TIMP-1 mRNA Levels Research Articles

Selective Regulation of MMP and TIMP mRNA Levels in Tree Shrew Sclera during Minus Lens Compensation and Recovery

In juvenile tree shrews, a minus-power lens placed in front of the eye produces increased axial elongation and a myopic shift in refractive state that compensates for the power of the lens. Scleral tissue remodeling and modulation of the mechanical properties of the sclera occur during lens compensation. In this study, the time course of changes in scleral mRNA levels of three MMPs and three TIMPs during compensation for a minus lens and during recovery was investigated, to determine which, if any, are temporally associated with changes in the mechanical properties of the sclera and the axial elongation rate. Competitive RT-PCR was used to measure the levels of mRNA for MT1-MMP, MMP-2, MMP-3, TIMP-1, TIMP-2, and TIMP-3 in the scleras of tree shrews that had received either 1, 2, 4, or 11 days of monocular -5-D lens treatment, or 11 days of -5-D lens treatment followed by 2 or 4 days of recovery. Relative to their control eyes, treated eye MT1-MMP and MMP-2 mRNA levels were significantly higher, and TIMP-3 levels were lower by 1 to 4 days of minus lens treatment. These differential effects were absent by 11 days of treatment when the treated eyes had compensated for the lens. The levels of all three TIMPs spiked upward in both eyes after 2 days of recovery. The differential changes in MT1-MMP, MMP-2, and TIMP-3 mRNA levels were all restricted to the treated eye and were temporally associated with the differential changes in axial elongation, refractive state, and the previously measured changes in creep rate. The observed changes in MT1-MMP, MMP-2, TIMP-2, and TIMP-3 mRNA are consistent with visually modulated MT1-MMP activation of MMP-2 and with MT1-MMP degradation of scleral extracellular matrix components. These data constitute further evidence that visual signals modulate gene expression of selected MMPs and TIMPs to control scleral remodeling, the mechanical properties of the sclera, axial elongation, and refractive state.

Enhanced matrix degradation after withdrawal of TGF‐β1 triggers hepatocytes from apoptosis to proliferation and regeneration

TGF-beta1 is a profibrogenic cytokine participating in deposition of extracellular matrix in fibrotic disorders. In liver, its anti-proliferative/apoptotic effect on hepatocytes promotes fibrosis. The tetracycline-controlled double-transgenic TA(LAP-2)/p(tet)TGF-beta1 mouse provides a model for reversible liver fibrosis. In livers of TGF-beta1-expressing mice, hepatocytes showed synchronous apoptosis detected by DNA laddering and active caspase-3 staining that disappeared when expression of transgenic TGF-beta1 was switched off. In these 'off' mice, perisinusoidal liver fibrosis resolved within 21 days accompanied by elevated proliferation of hepatocytes. Here, we have specified the intermediary stages (2-3 days off and 6 days off) in terms of (i) proliferation (by immunohistochemical staining of proliferating cell nuclear antigen and expression of cyclin D1 mRNA) and (ii) extracellular matrix remodelling processes (by measuring mRNA expression of matrix metalloproteinases-2 and -13 (mmp-2 and mmp-13) and tissue inhibitor of matrix metalloproteinases 1 (timp-1) and quantitative morphometric analysis. In summary, we show a rapidly declining timp-1 mRNA level together with lastingly high mmp-2 and mmp-13 mRNA levels after 2-3 days, suggesting that high matrix-degrading potential represents a prerequisite for the markedly enhanced proliferation of hepatocytes in the early stages after switching off transgenic TGF-beta1.

Temporospatial expression of matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases in mouse antigen-induced arthritis

Several lines of evidence speak for an important role of matrix metalloproteinases (MMPs) in the development of progressive joint destruction. To better understand the role of MMPs and their tissue inhibitors (TIMPs) in this process, we have used the antigen-induced arthritis model to study the temporospatial expression of several MMPs and TIMPs during the progression of arthritis. Arthritis was induced by a single intra-articular injection of methylated bovine serum albumin (mBSA) into one or both knee joints of adult mice previously immunised against mBSA. Samples were collected at 3, 7, 21 and 42 days after induction of arthritis for histology and RNA extraction, and analysed by Northern hybridisation, histochemistry and immunohistochemistry for production of several MMPs and TIMPs -1, -2 and -3. A systematic analysis of MMP and TIMP mRNA levels in mouse knee joints demonstrated a general upregulation of both MMPs and TIMPs during progression of arthritis. Upregulation of MMP-9, -13 and -14 coincided with the advancement of cartilage degeneration, but the expression patterns of MMP-9 and -13 also followed the course of synovial inflammation. TIMPs were steadily upregulated throughout the examination period. Immunohistochemical localisation of MMPs and TIMPs suggested the synovium to be the major source of MMP and TIMP production in arthritis, although articular cartilage chondrocytes also showed an increased production of both MMPs and TIMPs.

Radiation and Transforming Growth Factor-β Cooperate in Transcriptional Activation of the Profibrotic Plasminogen Activator Inhibitor-1 Gene

Radiation-induced fibrosis is an important side effect in the treatment of cancer. Profibrotic proteins, such as plasminogen activator inhibitor-1 (PAI-1), transforming growth factor-beta (TGF-beta), and tissue type inhibitor of metalloproteinases-1 (Timp-1), are thought to play major roles in the development of fibrosis via the modulation of extracellular matrix integrity. We did a detailed analysis of transcriptional activation of these profibrotic genes by radiation and TGF-beta. Irradiation of HepG2 cells led to a high increase in PAI-1 mRNA levels and a mild increase in Timp-1 mRNA levels. In contrast, TGF-beta1 and Smad7 were not increased. Radiation and TGF-beta showed strong cooperative effects in transcription of the PAI-1 gene. The TGF-beta1 gene showed a mild cooperative activation, whereas Timp-1 and Smad7 were not cooperatively activated by radiation and TGF-beta. Analysis using the proximal 800 bp of the human PAI-1 promoter revealed a dose-dependent increase of PAI-1 levels between 2 and 32 Gy gamma-rays that was independent of latent TGF-beta activation. Subsequent site-directed mutagenesis of the PAI-1 promoter revealed that mutation of a p53-binding element abolished radiation-induced PAI-1 transcription. In line with this, PAI-1 was not activated in p53-null Hep3B cells, indicating that p53 underlies the radiation-induced PAI-1 activation and the cooperativity with the TGF-beta/Smad pathway. Together, these data show that radiation and TGF-beta activate PAI-1 via partially nonoverlapping signaling cascades that in concert synergize on PAI-1 transcription. This may play a role in patient-to-patient variations in susceptibility toward fibrosis after radiotherapy.

Monocyte matrix and ADAM metalloproteinase expression in type 2 diabetes after aspirin therapy

The matrix metalloproteinase system (MMP and the TIMP inhibitors), and the ADAM metalloproteinases, have roles in maintaining vascular plaque stability and the shedding of cell surface molecules, such as TNF-α and adhesion molecules; aspirin suppresses MMP expression and ADAM activity from some cell lines in vitro. In a randomised prospective controlled study, we examined peripheral venous monocyte MMP-9, TIMP-1 and ADAM mRNA levels, and protein expression, in subjects with type 2 diabetes ( n = 10) and controls ( n = 14) before and after oral aspirin therapy (150 mg daily for 14 days) or no active intervention. Baseline monocyte TIMP-1 mRNA levels were significantly lower in the diabetes group ( p = 0.0014), although monocyte MMP-9 mRNA, and MMP-9 and TIMP-1 protein expression after culture did not differ significantly between groups. Plasma MMP-9 ( p = 0.027) and TIMP-1 ( p = 0.016) concentrations were significantly greater, and the ratio of plasma TIMP-1:MMP-9 concentrations significantly lower, in the diabetes group ( p = 0.023). ADAM mRNA levels did not differ significantly between groups and oral aspirin therapy had no significant effect on any variable. Type 2 diabetes is characterised by reduced monocyte TIMP-1 mRNA levels, and a lower plasma MMP-9 to TIMP-1 protein ratio compared to controls, a pattern that would promote coronary plaque instability if reproduced within vascular plaque. Monocyte ADAM mRNA levels do not differ between group and oral aspirin has no significant effect on these variables.

Expression of Matrix Metalloproteinases and Their Tissue Inhibitor during Viral Encephalitis

Matrix metalloproteinases (MMPs) participate in remodeling the extracellular matrix and facilitate entry of inflammatory cells into tissues. Infection of the murine central nervous system (CNS) with a neurotropic coronavirus induces encephalitis associated with increased levels of mRNA encoding MMP-3 and MMP-12. Whereas virus-induced MMP-3 expression was restricted to CNS resident astrocytes, MMP-12 mRNA was expressed by both inflammatory cells and CNS resident cells. Immunosuppression increased both MMP-3 and MMP-12 mRNA levels in CNS resident cells, suggesting that the presence of virus rather than inflammation induced protease up-regulation. MMP activity is partially regulated by a small family of genes encoding tissue inhibitors of matrix metalloproteinases (TIMPs); among the TIMPs, only TIMP-1 mRNA expression increased in the CNS following coronavirus infection. During inflammation TIMP-1 mRNA was most prominently expressed by infiltrating cells. By contrast, in the immunosuppressed host TIMP-1 mRNA was expressed by CNS resident cells. Analysis of cytokine and chemokine mRNA induction within the infected CNS of healthy and immunocompromised mice suggested a possible correlation between increased viral replication and increased levels of beta interferon, MMP-3, MMP-12, and TIMP-1 mRNA. CD4+ T cells which localize to the perivascular and subarachnoid spaces were identified as the primary source of TIMP-1 protein. By contrast, protein expression was undetectable in astrocytes or CD8+ T cells, the primary antiviral effectors that localize to the CNS parenchyma in response to infection. These data suggest that in contrast to the results seen with MMPs, inhibition of protease activity via TIMP-1 expression correlates with the differential tissue distribution of T-cell subsets during acute coronavirus-induced encephalitis.

Depletion of Polyamines and Increase of Transforming Growth Factor‐β1, c‐myc, Collagen‐Type I, Matrix Metalloproteinase‐1, and Metalloproteinase‐2 mRNA in Primary Human Gingival Fibroblasts

The polyamines spermidine, spermine, and putrescine are known to be deeply linked with growth processes, gene expression, and extracellular matrix synthesis. Their cellular content depends primarily on the activity of the enzyme ornithine decarboxylase. High levels of ornithine decarboxylase and polyamines have been found in proliferative, inflammatory, and neoplastic pathologies of the oral cavity and in gingival fluid. Difluoromethylornithine (DFMO) selectively inhibits ornithine decarboxylase, thus depleting polyamine content and preventing cell proliferation and synthesis activity. The aim of this study was to investigate whether DFMO treatment could modify the genes involved in cell proliferation and extracellular matrix turnover. Fibroblasts derived from non-inflamed gingiva were maintained in Dulbecco's modified Eagle's medium (DMEM) plus alpha-difluoromethylornithine for 4 days. At 0, 24, 48, 72, and 96 hours cell number was assessed, polyamine levels were quantified with high performance liquid chromatography (HPLC) method, and transforming growth factor-beta1 (TGF-beta1), c-myc, matrix metalloproteinases (MMP)-1 and 2, collagen type I (COL-I) and tissue inhibitor of matrix metalloproteinases (TIMP)-1 were evaluated by reverse transcription polymerase chain reaction (RT-PCR). Fibroblasts treated with DFMO significantly decreased cell proliferation, ornithine decarboxylase activity, and putrescine levels at all treatment times, spermidine after 72 and 96 hours, and spermine after 96 hours of culture. Total polyamines decreased (P < or =0.01) at 96 hours after DFMO treatment, while c-myc, TGF-beta1, MMP-1 and 2, COL-I mRNA significantly increased. Conversely, TIMP-1 did not show any significant change. The polyamines trend was not correlated to c-myc, TGF-beta1, MMP-1 and -2, and TIMP-1 mRNA levels. Transforming growth factor-beta1 and c-myc mRNA expression were related and correlated to MMP-1 and 2, COL-I and TIMP-1 mRNA trend after DFMO treatment. Our data show that as the polyamine content decreases, TGF-beta1, c-myc, MMP-1 and -2, and COL-I mRNA levels increase, therefore a negative regulatory role of the polyamines on the mRNA expression could be suggested.

Matrix metalloproteinases mediate β-adrenergic receptor-stimulated apoptosis in adult rat ventricular myocytes

Changes in the synthesis and activity of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) are associated with myocardial remodeling. Here we measured the expression and activity of MMPs and TIMPs, and tested the hypothesis that increased MMP activity plays a proapoptotic role in beta-adrenergic receptor (beta-AR)-stimulated apoptosis of adult rat ventricular myocytes (ARVMs). beta-AR stimulation (isoproterenol, 24 h) increased mRNA levels of MMP-2 and TIMP-1 while it decreased TIMP-2 mRNA levels as analyzed by real-time PCR. Western blot analysis, immunocytochemical analysis, in-gel zymography, and MMP-2 activity assay confirmed beta-AR-stimulated increases in MMP-2 protein levels and activity. Inhibition of MMPs using GM-6001 (a broad-spectrum inhibitor of MMPs), SB3CT (inhibitor of MMP-2), and purified TIMP-2 inhibited beta-AR-stimulated apoptosis as determined by TdT-mediated dUTP nick end labeling staining. Treatment with active MMP-2 alone increased the number of apoptotic cells. This increase in MMP-2-mediated apoptosis was inhibited by GM-6001 and SB3CT pretreatment. Coimmunoprecipitation studies indicated increased physical association of MMP-2 with beta1-integrins after beta-AR stimulation. Inhibition of MMP-2 using SB3CT or stimulation of beta1-integrin signaling using laminin inhibited the increased association of MMP-2 with beta1-integrins. beta-AR stimulation increased poly-ADP-ribose-polymerase cleavage, which was inhibited by inhibition of MMP-2. These data suggest the following: 1) beta-AR stimulation increases MMP-2 expression and activity and inhibits TIMP-2 expression; 2) inhibition of MMPs, most likely MMP-2, inhibits beta-AR-stimulated apoptosis; and 3) the apoptotic effects of MMP-2 may be mediated, at least in part, via its interaction with beta1 integrins and poly-ADP-ribose-polymerase cleavage.

Immunosuppression and gingival overgrowth: gene and protein expression profiles of collagen turnover in FK506‐treated human gingival fibroblasts

FK506 is an immunosuppressive agent that, unlike cyclosporin A (CsA), does not induce gingival overgrowth (GO). CsA-induced GO is caused by quantitative modifications of the extracellular matrix components, particularly collagen (COL). Up to now, clinical trials have only investigated FK506 in relation with GO, so we aimed at analysing the effect of FK506 on COL turnover using a molecular approach, to evaluate the expression of genes and proteins related to this process. Human gingival fibroblasts were incubated with FK506 or its vehicle (VH) for 24, 48 and 72 h. COL type I (COL-I), matrix metalloproteinases (MMP)-1 and 2, tissue inhibitor of MMP (TIMP)-1 and transforming growth factor (TGF)-beta1 mRNA were assayed by Reverse transcriptase polymerase chain reaction; COL-I protein levels were determined by dot blot, MMP-1 and MMP-2 activity by zymography. Fibroblast proliferation decreased 48 and 72 h after treatment. COL-I gene and protein expression, TGF-beta1 and TIMP-1 mRNA levels were not significantly affected, whereas MMP-1 gene and protein expression and MMP-2 mRNA levels rose significantly in treated fibroblasts compared with VH. These findings suggest that increased MMP-1 gene and protein expression may be important for regulating COL-I homeostasis in the gingival connective compartment of FK506-immunosuppressed subjects.

213. Regulation of TGFβ superfamily-related genes in the newborn mouse testis by activin

Several members of the transforming growth factor-β (TGFβ) superfamily of ligands are known to influence development of male germ cells. In our ongoing investigations of genetically modified mice with different bioactive levels of activin, we noted that testicular germ cell numbers at birth are increased in the absence of activin.1 In this study we compared expression levels of TGFβ-related genes in mice with 0, 1 or 2 copies of the activin βA subunit gene. Candidate genes were identified through hybridisation of total testis RNA from newborn (Day 0) wild type (WT; 2 copies) and activin βA null mice (KO; 0 copies) to a TGFβ SuperArray Simplicity™ membrane. Densitometric analysis of resulting signals revealed several genes that appeared to differ between genotypes. To validate these findings, pooled testes from newborn WT (2 independent groups), heterozygous (het; 2 groups) and activin βA KO (3 groups) mice were collected for RNA extraction. Quantitative real-time PCR analysis was performed using the Roche Light Cycler, with β-actin mRNA level used as the housekeeping reference. The mRNAs selected for analysis were: inhibin α, p15INK4b, Smad5, insulin-like growth factor 1 (IGF-1), ALK6 (BMPRI-B), and tissue inhibitor of metalloproteinase 1 (Timp1). The inhibin α transcript level was significantly decreased in the activin βA KO animal compared with WT, while the het sample showed an intermediate effect. Both Smad5 and IGF-1 transcripts appeared to increase in the KO animals compared with WT; however, the cyclin-dependent kinase inhibitor, p15INK4b, did not change significantly across the genotypes. Similarly, ALK-6 and Timp1 mRNA levels were also unaffected by genotype. These findings illustrate the impact of graded levels of activin A on specific genes in the fetal testis. (1)Mendis et al. (2004). Reprod. Fertil. Dev. 16(Suppl.), 103.

Expression of matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-1 in hepatic stellate cells during rat hepatic fibrosis and its intervention by IL-10.

To investigate the expression of matrix metallopr-oteinase-2 and tissue inhibitor of metalloproteinase-1 in hepatic fibrosis and the antifibrogenic role of exogenous interleukin-10 (IL-10). Hepatic fibrosis was induced by CCl(4) administration and 60 male Sprague-Dawley rats were randomly divided into normal control group (group N, 8 rats), CCl(4)-induced group (group C, 28 rats) and IL-10-treated group (group I, 24 rats). At the beginning of the 7(th) and 11(th) wk, rats in each group were routinely perfused with pronase E and type IV collagenase through portal vein catheter and the suspension was centrifuged by 11% Nycodenz density gradient to isolate hepatic stellate cells (HSCs). RT-PCR was used to analyze mRNA of MMP-2 and TIMP-1 from freshly isolated cells. Densitometric data were standardized with beta-actin signals. Immunocytochemistry was performed to detect MMP-2 and TIMP-1 expression in HSC cultured for 72 h. Compared to group N in the 7(th) wk, MMP-2 and TIMP-1 mRNA increased in group C (P = 0.001/0.001) and group I (P = 0.001/0.009). The level of MMP-2 and TIMP-1 mRNA in group I was significantly lower than that in group C (P = 0.001/0.001). In the 11(th) wk, MMP-2 mRNA in group I was still lower than that in group C (P = 0.005), but both dropped compared with that in the 7(th) week (P = 0.001/0.004). TIMP-1 mRNA in group I was still lower than that in group C (P = 0.001), and increased in group C (P = 0.001) while decreased in group I (P = 0.042) compared with that in the 7(th) wk. Same results were found by immunocytochemistry. Expression of MMP-2 and TIMP-1 is increased in hepatic fibrosis. IL-10 exhibits an antifibrogenic effect by suppressing MMP-2 and TIMP-1 expression.

Effects of augmentation of liver regeneration recombinant plasmid on rat hepatic fibrosis.

To investigate the effects of eukaryotic expression of plasmid on augmentation of liver regeneration (ALR) in rat hepatic fibrosis and to explore their mechanisms. Ten rats were randomly selected from 50 Wistar rats as normal control group. The rest were administered intraperitoneally with porcine serum twice weekly. After 8 wk, they were randomly divided into: model control group, colchicine group (Col), first ALR group (ALR1), second ALR group (ALR2). Then colchicine ALR recombinant plasmid were used to treat them respectively. At the end of the 4th wk, rats were killed. Serum indicators were detected and histopathological changes were graded. Expression of type I, III, collagen and TIMP-1 were detected by immunohistochemistry and expression of TIMP-1 mRNA was detected by semi-quantified RT-PCR. The histologic examination showed that the degree of the rat hepatic fibrosis in two ALR groups was lower than those in model control group. Compared with model group, ALR significantly reduced the serum levels of ALT, AST, HA, LN, PCIII and IV (P<0.05). Immunohistochemical staining showed that expression of type I, III, collagen and TIMP-1 in two ALR groups was ameliorated dramatically compared with model group (I collagen: 6.94+/-1.42, 5.80+/-1.66 and 10.83+/-3.58 in ALR1, ALR2 and model groups, respectively; III collagen: 7.18+/-1.95, 4.50+/-1.67 and 10.25+/-2.61, respectively; TIMP-1: 0.39+/-0.05, 0.20+/-0.06 and 0.53+/-0.12, respectively, P<0.05 or P<0.01). The expression level of TIMP-1 mRNA in the liver tissues was markedly decreased in two ALR groups compared with model group (TIMP-1 mRNA/beta-actin: 0.89+/-0.08, 0.65+/-0.11 and 1.36+/-0.11 in ALR1, ALR2 and model groups respectively, P<0.01). ALR recombinant plasmid has beneficial effects on rat hepatic fibrosis by enhancing regeneration of injured liver cells and inhibiting TIMP-1 expressions.

Treatment of experimental autoimmune encephalomyelitis with a neurotropic alphavirus vector expressing tissue inhibitor of metalloproteinase-2.

Prompted by our recent observations of increased MMP-8 and MMP-9 with simultaneous downregulation of tissue inhibitor of metalloproteinase-2 (TIMP-2) and TIMP-3 mRNA levels in the central nervous system (CNS) of mice with severe experimental autoimmune encephalomyelitis (EAE), we used Semliki Forest virus (SFV) to transfer and express recombinant murine TIMP-1-3 genes in the CNS. TIMP-1, TIMP-2 and TIMP-3 expression was confirmed in cultured cells and in the CNS of infected mice. Following intraperitoneal infection with 10(6) plaque-forming units (PFU) of SFV-TIMP, focal TIMP protein expression was achieved throughout the brain. Although already treatment with empty vector inhibited development of EAE to some extent, the expression of TIMP-2 by the virus significantly enhanced the inhibition. TIMP-3-administered mice also had lower disease grade, but the inhibition was not statistically significant. In contrast, SFV-TIMP-1 had no effect, similar to co-infection with TIMP-2 and TIMP-3. We found TIMP-2 expression also by non-infected CNS-resident cells surrounding the virus-positive areas, suggesting a bystander TIMP-2 induction. These data strengthen the view that matrix metalloproteinases are involved in the pathogenesis of EAE and provide clear evidence that virus-mediated delivery of their protein inhibitors can be effective in preventing the clinical disease. TIMPs might be candidates for novel treatment regimens in CNS autoimmune disorders, such as multiple sclerosis.

The balance between MMP-2/-9 and TIMP-1/-2 is shifted towards MMP in renal cell carcinomas and can be further disturbed by hydrogen peroxide

In malignant tumors the balance of matrix metalloproteinases (MMP) and tissue inhibitors of matrix metalloproteinases (TIMP) is disturbed. Radical oxygen species (ROS) and hydrogen peroxide potentially influence this balance. Therefore, we analyzed the balance of MMP and TIMP in renal cell carcinoma (RCC) specimens and cell lines. In RCC specimens MMP-2, MMP-9, TIMP-1, and TIMP-2 were immunohistochemically detected. Tumor-associated macrophages (TAM) as a potential source of ROS were characterized with an anti-CD68 antibody. Three RCC cell lines were treated with sublethal concentrations of hydrogen peroxide to simulate the effects of radical oxygen species. MMP-2 and MMP-9 protein expression was measured by zymography. mRNA expression of MMP-2, MMP-9, TIMP-1, and TIMP-2 was assessed by quantitative reverse transcriptase polymerase chain reaction. Tumor cell-derived reactive oxygen species were measured by FACS analysis and dihydrorhodamine 123 oxidation. In RCCs the MMP and TIMP expression profile was variable. The balance between MMP and TIMP was shifted towards MMP in comparison to matched normal controls. TAM were localized in a close vicinity to MMP expresssing tumor cells. As in RCC specimens, the expression of MMP and TIMP in the analyzed RCC cell lines varied. Hydrogen peroxide induced MMP-2 and -9 mRNA and protein expression, whereas TIMP-1 and TIMP-2 mRNA levels remained unaffected in cell lines. Thus, the ratio between MMP and TIMP was shifted towards MMP. Tumor cells did not increase the production of reactive oxygen species stimulation with phorbol ester or hydrogen peroxide. In RCC the balance between MMP and TIMP is disturbed. Oxidative stress potentially increases this imbalance. TAM might be one source of hydrogen peroxide thus supporting the invasive properties of RCCs.

Adiponectin specifically increased tissue inhibitor of metalloproteinase-1 through interleukin-10 expression in human macrophages.

Vascular inflammation and subsequent matrix degradation play an important role in the development of atherosclerosis. We previously reported that adiponectin, an adipose-specific plasma protein, accumulated to the injured artery and attenuated vascular inflammatory response. Clinically, high plasma adiponectin level was associated with low cardiovascular event rate in patients with chronic renal failure. The present study was designed to elucidate the effects of adiponectin on matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) in human monocyte-derived macrophages. Human monocyte-derived macrophages were incubated with the physiological concentrations of human recombinant adiponectin for the time indicated. Adiponectin treatment dose-dependently increased TIMP-1 mRNA levels without affecting MMP-9 mRNA levels. Adiponectin also augmented TIMP-1 secretion into the media, whereas MMP-9 secretion and activity were unchanged. Time course experiments indicated that TIMP-1 mRNA levels started to increase at 24 hours of adiponectin treatment and were significantly elevated at 48 hours. Adiponectin significantly increased interleukin-10 (IL-10) mRNA expression at the transcriptional level within 6 hours and significantly increased IL-10 protein secretion within 24 hours. Cotreatment of adiponectin with anti-IL-10 monoclonal antibody completely abolished adiponectin-induced TIMP-1 mRNA expression. Adiponectin selectively increased TIMP-1 expression in human monocyte-derived macrophages through IL-10 induction. This study identified, for the first time, the adiponectin/IL-10 interaction against vascular inflammation.

Disruption of the TIMP-1 gene product is associated with accelerated endometrial gland formation during early postnatal uterine development.

Postnatal uterine development is marked by periods of tissue remodeling. The objective of the present study was to examine the role of tissue inhibitor of metalloproteinase-1 (TIMP-1), a regulator of tissue remodeling events, during postnatal uterine development and to assess the phenotypic consequences of disruption of the TIMP-1 gene product during this time period. To accomplish this goal, wild-type and TIMP-1 null mice were sacrificed at Postnatal Days (PNDs) 5, 10, 15, 20, and 25 and uterine morphology, TIMP expression and matrix metalloproteinase (MMP) activity were assessed. In wild-type mice, TIMP-1 mRNA steady-state levels were highest at PND 5, after which expression decreased. TIMP-2 and TIMP-3 expression in wild-type mice showed no significant changes from PND 5 to 25. In TIMP-1 null mice, TIMP-2 and TIMP-3 expression patterns were similar to those in wild-type counterparts with the exception that, at PND 10, TIMP-2 and TIMP-3 expression was significantly lower in the null mice. Endometrial gland number and uterine histology were similar between genotypes at PNDs 5 and 10, but at PNDs 15 and 20, endometrial glands were more abundant in TIMP-1 null mice. Associated with the increased gland density in the null mice was an increase in total MMP activity above the levels expressed in wild-type mice. In summary, disruption of the TIMP-1 gene product is associated with reduced TIMP-2 and TIMP-3 steady-state mRNA levels, elevated MMP activity, and accelerated endometrial gland formation. We conclude that, during early postnatal uterine development, TIMP-1 may be critical for proper endometrial gland development.

Effect of cyclosporin A on human gingival fibroblast collagen turnover in relation to the development of gingival overgrowth: an in vitro study

In a significant number of cases (25–81%) immunosuppressant treatment with cyclosporin A (CsA) is associated with gingival overgrowth, seriously interfering with the functions of mastication and speech. In CsA-induced gingival enlargement, quantitative modifications of the extracellular matrix components occur, and collagen (COL) metabolism and matrix metalloproteinases (MMPs) have been suggested as being the main targets. Since the mechanisms at the basis of CsA-induced gingival overgrowth are not yet completely understood, our aim was to analyze the effect of CsA on COL turnover in cultured human gingival fibroblasts. Cultured human gingival fibroblasts from four healthy volunteers were incubated with CsA (800 ng/ml) or with its vehicle (VH) for variable intervals of time (24, 48, 72 h). Fibroblast morphology was studied by light and electron microscope. Collagen type I (COL-I), MMP-1, MMP-2, TIMP-1 and TGF-β1 mRNA were determined by RT-PCR; COL-I and MMP-1 by dot blot, and MMP-2 by zymography. Our results evidenced an up-regulation of COL-I and TGF-β1 gene expression 72 h after CsA treatment. MMP-1, MMP-2 and TIMP-1 mRNA levels are affected but not significantly. Protein analysis revealed COL-I increase at all the considered times and, 72 h after CsA treatment, reduced collagenolytic levels. Our data suggest that COL accumulation during CsA-induced gingival overgrowth may be mainly sustained by an altered COL-I degradation due to decreased MMP-1 activity. However, interindividual differences of collagenase levels after CsA treatment suggest that a genetic predisposition to develop gingival overgrowth may be relevant.

Differential expression and activity of matrix metalloproteinases during flow-modulated vein graft remodeling

Objective While shear stress closely regulates vascular remodeling, the mediators of this process have been only partially elucidated. The current study examined the role of the gelatinases in flow-mediated vein graft intimal hyperplasia. We hypothesized that matrix metalloproteinase (MMP)–2 and MMP-9 expression and protein levels, relative to tissue inhibitors of metalloproteinase (TIMP)–1 and TIMP-2, are upregulated in a flow-dependent manner during vein graft arterialization. Methods Bilateral common carotid interposition vein grafting was performed in rabbits. Reduction in flow was achieved through unilateral ligation of the internal carotid artery and three of four branches of the external carotid artery. At 28 days grafts were harvested and analyzed for intimal area; MMP-2 and MMP-9, and TIMP-1 and TIMP-2 messenger RNA content, via quantitative reverse transcription polymerase chain reaction; and MMP-2 and MMP-9, and TIMP-1 and TIMP-2 protein concentrations, via both bioactivity assay and zymography. Results Branch ligation resulted in a 10-fold difference in mean flow rate and accelerated development of intimal hyperplasia in a low-flow environment. Exposure of the vein graft to arterial hemodynamics induced a marked rise in MMP-9 mRNA levels, whereas only a modest increase in MMP-2 mRNA was observed. MMP-2 protein was 50 to 100 times more abundant than MMP-9, and was significantly upregulated in grafts that demonstrated enhanced intimal thickening. Immunohistochemistry demonstrated that MMP-2 was located throughout the myointima, whereas MMP-9 was localized almost exclusively to the region of endothelium. No differences in TIMP-1 and TIMP-2 mRNA or protein levels were detected between high-flow and low-flow grafts. Conclusion MMP-2 is the predominate gelatinase that regulates early vein graft remodeling. Despite a marked increase in MMP-9 gene expression, development of intimal hyperplasia after a reduction in wall shear rate correlates with an increase in MMP-2 protein levels. These data suggest differential regulatory mechanisms for proteases within the remodeling vein graft wall. Modulation of extracellular matrix biologic features may offer therapeutic strategies for the prevention of vein graft failure.

Decreased collagen-degrading activity could be a marker of prolonged mesangial matrix expansion.

Mesangial matrix expansion is caused by the overproduction and/or the impaired proteolytic degradation of the extracellular matrix. However, the relative contribution of these changes to the development of prolonged mesangial matrix expansion is still poorly understood. We aimed to elucidate the relative role of the matrix metalloproteinase (MMP)/tissue inhibitors of metalloproteinases (TIMPs) system in the development of prolonged mesangial matrix expansion. We prepared two rat models, showing reversible or prolonged mesangial matrix expansion, induced by a single injection or two consecutive injections of anti-Thy-1.1 monoclonal antibody 1-22-3, respectively. We analyzed the glomerular expression of type I and type IV collagens; MMP-2, -9, and -13; membrane type 1-MMP (MT1-MMP); TIMP-1; and urinary type I collagen-degrading activity in both models. There were no differences in glomerular mRNA levels of type I and type IV collagens between the reversible and the prolonged models. MMP-9 mRNA expression and protein level was lower in the prolonged model than in the reversible one, whereas there were no differences in mRNA levels of MMP-2, -13, MT1-MMP, or TIMP-1 between the two models. Urinary type I collagen-degrading activity in the prolonged model was lower than that in the reversible one. Furthermore, there was a significant correlation between the mesangial matrix expansion and urinary type I collagen-degrading activity. Impaired expression of MMP-9 may contribute to the development of prolonged mesangial matrix expansion. Analysis of urinary type I collagen-degrading activity may provide additional diagnostic information in mesangial proliferative glomerulonephritis. Mesangial matrix expansion is caused by the overproduction and/or the impaired proteolytic degradation of the extracellular matrix. However, the relative contribution of these changes to the development of prolonged mesangial matrix expansion is still poorly understood. We aimed to elucidate the relative role of the matrix metalloproteinase (MMP)/tissue inhibitors of metalloproteinases (TIMPs) system in the development of prolonged mesangial matrix expansion.

Tissue inhibitors of metalloproteinase expression in human breast cancer: TIMP-3 is associated with adjuvant endocrine therapy success.

Tissue inhibitors of matrix metalloproteinase (TIMPs) may be involved in tumour growth, apoptosis, angiogenesis, invasion, and the development of metastases. This study has evaluated the association of the expression levels of the TIMP forms 1, 2, 3, and 4, measured by quantitative real-time RT-PCR, with classical clinicopathological characteristics, ie age, menopausal status, tumour size, histological grade, number of involved lymph nodes, and steroid hormone receptor status, and with disease progression and treatment sensitivity in 273 breast cancer patients. The mRNA levels of TIMP-1 and TIMP-2 were not associated with any known clinicopathological tumour feature. TIMP-3 and TIMP-4 levels were significantly higher in steroid hormone receptor-positive samples, although the levels of TIMP-4 were much lower than those of the other TIMPs. Only TIMP-3 predicted relapse-free survival (RFS) time differently depending on post-surgical treatment as, in particular, the interaction of TIMP-3 with endocrine therapy (p = 0.008, HR = 0.24, 95% CI = 0.09-0.69) contributed significantly to RFS in multivariate Cox regression analysis. In subgroup analyses, the 107 patients treated with tamoxifen differed greatly in prognosis after dichotomization by the median TIMP-3 level (p = 0.0003). Thus, high tumour levels of the matrix metalloproteinases inhibitor and pro-apoptotic factor TIMP-3 are associated with successful tamoxifen treatment of patients with breast cancer.