Inhibition Of Metalloproteinase Activity Research Articles

A design-of-experiment approach for obtaining Symphytum officinale L. extracts for cosmetic purposes

Promotion of wound or bone healing and reduction of inflammation are commonly known therapeutic qualities of comfrey (Symphytum officinale). Given the presence of active constituents, including allantoin and phenolics, comfrey is widely used in cosmetic and pharmaceutical industries. The aim of this study was to optimize a three-solvent system (water, ethanol, 1,3-propanediol) to maximize the extraction efficiency of allantoin, rosmarinic acid, rabdosiin, and globoidnan A in accordance with the approach of the simplex centroid mixture design. Polynomial regression models of the mixture composition showed the impact of the extracts on cytotoxicity, metalloproteinase inhibition activity, and antioxidant properties response. Additionally, the physical properties, including surface tension and contact angle of the mixture composition, were measured. Based on the maximization of the extraction efficiency of all compounds, an optimum solvent composition was determined as 30.9% of water, 39.2% of ethanol, and 29.9% of propanediol. Such a mixture allowed extraction of 93.6%, 96.8%, 77.0%, and 97.2% of the total amount of allantoin, rosmarinic acid, rabdosiin, and globoidnan A, respectively. The optimal mixture composition for extraction of the compounds was not correlated with inhibition of metalloproteinase activity and cell viability, while a strong positive relationship with antioxidant properties was observed. The optimized solvent composition ensured good extraction efficiency, and the extract had high antioxidant properties. In the near future, in vivo studies with instrumental methods should be conducted to evaluate the practical application of the extracts in the cosmetic industry.

Beneficial Effects of Ursolic Acid and Its Derivatives-Focus on Potential Biochemical Mechanisms in Cardiovascular Conditions.

Ursolic acid (UA) is a natural pentacyclic triterpenoid found in a number of plants such as apples, thyme, oregano, hawthorn and others. Several in vitro and in vivo studies have presented its anti-inflammatory and anti-apoptotic properties. The inhibition of NF-κB-mediated inflammatory pathways and the increased scavenging of reactive oxygen species (ROS) in numerous ways seem to be the most beneficial effects of UA. In mice and rats, administration of UA appears to slow down the development of cardiovascular diseases (CVDs), especially atherosclerosis and cardiac fibrosis. Upregulation of endothelial-type nitric oxide synthase (eNOS) and cystathionine-λ-lyase (CSE) by UA may suggest its vasorelaxant property. Inhibition of metalloproteinases activity by UA may contribute to better outcomes in aneurysms management. UA influence on lipid and glucose metabolism remains inconsistent, and additional studies are essential to verify its efficacy. Furthermore, UA derivatives appear to have a beneficial impact on the cardiovascular system. This review aims to summarize recent findings on beneficial effects of UA that may make it a promising candidate for clinical trials for the management of CVDs.

Mushrooms as potential therapeutic agents in the treatment of cancer: Evaluation of anti-glioma effects of Coprinus comatus, Cantharellus cibarius, Lycoperdon perlatum and Lactarius deliciosus extracts

Cancer incidence rates are on the increase worldwide. The most common brain cancer in adults is glioblastoma. Currently available treatment modalities are limited and natural products such as mushrooms could enhance them. Apart from nutritional value, mushrooms are an excellent source of bioactive compounds and therefore could be used to treat various disorders. The aim of the study was to assess the anti-glioma potential of selected mushrooms on U87MG, LN-18 glioblastoma and SVGp12 normal human astroglial cell lines. The materials were Cantharellus cibarius, Coprinus comatus, Lycoperdon perlatum and Lactarius delicious. Aqueous, 70 % ethanol or 95 % ethanol extracts from mushrooms were used for analysis including assessment of antioxidant activity by DPPH assay, cell viability by MTT assay, DNA biosynthesis by thymidine incorporation assay, activity of metalloproteinase by gelatin zymography and cell cycle assay by flow cytometry. Mushroom extracts influenced the viability and DNA biosynthesis of cancer cells. Activity of ethanol mushroom extracts was stronger than that of aqueous extracts. Anti-glioma mechanism consisted in inhibition of cancer cell proliferation and induction of apoptosis associated with arrest of cells in subG1 or G2/M phase of cell cycle, and inhibition of metalloproteinases activity. Among investigated mushrooms, L. deliciosus and C. comatus showed the greatest anti-glioma potential.

Cyclosporin A activates human hepatocellular carcinoma (HepG2 cells) proliferation: implication of EGFR-mediated ERK1/2 signaling pathway.

One of the most common causes of cancer mortality worldwide is hepatocellular carcinoma (HCC). Extracellular signal-regulated kinase (ERK1/2) pathway has been shown to play an important role in the development and progression of HCC. Here, we demonstrate that the immunosuppressive agent cyclosporin A (CsA) has the ability to increase the cellular growth in HCC (HepG2 cells) via activation of ERK1/2 signaling cascade. It was found that ERK1/2 phosphorylation induced by CsA was highly reduced in the presence of the reactive oxygen species (ROS) scavenger polyethylene glycol-superoxide dismutase (PEG-SOD). Furthermore, it was observed that inhibition of metalloproteinase activity using TAPI-2 prevents ERK1/2 activation by CsA. Moreover, a disintegrin and metalloproteinase domain 17 (ADAM-17) activity was found to be critical for ERK phosphorylation by CsA. In addition, CsA-induced ERK phosphorylation was highly reduced in the presence of either neutralizing anti-heparin-binding-epidermal growth factor (HB-EGF) antibody or UO126 (MEK inhibitor). By using the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor AG1478, it was found that EGFR is critical for ERK phosphorylation induced by CsA. Furthermore, CsA-induced cell proliferation was strongly reduced in the presence of either PEG-SOD or TAPI-2 or neutralizing anti-ADAM17 antibody or neutralizing anti-HB-EGF antibody or AG1478 or UO126. Collectively, these data demonstrate that CsA has the ability to activate ERK1/2 signaling cascade that could be translated into an increase in HepG2 cell proliferation. Furthermore, these data support the role of ROS, ADAM-17, and EGFR in ERK1/2 signaling activation and subsequent cell proliferation induced by CsA in HepG2 cells.

Collagen peptides modulate the metabolism of extracellular matrix by human dermal fibroblasts derived from sun-protected and sun-exposed body sites.

Clinical data published in recent years have demonstrated positive effects of collagen hydrolysate (CH) on skin aging clinical signs. CH use as food supplement has a long history; however, few studies have addressed the underlying purpose of CH on the cellular and molecular biology of skin cells that could elucidate clinical improvement findings. Wide diversity of characteristics has been reported for dermal fibroblasts derived from different body sites and it is unknown whether collagen peptides could modulate differently cells from chronological aged and photoaged skin areas. This study investigated the influence of CH on the extracellular matrix metabolism and proliferation of human dermal fibroblasts (HDFs) derived from chronological aged (sun-protected) and photoaged (sun-exposed) body sites. CH treatment did not affect cellular proliferation of either cell cultures, but notably modulated cell metabolism in monolayer model, increasing the content of dermal matrix precursor and main protein, procollagen I and collagen I, respectively. These effects were confirmed in the human dermal equivalent model. The increase in collagen content in the cultures was attributed to stimulation of biosynthesis and decreased collagen I metabolism through inhibition of metalloproteinase activity (MMP) 1 and 2. Modulation of CH in dermal metabolism did not differ between cells derived from sun-protected and sun-exposed areas, although lower concentrations of CH seemed to be enough to stimulate sun-exposed-derived HDFs, suggesting more pronounced effect in these cells. This study contributes to understanding the biological effects of CH on skin cells and viability of its use as a functional ingredient in food supplements.

Tissue inhibitor of metalloproteinases 3-dependent microvascular endothelial cell barrier function is disrupted under septic conditions.

Sepsis is associated with dysfunction of microvascular endothelial cells (MVEC) leading to tissue edema and multiple organ dysfunction. Metalloproteinases can regulate MVEC function through processing of cell surface proteins, and tissue inhibitor of metalloproteinases 3 (TIMP3) regulates metalloproteinase activity in the lung following injury. We hypothesize that TIMP3 promotes normal pulmonary MVEC barrier function through inhibition of metalloproteinase activity. Naive Timp3(-/-) mice had significantly higher basal pulmonary microvascular Evans blue (EB) dye-labeled albumin leak vs. wild-type (WT) mice. Additionally, cecal-ligation/perforation (CLP)-induced sepsis significantly increased pulmonary microvascular EB-labeled albumin leak in WT but not Timp3(-/-) mice. Similarly, PBS-treated isolated MVEC monolayers from Timp3(-/-) mice displayed permeability barrier dysfunction vs. WT MVEC, evidenced by lower transendothelial electrical resistance and greater trans-MVEC flux of fluorescein-dextran and EB-albumin. Cytomix (equimolar interferon γ, tumor necrosis factor α, and interleukin 1β) treatment of WT MVEC induced significant barrier dysfunction (by all three methods), and was associated with a time-dependent decrease in TIMP3 mRNA and protein levels. Additionally, basal Timp3(-/-) MVEC barrier dysfunction was associated with disrupted MVEC surface VE-cadherin localization, and both barrier dysfunction and VE-cadherin localization were rescued by treatment with GM6001, a synthetic metalloproteinase inhibitor. TIMP3 promotes normal MVEC barrier function, at least partially, through inhibition of metalloproteinase-dependent disruption of adherens junctions, and septic downregulation of TIMP3 may contribute to septic MVEC barrier dysfunction.

Multifunctional Nanoparticles Facilitate Molecular Targeting and miRNA Delivery to Inhibit Atherosclerosis in ApoE(-/-) Mice.

The current study presents an effective and selective multifunctional nanoparticle used to deliver antiatherogenic therapeutics to inflamed pro-atherogenic regions without off-target changes in gene expression or particle-induced toxicities. MicroRNAs (miRNAs) regulate gene expression, playing a critical role in biology and disease including atherosclerosis. While anti-miRNA are emerging as therapeutics, numerous challenges remain due to their potential off-target effects, and therefore the development of carriers for selective delivery to diseased sites is important. Yet, co-optimization of multifunctional nanoparticles with high loading efficiency, a hidden cationic domain to facilitate lysosomal escape and a dense, stable incorporation of targeting moieties is challenging. Here, we create coated, cationic lipoparticles (CCLs), containing anti-miR-712 (∼1400 molecules, >95% loading efficiency) within the core and with a neutral coating, decorated with 5 mol % of peptide (VHPK) to target vascular cell adhesion molecule 1 (VCAM1). Optical imaging validated disease-specific accumulation as anti-miR-712 was efficiently delivered to inflamed mouse aortic endothelial cells in vitro and in vivo. As with the naked anti-miR-712, the delivery of VHPK-CCL-anti-miR-712 effectively downregulated the d-flow induced expression of miR-712 and also rescued the expression of its target genes tissue inhibitor of metalloproteinase 3 (TIMP3) and reversion-inducing-cysteine-rich protein with kazal motifs (RECK) in the endothelium, resulting in inhibition of metalloproteinase activity. Moreover, an 80% lower dose of VHPK-CCL-anti-miR-712 (1 mg/kg dose given twice a week), as compared with naked anti-miR-712, prevented atheroma formation in a mouse model of atherosclerosis. While delivery of naked anti-miR-712 alters expression in multiple organs, miR-712 expression in nontargeted organs was unchanged following VHPK-CCL-anti-miR-712 delivery.

Septic Murine Pulmonary Microvascular Endothelial Barrier Dysfunction in vitro is Regulated by TIMP3

Sepsis often causes dysfunction of pulmonary microvascular endothelial cells (PMECs) leading to pulmonary edema. Metalloproteinases can regulate endothelial function through processing of cell surface proteins, including adherens junctions proteins, which has been associated with increased permeability. Tissue inhibitor of metalloproteinases 3 (TIMP3) regulates metalloproteinase function in the lung following injury. Thus, we hypothesize that TIMP3 promotes PMEC stability via inhibition of metalloproteinase activity. To test this hypothesis, we used PMECs isolated from WT and Timp3‐/‐mice. We found that TIMP3 levels (mRNA and protein) were decreased in WT PMECs in a time‐dependent fashion under septic conditions (cytomix). Analysis of leak (transendothelial electrical resistance, dextran, and albumin flux) revealed that Timp3‐/‐PMECs had significantly higher permeability under resting conditions (PBS) vs. WT PMECs. Although cytomix treatment significantly increased WT trans‐PMEC permeability, there was no effect on Timp3‐/‐PMEC barrier function. Additionally, the increased basal Timp3‐/‐ PMEC permeability was associated with disrupted surface vascular endothelial‐cadherin localization, which was rescued by treatment with GM6001, a synthetic inhibitor of metalloproteinases. Together, our data suggest that TIMP3 supports normal PMEC barrier function and that septic downregulation of TIMP3 may be an important contributor to septic PMEC barrier dysfunction.

Inhibition of metalloproteinase activity in FANCA is linked to altered oxygen metabolism.

Bone marrow (BM) failure, increased risk of myelodysplastic syndrome, acute leukaemia and solid tumors, endocrinopathies and congenital abnormalities are the major clinical problems in Fanconi anemia patients (FA). Chromosome instability and DNA repair defects are the cellular characteristics used for the clinical diagnosis. However, these biological defects are not sufficient to explain all the clinical phenotype of FA patients. The known defects are structural alteration in cell cytoskeleton, altered structural organization for intermediate filaments, nuclear lamina, and mitochondria. These are associated with different expression and/or maturation of the structural proteins vimentin, mitofilin, and lamin A/C suggesting the involvement of metalloproteinases (MPs). Matrix metalloproteinases (MMP) are involved in normal physiological processes such as human skeletal tissue development, maturation, and hematopoietic reconstitution after bone marrow suppression. Current observations upon the eventual role of MPs in FA cells are largely inconclusive. We evaluated the overall MPs activity in FA complementation group A (FANCA) cells by exposing them to the antioxidants N-acetyl cysteine (NAC) and resveratrol (RV). This work supports the hypothesis that treatment of Fanconi patients with antioxidants may be important in FA therapy.

Understanding CpG Methylation in the Context of Osteoarthritis

Worldwide, osteoarthritis (OA) affects approxi‐ mately 40% of people older than 70 years and is therefore the most common form of arthritis. OA is a complex degenerative disease of the joint but is epitomized by focal degradation of articular cartilage [1]. Cartilage itself is a highly special‐ ized tissue composed of an extracellular matrix (ECM) rich in type‐II collagen and the proteo‐ glycan aggrecan. The tissue is sparsely populated by the sole resident cell, the chondrocyte, which maintains tissue integrity by balancing the syn‐ thesis and degradation of ECM components [2]. In OA, this balance is perturbed in favor of ECM degradation, mediated by proteolytic enzymes including MMP13 and ADAMTS5, which prob‐ ably degrade the type‐II collagen and aggrecan, respectively [3]. Inhibition of metalloproteinase activity should protect against cartilage destruc‐ tion, but has failed in clinical trials in part owing to nonselectivity and resulting adverse side effects [4]. Amongst other things, this has led to the sug‐ gestion that targeting the regulation of expression of specific proteases, or regulating factors, could be an important therapeutic option to slow or halt OA progression. There is an urgent need for such disease‐modifying, noninvasive treatments for OA since the only current therapy is joint replacement surgery. Epigenetic regulation involves three basic mechanisms: DNA methylation, histone modi‐ fications and ncRNAs. DNA methylation rep‐ resents the most stable epigenetic modification and involves the addition of a methyl group to the cytosine at CpG dinucleotides, converting cytosine to 5‐methylcytosine. This functions to disrupt the binding of transcription factors or enhance the binding of repressors, leading to an altered transcriptional state of the cell [5]. In terms of OA research, a number of studies have inves‐ tigated the role that epigenetics, especially DNA methylation, could play in OA, particularly in terms of regulating the balance between matrix synthesis and degradation in cartilage. These studies have focused on correlating DNA methyla‐ tion status with gene expression at a few candidate genes (loci), including those coding for aggrecan, collagens, MMPs, LEPTIN, SOX9 and RUNX2 [6]. Broadly, these works have shown a correlation between promoter methylation and the expression of the associated gene. Recently, we took this work one step further and identified a specific differen‐ tially methylated CpG locus within the MMP13 promoter whose methylation status regulates the expression of the gene by controlling the binding of the transcriptional regulator CREB [7]. These and ongoing ‘candidate’ gene studies will continue to identify differentially methylated regions that may be important in onset of the disease.

Heparin-Binding EGF-Like Growth Factor (HB-EGF) Mediates 5-HT-Induced Insulin Resistance Through Activation of EGF Receptor-ERK1/2-mTOR Pathway

Although an inverse correlation between insulin sensitivity and the level of Gq/11-coupled receptor agonists, such as endothelin-1, thrombin, and 5-hydroxytryptamine (5-HT), has been reported, its precise mechanism remains unclear. In this report, we provide evidence that 5-HT induced production of heparin-binding epidermal growth factor-like growth factor (HB-EGF) and caused insulin resistance in 3T3-L1 adipocytes, primary adipocytes, and C2C12 myotubes. In 3T3-L1 adipocytes, 5-HT stimulated HB-EGF production by promoting metalloproteinase-dependent shedding of transmembrane protein pro-HB-EGF. HB-EGF then bound and tyrosine-phosphorylated EGF receptors, which activated the mammalian target of rapamycin pathway through ERK1/2 phosphorylation. Mammalian target of rapamycin activation caused serine phosphorylation of insulin receptor substrate-1, which attenuated insulin-stimulated tyrosine phosphorylation of insulin receptor substrate-1 and glucose uptake. Pharmacological inhibition of either Gq/11-coupled receptors or metalloproteinases, as well as either inhibition or knockdown of HB-EGF or Gαq/11, restored insulin signal transduction impaired by 5-HT. Inhibition of metalloproteinase activity also abolished HB-EGF production and subsequent EGF receptor activation by other Gq/11-coupled receptor agonists known to cause insulin resistance, such as endothelin-1 and thrombin. These results suggest that transactivation of the EGF receptor through HB-EGF processing plays a pivotal role in 5-HT-induced insulin resistance.

Proangiogenic Factor PlGF Programs CD11b+ Myelomonocytes in Breast Cancer during Differentiation of Their Hematopoietic Progenitors

Tumor-mobilized bone marrow-derived CD11b(+) myeloid cells promote tumor angiogenesis, but how and when these cells acquire proangiogenic properties is not fully elucidated. Here, we show that CD11b(+) myelomonocytic cells develop proangiogenic properties during their differentiation from CD34(+) hematopoietic progenitors and that placenta growth factor (PlGF) is critical in promoting this education. Cultures of human CD34(+) progenitors supplemented with conditioned medium from breast cancer cell lines or PlGF, but not from nontumorigenic breast epithelial lines, generate CD11b(+) cells capable of inducing endothelial cell sprouting in vitro and angiogenesis in vivo. An anti-Flt-1 mAb or soluble Flt-1 abolished the generation of proangiogenic activity during differentiation from progenitor cells. Moreover, inhibition of metalloproteinase activity, but not VEGF, during the endothelial sprouting assay blocked sprouting induced by these proangiogenic CD11b(+) myelomonocytes. In a mouse model of breast cancer, circulating CD11b(+) cells were proangiogenic in the sprouting assays. Silencing of PlGF in tumor cells prevented the generation of proangiogenic activity in circulating CD11b(+) cells, inhibited tumor blood flow, and slowed tumor growth. Peripheral blood of breast cancer patients at diagnosis, but not of healthy individuals, contained elevated levels of PlGF and circulating proangiogenic CD11b(+) myelomonocytes. Taken together, our results show that cancer cells can program proangiogenic activity in CD11b(+) myelomonocytes during differentiation of their progenitor cells in a PlGF-dependent manner. These findings impact breast cancer biology, detection, and treatment.

Inhibition of Metalloproteinase Activity by Chinese Formulations Used to Treat Inflammatory Diseases

Matrix metalloproteinases (MMPs) are known to be involved in a number of pathological processes including cancer, atherosclerosis, arthritis and neurodegenerative disease among others. The drive to develop MMP inhibitors as therapeutics has beef put forward for years by pharmaceutical companies as well as academicians. In an attempt to screen for MMP inhibitors from Traditional Chinese Medicines (TCMs), a number of Chinese formulations used to treat inflammatory diseases such as nephritis and hepatitis have been studied. Strong inhibitory effects of three Chinese formulations toward the activity of MMP-16 have been discovered. These results suggest that these anti-inflammatory medicines contain some unknown MMP inhibitory compound(s) and provide reasonable molecular mechanisms for their therapeutic effects.

Matrix metalloproteinase inhibition impairs the processing, formation and mineralization of dental tissues during mouse molar development

Organotypic cultures of embryonic mouse tooth germs were used to investigate the developmental expression and roles of MMPs in the formation and mineralization of dentin and enamel matrices. The spatially and temporally regulated expression of MMP-2, MMP-9 and MMP-20 in developing mouse tooth germs in vivo was maintained in culture. The inhibition of metalloproteinases activity by marimastat altered the morphogenesis and mineralization of the tooth germs associated with an inhibition of the activation of both MMP-20 and MMP-2. MMP inhibition deregulated the molecular processing of two major dental matrix proteins, amelogenin and dentin sialoprotein (DSP). This coincided with their accumulation and the loss of their normal distribution within the extracellular matrix, resulting in a defective mineralization of dentin and enamel matrices. These findings demonstrate the critical role of MMPs in the processing and maturation of the dental matrix.

Inhibition of metalloproteinase activity by fruit extracts

While the metalloproteinase enzymes are essential for development and remodeling of tissues, aberrant over expression of these enzymes contributes to several pathologic conditions. In particular, metalloproteinase over expression in cancer plays a significant role in metastasis by providing a mechanism for invasion and spread. The data presented here indicate that water extracts of raspberries, blackberries and muscadine grapes inhibit the activities of metalloproteinases 2 and 9. This inhibition could contribute to the suppression of carcinogenesis by diets high in fruit content.

Inhibition of metalloproteinase activity by fruit extracts

While the metalloproteinase enzymes are essential for development and remodeling of tissues, aberrant over expression of these enzymes contributes to several pathologic conditions. In particular, metalloproteinase over expression in cancer plays a significant role in metastasis by providing a mechanism for invasion and spread. The data presented here indicate that water extracts of raspberries, blackberries and muscadine grapes inhibit the activities of metalloproteinases 2 and 9. This inhibition could contribute to the suppression of carcinogenesis by diets high in fruit content.

Metalloproteinase inhibitors improve the recovery and hemostatic function of in vitro–aged or –injured mouse platelets

AbstractPlatelet transfusions are a crucial component of support for patients with severe thrombocytopenia. Storage of platelet concentrates, however, is associated with a reduction in platelet posttransfusion recovery and hemostatic function. In this study, we established a model of mitochondrial injury that resembles platelet storage lesion. Mitochondrial injury, provoked by incubation of platelets with carbonyl cyanide m-chlorophenylhydrazone (CCCP), led to reduced posttransfusion recovery in mice, an effect that directly correlated with the duration of treatment. Damaged platelets were characterized by shape change, disruption of membrane asymmetry, surface expression of P-selectin, and profound proteolysis of GPIbα. Using our model, we identified a key role for endogenous metalloproteinase(s) in platelet clearance, as their inhibition markedly improved posttransfusion recovery of both the mitochondria-injured and in vitro-aged mouse platelets. Metalloproteinase inhibition also prevented proteolysis of GPIbα on damaged platelets, thereby improving the hemostatic function of these cells in vivo. We propose that inhibition of metalloproteinase activity during storage could significantly improve the effectiveness of platelet transfusions. Surface expression of GPIbα might be a powerful marker to determine the quality of platelet concentrates, because it reflects metalloproteinase activity in vitro. (Blood. 2003;102: 4229-4235)