Tissue Inhibitor Of Metalloproteinase-1 Gene Expression Research Articles

Extracellular matrix collagen biomarkers levels in patients with chronic thromboembolic pulmonary hypertension.

Limited data exist on changes in the extracellular matrix (ECM) collagen biomarkers levels during chronic thromboembolic pulmonary hypertension (CTEPH) development. This study aimed to investigate ECM collagen biomarkers levels in stable patients with CTEPH. Patients with CTEPH and healthy persons were enrolled. Serum levels of procollagen III N-terminal peptide (PIIINP), carboxyterminal propeptide of type I procollagen (PICP), matrix metalloproteinases (MMP2), MMP9, and tissue inhibitor of metalloproteinases 1(TIMP1) were measured by ELISA. Clinical data coincident with samples were collected. The pulmonary endarterectomy (PEA) and control pulmonary artery tissue samples were analyzed for genetic and immunohistochemical differences. The serum concentrations of PIIINP, PICP, MMP2, and MMP9 decreased significantly in CTEPH patients compared to healthy controls (P < 0.001 for each). CTEPH patients had higher serum concentrations of TIMP1 (median, 111.97 [interquartile range, 84.35-139.93]) compared to healthy controls (74.97 [44.03-108.45] ng/mL, P < 0.001). The MMP2 to TIMP1 ratio was lower in patients than in the controls (P < 0.001). After adjusting for the body mass index (BMI), the MMP2 to TIMP1 ratio correlated negatively with pulmonary vascular resistance (PVR) (r = - 0.327, P = 0.025). Increased TIMP1 (P = 0.04) gene expression was identified in tissues of CTEPH patients. Immunohistochemistry results of vascular walls substantiated qRT-PCR results. This study indicates that ECM collagen biomarkers levels were significantly different in stable patients with CTEPH and healthy controls with significantly increased TIMP1 and decreased MMP2 and MMP9. Differences in TIMP1 expression should be expected not only among healthy controls and patients serum, but also across pathological tissue regions. These findings suggest that the state of vascular remodeling in pulmonary vascular bed in stable patients may be represented by ECM collagen biomarkers levels. We conclude that TIMP1 may play an important role in pulmonary vascular reconstruction in stable CTEPH patients.

4121 The beneficial, anti-fibrotic effects of chemokine receptor 2 and 5 antagonists on fat-exposed mouse primary hepatic stellate cells (pHSCs)

OBJECTIVES/GOALS: Non-alcoholic steatohepatitis (NASH) is a leading cause of cirrhosis in the world for which no anti-fibrotic therapies exist. We hypothesized that BMS-22 and maraviroc (MVC), chemokine receptor 2 (CCR2) and 5 (CCR5) antagonists, respectively, would diminish the fibrogenic activity of "fat-exposed" murine pHSCs. METHODS/STUDY POPULATION: pHSCs were isolated from livers of 6 week old male mice following 4 weeks on a NASH-inducing choline-deficient high fat diet (CDAHFD, “fat-exposed”) or standard diet (SD) and passaged in vitro. Early passage (6-12) pHSCs were plate-adhered and TGF-b-treated (10ng/mL) to maximally activate their pro-fibrogenic genes, collagen 1α1 (Col1A1), tissue inhibitor of metalloproteinase 1 (TIMP1), or α-smooth muscle actin (ACTA2). CDAHFD and SD pHSCs were then treated for 48 hours with increasing doses of BMS-22 or MVC (range: 0.3-120ng/mL) to determine (1) the degree of attenuation of the pro-fibrogenic response as measured by qPCR of fibrogenic genes (Col1A1, TIMP1,ACTA2); (2) enhancement of a fibrolytic response as measured by qPCR of matrix metalloproteinases (MMP) 2, 9 and 13 genes; and (3) pHSC migration using the scratch assay. Cell viability and CCR2 and CCR5 gene expression in response to escalating doses of antagonists were also measured. RESULTS/ANTICIPATED RESULTS: Plate- and TGF-b activated CDAHFD pHSCs had a 2-fold greater, dose-dependent attenuation of their pro-fibrogenic activity in response to BMS-CCR2-22 and MVC, when compared with plate- and TGF-b activated SD pHSCs, as measured by reductions in collagen 1α1 (Col1A1) and α-smooth muscle actin (ACTA2) gene expression. TIMP1 gene expression was unaffected by drug treatment for 48 hours. Cell viability was not affected up to doses of 30ng/mL of each drug. pHSCs also demonstrated a dose-dependent increase in CCR2, CCR5 and MMP-9 gene expression in response to surface receptor antagonism. Migration assays comparing CDAHFD and SD pHSCs in response to escalating doses of MVC and BMS-22 are ongoing and expected to demonstrate a significantly decreased migratory capacity of CDAHFD pHSCs than SD pHSCs in response to therapy, reflecting the increased susceptibility of the “fat-exposed” pHSCs to anti-fibrotic therapy than normal pHSCs. DISCUSSION/SIGNIFICANCE OF IMPACT: Anti-fibrotic drugs that dampen pro-fibrogenic activities of “fat-exposed” pHSCs are urgently needed. CCR2 and CCR5 antagonists, BMS-22 and MVC, respectively, can selectively dampen the pro-fibrogenic response of fat-exposed pHSCs, and must be considered for future trials in human NASH. CONFLICT OF INTEREST DESCRIPTION: Dr. Jill Smith has a patent licensing agreement with Immune Therapeutics, Inc.

Evaluating the Effects of Platelet-Rich Plasma and Amniotic Viscous Fluid on Inflammatory Markers in a Human Coculture Model for Osteoarthritis

To assess the anti-inflammatory effects of platelet-rich plasma (PRP) and amniotic viscous fluid using a human coculture system of cartilage and synovial tissue from osteoarthritic patients. A coculture system was created using cartilage and synovium from 3 patients undergoing total knee arthroplasty. To induce inflammation, interleukin-1β was added to each coculture. Biologic agents tested included 2 PRP concentrations (PRPL and PRPH) and 2 different samples of amniotic viscous fluid (Amnion and Flograft). Amnion was also tested with PRP to check for any additive effects. Quantitative polymerase chain reaction was used to measure gene expression of factors involved in osteoarthritis, including disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS-5), tissue inhibitor of metalloproteinases 1 (TIMP-1), vascular endothelial growth factor (VEGF), aggrecan, type 1 collagen, and nitric oxide, at 0, 24, 48, and 72hours. A synthetic nonsteroidal medication, Ketorolac, was used for baseline comparison to the biologic agents. When comparing from time 0, both Amnion and Flograft resulted in significant decreases of ADAMTS-5 and TIMP-1 gene expression in cartilage and synovium for up to 72hours. Both amniotic preparations increased collagen-1 gene expression in cartilage and decreased VEGF expression in synovium. Amnion was not found to have any effect on nitric oxide concentration at any time point (P > .05), as opposed to both PRP concentrations (P < .05). All biologic agents showed differences in gene expression similar to Ketorolac in ADAMTS-5, TIMP-1, and VEGF expression. This study found that amniotic fluid had anti-inflammatory effects mostly similar to those of both PRPH and PRPL; however, no significant additive effects in reducing inflammatory gene expression were found when combining biologic agents. PRP and amniotic fluid may provide alternative treatment options to delay the progression of the disease without the systemic and intra-articular side effects of corticosteroids.

Metformin improves the angiogenic potential of human CD34⁺ cells co-incident with downregulating CXCL10 and TIMP1 gene expression and increasing VEGFA under hyperglycemia and hypoxia within a therapeutic window for myocardial infarction.

BackgroundCardiovascular disease (CVD) is the leading cause of morbidity and mortality in patients with diabetes mellitus (DM). To identify the most effective treatment for CVD, it is paramount to understand the mechanism behind cardioprotective therapies. Although metformin has been shown to reduce CVD in Type-2 DM clinical trials, the underlying mechanism remains unexplored. CD34+ cell-based therapies offer a new treatment approach to CVD. The aim of this study was to investigate the effect of metformin on the angiogenic properties of CD34+ cells under conditions mimicking acute myocardial infarction in diabetes.MethodsCD34+ cells were cultured in 5.5 or 16.5 mmol/L glucose ± 0.01 mmol/L metformin and then additionally ± 4 % hypoxia. The paracrine function of CD34+ cell-derived conditioned medium was assessed by measuring pro-inflammatory cytokines, vascular endothelial growth factor A (VEGFA), and using an in vitro tube formation assay for angiogenesis. Also, mRNA of CD34+ cells was assayed by microarray and genes of interest were validated by qRT-PCR.ResultsMetformin increased in vitro angiogenesis under hyperglycemia–hypoxia and augmented the expression of VEGFA. It also reduced the angiogenic-inhibitors, chemokine (C–X–C motif) ligand 10 (CXCL10) and tissue inhibitor of metalloproteinase 1 (TIMP1) mRNAs, which were upregulated under hyperglycemia–hypoxia. In addition metformin, increased expression of STEAP family member 4 (STEAP4) under euglycemia, indicating an anti-inflammatory effect.ConclusionsMetformin has a dual effect by simultaneously increasing VEGFA and reducing CXCL10 and TIMP1 in CD34+ cells in a model of the diabetic state combined with hypoxia. Therefore, these angiogenic inhibitors are promising therapeutic targets for CVD in diabetic patients. Moreover, our data are commensurate with a vascular protective effect of metformin and add to the understanding of underlying mechanisms.Electronic supplementary materialThe online version of this article (doi:10.1186/s12933-016-0344-2) contains supplementary material, which is available to authorized users.

Antifibrotic effects of ambrisentan, an endothelin-A receptor antagonist, in a non-alcoholic steatohepatitis mouse model.

To examine the effects of the endothelin type A receptor antagonist ambrisentan on hepatic steatosis and fibrosis in a steatohepatitis mouse model. Fatty liver shionogi (FLS) FLS-ob/ob mice (male, 12 wk old) received ambrisentan (2.5 mg/kg orally per day; n = 8) or water as a control (n = 5) for 4 wk. Factors were compared between the two groups, including steatosis, fibrosis, inflammation, and endothelin-related gene expression in the liver. In the ambrisentan group, hepatic hydroxyproline content was significantly lower than in the control group (18.0 μg/g ± 6.1 μg/g vs 33.9 μg/g ± 13.5 μg/g liver, respectively, P = 0.014). Hepatic fibrosis estimated by Sirius red staining and areas positive for α-smooth muscle actin, indicative of activated hepatic stellate cells, were also significantly lower in the ambrisentan group (0.46% ± 0.18% vs 1.11% ± 0.28%, respectively, P = 0.0003; and 0.12% ± 0.08% vs 0.25% ± 0.11%, respectively, P = 0.047). Moreover, hepatic RNA expression levels of procollagen-1 and tissue inhibitor of metalloproteinase-1 (TIMP-1) were significantly lower by 60% and 45%, respectively, in the ambrisentan group. Inflammation, steatosis, and endothelin-related mRNA expression in the liver were not significantly different between the groups. Ambrisentan attenuated the progression of hepatic fibrosis by inhibiting hepatic stellate cell activation and reducing procollagen-1 and TIMP-1 gene expression. Ambrisentan did not affect inflammation or steatosis.

Adiponectin Reduces Hepatic Stellate Cell Migration by Promoting Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) Secretion

Hepatic stellate cells (HSC) are central players in liver fibrosis that when activated, proliferate, migrate to sites of liver injury, and secrete extracellular matrix. Obesity, a known risk factor for liver fibrosis is associated with reduced levels of adiponectin, a protein that inhibits liver fibrosis in vivo and limits HSC proliferation and migration in vitro. Adiponectin-mediated activation of adenosine monophosphate-activated kinase (AMPK) inhibits HSC proliferation, but the mechanism by which it limits HSC migration to sites of injury is unknown. Here we sought to elucidate how adiponectin regulates HSC motility. Primary rat HSCs were isolated and treated with adiponectin in migration assays. The in vivo actions of adiponectin were examined by treating mice with carbon tetrachloride for 12 weeks and then injecting them with adiponectin. Cell and tissue samples were collected and analyzed for gene expression, signaling, and histology. Serum from patients with liver fibrosis was examined for adiponectin and tissue inhibitor of metalloproteinase-1 (TIMP-1) protein. Adiponectin administration into mice increased TIMP-1 gene and protein expression. In cultured HSCs, adiponectin promoted TIMP-1 expression and through binding of TIMP-1 to the CD63/β1-integrin complex reduced phosphorylation of focal adhesion kinase to limit HSC migration. In mice with liver fibrosis, adiponectin had similar effects and limited focal adhesion kinase phosphorylation. Finally, in patients with advanced fibrosis, there was a positive correlation between serum adiponectin and TIMP-1 levels. In sum, these data show that adiponectin stimulates TIMP-1 secretion by HSCs to retard their migration and contributes to the anti-fibrotic effects of adiponectin.

Impact of losartan and angiotensin II on the expression of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 in rat vascular smooth muscle cells.

The present study aimed to investigate the impact of losartan and angiotensin II (AngII) on the expression of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1), secreted by rat vascular smooth muscle cells (VSMCs). Rat VSMCs were isolated and cultured in different concentrations of AngII and losartan for 24 h and western blot analysis and quantitative polymerase chain reaction were performed to observe the subsequent impact on the gene and protein expression of MMP-9 and TIMP-1. AngII was shown to promote the protein and gene expression of MMP-9 in VSMCs in a concentration-dependent manner. No effect was observed on the expression of TIMP-1, therefore, an increase in the MMP-9/TIMP-1 ratio was observed. Losartan was shown to be able to inhibit MMP-9 protein and gene expression in a concentration-dependent manner, whilst promoting an increase in TIMP-1 expression, thus decreasing the ratio of MMP-9/TIMP-1. The combined action of losartan and AngII resulted in the same directional changes in MMP-9 and TIMP-1 expression as observed for losartan alone. The comparison of AngII, losartan and the combinatory effect on the expression of MMP-9 and TIMP-1 in VSMCs indicated that losartan inhibited the effects of AngII, therefore reducing the MMP-9/TIMP-1 ratio, which may contribute to the molecular mechanism of losartan in preventing atherosclerosis. In atherosclerosis, the development of the extracellular matrix of plaque is closely correlated with the evolution of AS. The balance between MMPs and TIMPs is important in maintaining the dynamic equilibrium between the ECM, and the renin-angiotensin-aldosterone system, which is involved in the pathologenesis of AS, and in which AngII has a central role.

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

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&lt;0.05), collateral size (21.2%, p&lt;0.05), and collateral number (5.7-fold, p&lt;0.05) during MCAO. Prophylactic TCT supplementation significantly induced TIMP1 expression 9.6-fold (p&lt;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).

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...

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

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

Differential gene expression of MMP-1, TIMP-1 and HGF in clinically involved and uninvolved skin in South Africans with SSc

To investigate the differential expression of MMP-1, tissue inhibitor of metalloproteinase-1 (TIMP-1) and hepatocyte growth factor (HGF) in clinically involved (affected) and uninvolved (unaffected) skin in patients with SSc. Punch biopsies from affected forearm and unaffected upper back skin of 16 black South Africans with dcSSc and skin samples of 15 ethnically matched healthy controls were studied. Quantitative mRNA expression of MMP-1, TIMP-1 and HGF was performed by relative reverse transcription quantitative PCR. Compared with controls, TIMP-1 expression was significantly upregulated in patients, a 796- and 397-fold difference for affected and unaffected skin (P < 0.00001 for both), respectively. Conversely, MMP-1 expression was significantly decreased in patients, a 10- and 12.5-fold difference for affected and unaffected skin (P = 0.0004 for both), respectively. HGF expression was up-regulated in both affected and unaffected skin, a 14- and 18-fold difference (P = 0.004 and P = 0.002), respectively. Within the patient group, HGF expression in affected skin of patients correlated significantly with the European scleroderma disease activity score (r = 0.60, P = 0.013). Perturbations in gene expression of TIMP-1, MMP-1 and HGF were evident in both affected and unaffected skin of the dcSSc patients. Targeting TIMP-1, which showed the greatest dysregulation, needs to be explored as a way of reducing collagen deposition and fibrosis in dcSSc.

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.

Effect of angiotensin II receptor blocker on glucose-induced mRNA expressions of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 in rat mesangial cells

The decreased degradation of extra-cellular matrix proteins plays an important role in the onset of diabetic nephropathy. Matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1), which are members of the matrix metalloproteinase family, are associated with this process. Angiotensin II (AII) plays an important role in the development of diabetic nephropathy also. This research aimed to investigate the effect of angiotensin II receptor blocker on glucose-induced mRNA expressions of MMP-9 and TIMP-1 in rat mesangial cells. Rat mesangial cells were cultured and divided into 5 groups: normal glucose (group NG), high glucose (group HG), group NG + AII, NG + AII + saralasin (group NG + AII + S, saralasin is the AII receptor blocker) and HG + saralasin (group HG + S). After the cells were incubated for 24 hours, AII concentrations in the supernatant were measured by radioimmunoassay and the expression of MMP-9 and TIMP-1 mRNA was assayed by reverse transcription-polymerase chain reaction (RT-PCR). AII concentrations were higher in group HG ((56.90 +/- 13.54) pg/ml) and group HG + S ((51.30 +/- 5.96) pg/ml) than in group NG ((37.89 +/- 8.62) pg/ml, P < 0.05), whereas there was no significant difference between group HG and group HG + S. The expression of MMP-9 mRNA and MMP-9/TIMP-1 mRNA ratio in group NG + AII (MMP-9, 0.33 +/- 0.04; MMP-9/TIMP-1, 0.40 +/- 0.06) and group HG (MMP-9, 0.36 +/- 0.02; MMP-9/TIMP-1, 0.45 +/- 0.03) were decreased more significantly than those in group NG (MMP-9, 0.72 +/- 0.02; MMP-9/TIMP-1, 1.21 +/- 0.07). These values in group NG + AII + S (MMP-9, 0.71 +/- 0.02; MMP-9/TIMP-1, 1.18 +/- 0.05) were higher than those in group NG + AII, and the values in group HG + S (MMP-9, 0.71 +/- 0.02; MMP-9/TIMP-1, 1.16 +/- 0.05) were higher than those in group HG (all were P < 0.05). TIMP-1 mRNA expression was increased more significantly in group NG + AII (0.81 +/- 0.03) and group HG (0.80 +/- 0.03) than in group NG (0.59 +/- 0.02), but it was lower in group NG + AII + S (0.60 +/- 0.01) than in group NG + AII and also lower in group HG + S (0.61 +/- 0.01) than in group HG (all were P < 0.05). High glucose stimulates AII production. Both high glucose and AII induce a decrease in MMP-9 mRNA expression and MMP-9/TIMP-1 mRNA ratio as well as an increase in TIMP-1 mRNA expression, which can be reversed by saralasin, suggesting that high glucose can aggravate impaired matrix degradation by altering gene expression of MMP-9 and TIMP-1 and that the effect of high glucose may be mediated by AII.

Effect of dehydroepiandrosterone on cartilage and synovium of knee joints with osteoarthritis in rabbits

The aim of this study was to investigate the effects of intra-articular injection of dehydroepiandrosterone (DHEA) on cartilage and synovium of knee joints with osteoarthritis (OA) in rabbits and the underlying mechanism. Forty rabbits underwent unilateral anterior cruciate ligament transaction and were divided into two groups. Rabbits were injected with 100 mumol/l DHEA dissolved in the dimethylsulphoxide (DMSO) in the knee joints 5 weeks after transaction, once a week for 5 weeks. Rabbits injected with DMSO under the same condition were served as a control. All rabbits were killed 1 week after the last injection. The knee joints were evaluated by gross morphology, histology, and gene expression analysis. Gross morphologic inspection and histological evaluation showed that the DHEA group appeared less damage in cartilage and synovium as compared with the control. Gene expression analysis revealed that the mRNA expression of matrix metalloproteinase-3 (MMP-3) in cartilage and synovium decreased significantly in the DHEA group and that of tissue inhibitor of metalloproteinase-1 (TIMP-1) increased. No significant difference of interleukin-1 beta (IL-1beta) mRNA expression was found in the cartilage between two groups while the mRNA expression of IL-1beta in the synovium was largely suppressed in the DHEA group. The study suggests that DHEA plays a protective role against cartilage degradation and synovium inflammation in rabbits with OA. This role may be achieved through the regulation of the MMP-3, TIMP-1, and IL-1beta gene expression in the cartilage and synovium.

Effects of Runing Recipe medicated serum on expressions of genes in breast cancer cells

To explore the mechanisms of the effects of Runing Recipe in anti-invasion and anti-recurrence of breast cancer by experimental research in vitro. SD female rats were randomly divided into Runing Recipe-treated group and its decomposed formulas Kidney-Warming Recipe and Liver-Soothing Recipe-treated groups, tamoxifen (TAM) -treated group, cyclophosphamide (CTX) -treated group, and normal control group to make medicated serums. Methods of matrigel basement membrane and real-time reverse transcription polymerase chain reaction were employed to investigate the gene expressions of vascular endothelial growth factor (VEGF), matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) after MDA-MB-435 cells were treated with the medicated serums. The gene expression of VEGF was dropped in CTX-treated, TAM-treated and Liver-Smoothing Recipe-treated groups. The gene expression of TIMP-1 was up-regulated in CTX-treated, Runing Recipe-treated and Kidney-Warming Recipe-treated groups; while MMP-9 was down-regulated in these groups. The mechanisms of Runing Recipe in inhibiting the cancer cell invasion may be related to down-regulating the gene expressions of VEGF and MMP-9, and up-regulating the gene expression of TIMP-1.

Sphingosine Kinase 1 (SPHK1) Is Induced by Transforming Growth Factor-β and Mediates TIMP-1 Up-regulation

Transforming growth factor-beta (TGF-beta) signaling plays a pivotal role in extracellular matrix deposition by stimulating collagen production and other extracellular matrix proteins and by inhibiting matrix degradation. The present study was undertaken to define the role of sphingosine kinase (SphK) in TGF-beta signaling. TGF-beta markedly up-regulated SphK1 mRNA and protein amounts and caused a prolonged increase in SphK activity in dermal fibroblasts. Concomitantly, TGF-beta reduced sphingosine-1-phosphate phosphatase activity. Consistent with the changes in enzyme activity, corresponding changes in sphingolipid levels were observed such that sphingosine 1-phosphate (S1P) was increased (approximately 2-fold), whereas sphingosine and ceramide were reduced after 24 h of TGF-beta treatment. Given the relatively early induction of SphK gene expression in response to TGF-beta, we examined whether SphK1 may be involved in the regulation of TGF-beta-inducible genes that exhibit compatible kinetics, e.g. tissue inhibitor of metalloproteinase-1 (TIMP-1). We demonstrate that decreasing SphK1 expression by small interfering RNA (siRNA) blocked TGF-beta-mediated up-regulation of TIMP-1 protein suggesting that up-regulation of SphK1 contributes to the induction of TIMP-1 in response to TGF-beta. The role of SphK1 as a positive regulator of TIMP-1 gene expression was further corroborated by using ectopically expressed SphK1 in the absence of TGF-beta. Adenovirally expressed SphK1 led to a 2-fold increase of endogenous S1P and to increased TIMP-1 mRNA and protein production. In addition, ectopic SphK1 and TGF-beta cooperated in TIMP-1 up-regulation. Mechanistically, experiments utilizing TIMP-1 promoter constructs demonstrated that the action of SphK1 on the TIMP-1 promoter is through the AP1-response element, consistent with the SphK1-mediated up-regulation of phospho-c-Jun levels, a key component of AP1. Together, these experiments demonstrate that SphK/S1P are important components of the TGF-beta signaling pathway involved in up-regulation of the TIMP-1 gene.

MMP-1 and TIMP-1 production in MG-63 cells stimulated withPrevotella nigrescenslipopolysaccharide

The purpose of this study is to monitor the secretion of matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1) produced by human osteosarcoma cell line (MG63) stimulated with Prevotella nigrescens lipopolysaccharides (LPS). and to compare the level of secretion before and after the treatment of calcium hydroxide on P. nigrescens LPS. LPS was extracted and purified from anaerobically cultured P. nigrescens. MG63 cells were stimulated by the LPS (0, 1, ) or LPS() pretreated with 12.5 mg/ml of for 3 days. Total RNA was isolated from the cell. and real-time quantitative polymerase chain reaction (PCR) was performed for quantification of MMP-1 and TIMP-1. The results were as follows. 1. MMP-1 mRNA expression at 48 hr was highly increased by stimulation with P. nigrescens LPS. The increase was dose-dependent. 2. When stimulated with ( of LPS. TIMP-1 mRNA expression was highly increased at 24 hr and 48 hr. However. TIMP-1 expression was suppressed at higher concentration (). 3. When P. nigrescens LPS was pretreated with . MMP-1 and TIMP-1 gene expression was downregulated. The results of this study suggest that transcriptional regulation of MMP-1 and TIMP-1 by P. nigrescens LPS could be one of the important mechanisms in bone resorption of periapical inflammation. The result of calcium hydroxide on MMP-1 and TIMP-1 gene expression suppression shows that calcium hydroxide detoxified bacterial LPS and thus should be used the medication of choice for intracanal dressings in root canal infected with black-pigmented bacteria.

Transgene-derived hepatocyte growth factor attenuates reactive renal fibrosis in aristolochic acid nephrotoxicity.

Hepatocyte growth factor (HGF) has been demonstrated to attenuate acute tubular necrosis and interstitial fibrosis in a variety of rodent models of kidney disease. We investigated how HGF could affect chronic toxic nephropathy/interstitial fibrosis caused by aristolochic acid (AA). Wild-type and HGF transgenic mice received daily intraperitoneal injections of AA for 14 days. At sacrifice, kidneys were removed and used for microscopy examination and in vitro studies. To determine the molecular mechanisms of anti-fibrotic effects of HGF, cultured murine tubular epithelial cells (mProx24) were employed. Significant tubular degeneration was observed in both the transgenic and the wild-type mice to the same degree after 2 weeks treatment with AA. Interstitial fibrosis subsequently developed in the wild-type mice 4 weeks after cessation of AA administration. However, the transgenic mice manifested less fibrotic changes. Decreased expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) and increased matrix metalloproteinase-9 activity could partially account for the attenuation of fibrogenesis in the transgenic mouse kidney. HGF at 10 and 100 ng/ml could block TIMP-1 gene expression in mProx24 induced by epidermal growth factor, but a decrease in the number of mProx24 via apoptosis induced by AA was blocked only by HGF at 100 ng/ml. Circulating transgene-derived HGF (2-10 ng/ml) could not prevent tubular degeneration caused by AA, but attenuated interstitial fibrogenesis during tubular regeneration. These findings suggest a possible therapeutic efficacy for renal interstitial fibrosis following tubular degeneration even of low-dose HGF.