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      Zoledronate upregulates MMP-9 and -13 in rat vascular smooth muscle cells by inducing oxidative stress

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          Bisphosphonates, including zoledronate, target osteoclasts and are widely used in the treatment of osteoporosis and other bone resorption diseases, despite side effects that include damaging the stomach epithelium. Beneficial and adverse effects on other organ systems, including the cardiovascular system, have also been described and could impact on the use of bisphosphonates as therapeutic agents. Vascular smooth muscle cells (VSMCs) are major constituents of the normal vascular wall and have a key role in intimal thickening and atherosclerosis, in part by secreting MMPs that remodel the extracellular matrix and cleave cell surface proteins or secreted mediators. In this study, we investigated the effects of zoledronate on MMP expression.


          Rat VSMCs were stimulated by PDGF (50 ng/mL) plus TNF-α (10 ng/mL) or left unstimulated for a further 24 hours in serum-free medium. In other series of experiments, cells were pre-treated either with SC-514 (50 μM) or with apocynin (20 nM) for 2 hours, then zoledronate (100 μM) was added into 2% fetal calf serum containing medium for 24 hours.

          Results and discussion

          Using isolated rat VSMCs in culture, zoledronate (100 μM) increased MMP-9 and -13 mRNA expressions but inhibited MMP-2 expression. MMP-9 and MMP-13 up-regulation was shown to depend on the NF-κB pathway; and this was activated by zoledronate. Furthermore, zoledronate elevated the levels of reactive oxygen species detected by either dichlorofluorescein in isolated VSMCs or lucigenin enhanced chemiluminescence in rat aortic rings in vitro. Apocynin, an inhibitor of NADPH oxidase, reversed NF-κB activation and MMP-9 and MMP-13 up-regulation by zoledronate.


          We conclude that zoledronate increases MMP-9 and MMP-13 expressions in rat VSMCs dependent upon stimulation of the NF-κB pathway by reactive oxygen species. Effects on MMP expression may contribute to the pharmacologic profile of bisphosphonates.

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          Most cited references 25

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          Reactive oxygen species produced by macrophage-derived foam cells regulate the activity of vascular matrix metalloproteinases in vitro. Implications for atherosclerotic plaque stability.

          Vulnerable areas of atherosclerotic plaques often contain lipid-laden macrophages and display matrix metalloproteinase activity. We hypothesized that reactive oxygen species released by macrophage-derived foam cells could trigger activation of latent proforms of metalloproteinases in the vascular interstitium. We showed that in vivo generated macrophage foam cells produce superoxide, nitric oxide, and hydrogen peroxide after isolation from hypercholesterolemic rabbits. Effects of these reactive oxygens and that of peroxynitrite, likely to result from simultaneous production of nitric oxide and superoxide, were tested in vitro using metalloproteinases secreted by cultured human vascular smooth muscle cells. Enzymes in culture media or affinity-purified (pro-MMP-2 and MMP-9) were examined by SDS-PAGE zymography, Western blotting, and enzymatic assays. Under the conditions used, incubation with xanthine/xanthine oxidase increased the amount of active gelatinases, while nitric oxide donors had no noticeable effect. Incubation with peroxynitrite resulted in nitration of MMP-2 and endowed it with collagenolytic activity. Hydrogen peroxide treatment showed a catalase-reversible biphasic effect (gelatinase activation at concentrations of 4 microM, inhibition at > or = 10-50 microM). Thus, reactive oxygen species can modulate matrix degradation in areas of high oxidant stress and could therefore contribute to instability of atherosclerotic plaques.
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            Interleukin-1 induction of collagenase 3 (matrix metalloproteinase 13) gene expression in chondrocytes requires p38, c-Jun N-terminal kinase, and nuclear factor kappaB: differential regulation of collagenase 1 and collagenase 3.

            To examine the mechanism of interleukin-1 (IL-1)-induced collagenase 3 (matrix metalloproteinase 13 [MMP-13]) gene expression in cultured chondrocytes for the purpose of better understanding how the gene is induced in these cells, and how it contributes to cartilage degradation in osteoarthritis. The transcriptional and posttranscriptional responses of the MMP-13 gene to IL-1 were assessed first. Then, direct inhibitors of mitogen-activated protein kinase (MAPK) signaling pathways and a constitutive repressor of nuclear factor kappaB (NF-kappaB) were used to assess the role of each pathway in IL-1-mediated induction of MMP-13. We found that IL-1 induction of MMP-13 requires p38 activity, c-Jun N-terminal kinase (JNK) activity and NF-kappaB translocation. These results suggest that both NF-kappaB and activator protein 1 transcription factors are necessary for IL-1 induction of MMP-13. We also compared the signaling pathways necessary for IL-1 to stimulate collagenase 1 (MMP-1) in articular chondrocytes and chondrosarcoma cells and found that IL-1 induction of MMP-1 requires different pathways from those required by MMP-13. In chondrosarcoma cells, MMP-1 induction depends on p38 and MEK (an MAPK kinase of the extracellular signal-regulated kinase pathway) and does not require JNK or NF-kappaB. In articular chondrocytes, inhibition of MEK had no effect, while inhibition of p38 gave variable results. These studies demonstrate, for the first time, that p38, JNK, and NF-kappaB are required for IL-1 induction of MMP-13. The results also highlight the differential requirements for signaling pathways in the induction of MMP-1 and MMP-13. Additionally, they demonstrate that induction of MMP-1 by IL-1 in chondrocytic cells depends on unique combinations of signaling pathways that are cell type-specific.
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              Amino-biphosphonate-mediated MMP-9 inhibition breaks the tumor-bone marrow axis responsible for myeloid-derived suppressor cell expansion and macrophage infiltration in tumor stroma.

              BALB-neuT mice expressing an activated rat c-erbB-2/neu transgene under the mouse mammary tumor virus long terminal repeat show enhanced hematopoiesis with hyperproduction of myeloid-derived suppressor cells (MDSC) because of vascular endothelial growth factor (VEGF) secreted by the tumor. Here, we show that both tumor and stromal cells express matrix metalloproteinase-9 (MMP-9), thereby increasing the levels of pro-MMP-9 in the sera of tumor-bearing mice. Treatment with amino-biphosphonates impaired tumor growth, significantly decreased MMP-9 expression and the number of macrophages in tumor stroma, and reduced MDSC expansion both in bone marrow and peripheral blood by dropping serum pro-MMP-9 and VEGF. We dissected the role of tumor-derived MMP-9 from that secreted by stromal leukocytes by transplanting bone marrow from MMP-9 knockout mice into BALB-neuT mice. Although bone marrow progenitor-derived MMP-9 had a major role in driving MDSC expansion, amino-biphosphonate treatment of bone marrow chimeras further reduced both myelopoiesis and the supportive tumor stroma, thus enhancing tumor necrosis. Moreover, by reducing MDSC, amino-biphosphonates overcome the tumor-induced immune suppression and improved the generation and maintenance of antitumor immune response induced by immunization against the p185/HER-2. Our data reveal that suppression of MMP-9 activity breaks the vicious loop linking tumor growth and myeloid cell expansion, thus reducing immunosuppression. Amino-biphosphonates disclose a specific MMP-9 inhibitory activity that may broaden their application above their current usage.

                Author and article information

                Drug Des Devel Ther
                Drug Des Devel Ther
                Drug Design, Development and Therapy
                Drug Design, Development and Therapy
                Dove Medical Press
                18 April 2016
                : 10
                : 1453-1460
                [1 ]Department of Pharmacology, Faculty of Pharmacy, Ege University, Izmir, Turkey
                [2 ]Bristol Heart Institute, University of Bristol, Bristol Royal Infirmary, Bristol, UK
                Author notes
                Correspondence: Mehmet Zuhuri Arun, Department of Pharmacology, Faculty of Pharmacy, Ege University, 35100 Bornova, Izmir, Turkey, Tel +90 232 311 3288, Fax +90 232 388 4687, Email mehmet.arun@ 123456ege.edu.tr
                © 2016 Arun et al. This work is published and licensed by Dove Medical Press Limited

                The full terms of this license are available at https://www.dovepress.com/terms.php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License ( http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed.

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