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Role of Thymidine Recent Advances in Angiogenesis and Antiangiogenesis, 2009 69 cells transfected with three enzymatically inactive TP mutants, K115E, L148R and R202S did not possess angiogenic activity in contrast to wild-type transfectants [13]. Also the role of TP substrate (thymidine) and enzymatic products were extensively studied. TP, its catalytic product 2dR-1P, and the subsequent metabolite 2dR promoted endothelial tubulogenesis in vitro, and the regeneration of a wounded monolayer of ECs without exerting any mitogenic effect. In vivo, 2dR promoted blood vessel formation in an avascular sponge and in the CAM assay [12,16]. TP, 2dR-1P and 2dR were also found to induce chemotaxis of human umbilical vein endothelial cells (HUVEC). Migration induced by 2dR-1P was blocked by an alkaline phosphatase inhibitor, suggesting that the chemotactic activity of 2dR-1P first requires its conversion to 2dR. In a co-culture assay, U937 and THP1 human monocyte cells, which constitutively express high levels of TP, and TPoverexpressing carcinoma cells strongly induced HUVEC migration. Cell-stimulated HUVEC migration was inhibited by the TP inhibitor 5-chloro- 6(1-imidazolylmethyl)uracil but not by a neutralizing antibody to TP, although the latter completely blocked migration induced by purified TP [23]. Together, these experimental data indicate that the angiogenesis-stimulating properties of TP (i) require its enzymatic activity and (ii) are mediated by the degradation product 2dR. So, although a receptor for TP has never been identified and TP is mainly retained intracellularly, the protein can elicit its angiogenic activity via the intracellular metabolism of thymidine and subsequent extracellular release of 2dR, which forms a chemotactic gradient. Interestingly, several of the biological effects induced by 2dR were found to be inhibited by its stereoisomer 2-deoxy-L-ribose [24-26]. Only recently, molecular mediators of TP and signaling pathways associated with TP-induced angiogenesis have been identified. Both TP and 2dR were found to stimulate the formation of focal adhesions and the phosphorylation of focal adhesion kinase (FAK) at tyrosine 397 in HUVEC and this effect was blocked by antibodies to either integrin 51 or v3. TP and 2dR also increased the cell surface expression of integrin 51 and the association of FAK and vinculin with 51 [27]. A complementary DNA microarray was used to identify genes that are induced during in vitro migration of TP-overexpressing DLD-1 colon cancer cells. Rho-associated coiled-coil domain kinase (ROCK1) was found to be significantly overexpressed in TP transfectants compared with mock-transfected cells. TP transfectants also showed higher cell motility than control cells. Also addition of recombinant TP increased cell migration of wild-type DLD-1 cells, and motility was blocked by a neutralizing antibody to TP and by Y-27632, a specific inhibitor of ROCK1. Moreover, actin fiber polymerization, which is a marker of activation of ROCK1, was higher in TP transfectants than in control cells [28]. Addition of TP to AGS and MKN-45 gastric carcinoma cell lines resulted in increased cancer cell invasion through matrigel, which was accompanied by actin filament remodeling and increased activation of the phosphatidylinosital-3-kinase (PI3K) pathway. In addition, TP-overexpressing MKN-45 cells showed increased activity of mammalian target of rapamycin (mTOR) and p70 ribosomal S6 kinase (p70 S6K ) compared with control cells, suggesting the involvement of the PI3K/mTOR/ p70 S6K pathway in TP-induced migration and invasion of gastric carcinoma cells [19]. Transfection of RT112 human bladder carcinoma cells with TP resulted in the secretion of vascular endothelial growth factor (VEGF), interleukin (IL)-8, and matrix metalloproteinase (MMP)-1 [29]. Secretion of these angiogenic factors was only evident after supplementing the culture medium with thymidine, which recapitulates the tumor microenvironment in which thymidine concentrations are raised due to the hydrolysis of DNA from necrotic cells in hypoxic areas. Thymidine was shown to increase cellular levels of heme-oxygenase (HO-1), a marker for oxidative stress, in RT112/TP cells. This effect was abrogated by thymine, which may reverse thymidine catabolism by capturing 2dR-1P. These data suggest that 2dR-1P is responsible for the TP-induced oxidative stress, possibly by generating oxygen radical species during the early stages of protein glycation [29]. A timedependent increase in HO-1 expression was also observed during monolayer regeneration of RT112/TP cells [16]. Increased mRNA expression and activity of MMP-9 were observed in KB/TP cell cultures and tumors compared with their mock-transfected counterparts and this was suggested to reflect the increased invasive potential of KB/TP cells/tumors [26]. KK47 bladder cancer cells that overexpress TP had higher levels of MMP-7 and MMP-9 than control cells, whereas overexpression of TP in prostate cancer cells resulted in increased expression of MMP-1 and MMP-7. Moreover, in bladder cancers from 72 randomly selected patients, the expression level of TP was found to correlate with that of urokinase-type plasminogen activator (uPA), MMP-1, MMP-9, plasminogen activator inhibtor (PAI)-1 and VEGF. Taken together, these data indicate that the TP-induced production and
80 Recent Advances in Angiogenesis and Antiangiogenesis, 2009, 80-84 CHAPTER 9 Role of Stromal Cells in Neovascularization of Multiple Myeloma Maria Fico¹, Giuseppe Mangialardi¹, Roberto Ria¹, Michele Moschetta¹, Domenico Ribatti² and Angelo Vacca¹ ¹Department of Biomedical Sciences and Human Oncology, University of Bari Medical School, I-70124 Bari, Italy ²Department of Human Anatomy and Histology, University of Bari Medical School, I-70124 Bari, Italy Address correspondence to: Dr. Angelo Vacca, Department of Internal Medicine and Clinical Oncology, Unit of Allergology and Clinical Immunology, Policlinico – Piazza Giulio Cesare, 11, 70124 Bari, Italy; Tel: +39-080-559.34.44; Fax: +39-080- 559.21.89; Email: a.vacca@dimo.uniba.it 1. INTRODUCTION Abstract: Angiogenesis plays a pivotal role in progression of both solid and hematologic tumors. We have focused on multiple myeloma (MM) and its bone marrow stromal cells which are not only a support for tumor cell survival, but also active inducers of angiogenesis by releasing a broad number of angiogenic cytokines. Also, stromal cells such as macrophages and mast cells can participate in blood vessels formation in MM through other processes, such as a vasculogenic mimicry. Finally, it has been discovered that hematopoietic stem and progenitor cells (HSPCs) are involved in the vasculogenesis of MM. Multiple myeloma (MM) is a disease caused by the accumulation of malignant plasma cells that usually, but not in every case, actively produces antibodies [1]. During cell maturation, after the switch into the lymph nodes, B cells express a large number of adhesion molecules that facilitate their homing in the bone marrow where they afterwards differentiate. Once in the bone marrow, the adhesion molecules mediate the homotropic interactions between plasma cells and B cells, and the heterotropic interactions between plasma cells and both extracellular matrix and bone marrow stromal cells [2]. In MM, the expression of these molecules changes in the disease course, especially when plasma cells pass from the bone marrow into the peripheral blood [3]. Therefore, MM progression is characterized by three separate events: loss of the capacity to enhance apoptosis, expansion of transformed plasma cells into the bone marrow, and dissemination of malignant cells along the body. The last two steps need angiogenesis to develop. 2. ANGIOGENESIS IN ACTIVE MULTIPLE MYELOMA The angiogenesis in MM allows the formation of new vessels that provide nutrients for transformed cells, thus supporting their high level of replication, and simultaneously facilitate the egress of MM cells into the blood circulation. These newly-formed vessels have not an ordered architecture, and show a discontinuous endothelial surface [4]. Transformed plasma cells participate to the angiogenic process by Domenico Ribatti (Ed.) All rights reserved - © 2009 Bentham Science Publishers Ltd. producing a large number of cytokines that act directly on endothelial cells (ECs) and stromal cells which, in turn, release enzymes and other cytokines that amplify angiogenesis. Adhesion of MM cells to the bone marrow stroma is mediated by different types of surface receptors. MM plasma cells express very lateactivating antigen-4 and -5 (VLA-4 and VLA-5), and β2-integrin lymphocyte function-associated antigen-1 (LFA-1) that allow the aggregation of homotropic cells ad the link to heterotropic cells [5]. These cellcell interactions induce the release of interleukin-6 (IL-6) and transforming growth factor-β (TGF-β) by bone marrow stromal cells [6,7], and of IL-1β, tumor necrosis factor-α (TNF-α), IL-6 and vascular endothelial growth factor (VEGF) by tumor cells [8- 11]. These growth factors stimulate clonal expansion of plasma cells and contribute to bone destruction [12]. In particular, IL-6 hides the pro-apoptotic signals of FAS antigen [13]. Moreover, IL-6 synthesized not only by tumor cells but also by bone marrow stromal cells exerts an angiogenic activity both directly and indirectly, through the release of VEGF [14]. In turn, the production of IL-6 by stromal cells is due to the activity of fibroblast growth factor-2 (FGF-2) released by the plasma cells. (Fig. 1) summarizes the interplay between various cells present in the bone marrow microenvironment and various growth factors promoting angiogenesis in MM. 3. ROLE OF MACROPHAGES AND THEIR VASCULOGENIC MIMICRY IN MULTIPLE MYELOMA
Page 2 and 3: Recent Advances in Angiogenesis and
Page 4 and 5: FOREWORD It has been known for a ve
Page 6 and 7: single proangiogenic protein. Howev
Page 8 and 9: v Vito Pistoia Head, Laboratory of
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Page 12 and 13: 10 Recent Advances in Angiogenesis
Page 16 and 17: The Role of Mesenchymal Stem Cells
Page 22 and 23: Thymus and Angiogenesis Recent Adva
Page 34 and 35: Role of Stromal Cells Recent Advanc
Page 40 and 41: Tumor Targeting with Transgenic End
Page 44 and 45: Tumor Vascular Disrupting Agents Re
Page 50 and 51: Tumour Vascular Disrupting Agents R
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