both in acute and chronic liver injury induced by carbon tetrachloride (CCl4). Compared with wild-type mice, TIMP-1 knockout mice were more susceptible to CCl4-induced acute and chronic liver injury, as shown by higher levels of serum alanine aminotransferase (ALT), greater number of apoptotic hepatocytes, and more extended necroinflammatory foci. TIMP-1 knockout mice also displayed greater degree of liver fibrosis after chronic CCl4 injection when compared with wild-type mice. In vitro treatment with TIMP-1 inhibited cycloheximide-induced cell death of primary mouse hepatocytes. Finally, up-regulation of TIMP-1 in the liver and serum after chronic CCl4 treatment was markedly diminished in hepatocyte-specific signal transducer and activator of transcription 3 (STAT3) knockout mice. In vitro treatment with interleukin-6 stimulated TIMP-1 production in primary mouse hepatocytes, but to a lesser extent in STAT3-deficient hepatocytes.ConclusionsTIMP-1 plays an important role in protecting against acute and chronic liver injury and subsequently inhibiting liver fibrosis induced by CCl4. In addition to activated stellate cells and Kupffer cells, hepatocytes are also responsible for TIMP-1 production during liver injury via a STAT3-dependent manner.

Sexually Dimorphic Diet-Induced Insulin Resistance in Obese Tissue Inhibitor of Metalloproteinase-2 (TIMP-2)-Deficient Mice

Circulating levels of matrix metalloproteinases (MMPs) and their endogenous inhibitors, tissue inhibitor of metalloproteinases (TIMPs), are altered in human obesity and may contribute to its pathology. TIMP-2 exerts MMP-dependent (MMP inhibition and pro-MMP-2 activation) and MMP-independent functions. To assess the role of TIMP-2 in a murine model of nutritionally induced obesity, weight gain in wild-type and TIMP-2 deficient [knockout (KO)] mice fed a chow or high-fat diet (HFD) was determined. The effects of diet on glucose tolerance and insulin sensitivity, as well as pancreatic β-cell and adipocyte physiology, were assessed. Chow-fed TIMP-2 KO mice of both sexes became obese but maintained relatively normal glucose tolerance and insulin sensitivity. Obesity was exacerbated on the HFD. However, HFD-fed male, but not female, TIMP-2 KO mice developed insulin resistance with reduced glucose transporter 2 and pancreatic and duodenal homeobox 1 levels, despite increased β-cell mass and hyperplasia. Thus, although β-cell mass was increased, HFD-fed male TIMP-2 KO mice develop diabetes likely due to β-cell exhaustion and failure. TIMP-2 mRNA, whose expression was greatest in sc adipose tissue, was down-regulated in HFD-fed wild-type males, but not females. Furthermore, HFD increased membrane type 1-MMP (MMP-14) expression and activity in male, but not female, sc adipose tissue. Strikingly, MMP-14 expression increased to a greater extent in TIMP-2 KO males and was associated with decreased adipocyte collagen. Taken together, these findings demonstrate a role for TIMP-2 in maintaining extracellular matrix integrity necessary for normal β-cell and adipocyte physiology and that loss of extracellular matrix integrity may underlie diabetic and obesogenic phenotypes.

Overexpression of TACE and TIMP3 mRNA in head and neck cancer: association with tumour development and progression

Background:TACE/ADAM17 is a transmembranous protease that cleaves membrane-bound growth factors like EGFR ligands. TACE-dependent proteolysis is regulated by its inhibitor, tissue inhibitor of metalloproteinases 3 (TIMP3). This study analyses the role of TACE and TIMP3 mRNA expression in squamous cell carcinomas of the head and neck (HNSCCs).Methods:We analysed TACE and TIMP3 mRNA expression in HNSCCs from 106 patients by RNA in situ hybridisation.Results:TACE mRNA was upregulated in HNSCCs compared with dysplastic (P<0.05) and normal epithelia (P<0.001), with strong hybridisation signals in 21.9% of invasive tumour tissues and 4.5% of dysplasia. Elevated mRNA levels were accompanied by increased amounts of TACE protein in HNSCCs. TIMP3 mRNA expression in HNSCC-associated stroma was significantly higher than in the stroma adjacent to dysplastic or normal epithelia. Expression of TACE mRNA in HNSCCs was associated with tumour stage (P=0.019) and regional lymph node metastasis (P=0.009). Furthermore, levels of TACE mRNA in HNSCCs correlated with the expression of TIMP3 mRNA in HNSCC-associated stroma. Concomitantly, patients expressing high levels of TACE and TIMP3 mRNA showed significantly reduced overall survival compared with those with low mRNA levels.Conclusion:Our results indicate an important role of TACE and TIMP3 during development and progression of HNSCCs.

Effects of Chinese herbal medicine Shenlong Decoction on mRNA expressions of matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-1 in lung tissue of rats with pulmonary fibrosis induced by bleomycin

To observe the expressions of matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in rats with pulmonary fibrosis (PF) induced by bleomycin, and to explore the mechanisms of Shenlong Decoction in preventing and treating PF. A total of 230 Wistar rats were divided into normal control group, untreated group, prednisone group, and low-, medium- and high-dose Shenlong Decoction groups. Wistar rats were intratracheally injected with bleomycin to induce PF. From the 2nd day, rats in the normal control and untreated groups were lavaged with normal saline (NS), and rats in the other groups were lavaged with prepared Shenlong Decoction by the same amount. Hematoxylin-eosin (HE) staining and Masson staining were used to observe pathological changes in lung tissue at different time points, and to evaluate whether the model was successfully induced. Expressions of MMP-2 and TIMP-1 mRNAs in rats' lung tissue were measured by reverse transcription-polymerase chain reaction (RT-PCR). Expressions of MMP-2 and TIMP-1 mRNAs in lung tissue of rats were observed from all groups at each time point. In comparison with the normal control group, on the 7th day, the transcription levels of MMP-2 and TIMP-1 mRNAs, especially the former, of the untreated group increased significantly (P<0.05 or P<0.01). On the 14th day, the transcription levels of MMP-2 and TIMP-1 mRNAs kept rising, especially the latter (P<0.05 or P<0.01). On the 28th day, the transcription level of MMP-2 decreased a little, while the transcription level of TIMP-1 mRNA did not stop increasing (P<0.05 or P<0.01). Compared with the untreated group, decrease of the transcription levels of MMP-2 and TIMP-1 mRNAs were observed in the treatment groups, especially the former, and this effect continued to the 28th day with the medium-dose Shenlong Decoction group decreasing most obviously (P<0.05 or P<0.01). Shenlong Decoction may inhibit the expression of MMP-2 mRNA by up-regulating the expression of TIMP-1 mRNA so as to slow the progression of PF.

Fosinopril and valsartan intervention in gene expression of Klotho, MMP-9, TIMP-1, and PAI-1 in the kidney of spontaneously hypertensive rats.

To determine the role of fosinopril and valsartan intervention in Klotho, matrix metalloproteinase-9 (MMP-9), tissue inhibitor of metalloproteinase-1 (TIMP-1) and plasminogen activator inhibitor (PAI-1) gene expression in hypertensive renal interstitial fibrosis (RIF) in the kidney tissue of spontaneously hypertensive rats (SHR). We randomly divided 20 male 22-week-old SHR into 4 groups (5 in each group): a hypertension group (SHR group), a fosinopril group [Fos group, 10 mg/( kg · d) gavage], a valsartan group [Val group, 10 mg/( kg · d) gavage], and a fosinopril plus valsartan group [Fos + Val group, fosinopril 10 mg/( kg · d) + valsartan 50 mg/( kg · d) gavage]. Another five 22-week-old male Wistar Kyoto rats (WKY) were used as controls. Through monitoring the weight of the rats, tail artery pressure, 24-hour urine protein by fosinopril and/or valsartan intervention after the 8-week trial. RT-PCR and Western blot were used to detect the mRNA and protein expression of Klotho, MMP-9, TIMP-1, and PAI-1 in the kidneys. RT-PCR showed that in the SHR group, Klotho mRNA and protein expression were significantly decreased(P<0.01), while mRNA and protein expression of MMP-9, TIMP-1, and PAI-1 were significantly higher compared with the WKY group(P<0.01). With fosinopril and / or valsartan intervention, Klotho mRNA expression in the Fos group (P<0.01), Fos + Val group (P<0.01), Val group (P<0.05), Klotho protein expression in the Fos group(P<0.05), Fos + Val group (P<0.05), Val group (P<0.01), were significantly increased compared with those in the SHR group. The mRNA and protein expression of MMP-9, TIMP-1, and PAI-1 in the Fos group, Val group, and Fos + Val group were significantly lower than those in the SHR group (P<0.01). The expression of Klotho mRNA had negative correlation with the expression of MMP-9 mRNA (r= -0.864, P<0.01), TIMP-1 mRNA (r=-0.725, P<0.01) and PAI-1 mRNA (r=-0.785, P<0.01). The Klotho protein expression had negative correlation with the expression of MMP-9 protein (r=-0.614, P<0.05), TIMP-1 protein (r=-0.579, P<0.05), and PAI-1 protein (r=-0.552, P<0.05). Anti-aging gene Klotho and the genes related with extracellular matrix degradation gene MMP-9, TIMP-1, PAI-1 are involved in hypertensive renal injury. The expression of Klotho and MMP-9, TIMP-1, and PAI-1 is closely correlated. Fosinopril and valsartan which increase the Klotho mRNA and protein expression can alter the expression of Klotho-MMPs/TIMPs, which may be the main mechanism to prevent interstitial fibrosis.

Thrombopoietin Protects the Brain and Improves Sensorimotor Functions: Reduction of Stroke-Induced MMP-9 Upregulation and Blood—Brain Barrier Injury

This study was conducted to determine the protective efficacy and mechanisms of thrombopoietin (TPO) intervention in experimental focal stroke. Male rats underwent 2 hours of left middle cerebral artery occlusion (MCAO) followed by 22 hours of reperfusion. Vehicle or TPO (0.03 to 1.00 μg/kg) was administered intravenously immediately after reperfusion. Brain infarct and swelling, neurologic deficits, matrix metalloproteinase-9 (MMP-9), tissue inhibitor of metalloproteinase-1 (TIMP-1), TPO and c-Mpl (TPO receptor) mRNA, MMP-9 enzyme activity and protein expression, and the integrity of the blood-brain barrier (BBB) were subsequently measured. MCAO reperfusion produced a large infarct and swelling after stroke. Thrombopoietin significantly reduced these in a dose-dependent manner. The most effective TPO dose, 0.1 μg/kg, when administrated immediately or 2 hours after reperfusion, significantly reduced infarct and swelling and ameliorated neurologic deficits after stroke. Stroke-induced increases in cortical MMP-9 mRNA, enzyme activity and protein expression, TIMP-1 mRNA, and Evans blue extravasation were reduced by TPO intervention. Thrombopoietin did not alter cortical TPO or c-Mpl mRNA expression, blood pressure, heart rate, blood hematocrit, or platelets. This is the first demonstration of TPO's efficacy in reducing ischemic brain injury and improving functional outcome, partly by inhibiting the stroke-induced increase in MMP-9 and the early, negative effects on the BBB.

Role of tumor necrosis factor–α in wound repair in human vocal fold fibroblasts

Tumor necrosis factor-alpha (TNF-alpha) is an inflammatory cytokine and apoptotic molecule that appears to be a mediator in inflammation and fibrosis. The objective of this investigation was to examine the effects of TNF-alpha on 3D Carbylan-GSX in vitro cultured human vocal fold fibroblasts (hVFFs), to provide insight into the mechanism responsible for the improved vocal fold wound healing that has been previous reported with Carbylan-GSX treatment. In vitro cell culture. hVFF were cultured in 3D Carbylan-GSX and on polystyrene with different dosages of TNF-alpha (0, 0.1, 1, 10, and 100 ng/mL) with and without 10% fetal bovine serum (FBS). hVFF response to TNF-alpha was characterized by morphology, proliferation rates, and gene transcript levels for matrix metalloproteinase 1 (MMP1), matrix metalloproteinase 2 (MMP2), tissue inhibitor of metalloproteinase 3 (TIMP3), collagen I, collagen III, fibronectin, and TNF-alpha receptor. In 3D Carbylan-GSX, TNF-alpha inhibited hVFF proliferation in a dose-dependent manner. TNF-alpha (0.1-100 ng/mL) was shown to significantly downregulate TIMP3 and extracellular matrix-related mRNA transcript levels for collagen III and fibronectin and to upregulate MMP1 and MMP2 expression, resulting in increased MMP/TIMP3 ratios. TNF-alpha receptor expression was significantly upregulated in Carbylan-GSX compared to control polystyrene. Responses were more marked in 10% FBS culture. After vocal fold injury, locally injected Carbylan-GSX can enhance the role of TNF-alpha in remodeling the lamina propria layer of the vocal fold, accelerating wound healing. Carbylan-GSX has potential as a new therapeutic approach that may lead to better treatment of vocal fold wound healing.

Modulation of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 in RAW264.7 cells by irradiation

Radiation-induced pulmonary injury is a severe complication affecting the quality of life of patients. Although the pathophysiology of the process is not fully understood, we hypothesized that it is potentially related to macrophages and their secretion of matrix metalloproteinase-9 (MMP-9). Macrophages are a type of inflammatory cell that synthesize hundreds of bioactive substances and enzymes. MMP-9 is closely involved in the maintenance of the basilar membrane, and leads to increased extracellular matrix deposition within the lung, which is a characteristic feature of radiation-induced lung fibrosis. We examined the role of ionizing radiation in modulating the production of MMP-9 in a macrophage cell line. RAW264.7 cells were irradiated with various doses of γ-rays, and then MMP-9 and tissue inhibitor of metalloproteinase-1 (TIMP-1) levels were determined at several time points. RT-PCR revealed a marked increase in the levels of MMP-9 mRNA, which peaked at 24 h post-irradiation and had begun to decline by 48 h. By contrast, TIMP-1 mRNA experienced only a slight increase at 24h post-irradiation, reaching significance at 48 h post-irradiation. Western blot analysis demonstrated an increased expression of MMP-9 protein in the irradiated cells, while TIMP-1 protein levels were not notably changed. Dexamethasone inhibited the increased expression of MMP-9 protein induced by ionizing radiation. These results indicate that MMP-9 expression by RAW264.7 cells, and an imbalance between MMP-9 and TIMP-1, may be involved in radiation-induced lung injury.

The effects of the selective mineralocorticoid receptor antagonist eplerenone on hepatic fibrosis induced by bile duct ligation in rat

The aim of this study was to examine the effects of eplerenone on hepatic fibrosis induced by bile duct ligation (BDL) in rat. Low- (1.0 mg/kg body weight, BW) and high- (4.0 mg/kg BW) dose eplerenone was administered orally for 21 days immediately following BDL. Fibrosis was assessed by measuring the fibrotic area after Sirius red staining. Immunostaining for alpha-smooth muscle actin (SMA), 4-hydroxy-2-nonenal (4-HNE) and 8-hydroxy-2-deoxyguanosine (8-OHdG) was also carried out. Gene expression levels of procollagen-I, transforming growth factor-beta1 (TGF-beta1), connective tissue growth factor (CTGF), tissue inhibitor of metalloproteinases-1 (TIMP-1) and matrix metalloproteinase-13 (MMP-13) in the liver were examined by real-time reverse transcriptase polymerase chain reaction. Plasma angiotensin II (ATII) concentration was measured via radioimmunoassay. The area of hepatic fibrosis and alpha-SMA positivity in the high-dose group was significantly decreased compared with that in the BDL group, but not in the low-dose group. 8-OHdG-positive cells in the low- and high-dose groups were significantly decreased compared with those in the BDL group. Immunostaining of 4-HNE in the high-dose group was significantly lower compared with that in the BDL group. Furthermore, TIMP-1 mRNA levels in the low- and high-dose groups were lower than that in the BDL group. The expression of TGF-beta1, CTGF, procollagen-1 and MMP-13 showed no differences. Plasma ATII concentration in the high-dose group was significantly decreased. Eplerenone attenuated the development of BDL-induced hepatic fibrosis by reducing oxidative stress, suppressing activated hepatic stellate cells and decreasing plasma ATII levels. Eplerenone may prove useful as an alternative treatment for antifibrosis therapy.

Effects of Liangxue Tongyu Formula on brain edema and expressions of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 in rats with intracerebral hemorrhage

To investigate the effects of Liangxue Tongyu Formula (LXTYF), a compound traditional Chinese herbal medicine, on brain edema in rats with intracerebral hemorrhage and to explore the mechanism. Intracerebral hemorrhage was induced by using the intrastriatal autologous blood injection. Rats were randomized into sham-operated (SO) group, intracerebral hemorrhage (ICH) group and LXTYF group. Rats in the LXTYF group were intragastrically administered with LXTYF every day while the other two groups were given normal saline. Brain water content was determined at 24, 48, 72, and 120 h after intracerebral hemorrhage. Matrix metalloproteinase-9 (MMP-9) level, and MMP-9 and tissue inhibitor of metalloproteinase-1 (TIMP-1) mRNA expressions in perihematoma area were detected by gelatin zymography and fluorescence quantitative real-time polymerase chain reaction at the four time points, respectively. Water content in the ICH group was highly elevated after intracerebral hemorrhage, and reached to the peak at 72 h. Compared with the ICH group, the LXTYF group had lower water contents at 48, 72 and 120 h after intracerebral hemorrhage (P<0.01). The difference in water content between the LXTYF and SO groups was significant only at 72 h (P<0.01). Although the pro-MMP-9 level and MMP-9 activity in the LXTYF and ICH groups were enhanced, they were still lower in the LXTYF group than in the ICH group (P<0.01 for 24, 48, 72 and 120 h, respectively). And there was no significant difference in them between the LXTYF group and the SO group at 120 h. Meanwhile, MMP-9 mRNA expressions were increased in the ICH and LXTYF groups, but the levels in the LXTYF group were significantly lower (P<0.01 for 48, 72 and 120 h, respectively) than those in the ICH group. Also, TIMP-1 mRNA expressions at 24, 48, 72 and 120 h after intracerebral hemorrhage were up-regulated in the LXTYF group, and there were significant differences in TIMP-1 expressions between the LXTYF group and ICH group after intracerebral hemorrhage (P<0.01). Liangxue Tongyu Formula ameliorates brain edema in rats after intracerebral hemorrhage by inhibiting MMP-9 expression and activity and up-regulating TIMP-1.

Hypermethylation and transcriptional downregulation of the TIMP3 gene is associated with allelic loss on 22q12.3 and malignancy in meningiomas.

The gene for the tissue inhibitor of metalloproteinase 3 (TIMP3) on 22q12.3 had been reported to be inactivated by promoter methylation in various types of cancers, with controversial findings in meningiomas. We performed direct sodium bisulfite sequencing in a series of 50 meningiomas, including 27 benign meningiomas [World Health Organization (WHO) grade I], 11 atypical meningiomas (WHO grade II) and 12 anaplastic meningiomas (WHO grade III), and found hypermethylation of TIMP3 in 67% of anaplastic meningiomas, but only 22% of atypical and 17% of benign meningiomas. Moreover, TIMP3 methylation scores were significantly inversely correlated with TIMP3 mRNA expression levels (P = 0.0123), and treatment of the meningioma cell line Ben-Men-1 with demethylating agents induced an increased TIMP3 mRNA expression. TIMP3 is located in the chromosomal band 22q12, the allelic loss of which occurs early in meningioma tumorigenesis and preferentially targets the NF2 tumor suppressor gene. In our tumor panel, all meningiomas with TIMP3 hypermethylation--except for a single case--exhibited allelic losses on 22q12.3. Thus, TIMP3 inactivation by methylation seems fairly exclusive to meningiomas with allelic losses on 22q12 but--in contrast to NF2 mutation--appears to be involved in meningioma progression as it is associated with a more aggressive, high-grade meningioma phenotype.

Effect of Latanoprost on the Expression of Matrix Metalloproteinases and Tissue Inhibitor of Metalloproteinase 1 on the Ocular Surface

To investigate the effect of topical latanoprost on the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinase 1 (TIMP-1) on the ocular surface. Tears were collected from 39 patients with glaucoma who used latanoprost, 0.005%, eyedrops (Xalatan) and 28 healthy volunteers. The MMP-9 concentration was measured. Conjunctival epithelial cells were collected from 10 eyes of 10 patients before and 1 to 3 months after starting to take topical latanoprost, 0.005%, and MMP-1, MMP-9, and TIMP-1 messenger RNA (mRNA) expression was analyzed. Both eyes of 48 mice were treated once a day with latanoprost, 0.005%, timolol gel, 0.5%, eyedrops, vehicle of Xalatan, or phosphate-buffered saline, and MMP-9 and TIMP-1 mRNA expression was analyzed. The median MMP-9 concentration in latanoprost-treated cases was 91.2 ng/mL (in controls, 19.7 ng/mL; P < .001). In latanoprost-treated cases, the relative ratio of MMP-9 to glyceraldehyde 3-phosphate dehydrogenase mRNA was significantly increased from 6.42 to 21.3 (P = .04, paired t test) and the relative amount of TIMP-1 was significantly decreased from 154 to 105 (P = .009). The relative amount of MMP-1 to GAPDH mRNA before and after latanoprost use was not significantly different (P = .16). In mice, MMP-9 expression was increased and TIMP-1 expression was decreased on the ocular surface at 8 weeks after latanoprost use. The topical use of latanoprost increases MMP-1 and MMP-9 and decreases TIMP-1 on the ocular surface. The use of topical latanoprost might not be recommended in patients with keratoconus or after laser-assisted in situ keratomileusis.

Expression of MMP-9, TIMP-1, and NF-κB in papillary thyroid carcinoma and its significance

The expressions of matrix metalloproteinnse-9 (MMP-9), tissue inhibitor of metalloproteinase-1 (TIMP-1) ,and NF-κB in 157 cases of papillary thyroid cancer and 30 cases of papillary thyroid tumors were detected by immunohistochemistry and in situ hybridization. The expressions of MMP-9 and NF-κB protein and mRNA in papillary thyroid cancers were significantly higher than those in benign tumors (all P <0.01), and increased according to the pathological grading (P<0.01). The expressions of MMP-9 and NF-κB protein and mRNA in papillary thyroid cancers with cervical lymph node metastasis were higher than those without metastasis (both P <0.01). The expressions of TIMP-1 protein and mRNA appeared an opposite direction of MMP-9 expression. Key words: Matrix metalloproteinase-9; Tissue inhibitor of metalloproteinnse-1; NF-κB; In situ hybridization; Immunohistochemistry

Effects of cocktail therapy on cell proliferation and Collagen I and TIMP-1 mRNA expression in rat hepatic stellate cells

AIM: To investigate the effects of cocktail therapy on cell proliferation and collagen I (Col I) and tissue inhibitor of metalloproteinase-1 (TIMP-1) mRNA expression in rat hepatic stellate cells. METHODS: Hepatic stellate cells (HSC-T6) cultured in vitro were exposed to taurine (taurine group), epigallocatechin gallate (EGCG; EGCG group), genistein (genistein group), and a combination of these three drugs (cocktail-treated group) for 24 h, respectively. Cell proliferation was determined by methyl thiazol tetrazolium (MTT) assay. The expression of Col Ⅰ and TIMP-1 mRNAs was measured by semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR). RESULTS: Cell proliferation was significantly inhibited in the taurine group, EGCG group, genistein group and cocktail-treated group compared with the control group (0.237 ± 0.007, 0.216 ± 0.009, 0.242 ± 0.008 and 0.130 ± 0.004 vs 0.452 ± 0.011, respectively; all P＜0.05). The reduced rate of cell proliferation was more significant in the cocktail-treated group than in other treatment groups (all P＜0.05). The expression levels of Col I and TIMP-1 mRNAs markedly decreased in all treatment groups when compared with the control group (all P＜0.05). The expression levels of Col Ⅰ and TIMP-1 mRNAs were significantly lower in the cocktail-treated group than in other treatment groups (P＜0.05). CONCLUSION: Cocktail therapy significantly inhibits cell proliferation and down-regulates Col Ⅰ and TIMP-1 mRNA expression in HSCs. Cocktail therapy may be more effective than monotherapy in the treatment of liver fibrosis.