Active Matrix Metalloproteinases Research Articles

Design, (Radio)Synthesis, and in Vitro and in Vivo Evaluation of Highly Selective and Potent Matrix Metalloproteinase 12 (MMP-12) Inhibitors as Radiotracers for Positron Emission Tomography.

Dysregulated levels of activated matrix metalloproteinases (MMPs) are linked to different pathologies, such as cancer, atherosclerosis, neuroinflammation, and arthritis. Therefore, imaging of MMPs with positron-emission tomography (PET) represents a powerful tool for the diagnosis of MMP-associated diseases. Moreover, to distinguish between the distinct functions and roles of individual MMPs in particular pathophysiological processes, their specific imaging must be realized with radiolabeled tracers, such as fluorine-18-labeled MMP inhibitors (MMPIs). Therefore, fluorinated dibenzofuransulfonamide-based MMPIs showing excellent inhibition of MMP-12 and selectivity for MMP-12 over other MMPs were prepared. MMP-12 is a key enzyme in diseases such as chronic obstructive pulmonary disease (COPD) and atherosclerosis. Because of their promising in vitro properties, three candidates (4, 9, and 19) were selected from this library, and radiofluorinated analogues ([18F]4, [18F]9, and [18F]19) were successfully synthesized. Initial in vitro serum stability and in vivo biodistribution studies of the radiolabeled MMPIs with PET demonstrated their potential benefit for preferable MMP-12 imaging.

EGFR-mediated matrix metalloproteinase-7 up-regulation promotes epithelial-mesenchymal transition via ERK1-AP1 axis during ovarian endometriosis progression.

Endometriosis, characterized by extrauterine development of endometrial glands and stroma, is associated with increased risk of ovarian cancer development. In the present study, we investigated the role of matrix metalloproteinase-7 (MMP-7) on epithelial-mesenchymal transition (EMT) during ovarian endometriosis ( N = 40) progression. We found that the expressions of EMT markers such as vimentin, slug, and N-cadherin were significantly elevated in late stages of ovarian endometriosis compared with those found in early stages. In addition, the activity and expression of ectopic MMP-7 were significantly higher in the late stages of endometriosis. In vitro studies revealed that increased expression of MMP-7 as well as epidermal growth factor (EGF), which was significantly elevated in severe stages of ovarian endometriosis, induced EMT in endocervical epithelial cells (End1/E6E7). Silencing the MMP-7 transcripts using small interfering RNA attenuated EMT responses, whereas treatment with recombinant active MMP-7 promoted EMT by cleaving E-cadherin. In addition, EGF receptor (EGFR) inhibitor treatments regressed endometriotic lesions and decreased MMP-7 activities in a mouse model of endometriosis. Chromatin immunoprecipitation assay identified EGFR-mediated ERK1 and activator protein 1 signaling for the transcriptional activation of MMP-7 in End1/E6E7 epithelial cells.-Chatterjee, K., Jana, S., DasMahapatra, P., Swarnakar, S. EGFR-mediated matrix metalloproteinase-7 up-regulation promotes epithelial-mesenchymal transition via ERK1-AP1 axis during ovarian endometriosis progression.

Levels of MMP-9 in patients with intracranial aneurysm: Relation with risk factors, size and clinical presentation

BackgroundIntracranial aneurysms are arterial anomalies affecting 2% to 3% of the general population in the world and these ruptures are associated with a high mortality. Some risk factors, such as age, gender, smoking, alcohol, hypertension and familial history are associated with the number of aneurysms and their size. In addition, inflammatory processes within the blood vessels of the brain can activate matrix metalloproteinase-9 (MMP-9), which degrades various components of the extracellular matrix, such as elastin. Thereby, this work has aimed at evaluating the relationship between plasma MMP-9 levels and the risk factors that are associated with intracranial aneurysm, as well as investigating the aneurysm statuses (ruptured and unruptured) and comparing them with the control volunteers. MethodsBetween August 2014 to June 2016, blood samples were collected from 282 patients (204 ruptured and 78 unruptured saccular intracranial aneurysms) and 286 control volunteers. The MMP-9 plasma levels were measured by ELISA. Statistical analyzes were performed with SPSS software when using parametric or nonparametric tests, after the normality tests. ResultsHigher levels of MMP-9 were found in the aneurysm groups as a whole and when they were stratified by rupture status, then compared with the control group (p < 0.0001). When stratifying them by diameter, those smaller than 7 mm presented high levels of MMP-9 (p < 0.0001), especially in the ruptured ones. As for risk factors, hypertension and smoking were the most important. However, hypertension was mostly associated with the ruptured aneurysms (p < 0.0001). ConclusionsHigh levels of MMP-9 were found in smaller ruptured and unruptured intracranial aneurysms (<7 mm) with strongest statistical associations than other sizes, especially when associated with smoking and hypertension.

The Dynamics of Impaired Blood-Brain Barrier Restoration in a Rat Model of Co-morbid Injury

Defect in brain microperfusion is increasingly recognized as an antecedent event to Alzheimer's disease (AD) and ischemia. Nevertheless, studies on the role of impaired microperfusion as a pathological trigger to neuroinflammation, Aβ deposition as well as blood-brain barrier (BBB) disruption, and the etiological link between AD and ischemia are lacking. In this study, we employ in vivo sequential magnetic resonance imaging (MRI) and computed tomography (CT) imaging in a co-morbid rat model of β-amyloid toxicity (Aβ) and ischemia (ET1) with subsequent histopathology of striatal lesion core and penumbra at 1, 7, and 28days post injury. Within 24h, cerebral injury resulted in increased BBB permeability due to the dissolution of β-dystroglycan (β-DG) and basement membrane laminin by active matrix metalloproteinase9 (MMP9). As a result, net flow of circulating IgG down a hydrostatic gradient into the parenchyma led to vasogenic edema and impaired perfusion, thus increasing the apparent hyperintensity in true fast imaging with steady-state free precession (true FISP) imaging and acute hypoperfusion in CT. This was followed by a slow recruitment of reactive astroglia to the affected brain and depolarization of aquaporin4 (AQP4) expression resulting in cytotoxic edema-in an attempt to resolve vasogenic edema. On d28, functional BBB was restored in ET1 rats as observed by astrocytic MMP9 release, β-DG stabilization, and new vessel formation. This was confirmed by reduced hyperintensity on true FISP imaging and normalized cerebral blood flow in CT. While, Aβ toxicity alone was not detrimental enough, Aβ+ET1 rats showed delayed differential expression of MMP9, late recruitment of astroglial cells, protracted loss of AQP4 depolarization, and thus delayed BBB restoration and cerebral perfusion.

Oleuropein potentiates anti-tumor activity of cisplatin against HepG2 through affecting proNGF/NGF balance

AimsOleuropein is considered as a new chemotherapeutic agent in human hepatocellular carcinoma (HCC) while, its exact underlying molecular mechanism still not yet explored. In addition, cisplatin is a standard anticancer drug against solid tumors with toxic side effects. Therefore, we conducted this study to assess antitumor activity of oleuropein either alone or in combination with cisplatin against HepG2, human HCC cell lines, via targeting pro-NGF/NGF signaling pathway. Main methodsHepG2 cells were treated with cisplatin (20, 50, 100 μM) and oleuropein (100, 200, 300 and 400 μM) as well as some of the cells were treated with 50 μM cisplatin and different concentrations of oleuropein. Gene expressions of nerve growth factor (NGF), matrix metalloproteinase-7 (MMP-7) and caspase-3 were evaluated by real time-PCR. In addition, protein levels of NGF and pro-form of NGF (pro-NGF) were measured by ELISA while, nitric oxide (NO) content was determined colorimetrically. Key findingsCisplatin treatment showed a significant elevation of NO content and pro-NGF protein level with a marked reduction of NGF protein level in addition to the upregulation of caspase-3 along with downregulation of MMP-7 gene expressions in a dose-dependent manner. However, the combination of 50 μM cisplatin and 200 μM oleuropein showed the most potent effect on the molecular level when compared with oleuropein or cisplatin alone. SignificanceOur results showed for the first time that the anti-tumor activity of oleuropein against HCC could be attributed to influencing the pro-NGF/NGF balance via affecting MMP-7 activity without affecting the gene expression of NGF. Concurrent treatment with both oleuropein and cisplatin could lead to more effective chemotherapeutic combination against HCC.

Effect of andecaliximab (anti-MMP9) on proteolysis of IL-7 in vitro, TCR diversity in mice, and serum IL-7 in gastric cancer patients in combination with chemotherapy.

101 Background: Andecaliximab (andeca) is a monoclonal antibody that selectively inhibits matrix metalloproteinase 9 (MMP9). IL-7 selectively enhances the proliferation and survival of naïve, memory, and effector T-cells (but not regulatory T-cells) in the periphery. Previous clinical trials with systemic recombinant IL-7 therapy increased TCR diversity. In the disease setting, elevated circulating IL-7 may be due to a compensatory increase in IL-7 production as demonstrated in mice upon inhibition of IL-7 signaling. Methods: An in vitro screen, in which recombinant active human MMP9 was incubated with &gt; 140 human recombinant folded proteins, identified IL-7 as the most efficient substrate of MMP9. Results: IL-7 proteolysis occurred between A128:L129. IL-7 proteolysis altered the global protein structure, evidenced by a loss of cooperative unfolding observed with intact IL-7 (intrinsic fluorescence, Tm = 59.3o C). Mouse MMP9 proteolyzed mouse IL-7 in vitro. In the orthotopic syngeneic NeuT mouse model, MMP9 inhibition reduced tumor growth (p = 0.0005). In a 7-day NeuT study, anti-MMP9 alone improved TCR diversity (decreased clonality) within tumor-infiltrating T-cells (Dunnett’s p = 0.0083). Further, anti-MMP9 and anti-PDL1 co-treatment promoted an increase in CD3+ cells (p = 0.01), CD4+ T cells (p = 0.006), and CD8+ T cells (p = 0.013) concomitant with a decrease in tumor-associated CD25+ FoxP3+ regulatory T cells (p = 0.04) in the tumor. In gastric cancer patient serum, pro-MMP9 (p &lt; 0.0001), active MMP9 (p &lt; 0.0001), and IL-7 (p &lt; 0.0001) were higher than healthy controls. Serum IL-7 levels were normalized upon treatment with andeca plus mFOLFOX6 (N = 40; FDR-corrected p &lt; 0.001) in a Phase I gastric cancer study. Conclusions: MMP9 proteolyzed IL-7 in vitro. Specific MMP9 inhibition in a mouse tumor model improved TCR diversity. Andeca +mFOLFOX6 therapy normalized serum IL-7 levels, which could be due to andeca, chemotherapy, or disease resolution. The functional implications of IL-7 proteolysis by MMP9 in gastric cancer are currently under investigation.

FOXO3a (Forkhead Transcription Factor O Subfamily Member 3a) Links Vascular Smooth Muscle Cell Apoptosis, Matrix Breakdown, Atherosclerosis, and Vascular Remodeling Through a Novel Pathway Involving MMP13 (Matrix Metalloproteinase 13).

Vascular smooth muscle cell (VSMC) apoptosis accelerates atherosclerosis and promotes breakdown of the extracellular matrix, but the mechanistic links between these 2 processes are unknown. The forkhead protein FOXO3a (forkhead transcription factor O subfamily member 3a) is activated in human atherosclerosis and induces a range of proapoptotic and other transcriptional targets. We, therefore, determined the mechanisms and consequences of FOXO3a activation in atherosclerosis and arterial remodeling after injury. Expression of a conditional FOXO3a allele (FOXO3aA3ER) potently induced VSMC apoptosis, expression and activation of MMP13 (matrix metalloproteinase 13), and downregulation of endogenous TIMPs (tissue inhibitors of MMPs). mmp13 and mmp2 were direct FOXO3a transcriptional targets in VSMCs. Activation of endogenous FOXO3a also induced MMP13, extracellular matrix degradation, and apoptosis, and MMP13-specific inhibitors and fibronectin reduced FOXO3a-mediated apoptosis. FOXO3a activation in mice with VSMC-restricted FOXO3aA3ER induced MMP13 expression and activity and medial VSMC apoptosis. FOXO3a activation in FOXO3aA3ER/ApoE-/- (apolipoprotein E deficient) mice increased atherosclerosis, increased necrotic core and reduced fibrous cap areas, and induced features of medial degeneration. After carotid artery ligation, FOXO3a activation increased VSMC apoptosis, VSMC proliferation, and neointima formation, all of which were reduced by MMP13 inhibition. FOXO3a activation induces VSMC apoptosis and extracellular matrix breakdown, in part, because of transcriptional activation of MMP13. FOXO3a activation promotes atherosclerosis and medial degeneration and increases neointima after injury that is partly dependent on MMP13. FOXO3a-induced MMP activation represents a direct mechanistic link between VSMC apoptosis and matrix breakdown in vascular disease.

The promoting effects of SNAI1 activating matrix metalloproteinase on choroidal neovascularization under hypoxia

Objective To investigate whether vascular endothelial cells in choroidal neovascularization whether hypoxia condition can up-regulate SNAI1 and activate matrix metalloproteinase (MMP)2 and MMP9 therefore to participate in choroidal neovascularization(CNV). Methods Sixteen SPF male C57 mice aged 6-8 weeks were divided into control group and model group.CNV models were induced by retinal laser photocoagulation, and flat-mount and frozen sections of retinal pigment epithelium (RPE)-choroid-sclera compound were prepared at 7 days after modeling.The CNV in flat-mount was examined by Isolectin B4 staining, and the location of SNAI1, MMP2 and MMP9 in frozen sections was determined by immunofluorescence technology.The expression of SNAI1, MMP2 and MMP9 at mRNA level in CNV was detected by real-time fluorescence quantitative PCR (real-time PCR). The use and care of experimental animals complied with Statement for the Use of Animals in Ophthalmic and Visual Research.The RF/6A cells were divided into normoxia group and hypoxia group and cultured for 24 hours in 5%CO2 condition and mix condition of 94%N2, 5%CO2 and 1%O2, respectively.The expression of SNAI1, MMP2 and MMP9 in the cells at mRNA and protein levels was detected by real-time PCR and Western blot assay, respectively.Small interfering RNA of SNAI1 (siSNAI1) was transfected into the cells, and then the expression of MMP2 in the cells at mRNA and protein levels was detected by real-time PCR and Western blot assay, respectively, and the migrating number of the cells was assayed by Transwell chamber assay. Results CD31 and SNAI1 positive-response cells were seen in RPE-choroid-sclera flat-mounts under the laser scanning confocal microscope.The relative expression levels of SNAI1 mRNA and MMP2 mRNA in RPE-choroid-sclera tissues were higher in the model group than those in the control group (SNAI1 mRNA: 1.291±0.060 vs.0.759±0.074, P=0.001; MMP2 mRNA: 1.610±0.424 vs.0.772±0.080, P=0.044). The expression of MMP9 mRNA was not significantly elevated between model group and control group (P>0.05). The relative expression level of MMP2 mRNA was higher in comparison with MMP9 mRNA in the model group (P 0.05). The relative expressions of MMP2 mRNA in the cells were 0.217±0.036 and 0.818±0.105, and those of MMP2 protein in the cells were 0.236±0.009 and 1.043±0.120 in the hypoxia+ siSNAI1 group and only hypoxia group, respectively, with significant differences between them (P=0.002, 0.003). The migrating number of the cells was (254.60±71.31)/field in the hypoxia+ siSNAI1 group, which was significantly less than (534.10±96.21) /field in the control group (P=0.029). Conclusions The hypoxic environment at CNV can activate MMP2 by up-regulating the expression of SNAI1, which promotes the migration of vascular endothelial cells and therefore participates in CNV formation, and the intervention of SNAI1 activation under the hypoxic condition can inhibit this process. Key words: Choroidal neovascularization; Matrix metalloproteinase; Small interfering RNA; Transcriptional factor SNAI1; Vascular endothelial cells; Disease model, C57 mice; Hypoxia

Association of chairside salivary aMMP-8 findings with periodontal risk assessment parameters in patients receiving supportive periodontal therapy.

PurposeThe aim of this retrospective cross-sectional study was to evaluate whether salivary findings of active matrix-metalloproteinase 8 (aMMP-8) chairside (point of care; POC) tests were associated with periodontal risk assessment parameters in patients receiving supportive periodontal therapy (SPT).MethodsA total of 125 patients receiving regular SPT were included, and their records were examined. The following inclusion criteria were used: a diagnosis of chronic periodontitis, at least 1 non-surgical periodontal treatment (scaling and root planning) with following regular SPT (minimum once a year), at least 6 remaining teeth, and clinical and aMMP-8 findings that were obtained at the same appointment. In addition to anamnestic factors (e.g., smoking and diabetes), oral hygiene indices (modified sulcus bleeding index [mSBI] and approximal plaque index), periodontal probing depth simultaneously with bleeding on probing, and dental findings (number of decayed, missing, and filled teeth) were recorded. Salivary aMMP-8 levels were tested using a commercial POC test system (Periomarker, Hager & Werken, Duisburg, Germany). Statistical analysis was performed using the t-test, Mann-Whitney U test, Fisher's exact test, and χ2 test, as appropriate (P<0.05).ResultsOnly the mSBI was significantly associated with positive salivary aMMP-8 findings (aMMP-8 positive: 27.8%±20.9% vs. aMMP-8 negative: 18.0%±14.5%; P=0.017). No significant associations were found between aMMP-8 and smoking, diabetes, periodontal parameters, or parameters related to the maintenance interval (P>0.05).ConclusionsSalivary aMMP-8 chairside findings were not associated with common parameters used for periodontal risk assessment in patients receiving SPT. The diagnostic benefit of POC salivary aMMP-8 testing in risk assessment and maintenance interval adjustment during SPT remains unclear.

A fluorogenic probe for sensitive detection of MMP-2/9 activities in clinical colorectal cancer tissues

Developing a novel fluorogenic probe which can be specifically recognized by active matrix metalloproteinase-2 and matrix metalloproteinase-9 (MMP-2/9) to rapidly and accurately detect the clinical colorectal cancer is significant but remains a great challenge. Herein, we have designed and synthesized a novel fluorescence resonance energy transfer (FRET)-based probe Dab-GPLGVRGY-FITC for specific detection of MMP-2/9 activity. The probe was prepared by conjugating fluorescein-N3 (FITC-N3) and fluorescent quencher dabcyl-NHS to MMP-2/9-responsive peptide substrate GPLGVRGY through copper (I)-catalyzed ″click″ and amidation reactions, respectively. To assess the specificity and sensitivity of the probe for active MMP-2/9 in vitro , the enzymatic assays were firstly performed with recombinant MMP-2/9. Fluorescent signal change of the mixed solution of probe and MMP-2 with and without treatment of MMP-2 specific inhibitor was recorded at 520 nm with fluorescence spectrometer. The capability of probe for specific detection of endogenous MMP-2/9 activity was further evaluated with human colorectal cancer cell LoVo and 30 clinical colorectal cancer tissue samples. The in vitro enzymatic assay results indicated that the probe showed good specificity and sensitivity for recombinant MMP-2/9 comparing to other proteins. The detection limit for recombinant MMP-2 is estimated to be 14 ng mL−1. Besides, the confocal laser scanning microscopy (CLSM) imaging results showed that enhanced green fluorescence signals were collected for LoVo cells upon treatment with probe. However, most of the fluorescence was readily competed away by pretreatment of the probe with inhibitor. In sharp contrast, only weak fluorescence was observed for normal gastric mucosa epithelial GES-1 cells. Additionally, the lysates of 15 clinical colorectal tissues displayed ~5 to 9-fold fluorescence enhancements after treatment of the probe in comparison with normal gastritis tissues. However, the fluorescence intensities of 15 peficancerous tissue lysates was only ~0.9 to 3-fold higher than normal tissues. Most notably, in comparison with normal gastritis tissue, strong green fluorescence was observed for both colorectal tissue and peficancerous tissue once the probe was sprayed onto the tissue surface. In summary, this probe shows good specificity and sensitivity for the detection of active MMP-2 both in cancer cell and clinical cancer tissue, which may have great potential for early diagnosis of MMP-2/9 active colorectal cancer in clinic.

Crosstalk Between Histone and DNA Methylation in Regulation of Retinal Matrix Metalloproteinase-9 in Diabetes.

PurposeDiabetes activates matrix metalloproteinase-9 (MMP-9), and MMP-9 via damaging retinal mitochondria, activates capillary cell apoptosis. MMP-9 promoter has binding sites for many transcription factors, and in diabetes its promoter undergoes epigenetic modifications, including histone modifications and DNA methylation. Enhancer of Zeste homolog 2 (Ezh2), which catalyzes dimethylation/trimethylation of histone 3 lysine 27 (H3K27me2 and me3), is also associated with DNA methylation. Our aim was to investigate link(s) between histone and DNA modifications in the regulation of MMP-9.MethodsUsing human retinal endothelial cells, and also retinal microvessels from diabetic rats, effect of hyperglycemia on H3K27me3, and recruitment of Ezh2 at the MMP-9 promoter were quantified by chromatin-immunoprecipitation technique. Role of H3K27 trimethylation in regulating DNA methylation-transcription of MMP-9 was determined by regulating Ezh2 by its specific siRNA and also a pharmacologic inhibitor.ResultsHyperglycemia elevated H3K27me3 levels and the recruitment of Ezh2 at the MMP-9 promoter, and increased the enzyme activity of Ezh2. Inhibition of Ezh2 attenuated recruitment of both DNA methylating (Dnmt1) and hydroxymethylating (Tet2) enzymes and 5 hydroxymethyl cytosine at the same region of the MMP-9 promoter, and prevented increase in MMP-9 transcription and mitochondrial damage.ConclusionsActivation of Ezh2 in diabetes, via trimethylation of H3K27, facilitates recruitment of the enzymes responsible for regulation of DNA methylation of the MMP-9 promoter, resulting in its transcriptional activation. Thus, a close crosstalk between H3K27 trimethylation and DNA methylation in diabetes plays a critical role in the maintenance of cellular epigenetic integrity of MMP-9.

Abstract 24034: MFG-E8 Fragment Medin and Arterial Aging

Background: Aging increases milk fat globule EGF-VIII (MFG-E8) expression in the rat, nonhuman primate and human aortic walls, facilitating the invasion, proliferation, and proinflammation of vascular smooth muscle cells (VSMCs). The MFG-E8 C-terminal fragment medin has been reported to be involved in the necrosis of VSMCs at the inflammatory sites of aortic dissection, however, its cleavage processes, aortic wall levels with aging and bio-role in VSMCs remains to be determined. Material and Methods: In this study, immunofluorescence, immunohistochemistry and western blot analysis demonstrate that MFG-E8 and its fragment medin ( Figure ), activated matrix metalloproteinase type II (MMP-2) and platelet derived growth factor receptor type-beta (PDGFR-β) protein levels were markedly increased in human grossly normal aortic walls from old (n=10, &gt;50 yrs) vs. younger donors (n=10, &lt;25 yrs). Importantly, exposure of medin peptide (20 to100nM) to primary cultured VSMCs isolated from young (8 mo) and old (30 mo) FXBN rat aortae significantly increased MMP-2 activation, PDGFR-β expression, and migratory capacity measured by a modified Boyden chamber in a dose-dependent manner in both also. Separate exposure of activated MMP-2 to recombinant human MFG-E8 protein and also to MFG-E8 enriched old human aortic protein markedly increased the cleavage product medin in both, which was substantially inhibited by an MMP inhibitor, GM6001. Exposure of medin to VSMCs did not significantly affect the expression of cell cycle related proteins. In addition, PDGF-BB treatment markedly activated MMP-2 in both young and old VSMCs, which was substantially reduced by a PDGFR-β inhibitor, RTK. Conclusion: Taken together, targeting MFG-E8 or its cleavage product medin is a novel approach to the prevention or treatment of large arterial aging or age-associated disease.

Expression of CD147 in laser-induced rat choroidal neovascularization model

Background The pathology of choroidal neovascularization (CNV) is not clear.CD147 plays a critical role in angiogenesis through activating matrix metalloproteinases (MMPs) and degradation of extracellular matrix (ECM). However, the study of CD147 in CNV is rare. Objective This study was to investigate the expression of CD147 and vascular endothelial growth factor (VEGF) in laser-induced rat CNV model. Methods Thirty clean BN rats were divided into normal control group (six rats) and laser group (24 rats) according to the random number table.CNV was induced on laser photocoagulation group rats using 532 nm multiple-wave length photocoagulation.The development of CNV was followed up by fundus fluorescence angiography (FFA) on 1 day, 7, 14 and 21 days post-photocoagulation.The expression of CD147 protein in retinal pigment epithelium(RPE)-choroid-sclera complex was examined by Western blot in normal control group and 1 day, 7, 14, 21 days post-photocoagulation group.The vitreous VEGF concentrations of those groups were detected by ELISA. Results Disk like fluorescein leakage was detected on 7 days after photocoagulation, and CNV was formed on 14 days after photocoagulation.The expressions of CD147 protein were 1.00±0.43, 0.97±0.53, 0.99±0.45, 1.56±0.67 and 2.27±0.54, and the concentrations of vitreous VEGF were (72.96±29.95), (79.36±10.46), (103.82±32.94), (166.05±21.54) and (195.64±39.90)pg/ml in normal control group and 1 day, 7, 14, 21 days post-photocoagulation group, with significant differences among the groups (CD147: F=10.95, P<0.01; VEGF: F=304.50, P<0.01). The expressions of CD147 and VEGF were significantly higher in the 14, 21 days post-photocoagulation groups than those in the normal control group (all at P<0.05). Conclusions The expression of CD147 increases as the induction of CNV, in accordance with the expression of VEGF.CD147 may play an important role in the early development of CNV. Key words: Choroidal neovascularization; CD147; Vascular endothelial growth factor; Matrix metalloproteinases

Sustained activation of P2X7 induces MMP-2-evoked cleavage and functional purinoceptor inhibition.

P2X7 purinoceptor promotes survival or cytotoxicity depending on extracellular adenosine triphosphate (ATP) stimulus intensity controlling its ion channel or P2X7-dependent large pore (LP) functions. Mechanisms governing this operational divergence and functional idiosyncrasy are ill-understood. We have discovered a feedback loop where sustained activation of P2X7 triggers release of active matrix metalloproteinase 2 (MMP-2), which halts ion channel and LP responses via the MMP-2-dependent receptor cleavage. This mechanism operates in cells as diverse as macrophages, dystrophic myoblasts, P2X7-transfected HEK293, and human tumour cells. Given that serum-born MMP-2 activity also blocked receptor functions, P2X7 responses in vivo may decrease in organs with permeable capillaries. Therefore, this mechanism represents an important fine-tuning of P2X7 functions, reliant on both cell-autonomous and extraneous factors. Indeed, it allowed evasion from the ATP-induced cytotoxicity in macrophages and human cancer cells with high P2X7 expression levels. Finally, we demonstrate that P2X7 ablation eliminated gelatinase activity in inflamed dystrophic muscles in vivo. Thus, P2X7 antagonists could be used as an alternative to highly toxic MMP inhibitors in treatments of inflammatory diseases and cancers.

BMAL1 facilitates trophoblast migration and invasion via SP1-DNMT1/DAB2IP pathway in recurrent spontaneous abortion.

The underlying mechanism about rhythms and epigenetics leading to aberrant trophoblast migration and invasion in recurrent spontaneous abortion (RSA) remains unknown. Brain and muscle ARNT-like protein 1 (BMAL1) is considered as a crucial role in fertility, and polymorphism of BMAL1 gene has been reported to be associated with risk of miscarriage. However, the functional role of BMAL1 in RSA is not fully understood. Previous study shows the descended expression of DNA 5′-cytosine-methyltransferases 1 (DNMT1) in the villous of early pregnancy loss. Thus, understanding of the regulation of DNMT1 expression may be of significance for the elucidation of the process of RSA. Using HTR-8/SVneo and JEG-3 cell lines, we certified the induction of specificity protein 1 (SP1) to DNMT1 and DAB2 interaction protein (DAB2IP), respectively, both of which further activated matrix metallo-proteinase 2/9 (MMP2/9), bringing out changes in trophoblast migration and invasion. Notably, BMAL1 functioned as a positive upstream factor of SP1 only in HTR-8/SVneo cells but not in JEG-3 cells, inducing SP1-DNMT1/DAB2IP pathway and facilitating migration and invasion of trophoblasts. In addition, progesterone might restore the down-regulation of BMAL1 and downstream pathway in a dose-dependent manner. Last but not least, the decreased abundance of BMAL1 was correlated positively with that of SP1, DNMT1, DAB2IP, MMP2 and MMP9 in human villous specimens of RSA. Our results demonstrate that the induction of BMAL1 to SP1 contributes to the expression of DNMT1 and DAB2IP, respectively, activating trophoblast migration and invasion. The deregulation of the BMAL1-mediated pathway in RSA can be rescued by progesterone.

Blocking the ERK1/2 signal pathway can inhibit S100A12 induced human aortic smooth muscle cells damage.

Increased levels of S100A12 and activated matrix metalloproteinase 2/9 (MMP-2/9) produced by human aortic smooth muscle cells (HASMCs) have recently implicated in the development of thoracic aortic disease. In the present study, we investigated the effect of S100A12 on HASMCs and identified the intracellular signal pathways involved by Western blot. The results were shown that up-expression of S100A12 in HASMCs induced cell apoptosis and inhibited cell proliferation. Additionally, S100A12 significantly increased the expression of MMP-2, MMP-9, and VCAM-1 in HASMCs at translational levels. Furthermore, our results also showed that S100A12 induced HASMCs damage by increased related proteins expression was mediated by the activation of ERK1/2 signal pathway, whereas p38 MAPK had no effect on those processes. Blocked the activation of ERK1/2 could decrease S100A12 induced the apoptosis and inhibited cell proliferation of HASMCs. In conclusion, these results indicated that S100A12 could increase the expression of MMP-2, MMP-9, and vascular cell adhesion molecule 1 (VCAM-1) in HASMCs via activation of ERK1/2 signal pathway, which leads to injury of HASMCs. Therefore, antagonists of ERK1/2 may be useful for treating thoracic aortic dissection.

Glutaminase and MMP-9 Downregulation in Cortex and Hippocampus of LPA1 Receptor Null Mice Correlate with Altered Dendritic Spine Plasticity.

Lysophosphatidic acid (LPA) is an extracellular lipid mediator that regulates nervous system development and functions acting through G protein-coupled receptors (GPCRs). Here we explore the crosstalk between LPA1 receptor and glutamatergic transmission by examining expression of glutaminase (GA) isoforms in different brain areas isolated from wild-type (WT) and KOLPA1 mice. Silencing of LPA1 receptor induced a severe down-regulation of Gls-encoded long glutaminase protein variant (KGA) (glutaminase gene encoding the kidney-type isoforms, GLS) protein expression in several brain regions, particularly in brain cortex and hippocampus. Immunohistochemical assessment of protein levels for the second type of glutaminase (GA) isoform, glutaminase gene encoding the liver-type isoforms (GLS2), did not detect substantial differences with regard to WT animals. The regional mRNA levels of GLS were determined by real time RT-PCR and did not show significant variations, except for prefrontal and motor cortex values which clearly diminished in KO mice. Total GA activity was also significantly reduced in prefrontal and motor cortex, but remained essentially unchanged in the hippocampus and rest of brain regions examined, suggesting activation of genetic compensatory mechanisms and/or post-translational modifications to compensate for KGA protein deficit. Remarkably, Golgi staining of hippocampal regions showed an altered morphology of glutamatergic pyramidal cells dendritic spines towards a less mature filopodia-like phenotype, as compared with WT littermates. This structural change correlated with a strong decrease of active matrix-metalloproteinase (MMP) 9 in cerebral cortex and hippocampus of KOLPA1 mice. Taken together, these results demonstrate that LPA signaling through LPA1 influence expression of the main isoenzyme of glutamate biosynthesis with strong repercussions on dendritic spines maturation, which may partially explain the cognitive and learning defects previously reported for this colony of KOLPA1 mice.

Mesenchymal stem cells correct impaired diabetic wound healing by decreasing ECM proteolysis.

Impaired diabetic wound healing is associated with a dermal extracellular matrix protein profile favoring proteolysis; within the healing diabetic wound, this is represented by an increase in activated matrix metalloproteinase (MMPs). Treatment of diabetic wounds with mesenchymal stem cells (MSCs) has been shown to improve wound healing; however, there has not yet been an assessment of their ability to correct dysregulation of MMPs in diabetic wounds. Furthermore, there has been no prior assessment of the role of microRNA29b (miR-29b), an inhibitory regulatory molecule that targets MMP-9 mRNA. Using in vitro models of fibroblast coculture with MSCs and in vivo murine wound healing models, we tested the hypothesis that MSCs correct dysregulation of MMPs in a microRNA-29b-dependent mechanism. In this study, we first demonstrated that collagen I and III protein content is significantly reduced in diabetic wounds, and treatment with MSCs significantly improves collagen I content in both nondiabetic and diabetic wounds. We then found that MMP-9 gene expression and protein content were significantly upregulated in diabetic wounds, indicating elevated proteolysis. Treatment with MSCs resulted in a decrease in MMP-9 gene expression and protein content level in diabetic wounds 3 and 7 days after wounding. Zymographic analysis indicated that MSC treatment also decreased the amount of activated MMP-9 present in diabetic wounds. Furthermore, miR-29b expression was inversely associated with MMP-9 gene expression; miR-29b expression was decreased in diabetic wounds and diabetic fibroblast. Following treatment of diabetic wounds with MSCs, as well as in diabetic fibroblasts cocultured with MSCs, miR-29b was significantly increased. These findings suggest a potential mechanism through which MSCs enhance diabetic wound healing by improving collagen I content in diabetic wounds through decreasing MMP-9 expression and increasing miR-29b expression.

CCR7 Mediates TGF-β1-Induced Human Malignant Glioma Invasion, Migration, and Epithelial-Mesenchymal Transition by Activating MMP2/9 Through the Nuclear Factor KappaB Signaling Pathway.

Chemokine receptor 7 (CCR7) has emerged as an inducer of invasion, migration, and epithelial-mesenchymal transition (EMT) in cancer. In this research, human malignant glioma cells were stimulated with transforming growth factor beta 1 (TGF-β1) and siCCR7. The data show that CCR7 mediates TGF-β1-induced EMT, migration, and invasion in U251 and U87 cells and that these effects of TGF-β1 were reversed by treatment with siCCR7 or a CCR7 neutralizing antibody. Importantly, the TGF-β1-mediated increase in nuclear factor kappaB (NF-κB) activity in human glioma cells was reduced by treatment with siCCR7 or a CCR7 neutralizing antibody. Furthermore, CCR7 was shown to mediate TGF-β1-induced glioma cancer cell migration by activating matrix metalloproteinase 2 (MMP2)/9. Our results indicate that CCR7 mediates TGF-β1-induced MMP2/9 expression through NF-κB signaling, thus facilitating glioma cell migration, invasion, and EMT, all of which progressively increase with glioblastoma progression. These findings indicate that CCR7 is a potential therapeutic target for malignant glioma.

Overexpression of carbonic anhydrase IX induces cell motility by activating matrix metalloproteinase-9 in human oral squamous cell carcinoma cells.

Oral cancer is a solid malignant tumor that is prone to occur following hypoxia. There are no clear studies showing a link between hypoxia and oral carcinogenesis. Carbonic anhydrase IX (CAIX), which is a hypoxia-induced transmembrane protein, is highly expressed in various types of human cancer. However, the effects of CAIX on the metastasis of human oral cancer cells and the underlying molecular mechanisms have not been clarified. In this study, we observed that CAIX overexpression increased the migratory and invasive abilities of SCC-9 and SAS cells. In addition, CAIX overexpression increased the mRNA and protein expression of matrix metalloproteinase-9 (MMP-9) and the phosphorylation of focal adhesion kinase (FAK), steroid receptor coactivator (Src), and extracellular signal-regulated kinase 1/2 signaling proteins. CAIX overexpression also increased the binding capacity of nuclear factor-κB (NF-κB), c-Jun, and c-Fos on the MMP-9 gene promoter. In addition, treatment with MMP-9 short hairpin RNA, an MMP inhibitor (GM6001), an FAK mutant, or an MEK inhibitor (U0126) inhibited CAIX-induced cell motility in SCC-9 cells. Moreover, data sets from The Cancer Genome Atlas demonstrated that CAIX expression was significantly associated with advanced progression and poor survival in oral cancer. In conclusion, it can be inferred that CAIX overexpression induces MMP-9 gene expression, which consequently induces the metastasis of oral cancer cells.