MMP-9 Synthesis Research Articles

Temporal Profile of Matrix Metalloproteinases and Their Inhibitors in a Human Endothelial Cell Culture Model of Cerebral Ischemia

Background: Matrix metalloproteinases (MMPs) are key players in proteolytic blood-brain barrier (BBB) disruption during ischemic stroke, leading to vascular edema, hemorrhagic transformation and infiltration by leukocytes. Their effect is dampened by the endogenous tissue inhibitors of metalloproteinases (TIMPs). The respective cellular source of specific MMPs and TIMPs during BBB breakdown is still under investigation. Methods: We analyzed the MMP and TIMP release of human brain microvascular endothelial cells (BMECs) under oxygen glucose deprivation (OGD). Cultured human BMECs (the hCMEC/D3 cell line) were subjected to OGD (6, 12, 18 and 24 h). Gene expression of MMP-2, MMP-9, TIMP-1 and TIMP-2 were serially measured by quantitative real time-PCR and compared to ELISA-detected cell culture medium levels. Results: OGD induced a significant and long-lasting increase in MMP-2 gene expression, reaching a plateau after 12 h. Medium protein levels of MMP-2 were correspondingly elevated at 12 h of OGD. The MMP-9 synthesis rate was detectable at very low levels and remained unaffected by OGD. TIMP-1 gene expression and secretion declined under OGD, whereas both expression and secretion of TIMP-2 remained stable. Contrary to the respective gene expression rate, medium levels of MMP-2, TIMP-1 and TIMP-2 started a simultaneous decline after 12 h of OGD. This is most likely due to an impaired synthesis and enhanced consumption rate under OGD. Conclusions: The objective of our study was to determine the contribution of human BMECs to the MMP metabolism under in vitro OGD conditions simulating ischemic stroke. Our results suggest that human BMECs switch to a proinflammatory state by means of an enhanced production of MMP-2, attenuated release of TIMP-1, and unaffected production of TIMP-2. Thus, human BMECs might participate in the MMP-mediated BBB breakdown during ischemic stroke. However, our data does not support human BMECs to be a source of MMP-9.

Estrogen Receptor Activation, TNF-α, and Endothelial Dysfunction

Studying the pathology of atherosclerosis in humans is problematic due to the slow evolution of this disease. However, 2 decades ago an experimental model, transgenic apolipoprotein E knockout mice characterized by accelerated atherogenesis, was developed. The influence of estrogens on atherogenesis is complex. At the early stages of atherosclerosis, estrogens have a beneficial influence by diminishing plaque formation. At the later stages, when atherosclerotic plaques have been formed, estrogens augment the expression of metalloproteinases (MMPs) and increase the risk of plaque rupture. Furthermore, estrogens have thrombogenic properties. The effects of estrogens on cellular functions may be expressed through genetic, nongenetic, direct, and indirect action. The modifications of the expression of various genes which are affected by estrogens are mediated via 2 types of estrogen receptors (ERa or ER1 and ERb or ER2). The existence of ERs was reported in the early 1960s and the encoding of complementary DNA of ERs was achieved in the mid1980s. During the past decade, Thomas et al discovered another ER, GPR30 (G protein-coupled, 7-transmembrane receptor). This receptor is expressed in smooth muscle cells of arteries and veins. The cells of breast, ovaries, and endometrium also have ERs. Tumor necrosis factor a (TNF-a) is involved in atherogenesis by weakening endothelium-dependent and nitric oxide (NO)-mediated vasodilation, diminishing the production of NO, and enhancing the catabolism of NO. Women have lower TNF-a levels and a lower incidence of heart dysfunction and sepsis-related morbidity and mortality compared to men. In this issue of Angiology, Professor Palombo’s group reports that ER activation exerts a protective effect on endothelial cells by inhibiting TNF-a-induced expression of MMP-9 and suppression of endothelial NO synthase (eNOS) expression. The MMP-9, expressed in atherosclerotic plaques, contributes to the deterioration of many types of collagen. Estrogens seem to regulate the synthesis of MMP-9 through the inhibitors of MMPs. The NO regulates a number of mechanisms, including vascular tone, myocardial contractility, leucocyte–endothelial interactions, integrity and permeability of endothelium, proliferation of vascular smooth muscle cells (VSMCs), and antithrombotic action (inhibits platelet activation). The release of NO by a healthy endothelium induces relaxation of smooth muscle cells and inhibits platelet activation. Estrogens activate eNOS through 2 signaling pathways, the mitogen-activated protein kinase and the phosphatidylinositol 3-kinase/protein kinase B leading to increase NO release. The endothelium modulates vessel tone as well as platelet activity. The endothelium expresses both types of ERs and is a target for sex hormones. Opposite to premenopausal women, postmenopausal women have elevated TNF-a serum levels (increased TNF-a expression induces the production of reactive oxygen species, resulting in endothelial dysfunction). In vitro data showed that estrogen inhibits TNF gene transcription via ERs. Furthermore, others showed an additional capability of estrogens to inhibit the expression of VSMCs in cultured human endothelial cells and animal models of atherosclerosis. The mechanisms described above support that the ER activators such as apigenin may diminish endothelial dysfunction caused by TNF-a. Also, Choi has documented that treatment with apigenin decreased the TNF-a-induced production of NO in osteoblasts suggesting that apigenin may be a new pharmacological tool for the treatment of osteoporosis. Similar results were reported after evaluation of the antiinflammatory properties of apigenin in an in vitro model. They reported inhibition of gene expression of TNF and inducible NOS. However, Collins-Burow, after evaluation of the estrogenic and antiestrogenic activity of flavonoids in the ER-positive MCF-7 human breast cancer cell line, found that several flavonoids, including apigenin, did not appear to correlate with binding to ER. Therefore, their suppression of

The carotenoid lutein enhances matrix metalloproteinase-9 production and phagocytosis through intracellular ROS generation and ERK1/2, p38 MAPK, and RARβ activation in murine macrophages

Early studies have demonstrated the ability of dietary carotenoids to enhance immune response, but the mechanism underlying their influence on macrophage activity remains unclear. Here, we investigated the effects of carotenoids on macrophage activity. Carotenoids, including lutein and lycopene, enhanced MMP-9 activity in RAW264.7 macrophages. Lutein was chosen as a representative and analyzed further in this study. It increased the synthesis, activity, and release of MMP-9 in murine RAW264.7 and primary-cultured peritoneal macrophages. MMP-9 induction by lutein was through the transcriptional regulation of mmp-9. It was blunted by the MAPK inhibitors targeting ERK1/2 and p38 MAPK, the reagents that inhibit free radical signaling, and the inhibitors and siRNA targeting RARβ. Moreover, lutein induced Nox activation and intracellular ROS production at an early stage of treatment. This carotenoid also caused ERK1/2 and p38 MAPK activation, RARβ expression, and RAR interaction with its responsive element in the promoter region. These findings suggest the involvement of ROS, MAPKs, and RARβ activation in lutein-driven MMP-9 expression and release. Interestingly, lutein enhanced the phagocytic activity of macrophages, and the secreted MMP-9 appeared to be involved in this process. In summary, we provide evidence here for the first time that the carotenoid lutein induces intracellular ROS generation and MAPK and RARβ activation in macrophages, leading to an increase in MMP-9 release and macrophage phagocytosis. Our results demonstrate that lutein exerts an immunomodulatory effect on macrophages.

Abstract 203: Smooth Muscle Cells from Abdominal Aortic Aneurysms are Unique and Can Independently and Synergistically Degrade Insoluble Elastin

Background The purpose of this study was to further elucidate the role of the vascular smooth muscle cell (SMC) in abdominal aortic aneurysm (AAA) disease. We hypothesized that that AAA-SMC are unique and actively participate in the process of degrading the aortic matrix. Methods and Results Whole-genome expression profiles of SMC from AAA, normal abdominal aorta (NAA) and carotid endarterectomy (CEA) were compared. We quantified elastolytic activity by culturing SMC in [3H]elastin-coated plates and measuring solubilized tritium in the media after 7 days. MMP-2 and MMP-9 production was assessed using real-time PCR, zymography and western blotting. Each SMC type exhibited a unique gene expression pattern. AAA-SMC had greater elastolytic activity than NAA (+68%, p<0.001) and CEA-SMC (+45%, p<0.001). Zymography showed an increase of active-MMP-2 (62kD) in media from AAA-SMC. AAA-SMC demonstrated 2-fold greater expression of MMP-2 mRNA (p<0.05) and 7.3-fold greater MMP-9 expression (p<0.01) than NAA-SMC. Culture with U937 monocytes caused a synergistic increase of elastolysis by AAA-SMC (41%, p<0.001) but not NAA or CEA (p=0.99). Co-culture with U937 caused a large increase in MMP-9 mRNA in AAA and NAA-SMC (p<0.001). MMP-2 mRNA expression was not affected. Western blots of culture media showed a 4-fold increase of MMP-9 (92kD) protein in AAA-SMC/U937 compared to NAA-SMC/U937 (p<0.001) and a large increase in active-MMP2 (62kD) which was less apparent in NAA/U937 media (p<0.01). Conclusions AAA-SMC have a unique gene expression profile and a pro-elastolytic phenotype that is augmented by macrophages. This may occur via a failure of post-transcriptional control of MMP-9 synthesis.

Abstract 5056: Up-regulated expression and activation of invadopodia-associated proteases in esophageal squamous cell carcinoma (ESCC).

Abstract To invade adjacent tissues, cancer cells form special membrane protrusions with high proteolytic activity, named invadopodia. To better understand the role of invadopodia-associated proteolytic systems in the growth of esophageal cancer, we studied the expression of two serine proteases, FAP-alpha and DPPIV, and three metalloproteinases, MMP-2, MMP-9 and MT1-MMP, in a primary ESCC. The enzyme profile was evaluated in relation to the synthesis of cortactin as an important control factor in invadopodia formation and function. The expression and activity of proteases and cortactin in cancer tissues paired with non-cancer tissues were analyzed by RT-PCR, WB and zymography. Tissue samples were obtained from 24 patients who were undergoing surgical resection for esophageal carcinoma. All the tumors had been classified as ESCC and diagnosed as T3 or T4, N1-N3, and M0-M1 according to the TNM classification. The non-cancer tissue samples were taken from the distal area (5-10 cm from the macroscopic changes) and confirmed in histological examinations to be R0. We found an average 3-fold increase in DPPIV expression and an average five-fold higher level of FAP-alpha expression in ESCC over the control tissues. Up-regulation of DPPIV positively correlates with the FAP-alpha expression ratio. FAP-alpha transcripts and DPPIV mRNAs were observed in all the analyzed cancer and non-cancer tissue samples. We found a much higher level of the active MT1-MMP form in cancers relative to paired non-cancer tissues. The correlation between the level of MT1-MMP active form and MMP-2 activity observed in this study shows a tight connection between the two enzymes. We observed enhanced synthesis and activation of both MMP-2 and MMP-9 in cancer tissues. The cancer to normal ratios of the two active gelatinases correlated positively with the expression levels of both serine proteases. We examined the levels of CTTN in the cancer and non-cancer tissues of esophagus in order to find out if CTTN expression is associated with the expression and/or activity of invadopodia proteases. The increased level of CTTN was identified in 35% of ESCC tissues but the differences between groups were not statistically significant. No significant correlation was found between CTTN expression level and any other factors evaluated in our study. In conclusion, we showed that invadopodia-associated proteases, including FAP-alpha, DPPIV, MT1-MMP, MMP-2 and MMP-9, are significantly up-regulated in ESCC and that it might contribute to a more aggressive esophageal cancer. The presence of FAP-alpha in the cancer-free surgical margin suggests that alterations in the stromal environment may extend much father into surrounding tumor tissues than is usually considered. Citation Format: Katarzyna Augoff, Anita Hryniewicz-Jankowska, Renata Tabola, Leszek Czapla, Piotr Szelachowski, Krzysztof Grabowski, Aleksander F. Sikorski. Up-regulated expression and activation of invadopodia-associated proteases in esophageal squamous cell carcinoma (ESCC). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5056. doi:10.1158/1538-7445.AM2013-5056

Different Effects of the Immunomodulatory Drug GMDP Immobilized onto Aminopropyl Modified and Unmodified Mesoporous Silica Nanoparticles upon Peritoneal Macrophages of Women with Endometriosis

The aim of the present work was to compare in vitro the possibility of application of unmodified silica nanoparticles (UMNPs) and modified by aminopropyl groups silica nanoparticles (AMNPs) for topical delivery of immunomodulatory drug GMDP to the peritoneal macrophages of women with endometriosis. The absence of cytotoxic effect and high cellular uptake was demonstrated for both types of silica nanoparticles. The immobilization of GMDP on the UMNPs led to the suppression of the stimulatory effect of GMDP on the membrane expression of scavenger receptors SR-AI and SR-B, mRNAs expression of NOD2 and RAGE, and synthesis of proteolytic enzyme MMP-9 and its inhibitor TIMP-1. GMDP, immobilized onto AMNPs, enhanced the initially reduced membrane expression of SRs and increased NOD2, RAGE, and MMP-9 mRNAs expression by macrophages. Simultaneously high level of mRNAs expression of factors, preventing undesirable hyperactivation of peritoneal macrophages (SOCS1 and TIMP-1), was observed in macrophages incubated in the presence of GMDP, immobilized onto AMNPs. The effect of AMNPs immobilized GMDP in some cases exceeded the effect of free GMDP. Thus, among the studied types of silica nanoparticles, AMNPs are the most suitable nanoparticles for topical delivery of GMDP to the peritoneal macrophages.

Stimulation of MMP-1 and CCL2 by NAMPT in PDL Cells

Periodontitis is an inflammatory disease caused by pathogenic microorganisms and characterized by the destruction of the periodontium. Obese individuals have an increased risk of periodontitis, and elevated circulating levels of adipokines, such as nicotinamide phosphoribosyltransferase (NAMPT), may be a pathomechanistic link between both diseases. The aim of this in vitro study was to examine the regulation of periodontal ligament (PDL) cells by NAMPT and its production under inflammatory and infectious conditions. NAMPT caused a significant upregulation of 9 genes and downregulation of 3 genes, as analyzed by microarray analysis. Eight of these genes could be confirmed by real-time PCR: NAMPT induced a significant upregulation of EGR1, MMP-1, SYT7, ITPKA, CCL2, NTM, IGF2BP3, and NRP1. NAMPT also increased significantly the MMP-1 and CCL2 protein synthesis. NAMPT was significantly induced by interleukin-1β and the periodontal microorganism P. gingivalis. NAMPT may contribute to periodontitis through upregulation of MMP-1 and CCL2 in PDL cells. Increased NAMPT levels, as found in obesity, may therefore represent a mechanism whereby obesity could confer an increased risk of periodontitis. Furthermore, microbial and inflammatory signals may enhance the NAMPT synthesis in PDL cells and thereby contribute to the increased gingival and serum levels of this adipokine, as found in periodontitis.

PIP, Not FiO<sub>2</sub> Regulates Expression of MMP-9 in the Newborn Rabbit VILI with Different Mechanical Ventilation Strategies

Background: Results from experimental and clinical studies have shown that mechanical ventilation or/and hyperoxia may aggravate a pre-existing lung injury or even cause lung injury in healthy lungs by affecting the expression of MMP-9, but the MMP-9 effects are controversial. How are MMP-9 regulated when multicausative factors of injury such as different FiO2, PIP, and respiratory time (RT) impose simultaneously on lungs? Methods: Newborn New Zealand white rabbits were randomly allocated to an unventilated air control group or to one of the 2 × 3 × 3 ventilation strategies by using a factorial design, with different FiO2, PIP, and RT. Then, lung wet-to-dry ratio (W/D), lung histopathology scores, transmission electron microscope, and cells in BALF were analyzed in these different groups. MMP-9 levels were studied by immunohistochemistry and ELISA. Results: MMP-9 levels were significantly different among 3 PIP ventilation regimes (F = 7.215) and MPIP group was the highest among 3 PIP groups. The lung histopathology score in 100% oxygen was significantly higher than in 45% oxygen group (F = 9.037) and MPIP group was the lowest among 3 PIP groups (F = 57.515) and RT 6 h was more serious than RT 1 h. MMP-9 positively correlated with monocytes, but negatively correlated with neutrophils and lung injury histopathology scores. Conclusions: Different PIP and FiO2 exert simultaneously on newborn lung in newborn rabbits ventilation, only mechanical stretch stimulation affects MMP-9 synthesis. Advisable mechanical stretch can promote MMP-9 expression and has protective role in lung in VILI. HPIP causes barotraumas and LPIP induces atelectrauma.

IL-13 Promotes Collagen Accumulation in Crohn’s Disease Fibrosis by Down-Regulation of Fibroblast MMP Synthesis: A Role for Innate Lymphoid Cells?

BackgroundFibrosis is a serious consequence of Crohn’s disease (CD), often necessitating surgical resection. We examined the hypothesis that IL-13 may promote collagen accumulation within the CD muscle microenvironment.MethodsFactors potentially modulating collagen deposition were examined in intestinal tissue samples from fibrotic (f) CD and compared with cancer control (C), ulcerative colitis (UC) and uninvolved (u) CD. Mechanisms attributable to IL-13 were analysed using cell lines derived from uninvolved muscle tissue and tissue explants.ResultsIn fCD muscle extracts, collagen synthesis was significantly increased compared to other groups, but MMP-2 was not co-ordinately increased. IL-13 transcripts were highest in fCD muscle compared to muscle from other groups. IL-13 receptor (R) α1 was expressed by intestinal muscle smooth muscle, nerve and KIR+ cells. Fibroblasts from intestinal muscle expressed Rα1, phosphorylated STAT6 in response to IL-13, and subsequently down-regulated MMP-2 and TNF-α-induced MMP-1 and MMP-9 synthesis. Cells with the phenotype KIR+CD45+CD56+/−CD3− were significantly increased in fCD muscle compared to all other groups, expressed Rα1 and membrane IL-13, and transcribed high levels of IL-13. In explanted CD muscle, these cells did not phosphorylate STAT6 in response to exogenous IL-13.ConclusionsThe data indicate that in fibrotic intestinal muscle of Crohn’s patients, the IL-13 pathway is stimulated, involving a novel population of infiltrating IL-13Rα1+, KIR+ innate lymphoid cells, producing IL-13 which inhibits fibroblast MMP synthesis. Consequently, matrix degradation is down-regulated and this leads to excessive collagen deposition.

Locally Applied Leptin Induces Regional Aortic Wall Degeneration Preceding Aneurysm Formation in Apolipoprotein E–Deficient Mice

Leptin promotes atherosclerosis and vessel wall remodeling. As abdominal aortic aneurysm (AAA) formation involves tissue remodeling, we hypothesized that local leptin synthesis initiates and promotes this process. Human surgical AAA walls were analyzed for antigen and mRNA levels of leptin and leptin receptor, as well as mRNA for matrix metalloproteinases (MMP)-9 and MMP-12. Leptin and leptin receptor antigen were evident in all AAAs, and leptin, MMP-9, and MMP-12 mRNA was increased relative to age-matched nondilated controls. To simulate in vivo local leptin synthesis, ApoE(-/-) mice were subjected to a paravisceral periaortic application of low-dose leptin. Leptin-treated aortas exhibited decreased transforming growth factor-β and increased MMP-9 mRNA levels 5 days after surgery, and leptin receptor mRNA was upregulated by day 28. Serial ultrasonography demonstrated accelerated regional aortic diameter growth after 28 days, correlating with local medial degeneration, increased MMP-9, MMP-12, and periadventitial macrophage clustering. Furthermore, the combination of local periaortic leptin and systemic angiotensin II administration augmented medial MMP-9 synthesis and aortic aneurysm size. Leptin is locally synthesized in human AAA wall. Paravisceral aortic leptin in ApoE(-/-) mice induces local medial degeneration and augments angiotensin II-induced AAA, thus suggesting novel mechanistic links between leptin and AAA formation.

Arrabidaea chica extract improves gait recovery and changes collagen content during healing of the Achilles tendon

IntroductionTendon lesions are still a serious clinical problem. The leaves of the Bignoniaceae Arrabidaea chica (Humb. & Bonpl.) B. Verlot. (syn. Bignonia chica (Bonpl.)) have been used in traditional medicine and described in the literature for its healing properties. However, no study has shown the effects of A. chica during tendon healing. The aim of this study was to investigate the healing properties of the A. chica leaves extract on tendons after partial transection. MethodsA partial transection in the tension region of the Achilles tendon of rats was performed with subsequent posterior topical application of A. chica extract (2.13g/mL in 0.85% saline solution) at the site of the injury. The animals (n=154) were separated into 7 groups: N – rats with tendons without transection; S7, S14 and S21 – rats with tendons treated with topical applications of saline for 7 days and sacrificed on the 7th, 14th and 21st days after surgery, respectively; A7, A14 and A21 – rats with tendons treated with topical applications of the plant extract. The transected regions of the tendons were analyzed through biochemical, morphological and functional analyses. To evaluate the type and concentration of collagen, Western blotting for collagen types I and III was performed, and the hydroxyproline concentration was determined. The participation of metalloproteinases (MMP)-2 and -9 during tendon remodelling was investigated through zymography. Gait recovery was analyzed using the catwalk system. The organization of the extracellular matrix and morphometry were detected in sections stained with haematoxylin–eosin. ResultsThe application of A. chica extract in the region of tendon injury led to an increase in the amount of hydroxyproline (mg/g tissue) on the 7th (91.5±18.9) and 21st (95.8±11.9) days after the tendon lesion relative to the control groups treated with saline (S7: 75.2±7.2; and S21: 71.9±7.9). There were decreases in collagen types I and III (as determined by densitometry) in the groups treated with the plant extract 7 days after injury (type I: 103.9±15.9; type III: 206.3±8.1) compared to the saline-treated groups (type I: 165.2±31.1; type III: 338.6±48.8). The plant extract stimulated the synthesis of MMP-2 on the 21st day after the lesion and decreased the amount of latent and active isoforms of MMP-9 on the 14th day. Analysis by the catwalk system (max contact intensity) showed that the A. chica extract improved the gait of rats on the 7th day of the healing process when compared to the saline group. ConclusionsThe use of A. chica extract during the healing process of the tendon leads to an increase in collagen content and improved gait recovery. Further studies will be performed to analyze the effect of this plant extract on the organization of the collagen bundles of tendons after lesions and to study its probable anti-inflammatory effect.

Secretion of matrix metalloproteinase-9 from astrocytes by inhibition of tonic P2Y14-receptor-mediated signal(s).

Glial cells have various important roles in regulation of brain functions. For such events, extracellular nucleotides/P2 receptors have central roles. Although there have been huge amount of literature about activation of P2 receptors and glial functions, little is known about what happens in glia or the brain if glial P2 receptor is inhibited. Here we show that the inhibition of P2 receptors in astrocytes, the most abundant glial cells and cause a constitutive release of nucleotides, resulted in secretion of metalloproteinase-9 (MMP-9), a metal-dependent endopeptidase that degrades extracellular matrix molecules and is important in regulation of brain remodeling. When cultured astrocytes were treated with apyrase (ecto-nucleotidase), reactive blue 2 (P2 receptor antagonist), and pertussis toxin, they secreted MMP-9, suggesting that Gi-coupled P2Y receptor-mediated signals constitutively suppress the production of MMP-9. Among Gi-coupled P2Y receptors, we found that an inhibition of P2Y(14) receptor, a receptor for nucleotide-sugars such as UDP-glucose, is responsible for the production of MMP-9 by pharmacological and molecular biochemical analysis. As for the mechanisms, the inhibition of P2Y(14) receptors resulted in the release of tumor necrosis factor (TNF)-α which then acted on astrocytes to induce MMP-9. Taken together, our results suggest that the constitutive releases of nucleotide-sugars in astrocytes should play an important role in maintaining the normal status of the cell, through Gi-coupled P2Y(14) receptors, and when the signal is removed, the cells start to release TNF-α, which then acts on astrocytes in a feedback fashion to boost MMP-9 synthesis and secretion.

Letter to the editor: a meta-analysis of MMP-2 expression on the survival of patients with colorectal cancer

Dear Editor: Colorectal cancer (CRC) is one of the most common malignancies in the world and causes serious damage to human health. Despite diagnostic and therapeutic improvements, the prognosis for CRC patients is generally poor. Prognostic factors for survival are useful in the management of CRC patients and to individualize treatment and improve prognosis, and these factors are useful when choosing the treatment on an individual basis, principally disease stage, and performance status. The biological factors involved in carcinogenesis should also be considered as potential survival prognostic factors. Matrix metalloproteinases (MMPs) are able to degrade extracellular matrix components and promote the formation of metastasis. MMPs have an important role in tumor invasion and angiogenesis. MMP-2 is considered to play a critical role in metastasis, and the synthesis and secretion of MMP-2 can be stimulated by a variety of stimuli, including cytokines, during various pathological processes such as tumor invasion, atherosclerosis, and inflammation. Therefore, MMP-2 should be a promising prognostic marker and enhanced expression of MMP-2 in cancer cells has been noticed to be a significant factor to predict poor survival in CRC. A number of studies investigated the relationship between TS expression and survival in CRC patients; however, the results are inconsistent. The aim of this report was to review published studies that investigated the relationship between MMP-2 expression and CRC survival, and to use standard meta-analysis techniques to derive a more precise estimate of the prognostic significance of MMP-2 expression. A computerized bibliography was extracted from the electronic database PubMed, EMBASE, and CBM database until January 31, 2012. The search strategy included the terms: (“colorectal cancer” or “rectal cancer” or “colon cancer”) and (“MMP-2” or “matrix metalloproteinase-2”). All studies matching the eligibility criteria were retrieved and bibliographies checked for other relevant publications. Review articles and bibliographies of other relevant studies identified were hand-searched to identify additional studies. To be eligible for inclusion in this systematic review, a study must meet the following criteria: (1) patients with CRC, (2) it assesses the relationship between MMP-2 and survival, and has been published as a full paper, (3) it has a follow-up time exceeding 3 years, (4) hazard ratio (HR@@) and its 95 % confidence interval (95 % CI) comparing patients with high MMP-2 expression with patients with low expression were described or statistically extractable from data in the article. When duplicate articles were published, only the newest or most informative single article was selected. Only studies providing information on survival were included; studies investigating response rates only, without survival analysis, were excluded. Care was taken to include only primary data or data that superseded earlier work. Two investigators independently extracted data and reached consensus on the following characteristics of the selected studies. To minimize the bias and to improve the reliability, 2 reviewers checked all potentially relevant studies independently. Potential disagreements were resolved by consensus. The following characteristics were extracted from eligible studies: name of first author, year of publication, sample size, test method, cutoff value, tumor stage, histologic Y. Gao : S. Lin : P. Yuan :M. Wang Department of Pathology, the Fourth Affiliated Hospital of China Medical University, Shenyang 110013, China

MiR-218 reverses high invasiveness of glioblastoma cells by targeting the oncogenic transcription factor LEF1

The invasive behavior of glioblastoma multiforme (GBM) cells is one of the most important reasons for the poor prognosis of this cancer. For invasion, tumor cells must acquire an ability to digest the extracellular matrix and infiltrate the normal tissue bordering the tumor. Preventing this by altering effector molecules can significantly improve a patient's prognosis. Accumulating evidence suggests that miRNAs are involved in multiple biological functions, including cell invasion, by altering the expression of multiple target genes. The expression levels of miR-218 correlate with the invasive potential of GBM cells. In this study, we found that miR-218 expression was low in glioma tissues, especially in GBM. The data showed an inverse correlation in 60 GBM tissues between the levels of miR-218 and MMP mRNAs (MMP-2, -7 and -9). Additionally, ectopic expression of miR-218 suppressed the invasion of GBM cells whereas inhibition of miR-218 expression enhanced the invasive ability. Numerous members of the MMP family are downstream effectors of the Wnt/LEF1 pathway. Target prediction databases and luciferase data showed that LEF1 is a new direct target of miR-218. Importantly, western blot assays demonstrated that miR-218 can reduce protein levels of LEF1 and MMP-9. We, therefore, hypothesize that miR-218 directly targets LEF1, resulting in reduced synthesis of MMP-9. Results suggest that miR-218 is involved in the invasive behavior of GBM cells and by targeting LEF1 and blocking the invasive axis, miR-218-LEF1-MMPs, it may be useful for developing potential clinical strategies.

Abstract 100: Periaortic Leptin Induces Local Medial Degeneration in ApoE -/- Mice Preceding Aneurysm Formation

Background: Leptin (Ob) is a proatherogenic hormone involved in vessel wall remodeling. We hypothesized that Ob locally synthesized in the abdominal aorta regulates vascular remodeling mediators (TGFβ, MMPs) thereby sensitizing the aorta to subsequent aneurysm formation. Methods: Human abdominal aortic aneurysm (AAA) wall (n=10), and non-dilated aortic (n=3) samples underwent real time qPCR mRNA analysis for Ob and Ob receptor (ObR), and immunohistochemical analysis (SM-αactin, CD68, Ob, and ObR). Plasma samples obtained prior to AAA surgery were analyzed for Ob and ObR antigen (n=20). A novel mouse model was utilized to elucidate the role of Ob in AAA formation. Local Ob synthesis was simulated by point application of 20μg Ob slow release (n=10), or placebo (n=10) compound at the peri-vascular space of the para-visceral aorta in ApoE -/- mice on a Western diet. Serial ultrasound examinations on days 0, 14, 28, 35, and 42 assessed aortic diameter at reference locations. A parallel group (n=10), randomized to Ob or placebo treatment was euthanized on day 5 for aortic mRNA analysis. Results: Increased Ob mRNA levels and immunoreactivity were evident in human AAA wall samples vs controls. Plasma Ob and ObR levels were within normal range. Ob treated mice demonstrated augmented growth rate of luminal aortic diameter in the para-visceral segment on day 28 (p=0.0001). Aortas analyzed 5 days after Ob deployment revealed suppressed TGFβ mRNA (p=0.002), while MMP-9 transcript was up-regulated (p=0.047). Ob treated mice euthanized on day 45 demonstrated localized aortic derangement of elastic fibers and loss of SM-αActin (p=0.07), alongside with increased MMP-9 (p=0.02), MMP-12 (p=0.001) antigen, and augmented adventitial macrophage infiltration. Conclusions: Ob is locally synthesized in human AAA wall. Peri-aortic Ob modulates the vessel wall in ApoE -/- mice, inducing medial degeneration at the para-visceral region. Mechanistically, the observed derangement of ECM components was associated with suppressed TGFβ mRNA, and up-regulated MMP-9 synthesis. These data suggest novel mechanistic links between Ob and human AAA formation.

Long term potentiation affects intracellular metalloproteinases activity in the mossy fiber — CA3 pathway

Matrix Metalloproteinases (MMPs) are a family of endopeptidases known to process extracellular proteins. In the last decade, studies carried out mainly on the Schaffer collateral-CA1 hippocampal projection have provided solid evidence that MMPs regulate synaptic plasticity and learning. Recently, our group has shown that MMP blockade disrupts LTP maintenance also in the mossy fiber-CA3 (mf-CA3) projection (Wojtowicz and Mozrzymas, 2010), where LTP mechanisms are profoundly different (NMDAR-independent and presynaptic expression site). However, how plasticity of this pathway correlates with activity and expression of MMPs remains unknown. Interestingly, several potential MMP substrates (especially of gelatinases) are localized intracellularly but little is known about MMP activity in this compartment. In the present study we have asked whether LTP is associated with the expression and activity of gelatinases in apparent intra- and extracellular compartments along mf-CA3 projection. In situ zymography showed that LTP induction was associated with increased gelatinases activity in the cytoplasm of the hilar and CA3 neurons. Using gelatin zymography, immunohistochemistry and immunofluorescent staining we found that this effect was due to de novo synthesis and activation of MMP-9 which, 2-3h after LTP induction was particularly evident in the cytoplasm. In contrast, MMP-2 was localized preferentially in the nuclei and was not affected by LTP induction. In conclusion, we demonstrate that LTP induction in the mf-CA3 pathway correlates with increased expression and activity of MMP-9 and provide the first evidence that this increase is particularly evident in the neuronal cytoplasm and nucleus.

The involvement of oxidants and NF-κB in cytokine-induced MMP-9 synthesis by bone marrow-derived osteoprogenitor cells

The activity of immune cells affects the balance between bone mineralization and resorption carried out by the opposing actions of osteoblasts and osteoclasts, respectively. This study was aimed at determining the possible interaction between inflammatory conditions and collagen type I degrading MMP (mainly MMP-2 and MMP-9) synthesis and secretion in rat osteoprogenitors. The study was performed using primary rat bone marrow-derived osteoprogenitors during their advanced osteogenesis. Biochemical, immunohistochemical, and molecular biology techniques were used to investigate the influence of pro-inflammatory cytokines on MMP-2 and MMP-9 synthesis and secretion in osteoprogenitors. Results indicated that both synthesis and secretion of MMPs (MMP-1, -2, -8, -9, and -13) were significantly induced after pro-inflammatory cytokine treatments, except MMP-2, whose levels remained unchanged. NF-κB (nuclear factor kappa-light chain enhancer of activated B cells) inhibition assays showed that induced MMP-9 secretion by inflammatory cytokines was mediated by activation of NF-κB via the classical pathway and that oxidants play a significant role in this signal transduction pathway. In contrast, no such effect was observed for synthesis of MMP-2. These results indicate the possibility that inflammatory processes may trigger osteoblasts to absorb bone by secreting elevated levels of MMPs capable of degrading collagen type I, especially MMP-9 which is upregulated due to increased NF-κB transcription activity.

Patterns of matrix metalloproteinases and transforming growth factor-beta 1 expression during peritoneal repair in chlorhexidine induced peritoneal fibrosis mice

Recovery from peritoneal fibrosis (PF) involves the digestion of accumulated collagens and remodeling. Matrix metalloproteinases (MMPs) may play an important role in repair. The role of MMP-13, an important component in the MMP cascade, in PF is still unclear. We examined the sequential expression of MMP synthesis during repair in a PF mouse. Forty-eight mice at 8 weeks of age were given an intraperitoneal injection of 0.1% chlorhexidine gluconate (CG) for 3 weeks. Control mice were injected with the same dosages of 15% ethanol dissolved in saline. These mice were sacrificed, and anterior abdominal walls were obtained on days 21, 28, 35, 42, 49, and 56. Gene expressions of MMP-2 and -13, tissue inhibition of metalloproteinase-1 (TIMP-1) and -2, MT1-MMP, transforming growth factor-beta 1, and collagen types I and III were analyzed by real-time polymerase chain reaction. MMP-13 enzyme activity was also measured. In immunohistological evaluation, MMP-13 expressing cells were examined. Thickening of the peritoneum and marked infiltration of monocytes were induced by CG, and the alterations remained until 7 days after cessation of CG. Then tissue repair rapidly advanced. Synthesis of collagen types I and III, MMP-2, TIMP-1 and -2, and transforming growth factor-beta 1 in the injured peritoneum was significantly increased until day 28. These increments were preceded by an increase of MMP-13 synthesis and activity after cessation of CG. Some infiltrating macrophages in the thickened peritoneum showed MMP-13 expression early after cessation of CG. MMP-13 was synthesized by infiltrating monocytes early in the repair process in the CG-induced PF mouse. After cessation of stimulant, increase of MMP-13 synthesis may act as an inducer of an efficient degradation cascade in collagen-rich peritoneal tissue. 腹膜纖維化的復原涉及膠原蛋白堆積的消化與其後的重塑，其中，基質金屬蛋白酶(MMPs)在組織修復期間可能佔有重要的角色。作為MMP級聯中的重要一環，MMP-13在腹膜纖維化上的角色仍然未明。本研究透過腹膜纖維化(PF)的小鼠，調查了在腹膜修復期間MMP生成的表現順序。 研究材料為48隻年齡8週的小鼠，先接受每天共3週的0.1%葡萄糖酸氯己定(CG)腹膜內注射；另外對照組的注射則採用相同容量or劑量含15%乙醇的鹽水。其後陸續宰殺小鼠以取得第21、28、35、42、49及56天的前腹壁檢體，並採用實時PCR測量以下的基因表現：MMP-2-13、TIMP-1-2、MT1-MMP、TGF-β(1)、及I III型膠原蛋白。此外亦同時測量MMP-13之酵素活動，並對表現MMP-13的細胞作免疫組織學評估。 在CG誘導下，可見腹膜增厚及單核球的明顯浸潤，直至停止CG後7天，其後可見組織修復的快速進行。在停止CG後的受損腹膜中，首先可見MMP-13生成與活動的增加，其後亦可見I III型膠原蛋白、MMP-2、TIMP-1-2、及TGF-β(1)生成的明顯增加，直至第28天。在停止CG後的早期，可見增厚腹膜中有若干巨噬細胞具MMP-13表現。 在CG所致的PF小鼠中，腹膜修復早期已可見浸潤的單核球產生MMP-13。在停止刺激後，在富含膠原蛋白的腹膜環境中，MMP-13似乎可促使一個高效降解級聯的進行。

Potent synergistic effect of IL-3 and TNF on matrix metalloproteinase 9 generation by human eosinophils

TNF (designated as TNF-α under previous nomenclature) is the preeminent activator of MMP-9 generation from a variety of cells including eosinophils. We have previously established that TNF strongly synergizes with IFN-γ and IL-4 for eosinophil synthesis of Th1- and Th2-type chemokines respectively. Thus, we sought to determine if TNF-induced synthesis of MMP-9 would be enhanced by the presence of Th1, Th2, or the eosinophil-associated common beta chain (βc) cytokines. Human blood eosinophils were cultured with TNF alone or in combination with either IFN-γ, IL-4, IL-3, IL-5, or GM-CSF. Concentrations and activities of MMP-9 in eosinophil culture supernates were measured by ELISA and gelatin zymography, mRNA transcription and stabilization by quantitative real-time PCR, and signaling events by immunoblotting and intracellular flow cytometric analysis. Individually, TNF, GM-CSF, or IL-3, but not IL-4 or IFN-γ, induced relatively small (<0.2ng/ml) but statistically significant quantities of MMP-9. Remarkable synergistic synthesis of MMP-9 (ng/ml levels) occurred in response to TNF plus IL-3, GM-CSF or IL-5, in the order of IL-3>GM-CSF>IL-5. Zymography revealed that eosinophils release MMP-9 in its pro-form. Eosinophil stimulation with the combination of IL-3 plus TNF led to increased steady-state levels of MMP-9 mRNA, prolonged mRNA stabilization, and enhanced activation of ERK1/2 phosphorylation. Inhibition of NF-κB, MEK kinase, or p38 MAP kinase, but not JNK signaling pathways, diminished IL-3/TNF-induced MMP-9 mRNA and protein production. Thus, the synergistic regulation of eosinophil MMP-9 by IL-3 plus TNF likely involves cooperative interaction of multiple transcription factors downstream from ERK, p38, and NF-κB activation as well as post-transcriptional regulation of MMP-9 mRNA stabilization. Our data indicate that within microenvironments rich in βc-family cytokines and TNF, eosinophils are an important source of proMMP-9 and highlight a previously unrecognized role for synergistic interaction between TNF and βc-family cytokines, particularly IL-3, for proMMP-9 synthesis.

Inhibitory Effects of Pentosan Polysulfate Sodium on MAP-Kinase Pathway and NF-κB Nuclear Translocation in Canine Chondrocytes &lt;i&gt;In Vitro&lt;/i&gt;

Pentosan polysulfate sodium (PPS) has a heparin-like structure and is purificated from the plant of European beech wood. PPS has been used for the treatment of interstitial cystitis for human patients. Recent years, it was newly recognised that PPS reduce pain and inflammation of OA. The molecular biological mechanism of PPS to express its clinical effects is not fully understood. The purpose of the present study is to investigate a mechanism of action of PPS on inflammatory reaction of chondrocytes in vitro. It was evaluated that effects of PPS on interleukin (IL)-1β-induced phosphorylation of mitogen-actiated protein kinases (MAPKs), such as p38, extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), nuclear translocation of nuclear factor-kappa B (NF-κB), and matrix metalloproteinase (MMP)-3 production in cultured articular chondrocytes. As a result, in the presence of PPS existence, IL-1β-induced phosphorylation of p38 and ERK were certainly inhibited, while JNK phosphorylation was not affected. Nuclear translocation of NF-κB and MMP-3 production were suppressed by PPS pretreatment prior to IL-1β stimulation. In conclusion, it is strongly suggested that PPS treatment prevents inflammatory intracellular responses induced by IL-1 β through inhibition of phosphorylation of certain MAPKs, p38 and ERK and then nuclear translocation of NF-κB in cultured chondrocytes. These PPS properties may contribute to suppressive consequence of catabolic MMP-3 synthesis. These data might translate the clinical efficacy as PPS treatment could inhibit the cartilage catabolism and related clinical symptoms of OA in dogs.