Detection Of Matrix Metalloproteinase Research Articles

NG2-Glia Cause Diabetic Blood-Brain Barrier Disruption by Secreting MMP-9.

Disorders of the blood-brain barrier (BBB) arising from diabetes mellitus are closely related to diabetic encephalopathy. Previous research has suggested that neuron-glia antigen 2 (NG2)-glia plays a key role in maintaining the integrity of the BBB. However, the mechanism by which NG2-glia regulates the diabetic BBB remains unclear. Type 2 diabetes mellitus (T2DM) db/db mice and db/m mice were used. Evans-Blue BBB permeability tests and transmission electron microscopy techniques were applied. Tight junction proteins were assessed by immunofluorescence and transmission electron microscopy. NG2-glia number and signaling pathways were evaluated by immunofluorescence. Detection of matrix metalloproteinase-9 (MMP-9) in serum was performed using enzyme-linked immunosorbent assay (ELISA). In T2DM db/db mice, BBB permeability in the hippocampus significantly increased from 16 weeks of age, and the structure of tight junction proteins changed. The number of NG2-glia in the hippocampus of db/db mice increased around microvessels from 12 weeks of age. Concurrently, the expression of MMP-9 increased in the hippocampus with no change in serum. Sixteen- week-old db/db mice showed activation of the Wnt/β-catenin signaling in hippocampal NG2-glia. Treatment with XAV-939 improved structural and functional changes in the hippocampal BBB and reduced MMP-9 secretion by hippocampal NG2-glia in db/db mice. It was also found that the upregulation of β-catenin protein in NG2-glia in the hippocampus of 16-week-old db/db mice was significantly alleviated by treatment with XAV-939. The results indicate that NG2-glia can lead to structural and functional disruption of the diabetic BBB by activating Wnt/β-catenin signaling, upregulating MMP-9, and degrading tight junction proteins.

Cardiac Fibroblasts Enhance MMP2 Activity to Suppress Gap Junction Function in Cardiomyocytes.

Although it is crucial to promptly restore blood perfusion to revive the ischemic myocardium, reperfusion itself can paradoxically contribute to the electrical instability and arrhythmias of the myocardium. Several studies have revealed that cardiac fibroblasts can impact cardiac electrophysiology through various mechanisms including the deposition of extracellular matrix, release of chemical mediators, and direct electrical coupling with myocytes. Previously, we have shown that hypoxia/reoxygenation (H/R)-treated rat fibroblasts conditional medium (H/R-FCM) could decrease the spontaneous beating frequency of rat neonatal cardiomyocytes and downregulate the expression of gap junction proteins. However, the specific mechanism by which H/R-FCM affects the gap junctions requires further investigation. H/R-FCM was obtained by culturing confluent rat cardiac fibroblasts (RCF) for 4h under hypoxic conditions. Gap junction function, hemichannel activity, and expression of Cx43 were examined upon treatment with H/R-FCM. Gelatin zymography was performed to detect matrix metalloproteinase (MMP) activity in the conditioned medium. The effect of H/R-FCM and MMP2 inhibitors on cardiac electrophysiology and arrhythmias was investigated with an isolated rat ischemia/reperfusion (I/R) model. H/R-FCM treatment impaired gap junction function, downregulated Cx43 expression, and increased hemichannel activity in rat cardiomyocytes (H9c2). The adverse effect of H/R-FCM on gap junction, which was confirmed by the cardiomyocyte H/R model, was involved in the activation of MMP2. MMP2 inhibition could partially attenuate the detrimental effects of I/R on myocardial electrophysiological indices and arrhythmia susceptibility. Our study indicates that inhibition of MMP2 may be a promising therapeutic target for the treatment of reperfusion arrhythmia.

MIA/CD-RAP Regulates MMP13 and Is a Potential New Disease-Modifying Target for Osteoarthritis Therapy.

Melanoma inhibitory activity/cartilage-derived retinoicacid-sensitive protein (MIA/CD-RAP) is a protein expressed and secreted by chondrocytes and cartilaginous tissues. MIA/CD-RAP-deficient mice develop milder osteoarthritis than wildtype mice. In this study, we investigated MIA/CD-RAP downstream targets to explain this reduced disease development. As a possible mediator, we could detect matrix metalloproteinase 13 (MMP13), and the influence of MIA/CD-RAP on MMP13 regulation was analyzed in vitro using SW1353 chondrosarcoma cells and primary chondrocytes. The femoral head cartilage of WT and MIA/CD-RAP -/- mice were cultured ex vivo to further investigate MMP13 activity. Finally, osteoarthritis was surgically induced via DMM in C57BL/6 mice, and the animals were treated with an MIA/CD-RAP inhibitory peptide by subcutaneously implanted pellets. MMP13 was regulated by MIA/CD-RAP in SW1353 cells, and MIA/CD-RAP -/- murine chondrocytes showed less expression of MMP13. Further, IL-1β-treated MIA/CD-RAP -/- chondrocytes displayed less MMP13 expression and activity. Additionally, MIA/CD-RAP-deficient ex vivo cultured cartilage explants showed less MMP13 activity as well as reduced cartilage degradation. The mice treated with the MIA/CD-RAP inhibitory peptide showed less osteoarthritis development. Our findings revealed MIA/CD-RAP as a new regulator of MMP13 and highlighted its role as a potential new target for osteoarthritis therapy.

Antioxidation Function of EGCG by Activating Nrf2/HO-1 Pathway in Mice with Coronary Heart Disease.

Objective To explore the effect and mechanism of epigallocatechin gallate (EGCG) in mice with coronary heart disease (CHD). Methods Firstly, a CHD model of mouse was established by feeding mice high-fat diet and randomly divided into four groups, including Model group (0.5% sodium cholate) and 10 mg/kg EGCG, 20 mg/kg EGCG, and 40 mg/kg EGCG groups. After oral administration of sodium cholate or EGCG, HE staining was conducted to assess the pathological changes of mouse cardiac tissues in each group of mice, biochemical kits to measure the levels of blood lipid and oxidative stress substance activity, and western blot to detect matrix metalloproteinase 2 (MMP-2), vascular endothelial growth factor (VEGFA), as well as expression levels of protein related to Nrf2/HO-1/NQO1 pathway in cardiac tissues. Results The mice in the CHD model appeared to have myocardial pathological damage with elevated serum total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and decreased high-density lipoprotein cholesterol (HDL-C). Of note, administration of EGCG significantly attenuated myocardial injuries and improved blood lipid levels in mice in a concentration-dependent manner. The advent of EGCG significantly decreased the expression of VEGFA and MMP-2 and increased the activity of superoxide dismutase (SOD), when reducing the content of reactive oxygen species (ROS) in the myocardial tissue and upregulating the expression of HO-1, NQO1, and Nrf2. Conclusion EGCG may reduce atherosclerotic plaque and alleviate pathological damage in the cardiac tissue of CHD mice as well as improve blood lipid levels with antioxidative effect. The mechanism of its effect may be related to the activation of the Nrf2/HO-1/NQO1 antioxidant pathway in vivo of the CHD mice.

Initial immune response to a FRET-based MMP sensor-immobilized silk fibroin hydrogel in vivo

To investigate the initial immune response to biodegradable silk fibroin (SF) hydrogels in vivo, a Förster/fluorescence resonance energy transfer (FRET)-based sensor was developed to detect matrix metalloproteinase (MMP) activity (FRET-MMPS) and immobilized to SF hydrogel. FRET-MMPS immobilized to SF hydrogel in vitro displayed intra-molecular FRET more than inter-molecular FRET, and MMP activity was detected through a decrease in FRET signal intensity. Then, the SF hydrogel modified with FRET-MMPS was implanted into mice subcutaneously, and it was observed that the FRET signal intensity decreased significantly soon (< 3 h) after implantation. Although the intensity exhibited a sharp decrease toward 24 h post-implantation, histological evaluation proved that bulk-level hydrogel degradation, such as breakdown, was mainly caused by macrophages and foreign body giant cells on a timescale of weeks. These results indicated that, immediately upon implantation, active MMPs reached the SF hydrogel and began cleaving SF networks, which might result in the loosening of the networks and then enabled immune cells, such as macrophages, to start the bulk-level hydrogel degradation. The sensor clarified the initial immune response to SF hydrogels and will provide clues for designing the biodegradation behaviors of scaffolds for regenerative medicine. Statement of significanceSilk fibroin (SF) materials are degraded gradually by the immune response. Immune cells, such as macrophages, break down implanted SF materials on a timescale of weeks or months, but the initial (< 24 h) immune response to SF materials remains unclear. In this study, SF hydrogels modified with Förster/fluorescence resonance energy transfer (FRET)-based matrix metalloproteinase (MMP) sensors were implanted in mice and within 3 h post-implantation, the SF hydrogels were degraded by MMPs. Although this molecular-level biodegradation was not correlated with the hydrogel breakdown, the MMPs were likely to loosen the SF networks to enable immune cells to infiltrate and degrade the hydrogel. This is the first study to unveil the initial stage of immune response to biomaterials.

In Situ Protease Secretion Visualization and Metastatic Lymph Nodes Imaging via a Cell Membrane-Anchored Upconversion Nanoprobe.

Matrix metalloproteinase (MMP) secretion is highly associated with tumor invasion and metastasis; therefore, monitoring MMP secretion is important for disease progression study and therapy choosing. Though working well for intracellular MMP imaging, the performance of current MMP detection probes is impaired in secretion monitoring due to the diffusion of MMP in an extracellular environment after secretion and low secreted amount. Here, we design a cell membrane-anchored ratiometric upconversion nanoprobe (UCNPs-Cy3/Pep-QSY7/Ab) for in situ MMP secretion visualization. Anti-EGFR is functionalized on the nanoprobe to provide specific recognition to tumor cells and guarantee fast response to MMP2 in the local place of secretion. MMP-responsive cleavage of Pep-QSY7 results in Cy3 luminescence recovery at 580 nm, which is ratioed over an internal standard of UCNP emission at 654 nm for MMP2 detection. The presented cell membrane-anchored ratiometric upconversion nanoprobe demonstrated that satisfactory results for in situ monitoring of MMP2 secretion from MDA-MB-231 cells and MCF-7 cells, as well as in vivo imaging of metastatic lymph nodes, would provide a universal platform for protease secretion study and contribute to tumor invasiveness assessment.

Distance-dependent visual fluorescence immunoassay on CdTe quantum dot-impregnated paper through silver ion-exchange reaction.

Apaper-based visual fluorescence immunoassay is presentedfor the detection of matrix metalloproteinase-7 (MMP7) that is related to renal cancer. The method is based on thedistance-dependent fluorescence quenching of CdTe quantum dots (QDs) on a nitrocellulose membrane by Ag+ followinga sandwich-type immunoreaction on microtiter wells using silver nanoparticle (AgNP)-labeled secondary antibody- and primary antibody-coated microtiter wells. The silver nanoparticles captured in the well are dissolved with HNO3, while the quenching effect of QDs is based on silver ion-exchange reaction under 365-nm excitation light irradiation. Increasingconcentration of released Ag+, thus higher concentration of the protein, leads to an increased distance of quenching on the nitrocellulose membrane. The paper-based immunoassay by combination of AgNP-assisted ion-exchange reaction with QD gives good distance-dependent responses and allows the detection of MMP7 at a concentration as low as 7.3pgmL-1. The coefficients of variation are less than 6.9% and 12.4% for intra-assay and inter-assay, respectively. High specificity and long-term stability are achieved during the assay. Importantly, the testing of human serum samples using our strategy shows well-matched results with commercial human MMP7 ELISA kits. Graphical abstract A distance-dependent visual immunoassay is developed for the determination of serum matrix metalloproteinase-7 on CdTe quantum dot-impregnated paper with silver ion-exchange reaction.

Persistent Chemiluminescent Glow of Phenoxy-dioxetane Luminophore Enables Unique CRET-Based Detection of Proteases.

Chemiluminescence is being considered an effective imaging modality as it offers low background and high sensitivity. Recent discovery by our group has led to development of new phenoxy-dioxetane chemiluminescence luminophores, which are highly bright under physiological conditions. However, the current scope of probes based on these luminophores is limited, as they can only be turned on by phenol protecting group removal. Here we present a new chemiluminescence resonance energy transfer (CRET) system, Glow-CRET, in which light emission is triggered by proteolytic cleavage of a peptide substrate that links a dioxetane luminophore and a quencher. In order to compose such system, a new phenoxy-dioxetane luminophore, 7-HC-CL, was developed. This luminophore exhibits intense and persistent glow chemiluminescence; it undergoes very slow chemiexcitation, and it has the highest chemiluminescence quantum yield ever reported under physiological conditions. Based on 7-HC-CL, a Glow-CRET probe for matrix metalloproteinases, MMP-CL, was synthesized. Incubation of MMP-CL with its cognate protease resulted in 160-fold increase in chemiluminescence signal. MMP-CL was also able to detect matrix metalloproteinase activity in cancer cells with significantly higher signal-to-background ratio than an analogous fluorescence resonance energy transfer (FRET)-based probe. This work is expected to open new horizons in chemiluminescence imaging, as it enables to use the dioxetanes in ways that had not been possible. We anticipate that 7-HC-CL and future derivatives will be utilized not only for the construction of further Glow-CRET probes, but also for other applications, such as chemiluminescence tagging of proteins.

N-Acetylcysteine potentiates the haemodynamic-improving effect of sildenafil in a rabbit model of acute pulmonary thromboembolism via the p38 MAPK pathway.

The current study aimed to investigate the effects of sildenafil and N-acetylcysteine (NAC) on the haemodynamics in a rabbit model of acute pulmonary thromboembolism (APT). We developed an APT model using healthy male China big-ear rabbits (2.7±0.4kg). The rabbits were divided into five groups subjected to various interventions. We recorded the haemodynamic parameters and assessed the oxidative stress and lipid peroxidation response in the groups. Additionally, we detected apoptosis-associated molecules, FoxO1, Bad and Bcl-2, in the lung tissue. Gelatine zymography was used to detect matrix metalloproteinase (MMP) activity in bronchoalveolar lavage (BLA). Pulmonary artery endothelial cells were isolated, and their apoptosis rates and MMP activity were assayed. N-acetylcysteine potentiated the haemodynamic-improving effect of sildenafil and significantly inhibited the oxidative stress response. N-acetylcysteine combined with sildenafil decreased MMP-2 and MMP-9 activity and NO consumption and inhibited apoptosis of pulmonary arterial endothelial cells. Moreover, NAC combined with sildenafil inhibited the expression of MCP-1 and p-p38 MAPK. Thus, NAC potentiates the haemodynamic-improving effect of sildenafil in a rabbit model of acute pulmonary thromboembolism via the MCP-1 and p38 MAPK signalling pathway. This study may provide a promising treatment method for APT.

Assessment of MMP-2/-9 expression by fluorescence endoscopy for evaluation of anastomotic healing in a murine model of anastomotic leakage.

BackgroundDisturbance of intestinal wound closure leads to insufficient anastomotic healing and is associated with considerable morbidity following colorectal resections. Matrix metalloproteinases (MMPs) play a crucial role in regulation of wound closure. Here fluorescence endoscopy was evaluated for assessment of MMP-2/-9 expression during failed intestinal anastomotic healing.MethodsDistal colonic anastomoses were performed as a model for disturbed healing in 36 Balb/c mice. Healing was evaluated endoscopically, macroscopically, and histologically after 1, 3 and 5 days. For detection of MMP-2/-9 expression fluorescence endoscopy (FE) was used following i.v.-administration of a Cy5.5-labeled MMP-2/-9 specific tracer. FE was complemented by quantification of the fluorescence signal using the MS-FX PRO Optical Imaging System. An overall leakage score was calculated and correlated with the results of FE.ResultsWith increasing incidence of anastomotic leakage from POD1 (17%) to POD5 (83%) the uptake of the MMP tracer gradually increased (signal-to-noise ratio (SNR), POD1: 17.91 ± 1.251 vs. POD3: 30.56 ± 3.03 vs. POD5: 44.8 ± 4.473, P<0.0001). Mice with defective anastomotic healing showed significantly higher uptake compared to non-defective (SNR: 37.37± 3.63 vs. 26.16± 3.635, P = 0.0369). White light endoscopy and FE allowed evaluation of anastomotic healing and visualization of mucosal MMPs in vivo. Using FE based detection of MMPs in the anastomosis, an overall positive predictive value of 71.4% and negative predictive value of 66.6% was calculated for detection of anastomotic leakage.ConclusionDuring disturbed anastomotic healing increased expression of MMP-2/-9 was observed in the anastomotic tissue. Fluorescence endoscopy for detection of MMP-2/-9 during the healing process might be a promising tool for early identification of anastomotic leakage.

Optical imaging of ovarian cancer using a matrix metalloproteinase-3-sensitive near-infrared fluorescent probe

Epithelial ovarian cancer (EOC) is the seventh most common cancer among women worldwide. The 5-year survival rate for women with EOC is only 30%-50%, which is largely due to the typically late diagnosis of this condition. EOC is difficult to detect in its early stage because of its asymptomatic nature. Recently, near-infrared fluorescent (NIRF) imaging has been developed as a potential tool for detecting EOC at the molecular level. In this study, a NIRF-sensitive probe was designed to detect matrix metalloproteinase (MMP) activity in ovarian cancer cells. A cyanine fluorochrome was conjugated to the amino terminus of a peptide substrate with enzymatic specificity for MMP-3. To analyze the novel MMP-3 probe, an in vivo EOC model was established by subcutaneously implanting SKOV3 cells, a serous-type EOC cell line, in mice. This novel MMP-3-sensitive probe specifically reacted with only the active MMP-3 enzyme, resulting in a significantly enhanced NIRF emission intensity. Histological analysis demonstrated that MMP-3 expression and activity were enhanced in the stromal cells surrounding the ovarian cancer cells. These studies establish a molecular imaging reporter for diagnosing early-stage EOC. Additional studies are required to confirm the early-stage activity of MMP-3 in EOC and its diagnostic and prognostic significance.

Noninvasive Detection and Imaging of Matrix Metalloproteinases for Cancer Diagnosis

Matrix metalloproteinases (MMPs) are important cancer biomarkers and the sensitive detection of MMPs is of great importance to improve clinical diagnosis and therapy of cancer at its early stage. Molecular imaging, including fluorescent optical imaging, radiolabeled imaging, and magnetic resonance imaging, is a powerful tool to visualize and characterize biological processes at the cellular and molecular level. The recognition of MMPs via imaging strategies by utilizing MMP-responsive probes has been a hot pursuit in recent years. Probes designed for MMP detection commonly have two features: (1) off-then-on state in detection signal response to the appearance of MMPs, which has been applied in optical imaging and magnetic resonance imaging; (2) specific retention upon sensing MMPs, which has been applied in radiolabeled imaging. The development of theory and technology in the field of biomarker probes will be beneficial to the accurate diagnosis and effective treatment of cancer.

The Transient Receptor Potential Melastatin 7 Channel Regulates Pancreatic Cancer Cell Invasion through the Hsp90α/uPA/MMP2 pathway

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with a very poor prognosis. There is an urgent need to better understand the molecular mechanisms that regulate PDAC cell aggressiveness. The transient receptor potential melastatin 7 (TRPM7) is a nonselective cationic channel that mainly conducts Ca2+ and Mg2+. TRPM7 is overexpressed in numerous malignancies including PDAC. In the present study, we used the PANC-1 and MIA PaCa-2 cell lines to specifically assess the role of TRPM7 in cell invasion and matrix metalloproteinase secretion. We show that TRPM7 regulates Mg2+ homeostasis and constitutive cation entry in both PDAC cell lines. Moreover, cell invasion is strongly reduced by TRPM7 silencing without affecting the cell viability. Conditioned media were further studied, by gel zymography, to detect matrix metalloproteinase (MMP) secretion in PDAC cells. Our results show that MMP-2, urokinase plasminogen activator (uPA), and heat-shock protein 90α (Hsp90α) secretions are significantly decreased in TRPM7-deficient PDAC cells. Moreover, TRPM7 expression in human PDAC lymph node metastasis is correlated to the channel expression in primary tumor. Taken together, our results show that TRPM7 is involved in PDAC cell invasion through regulation of Hsp90α/uPA/MMP-2 proteolytic axis, confirming that this channel could be a promising biomarker and possibly a target for PDAC metastasis therapy.

Disperse-and-Collect Approach for the Type-Selective Detection of Matrix Metalloproteinases in Porous Silicon Resonant Microcavities.

We report on the design and testing of photonic biosensors for the type-selective detection of different types of matrix metalloproteinases (MMPs). The ability to detect a panel of different MMP types has important implications for prognosis of wound healing. We combine the immunocapture of MMPs on dispersed magnetic nanoparticles modified with antibodies specific for target MMPs (immuno-magNPs) with subsequent MMP detection upon fluorogenic peptide cleavage in porous silicon resonant microcavity (pSiRM) architectures. We report fast, sensitive, and type-selective detection of MMPs directly in wound fluid. This study sets the scene for downstream developments of multiparametric biosensors as point-of-care (POC) prognostic tools that may step-change chronic wound management.

Inhibitory effect of trans-ferulic acid on proliferation and migration of human lung cancer cells accompanied with increased endogenous reactive oxygen species and β-catenin instability.

BackgroundTrans-ferulic (FA) acid exhibits antioxidant effects in vitro. However, the underlying mechanism of trans-FA activity in cellular physiology, especially cancer physiology, remains largely unknown. This study investigated the cellular physiological effects of trans-FA on the H1299 human lung cancer cell line.MethodsThe 2,2-diphenyl-1-picrylhydrazyl assay was used to determine free radical scavenging capability. Assessment of intracellular reactive oxygen species (ROS) was evaluated using oxidized 2ʹ,7ʹ-dichlorofluorescin diacetate and dihydroethidium staining. Trypan blue exclusion, colony formation, and anchorage-independent growth assays were used to determine cellular proliferation. Annexin V staining assay was used to assess cellular apoptosis by flow cytometry. Wound healing and Boyden’s well assays were used to detect the migration and invasion of cells. Gelatin zymography was used to detect matrix metalloproteinase (MMP-2 and MMP-9) activity. Western blotting was used to detect expression levels of various signaling pathway proteins.ResultsDPPH assay results indicated that trans-FA exerted potent antioxidant effects. However, trans-FA increased intracellular ROS levels, including hydrogen peroxide and superoxide anion, in H1299 cells. Trans-FA treatment inhibited cellular proliferation and induced moderate apoptotic cell death at the highest concentration used (0.6 mM). Furthermore, trans-FA moderately inhibited the migration of H1299 cells at the concentrations of 0.3 and 0.6 mM and attenuated MMP-2 and MMP-9 activity. Trans-FA caused the phosphorylation of β-catenin, resulting in proteasomal degradation of β-catenin. Conversely, trans-FA treatment increased the expression of pro-apoptotic factor Bax and decreased the expression of pro-survival factor survivin.ConclusionVarious concentrations (0.06–0.6 mM) of trans-FA exert both anti-proliferation and anti-migration effects in the human lung cancer cell line H1299.Electronic supplementary materialThe online version of this article (doi:10.1186/s13020-016-0116-7) contains supplementary material, which is available to authorized users.

Adipose-derived mesenchymal stem cells promote cell proliferation and invasion of epithelial ovarian cancer

Adipose-derived mesenchymal stem cell (ADSC) is an important component of tumor microenvironment. However, whether ADSCs have a hand in ovarian cancer progression remains unclear. In this study, we investigated the impact of human ADSCs derived from the omentum of normal donors on human epithelial ovarian cancer (EOC) cells in vitro and in vivo. Direct and indirect co-culture models including ADSCs and human EOC cell lines were established and the effects of ADSCs on EOC cell proliferation were evaluated by EdU incorporation and flow cytometry. Transwell migration assays and detection of MMPs were performed to assess the invasion activity of EOC cells in vitro. Mouse models were established by intraperitoneal injection of EOC cells with or without concomitant ADSCs to investigate the role of ADSCs in tumor progression in vivo. We found that ADSCs significantly promoted proliferation and invasion of EOC cells in both direct and indirect co-culture assays. In addition, after co-culture with ADSCs, EOC cells secreted higher levels of matrix metalloproteinases (MMPs), and inhibition of MMP2 and MMP9 partially relieved the tumor-promoting effects of ADSCs in vitro. In mouse xenograft models, we confirmed that ADSCs promoted EOC growth and metastasis and elevated the expression of MMP2 and MMP9. Our findings indicate that omental ADSCs play a promotive role during ovarian cancer progression.

Correlation Between MMP-7 and bFGF Expressions in Non-small Cell Lung Cancer Tissue and Clinicopathologic Features.

The present study intends to investigate the correlation between clinicopathologic features of non-small cell lung cancer and matrix metalloproteinase-7 (MMP-7) and basic fibroblast growth factor (bFGF) and to investigate the roles of MMP-7 and bFGF in detecting the course of disease of non-small cell lung cancer. Ninety cases of paraffin-embedded tissue samples from patient with primary non-small cell lung cancer and fifty cases of lung tissue samples from normal subjects were included in the present study. Immunohistochemical S-P method was used to detect proteins MMP-7 and bFGF. (1) The positive rate of MMP-7 protein was 14% in normal lung tissue section and 68.89% in non-small cell lung cancer tissue section, and the difference was statistically significant (χ (2)=38.774, P=0.000<0.05). There were 43 cases (43/56) with positive expression in patients with squamous cell carcinoma and 22 cases (22/34) with positive expression in patients with adenocarcinoma, and the difference was not statistically significant (χ (2)=1.539, P=0.215>0.05). There were 14 cases (14/51) with positive expression in patients with moderate- and well-differentiated lung carcinoma and 36 cases (36/39) with positive expression in patients with poor-differentiated lung carcinoma, and the difference was statistically significant (χ (2)=35.068, P=0.000<0.05). There were 37 cases (37/42) with positive expression in patients with lymphatic metastasis and 26 cases (26/48) with positive expression in patients without lymphatic metastasis, and the difference was statistically significant (χ (2) =12.279, P=0.000<0.05). (2) The mean intratumoral microvessel density (iMVD) was 46.2±6.77 in the field of lung cancer tissue at high magnification under MMP-7-positive condition and 30.8±7.54 in the field of lung cancer tissue at high magnification under MMP-7-negative condition, and the difference was statistically significant (t=9.641, P=0.000<0.05). (3) The positive rate of bFGF was 12% in normal tissue section and 63.3% in non-small cell lung cancer tissue section, and the difference was statistically significant (χ (2)=34.222, P=0.000<0.05). There were 41 cases (41/56) with positive expression in patients with squamous cell carcinoma and 20 cases (20/34) with positive expression in patients with adenocarcinoma, and the difference was not statistically significant (χ(2)=2.006, P=0.157>0.05). There were 29 cases (29/51) with positive expression in patients with moderate- and well-differentiated lung carcinoma and 35 cases (35/39) with positive expression in patients with poor-differentiated lung carcinoma, and the difference was statistically significant (χ (2)=10.085, P=0.001<0.05). There were 37 cases (37/42) with positive expression in patients with lymphatic metastasis and 25 cases (25/48) with positive expression in patients without lymphatic metastasis, and the difference was statistically significant (χ (2) =13.554, P=0.001<0.05). (4) The (iMVD) was 45.8±7.16 in the field at high magnification under bFGF-positive condition and 31.2±6.46 in the field at high magnification under bFGF-negative condition, and the difference was statistically significant (t=9.654, P=0.001<0.05). (5) A correlation was demonstrated between MMP-7 and bFGF in non-small cell lung cancer (r=0.353, P=0.000<0.05). Both MMP-7 and bFGF are participated in the progression of non-small cell lung cancer and exert a synergistic effect during physiological processes including pathogenesis, invasion, and metastasis of non-small cell lung cancer. Therefore, a combined detection of MMP-7 and bFGF for non-small cell lung cancer contributes to predict the progression and prognosis of non-small cell lung cancer, with significant clinical value.

Quantitative detection of matrix metalloproteinase-2 in normal coronal dentine of young people

To compare the distribution and concentration of matrix metalloproteinases-2 (MMP-2) in different dentin depth of premolar and molar of young people. Freshly extracted human premolars and molars (aged between 20-30) were sectioned to 1.5 mm thick slices along the longitudinal axis of the tooth separately. Enamel and pulp of each slice was removed, and then the premolar and molar slices were respectively divided into two subgroups according to superficial or deep dentin and pulverized to fine powder. After dentin protein was extracted, the concentrations of MMP-2 in different tooth were detected using fluorescent microsphere immunoassay. The content of MMP-2 in superficial layer dentin of premolar was (0.022 ± 0.006) ng/mg. The content of MMP-2 in deep layer dentin of premolar was (2.087 ± 0.090) ng/mg. The content of MMP-2 in superficial layer dentin of molar was (0.336 ± 0.037) ng/mg. The content of MMP-2 in deep layer dentin of molar was (3.312 ± 0.308) ng/mg. MMP-2 exists in human coronal dentin. In the same type of teeth of young people, the concentration of MMP-2 in deep dentin was significant higher than those in superficial dentin. In the same dentin depth, the concentration of MMP-2 in molar was significant higher than those in premolar.

In Vivo Imaging of Matrix Metalloproteinase 12 and Matrix Metalloproteinase 13 Activities in the Mouse Model of Collagen‐Induced Arthritis

To develop enzyme-activatable Förster resonance energy transfer (FRET) substrate probes to detect matrix metalloproteinase 12 (MMP-12) and MMP-13 activities in vivo in mouse models of inflammatory arthritis. Peptidic FRET probes activated by MMP-12 and MMP-13 were reverse designed from inhibitors selected from a phosphinic peptide inhibitor library. Selectivity of the probes was demonstrated in vitro using MMP-1, MMP-2, MMP-3, MMP-12, and MMP-13. In vivo activation of the probes was tested in the zymosan-induced mouse model of inflammation, and probe specificity was evaluated by the MMP inhibitor GM6001 and specific synthetic inhibitors of MMP-12 and MMP-13. The probes were used to monitor these enzyme activities in the collagen-induced arthritis (CIA) model in vivo. The MMP-12 and MMP-13 activity probes (MMP12ap and MMP13ap, respectively) discriminated between the activities of the 2 enzymes. The in vivo activation of these probes was inhibited by GM6001 and by their respective specific inhibitors. In the CIA model, MMP12ap activation peaked 5 days after disease onset and showed strong correlation with disease severity during this time (r = 0.85, P < 0.0001). MMP13ap activation increased gradually after disease onset and correlated with disease severity over a longer period of 15 days (r = 0.58, P < 0.0001). We generated two selective FRET probes that can be used to monitor MMP-12 and MMP-13 activities in live animals. MMP12ap follows the initial stage of inflammation in CIA, while MMP13ap follows the progression of the disease. The specificity of these probes is useful in monitoring the efficacy of MMP inhibitors.

Evaluation of matrix metalloproteinase-2 and -9 in the cerebrospinal fluid of dogs with intracranial tumors

To identify matrix metalloproteinase (MMP)-2 and -9 in CSF from dogs with intracranial tumors. CSF from 55 dogs with intracranial tumors and 37 control dogs. Latent and active MMP-2 and -9 were identified by use of gelatin zymography. The presence of MMPs in the CSF of dogs with intracranial tumors was compared with control dogs that were clinically normal and with dogs that had idiopathic or cryptogenic epilepsy or peripheral vestibular disease. Relationships between MMP-9 and CSF cell counts and protein were also investigated. Latent MMP-2 was found in CSF samples from all dogs, although active MMP-2 was not detected in any sample. Latent MMP-9 was detected in a subset of dogs with histologically documented intracranial tumors, including meningiomas (2/10), gliomas (3/10), pituitary tumors (1/2), choroid plexus tumors (5/6), and lymphoma (4/4), but was not detected in any control samples. Dogs with tumors were significantly more likely than those without to have detectable MMP-9 in the CSF, and the presence of MMP-9 was associated with higher CSF nucleated cell counts and protein concentration. Latent MMP-9 was detected in most dogs with choroid plexus tumors or lymphoma but in a smaller percentage of dogs with meningiomas, gliomas, or pituitary tumors. Detection of MMP in CSF may prove useful as a marker of intracranial neoplasia or possibly to monitor response of tumors to therapeutic intervention.