Structural characterisation of inhibitory and non-inhibitory MMP-9-TIMP-1 complexes and implications for regulatory mechanisms of MMP-9.

MMP-9 plays a number of important physiological functions but is also responsible for many pathological processes, including cancer invasion, metastasis, and angiogenesis. It is, therefore, crucial to understand its enzymatic activity, including activation and inhibition mechanisms. This enzyme may also be partially involved in the "cytokine storm" that is characteristic of COVID-19 disease (SARS-CoV-2), as well as in the molecular mechanisms responsible for lung fibrosis. Due to the variety of processing pathways involving MMP-9 in biological systems and its uniqueness due to the O-glycosylated domain (OGD) and fibronectin-like (FBN) domain, specific interactions with its natural TIMP-1 inhibitor should be carefully studied, because they differ significantly from other homologous systems. In particular, earlier experimental studies have indicated that the newly characterised circular form of a proMMP-9 homotrimer exhibits stronger binding properties to TIMP-1 compared to its monomeric form. However, molecular structures of the complexes and the binding mechanisms remain unknown. The purpose of this study is to fill in the gaps in knowledge. Molecular modelling methods are applied to build the inhibitory and non-inhibitory MMP-9-TIMP-1 complexes, which allows for a detailed description of these structures and should allow for a better understanding of the regulatory processes in which MMP-9 is involved.

TIMP1 down-regulation enhances gemcitabine sensitivity and reverses chemoresistance in pancreatic cancer.

The therapeutic effect of gemcitabine (GEM) in pancreatic ductal adenocarcinoma (PDAC) is limited due to low drug sensitivity and high drug resistance. Tissue inhibitor of matrix metalloprotease 1 (TIMP1) is reportedly associated with GEM resistance in PDAC. However, the effect of TIMP1 down-regulation in combination with GEM treatment is unknown. We analyzed the expression of TIMP1 in human PDAC tissue using western blot, quantitative real-time polymerase chain reaction (qRT-PCR), and immunohistochemistry. TIMP1 was highly expressed in PDAC specimens. Kaplan-Meier survival analysis suggested that a higher level of TIMP1 was correlated with poorer overall survival in 103 PDAC patients. The mRNA and protein expression profiles of TIMP1 were explored in the HTERT-HPNE human pancreatic ductal epithelium cell line, five PDAC cell lines (MIA PaCa-2, PANC-1, BxPC-3, Capan2, and SW1990), and two GEM-resistant PDAC cell lines (MIA PaCa-2R and PANC-1R). Compared with HTERT-HPNE, TIMP1 was highly expressed in the PDAC cell lines. In addition, TIMP1 was upregulated in GEM-resistant PDAC cell lines compared with their parental cells. When TIMP1 was knocked-down using short hairpin RNA, GEM-induced cytotoxicity and apoptosis were increased, while colony formation was repressed in MIA PaCa-2, PANC-1, and their GEM-resistant cells. When Bax was activated by BAM7 or Bcl-2 was inhibited by venetoclax, CCK-8 assays demonstrated that GEM sensitivity was restored in GEM-resistant cells. When Bax was down-regulated by siRNA, CCK-8 assays verified that GEM sensitivity was decreased in PDAC cells. The observations that TIMP1 knockdown enhanced GEM sensitivity and reversed chemoresistance by inducing cells apoptosis indicated cooperative antitumor effects of shTIMP1 and GEM therapy on PDAC cells. The combination may be a potential strategy for PDAC therapy.

Homocysteine Disrupts Balance between MMP-9 and Its Tissue Inhibitor in Diabetic Retinopathy: The Role of DNA Methylation.

High homocysteine is routinely observed in diabetic patients, and this non-protein amino acid is considered as an independent risk factor for diabetic retinopathy. Homocysteine biosynthesis from methionine forms S-adenosyl methionine (SAM), which is a major methyl donor critical in DNA methylation. Hyperhomocysteinemia is implicated in increased oxidative stress and activation of MMP-9, and in diabetic retinopathy, the activation of MMP-9 facilitates capillary cell apoptosis. Our aim was to investigate the mechanism by which homocysteine activates MMP-9 in diabetic retinopathy. Human retinal endothelial cells, incubated with/without 100 µM homocysteine, were analyzed for MMP-9 and its tissue inhibitor Timp1 expressions and interactions, and ROS levels. Timp1 and MMP-9 promoters were analyzed for methylated and hydroxymethylated cytosine levels (5mC and 5hmC respectively) by the DNA capture method, and DNA- methylating (Dnmt1) and hydroxymethylating enzymes (Tet2) binding by chromatin immunoprecipitation. The results were confirmed in retinal microvessels from diabetic rats receiving homocysteine. Homocysteine supplementation exacerbated hyperglycaemia-induced MMP-9 and ROS levels and decreased Timp1 and its interactions with MMP-9. Homocysteine also aggravated Dnmts and Tets activation, increased 5mC at Timp1 promoter and 5hmC at MMP-9 promoter, and suppressed Timp1 transcription and activated MMP-9 transcription. Similar results were obtained from retinal microvessels from diabetic rats receiving homocysteine. Thus, hyperhomocysteinemia in diabetes activates MMP-9 functionally by reducing Timp1-MMP-9 interactions and transcriptionally by altering DNA methylation-hydroxymethylation of its promoter. The regulation of homocysteine could prevent/slow down the development of retinopathy and prevent their vision loss in diabetic patients.

Induced Pluripotent Stem Cells Attenuate Endothelial Leakage in Acute Lung Injury via Tissue Inhibitor of Metalloproteinases-1 to Reduce Focal Adhesion Kinase Activity.

Induced pluripotent stem cells (iPSCs) can reduce the severity of endotoxin-induced acute lung injury (ALI). However, the interaction between iPSCs and vascular endothelium remains unclear. In this study, we investigated the effects of iPSCs in moderating pulmonary endothelial leakage in endotoxin-induced ALI. Murine iPSCs were delivered intravenously to male C57BL/6 mice (8-12 weeks old) 4 hours after intratracheal lipopolysaccharide (LPS) delivery. Histology, blood and bronchoalveolar lavage fluid (BALF) cytokine and junctional protein assays, and regulatory signaling pathway assays were performed 24 hours later. Human umbilical vein endothelial cells (HUVECs) were used as a model of junctional protein-expressing cells and stimulated with LPS. Our results showed that iPSC treatment alleviated histological signs of ALI, protein leakage, and proinflammatory cytokines. iPSC therapy restored vascular endothelial cadherin (VE-cadherin) expression in ALI mouse lungs. In HUVECs, human iPSCs (hiPSCs) restored disrupted VE-cadherin expression and reduced the activity of Snail and focal adhesion kinase (FAK) phosphorylation in Tyr397 in response to LPS. iPSC-conditioned medium contained extra antiangiogenic factor of tissue inhibitor of metalloproteinases-1 (TIMP-1) compared with control medium. TIMP-1 inhibition diminished the beneficial effects of iPSC-conditioned medium in ALI mice. Our study suggested that iPSCs attenuate endothelial cell leakage in endotoxin-induced ALI via a mechanism involving TIMP-1 and the FAK/Snail pathway. Stem Cells 2019;37:1516-1527.

Metalloproteinases and their Inhibitors under the Course of Immunostimulation by CPG-ODN and Specific Antigen Inhalation in Equine Asthma.

OBJECTIVES: Inhalation of immunostimulatory bacterial DNA segments (cytosine-phosphate-guanosine-oligodeoxynucleotides, CpG-ODN) normalizes clinical and cytologic parameters in severe equine asthma. We hypothesized that CpG-ODN inhalation also reduces the misbalance of elastinolytic activity in asthmatic horses. METHODS: Twenty asthmatic horses diagnosed by clinical examinations using a scoring system were included. All horses inhaled CpG-ODNs for 14 days in 2-day intervals. Matrix metalloproteinase (MMP-2/-9) and tissue inhibitors of metalloproteinase (TIMP-1/-2) concentrations were measured in tracheal aspirates using equine sandwich ELISAs before and 2 and 6 weeks after CpG-ODN inhalation. RESULTS: MMP and TIMP concentrations correlated with the results of clinical scoring in all stages of equine asthma. Inhalation therapy led to significant reductions in clinical scores. MMP-2, MMP-9, and TIMP-2 concentrations were significantly reduced immediately, and all MMP and TIMP concentrations 6 weeks after therapy. DISCUSSION: In equine asthma, overexpression of MMPs contributes to pathological tissue destruction, while TIMPs counteract MMPs with overexpression leading to fibrosis formation. The results of this study show that CpG-ODN inhalation may be an effective therapy to address a misbalance in equine asthma. CONCLUSIONS: Misbalance of elastinolytic activity seems to improve by CpG-ODN inhalation for at least 6 weeks posttherapy, which may reduce the remodeling of the extracellular matrix. Further studies should evaluate this effect in comparison to glucocorticoid inhalation therapy. SIGNIFICANCE: CpG-ODN inhalation may be an effective therapy in the prevention of pulmonary fibrosis formation in equine asthma.

Bone-derived Nestin-positive mesenchymal stem cells improve cardiac function via recruiting cardiac endothelial cells after myocardial infarction.

BACKGROUND: Bone-derived mesenchymal stem cell (BMSC) transplantation has been reported to be effective for the treatment of ischemic heart disease, but whether BMSCs are the optimal cell type remains under debate. Increasing numbers of studies have shown that Nestin, an intermediate filament protein, is a potential marker for MSCs, which raises the question of whether Nestin+ cells in BMSCs may play a more crucial role in myocardial repair. METHODS: Nestin+ cells were isolated using flow cytometry by gating for CD45- Ter119- CD31- cells from the compact bone of Nestin-GFP transgenic mice, expressing GFP driven by the Nestin promoter. Colony-forming and proliferative curve assays were conducted to determine the proliferative capacity of these cells, while qRT-PCR was used to analyze the mRNA levels of relative chemokines and growth factors. Cardiac endothelial cell (CEC) recruitment was assessed via a transwell assay. Moreover, permanent ligation of the left anterior descending (LAD) coronary artery was performed to establish an acute myocardial infarction (AMI) mouse model. After cell transplantation, conventional echocardiography was conducted 1 and 4 weeks post-MI, and hearts were harvested for hematoxylin-and-eosin (HE) staining and immunofluorescence staining 1 week post-MI. Further evaluation of paracrine factor levels and administration of a neutralizing antibody (TIMP-1, TIMP-2, and CXCL12) or a CXCR4 antagonist (AMD3100) in MI hearts were performed to elucidate the mechanism involved in the chemotactic effect of Nestin+ BMSCs in vivo. RESULTS: Compared with Nestin- BMSCs, a greater proliferative capacity of Nestin+ BMSCs was observed, which further exhibited moderately high expression of chemokines instead of growth factors. More CEC recruitment in the Nestin+ BMSC-cocultured group was observed in vitro, while this effect was obviously abolished after treatment with neutralizing antibodies against TIMP-1, TIMP-2, or CXCL12, and more importantly, blocking the CXCL12/CXCR4 axis with a AMD3100 significantly reduced the chemotactic effect of Nestin+ BMSCs. After transplantation into mice exposed to myocardial infarction (MI), Nestin+ BMSC-treated mice showed significantly improved survival and left ventricular function compared with Nestin- BMSC-treated mice. Moreover, endogenous CECs were markedly increased, and chemokine levels were significantly higher, in the infarcted border zone with Nestin+ BMSC treatment. Meanwhile, neutralization of each TIMP-1, TIMP-2, or CXCL12 in vivo could reduce the left ventricular function at 1 and 4 weeks post-MI; importantly, the combined use of these three neutralizing antibodies could make a higher significance on cardiac function. Finally, blocking the CXCL12/CXCR4 axis with AMD3100 significantly reduced the left ventricular function and greatly inhibited Nestin+ BMSC-induced CEC chemotaxis in vivo. CONCLUSIONS: These results suggest that Nestin+ BMSC transplantation can improve cardiac function in an AMI model by recruiting resident CECs to the infarcted border region via the CXCL12/CXCR4 chemokine pathway. And we demonstrated that Nestin+BMSC-secreted TIMP-1/2 enhances CXCL12(SDF1α)/CXCR4 axis-driven migration of endogenous Sca-1+ endothelial cells in ischemic heart post-AMI. Taken together, our results show that Nestin is a useful marker for the identification of functional BMSCs and indicate that Nestin+ BMSCs could be a better therapeutic candidate for cardiac repair.

Targeting a Designer TIMP-1 to the Cell Surface for Effective MT1-MMP Inhibition: A Potential Role for the Prion Protein in Renal Carcinoma Therapy.

Renal carcinoma cells express Membrane Type 1-Matrix Metalloproteinase (MT1-MMP, MMP-14) to degrade extracellular matrix components and a range of bioactive molecules to allow metastasis and cell proliferation. The activity of MT1-MMP is modulated by the endogenous inhibitors, Tissue Inhibitor of Metalloproteinases (TIMPs). In this study, we describe a novel strategy that would enable a "designer" TIMP-1 tailored specifically for MT1-MMP inhibition (V4A/P6V/T98L; Kiapp 1.66 nM) to be targeted to the plasma membrane for more effective MT1-MMP inhibition. To achieve this, we fuse the designer TIMP-1 to the glycosyl-phosphatidyl inositol (GPI) anchor of the prion protein to create a membrane-tethered, high-affinity TIMP variant named "T1Pr αMT1" that is predominantly located on the cell surface and co-localised with MT1-MMP. Confocal microscopy shows that T1Pr αMT1 is found throughout the cell surface in particular the membrane ruffles where MT1-MMP is most abundant. Expression of T1Pr αMT1 brings about a complete abrogation of the gelatinolytic activity of cellular MT1-MMP in HT1080 fibrosarcoma cells whilst in renal carcinoma cells CaKi-1, the GPI-TIMP causes a disruption in MMP-mediated proteolysis of ECM components such as fibronectin, collagen I and laminin that consequently triggers a downstream senescence response. Moreover, the transduced cells also suffer from an impairment in proliferation and survival in vitro as well as in NOD/SCID mouse xenograft. Taken together, our findings demonstrate that the GPI anchor of prion could be exploited as a targeting device in TIMP engineering for MT1-MMP inhibition with a potential in renal carcinoma therapy.

Overexpression of TIMP-1 and Sensitivity to Topoisomerase Inhibitors in Glioblastoma Cell Lines.

The multifunctional protein - tissue inhibitor of metalloproteinases-1 (TIMP-1) - has been associated with a poor prognosis in several types of cancers including glioblastomas. In addition, TIMP-1 has been associated with decreased response to chemotherapy, and especially the efficacy of the family of topoisomerase (TOP) inhibitors has been related to TIMP-1. As a second line treatment of glioblastomas, the vascular endothelial growth factor (VEGF) antibody bevacizumab is administered in combination with the TOP1 inhibitor irinotecan and glioblastoma cell levels of TIMP-1 could therefore potentially influence the efficacy of such treatment. In the present study, we aimed to investigate whether a high TIMP-1 expression in glioblastoma cell lines would affect the sensitivity to TOP inhibitors, and whether TIMP-1 overexpressing cells would have alterered growth and invasion. We established TIMP-1 overexpressing subclones from two human glioblastoma cell lines. TIMP-1 overexpressing U87MG cells were significantly more resistant than low TIMP-1 expressing clones and parental cells when exposed to SN-38 (TOP1 inhibitor) or epirubicin (TOP2 inhibitor). No significant differences were observed for the TIMP-1 transfected A172 cells. Implantation of both U87MG and A172 spheroids into organotypic brain slice cultures revealed a reduced growth of TIMP-1 overexpressing U87MG spheroids, however, no significant differences in invasion were observed. The present study suggests that TIMP-1 overexpression reduces the effect of TOP inhibitors in glioblastoma. TIMP-1 also appeared to reduce spheroid growth, but did not influence invasion. Whether TIMP-1 plays a role in irinotecan resistance and has a predictive potential in glioblastoma patients remains to be elucidated.

Capsaicin Inhibits Dimethylnitrosamine-Induced Hepatic Fibrosis by Inhibiting the TGF-ß1/Smad Pathway via Peroxisome Proliferator-Activated Receptor Gamma Activation.

Capsaicin (CPS) exerts many pharmacological effects, but any possible influence on liver fibrosis remains unclear. Therefore, we evaluated the inhibitory effects of CPS on dimethylnitrosamine (DMN) and TGF-ß1-induced liver fibrosis in rats and hepatic stellate cells (HSCs). CPS inhibited DMN-induced hepatotoxicity, NF-κB activation, and collagen accumulation. CPS also suppressed the DMN-induced increases in α-SMA, collagen type I, MMP-2, and TNF-α. In addition, CPS inhibited DMN-induced TGF-ß1 expression (from 2.3 ± 0.1 to 1.0 ± 0.1) and Smad2/3 phosphorylation (from 1.5 ± 0.1 to 1.1 ± 0.1 and from 1.6 ± 0.1 to 1.1 ± 0.1, respectively) by activating Smad7 expression (from 0.1 ± 0.0 to 0.9 ± 0.1) via PPAR-γ induction (from 0.2 ± 0.0 to 0.8 ± 0.0) (p < 0.05). Furthermore, in HSCs, CPS inhibited the TGF-ß1-induced increases in α-SMA and collagen type I expression, via PPAR-γ activation. These results indicate that CPS can ameliorate hepatic fibrosis by inhibiting the TGF-ß1/Smad pathway via PPAR-γ activation.

Higher levels of TIMP-1 expression are associated with a poor prognosis in triple-negative breast cancer.

BACKGROUND: Tissue inhibitor of metalloproteinases-1 (TIMP-1) is a multifunctional protein that can directly regulate apoptosis and metastasis. In this study, we investigated the functional and molecular mechanisms by which TIMP-1 influences triple-negative breast cancer (TNBC). METHODS: The expression level of TIMP-1 in breast cancer tissues was analyzed using the ONCOMINE microarray database. The overall survival of patients with distinct molecular subtypes of breast cancer stratified by TIMP-1 expression levels was evaluated using Kaplan-Meier analysis. Bisulfate sequencing PCR (BSP) was used to analyze the methylation status of the TIMP-1 promoter. Real-time-PCR (RT-PCR), Western blot and ELISA assays were used to evaluate gene and protein expression in cell lines and human tissue specimens. In addition, TIMP-1 function was analyzed using a series of in vitro and in vivo assays with cells in which TIMP-1 was inhibited using RNAi or neutralizing antibodies. RESULTS: We found that serum TIMP-1 levels were strongly enhanced in patients with TNBC and that elevated TIMP-1 levels were associated with a poor prognosis in TNBC. However, TIMP-1 levels were not significantly associated with overall survival in other subtypes of breast cancer or in the overall population of breast cancer patients. We also report the first evidence that the TIMP-1 promoter is hypomethylated in TNBC cell lines compared with non-TNBC cell lines, suggesting that aberrant TIMP-1 expression in TNBC results from reduced DNA methylation. RNAi-mediated silencing of TIMP-1 in TNBC cells induced cell cycle arrest at the G1 phase and reduced cyclin D1 expression. In addition, mechanistic analyses revealed that the p-Akt and p-NF-κB signaling pathways, but not the GSK-3ß and MAPK1/2 pathways, are associated with TIMP-1 overexpression in TNBC cells. Moreover, neutralizing antibodies against TIMP-1 significantly decreased the rate of tumor growth in vivo. CONCLUSIONS: Our findings suggest that TIMP-1 is a biomarker indicative of a poor prognosis in TNBC patients and that targeting TIMP-1 may provide an attractive therapeutic intervention specifically for triple-negative breast cancer patients.

Extracellular matrix metalloproteinase inducer (EMMPRIN) remodels the extracellular matrix through enhancing matrix metalloproteinases (MMPs) and inhibiting tissue inhibitors of MMPs expression in HPV-positive cervical cancer cells.

UNLABELLED: PUPOSE OF INVESTIGATION: To study the expression of extracellular matrix metalloproteinase inducer (EMMPRIN), matrix metalloproteinases (MMPs), and tissue inhibitors of MMP (TIMPs) in uterine cervical cancer cell lines in vitro. MATERIALS AND METHODS: EMMPRIN, MMPs, and TIMPs expression were assessed by Western blot and real-time RT-PCR from cervical carcinoma SiHa, HeLa, and C33-A cells. RESULTS: EMMPRIN recombinant significantly increased MMP-2, MMP-9 protein and mRNA expression in SiHa and Hela cells, but not in C33-A cells by Western blot analysis and real-time RT-PCR. EMMPRIN recombinant significantly inhibited TIMP-1 protein and mRNA levels in SiHa and Hela cells, but not in C33-A cells. There was no difference on the TIMP-2 expression in those cells with the treatment of EMMPRIN recombinant. EMMPRIN RNAi decreased MMP-2 and MMP-9 and increased TIMP-1 expression in SiHa and HeLa cells, but not in C33-A cells. There was no change on the expression of TIMP-2 mRNA levels in SiHa, HeLa and C33-A cells transfected with siEMMPRIN. CONCLUSION: EMMPRIN may induce MMP-2 and MMP-9, and downregulate TIMP-1 in HPV-positive cervical cancer cells in vitro.

Influence of Expression Plasmid of Connective Tissue Growth Factor and Tissue Inhibitor of Metalloproteinase-1 shRNA on Hepatic Precancerous Fibrosis in Rats.

BACKGROUND: In this study, influence caused by expression plasmids of connective tissue growth factor (CTGF) and tissue inhibitor of metalloproteinase-1 (TIMP-1) short hairpin RNA (shRNA) on mRNA expression of CTGF,TIMP-1,procol-α1 and PCIII in hepatic tissue with hepatic fibrosis, a precancerous condition, in rats is analyzed. MATERIALS AND METHODS: To screen and construct shRNA expression plasimid which effectively interferes RNA targets of CTGF and TIMP-1 in rats. 50 cleaning Wistar male rats are allocated randomly at 5 different groups after precancerous fibrosis models and then injection of shRNA expression plasimids. Plasmid psiRNA-GFP-Com (CTGF and TIMP-1 included), psiRNA-GFP-CTGF, psiRNA-GFP-TIMP-1 and psiRNA- DUO-GFPzeo of blank plasmid are injected at group A, B, C and D, respectively, and as model control group that none plasimid is injected at group E. In 2 weeks after last injection, to hepatic tissue at different groups, protein expression of CTGF, TIMP-1, procol-α1and PC III is tested by immunohistochemical method and,mRNA expression of CTGF,TIMP-1,procol-α1 and PCIII is measured by real-time PCR. One-way ANOVA is used to comparison between-groups. RESULTS: Compared with model group, there is no obvious difference of mRNA expression among CTGF,TIMP-1,procol-α1,PC III and of protein expression among CTGF, TIMP-1, procol-α1, PC III in hepatic tissue at group injected with blank plasmid. Expression quantity of mRNA of CTGF, TIMP-1, procol-α1 and PCIII at group A, B and C decreases, protein expression of CTGF, TIMP-1, procol-α1, PC III in hepatic tissue is lower, where the inhibition of combination RNA interference group (group A) on procol-α1 mRNA transcription and procol-α1 protein expression is superior to that of single interference group (group B and C) (P<0.01 or P<0.05). CONCLUSIONS: RNA interference on CTGF and/or TIMP-1 is obviously a inhibiting factor for mRNA and protein expression of CTGF, TIMP-1, procol-α1 and PCIII. Combination RNA interference on genes of CTGF and TIMP-1 is superior to that of single RNA interference, and this could be a contribution for prevention of precancerous condition.

Expression of matrix metalloproteinases in bovine luteal cells induced by prostaglandin F2α, interferon γ and tumor necrosis factor α.

We recently demonstrated that luteal cells flow out from the ovary via lymphatic vessels during luteolysis. However, the regulatory mechanisms of the outflow of luteal cells are not known. Matrix metalloproteinases (MMPs) can degrade the extracellular matrix and basal membrane, and tissue inhibitors of matrix metalloproteinases (TIMPs) inhibit the activity of MMPs. To test the hypothesis that MMP expression in luteal cells is regulated by luteolytic factors, we investigated the effects of prostaglandin F2α (PGF), interferon γ (IFNG) and tumor necrosis factor α (TNF) on the mRNA expression of MMPs and TIMPs in cultured luteal cells. Luteal cells obtained from the CL at the mid-luteal stage (days 8-12 after ovulation) were cultured with PGF (0.01, 0.1, 1 µM), IFNG (0.05, 0.5, 5 nM) and TNF (0.05, 0.5, 0.5 nM) alone or in combination for 24 h. PGF and IFNG significantly increased the expression of MMP-1 mRNA. In addition, 1 µM PGF in combination with 5 nM IFNG stimulated MMP-1 and MMP-9 mRNA expression significantly more than either treatment alone. In contrast, IFNG significantly decreased the level of MMP-14 mRNA. The mRNA expression of TIMP-1, which preferentially inhibits MMP-1, was suppressed by 5 nM INFG. One µM PGF and 5 nM IFNG suppressed TIMP-2 mRNA expression. These results suggest a new role of MMPs: luteal MMPs stimulated by PGF and IFNG break down the extracellular matrix surrounding luteal cells, which accelerates detachment from the CL during luteolysis, providing an essential prerequisite for outflow of luteal cells from the CL to lymphatic vessels.

Cannabinoids inhibit angiogenic capacities of endothelial cells via release of tissue inhibitor of matrix metalloproteinases-1 from lung cancer cells.

Cannabinoids inhibit tumor neovascularization as part of their tumorregressive action. However, the underlying mechanism is still under debate. In the present study the impact of cannabinoids on potential tumor-to-endothelial cell communication conferring anti-angiogenesis was studied. Cellular behavior of human umbilical vein endothelial cells (HUVEC) associated with angiogenesis was evaluated by Boyden chamber, two-dimensional tube formation and fibrin bead assay, with the latter assessing three-dimensional sprout formation. Viability was quantified by the WST-1 test. Conditioned media (CM) from A549 lung cancer cells treated with cannabidiol, Δ(9)-tetrahydrocannabinol, R(+)-methanandamide or the CB2 agonist JWH-133 elicited decreased migration as well as tube and sprout formation of HUVEC as compared to CM of vehicle-treated cancer cells. Inhibition of sprout formation was further confirmed for cannabinoid-treated A549 cells co-cultured with HUVEC. Using antagonists to cannabinoid-activated receptors the antimigratory action was shown to be mediated via cannabinoid receptors or transient receptor potential vanilloid 1. SiRNA approaches revealed a cannabinoid-induced expression of tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) as well as its upstream trigger, the intercellular adhesion molecule-1, to be causally linked to the observed decrease of HUVEC migration. Comparable anti-angiogenic effects were not detected following direct exposure of HUVEC to cannabinoids, but occurred after addition of recombinant TIMP-1 to HUVEC. Finally, antimigratory effects were confirmed for CM of two other cannabinoid-treated lung cancer cell lines (H460 and H358). Collectively, our data suggest a pivotal role of the anti-angiogenic factor TIMP-1 in intercellular tumor-endothelial cell communication resulting in anti-angiogenic features of endothelial cells.

Activated hepatic stellate cells are dependent on self-collagen, cleaved by membrane type 1 matrix metalloproteinase for their growth.

Stellate cells are distributed throughout organs, where, upon chronic damage, they become activated and proliferate to secrete collagen, which results in organ fibrosis. An intriguing property of hepatic stellate cells (HSCs) is that they undergo apoptosis when collagen is resolved by stopping tissue damage or by treatment, even though the mechanisms are unknown. Here we disclose the fact that HSCs, normal diploid cells, acquired dependence on collagen for their growth during the transition from quiescent to active states. The intramolecular RGD motifs of collagen were exposed by cleavage with their own membrane type 1 matrix metalloproteinase (MT1-MMP). The following evidence supports this conclusion. When rat activated HSCs (aHSCs) were transduced with siRNA against the collagen-specific chaperone gp46 to inhibit collagen secretion, the cells underwent autophagy followed by apoptosis. Concomitantly, the growth of aHSCs was suppressed, whereas that of quiescent HSCs was not. These in vitro results are compatible with the in vivo observation that apoptosis of aHSCs was induced in cirrhotic livers of rats treated with siRNAgp46. siRNA against MT1-MMP and addition of tissue inhibitor of metalloproteinase 2 (TIMP-2), which mainly inhibits MT1-MMP, also significantly suppressed the growth of aHSCs in vitro. The RGD inhibitors echistatin and GRGDS peptide and siRNA against the RGD receptor αVß1 resulted in the inhibition of aHSCs growth. Transduction of siRNAs against gp46, αVß1, and MT1-MMP to aHSCs inhibited the survival signal of PI3K/AKT/IκB. These results could provide novel antifibrosis strategies.

Hepatic apoptosis can modulate liver fibrosis through TIMP1 pathway.

Apoptotic injury participates in hepatic fibrosis, but the molecular mechanisms are not well understood. The present study aimed to investigate the role of inducible TIMP1 in the pathogenesis of hepatic apoptosis-fibrosis. Apoptosis was induced with GCDC, LPS, and alcohol in precision-cut liver slices or bile duct ligation (BDL) in rats, as reflected by caspase-3 activity, TUNEL assay, and apoptosis-related gene profiles. The hepatic fibrosis was detected with Picrosirius staining, hydroxyproline determination, and expression profiling of fibrosis-related genes. Levels of TIMP1 were upregulated by the hepatic apoptosis, but downregulated by caspase inhibitor. The inducible TIMP1 was apoptosis-dependent. Once TIMP1 was inhibited with treatment of TIMP1-siRNA, the fibrotic response was reduced as demonstrated by hydroxyproline assay. In addition, the expression of fibrosis-related genes aSMA, CTGF, and TGFb2r were down-regulated subsequent to the treatment of TIMP1-siRNA. TIMP1 could mediate the expression of fibrosis-related genes. TIMP1 was transcriptionally regulated by nuclear factor c-Jun as demonstrated by EMSA and ChIP assay. The treatment of c-Jun siRNA could significantly decrease the expression of TIMP1 induced by alcohol, GCDC, or LPS treatment. Hepatic apoptosis induces the expression of TIMP1. Inducible TIMP1 can modulate the expression of fibrosis-related genes in liver. TIMP1 pathway is a potential target for therapeutic intervention of fibrotic liver diseases.

Inhibition of myelin-cleaving poteolytic activities by interferon-beta in rat astrocyte cultures. Comparative analysis between gelatinases and calpain-II.

BACKGROUND: Proteolytic enzymes have been implicated in the pathogenesis of Multiple Sclerosis (MS) for both their ability to degrade myelin proteins and for their presence in MS plaques.In this study we investigated whether interferon-beta (IFN-ß) could differently modulate the activity and the expression of proteolytic activities against myelin basic protein (MBP) present in lipopolysaccharide (LPS)-activated astrocytes. METHODOLOGY/PRINCIPAL FINDINGS: Rat astrocyte cultures were activated with LPS and simultaneously treated with different doses of IFN-ß. To assess the presence of MBP-cleaving proteolytic activity, culture supernatants and cellular extracts collected from astrocytes were incubated with exogenous MBP. A MBP-degrading activity was found in both lysates and supernatants from LPS-activated astrocytes and was dose-dependently inhibited by IFN-ß. The use of protease inhibitors as well as the zymographic analysis indicated the presence of calpain II (CANP-2) in cell lysates and gelatinases A (MMP-2) and B (MMP-9) in cell supernatants. RT-PCR revealed that the expression of CANP-2 as well as of MMP-2 and MMP-9 was increased in LPS-activated astrocytes and was dose-dependently inhibited by IFN-ß treatment. The expression of calpastatin, the natural inhibitor of CANPs, was not affected by IFN-ß treatment. By contrast, decreased expression of TIMP-1 and TIMP-2, the natural inhibitors of MMP-9 and MMP-2, respectively, was observed in IFN-ß-treated astrocytes compared to LPS-treated cells. The ratio enzyme/inhibitor indicated that the effect of IFN-ß treatment is more relevant to CANP-2 than on MMPs. CONCLUSIONS/ SIGNIFICANCE: These results suggest that the neuroinflammatory damage during MS involves altered balance between multiple proteases and their inhibitors and indicate that IFN-ß is effective in regulating different enzymatic systems involved in MS pathogenesis.

Antiphotoaging effect of chitooligosaccharides on human dermal fibroblasts.

BACKGROUND/PURPOSE: In the present study, the effect of 3-5 kDa chitooligosaccharide (COS) on homeostasis between the expression of collagen-degrading matrix metalloproteinases (MMPs) and collagen synthesis was investigated using ultraviolet (UV)-A irradiated dermal fibroblasts. METHODS: UV protection imparted by 3-5 kDa COS was measured by examining the UV absorption spectrum. Collagenase MMP secretion was examined using an enzyme-linked immunosorbent assay. The levels of collagenases and collagen synthetic markers were determined by employing the reverse transcriptase-polymerase chain reaction and Western blot analysis. RESULTS: The 3-5 kDa COS not only absorbed UV-A and UV-B light but also inhibited collagenase (MMP-1, MMP-8, and MMP-13) and gelatinase (MMP-2 and MMP-9) MMP expression. The suppression of MMP expression was found to be due to an increase in expression of the tissue inhibitors of MMP (TIMP)-1 and TIMP-2. Treatment with 3-5 kDa COS enhanced collagen synthetic markers such as procollagen, type I, III, and IV collagens in UV-A-irradiated dermal fibroblasts. Furthermore, the effects of 3-5 kDa COS on collagen degradation and collagen synthesis in UV-A irradiated dermal fibroblasts were regulated via the inhibition of activating protein-1 (AP-1) signaling. CONCLUSION: Our results suggest that 3-5 kDa COS can be used to develop as topical applications for antiphotoaging cosmeceuticals as it enhances collagen synthesis.

Inhibitory effect of genistein on the invasive potential of human cervical cancer cells via modulation of matrix metalloproteinase-9 and tissue inhibitors of matrix metalloproteinase-1 expression.

BACKGROUND: One of the most challenging stumbling blocks for the treatment of cancer is the ability of cancer cells to break the natural barriers and spread from its site of origin to non-adjacent regional and distant sites, accounting for high cancer mortality rates. Gamut experimental and epidemiological data advocate the use of pharmacological or nutritional interventions to inhibit or delay various stage(s) of cancer such as invasion and metastasis. Genistein, a promising chemopreventive agent, has gained considerable attention for its powerful anti-carcinogenic, anti-angiogenic and chemosensitizing activities. METHODS: In this study, the cytotoxic potential of genistein on HeLa cells by cell viability assay and the mode of cell death induced by genistein were determined by nuclear morphological examination, DNA laddering assay and cell cycle analysis. Moreover, to establish its inhibitory effect on migration of HeLa cells, scratch wound assay was performed and these results were correlated with the expression of genes involved in invasion and migration (MMP-9 and TIMP-1) by RT-PCR. RESULTS: The exposure of HeLa cells to genistein resulted in significant dose- and time-dependent growth inhibition, which was found to be mediated by apoptosis and cell cycle arrest at G(2)/M phase. In addition, it induced migration-inhibition in a time-dependent manner by modulating the expression of MMP-9 and TIMP-1. CONCLUSION: Our results signify that genistein may be an effective anti-neoplastic agent to prevent cancer cell growth and invasion and metastasis. Therefore therapeutic strategies utilizing genistein could be developed to substantially reduce cancer morbidity and mortality.

Tumor cell-derived Timp-1 is necessary for maintaining metastasis-promoting Met-signaling via inhibition of Adam-10.

In many different tumor entities, increased expression of tissue inhibitor of metalloproteinases-1 (Timp-1) is associated with poor prognosis. We previously reported in mouse models that elevated systemic levels of Timp-1 induce a gene expression signature in the liver microenvironment increasing the susceptibility of this organ to tumor cells. This host effect was dependent on increased activity of the hepatocyte growth factor (Hgf)/hepatocyte growth factor receptor (Met) signaling pathway. In a recent study we showed that Met signaling is regulated by Timp-1 as it inhibits the Met sheddase A disintegrin and metalloproteinase-10 (Adam-10). The aim of the present study was to elucidate whether the metastatic potential of tumor cells benefits from autocrine Timp-1 as well and involves Adam-10 and Met signaling. In a syngeneic murine model of experimental liver metastasis Timp-1 expression and Met signaling were localized within metastatic colonies and expressed by tumor cells. Knock down of tumor cell Timp-1 suppressed Met signaling in metastases and inhibited metastasis formation and tumor cell-scattering in the liver. In vitro, knock down of tumor cell Timp-1 prevented Hgf-induced Met phosphorylation. Consequently, knock down of Met sheddase Adam-10 triggered auto-phosphorylation and responsiveness to Hgf. Accordingly, Adam-10 knock down increased Met phosphorylation in metastatic foci and induced tumor cell scattering into the surrounding liver parenchyma. In conclusion, these findings show that tumor cell-derived Timp-1 acts as a positive regulator of the metastatic potential and support the concept that proteases and their natural inhibitors, as members of the protease web, are major players of signaling during normal homeostasis and disease.