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113 Recent Advances in Angiogenesis and Antiangiogenesis, 2009 Taraboletti and Bonezzi effect on angiogenesis is extremely complex and context-dependent. Like other matricellular proteins, it is composed of a succession of different functional domains, each able to specifically interact with cell receptors, soluble cytokines and growth factors, extracellular matrix components, and proteases. The local presence of these receptors/ligands and the bioavailability of each TSP-1 domain will therefore dictate the pattern of molecular interactions, hence the biological effect of TSP-1 in a given biological setting. Although TSP-1 is commonly considered an inhibitor of angiogenesis, it can also stimulate it, through biologically active pro-angiogenic domains, mostly mapped in the N-terminal domain [15-20]. Thus, TSP-based antiangiogenic therapies could be designed either to mimic the molecule’s antiangiogenic properties or to antagonize its proangiogenic activity. TSP-1 can affect angiogenesis both directly and indirectly. As a direct inhibitor, it interacts with specific receptors on endothelial cells to affect cell viability and functions related to angiogenesis. As an indirect inhibitor, it binds to and influences the activity/bioavailability of various mediators of angiogenesis, such as angiogenic factors, cytokines and proteases. Although this review will mostly focus on the effects of TSP on endothelial cells, TSP-1 is also active on other cell types involved in angiogenesis, including smooth muscle cells, monocytes/macrophages and T cells [21, 22]. Here we focus on TSP-1 and tumor-associated angiogenesis. A brief description of membrane receptors or soluble TSP-1 ligands is provided, followed by a list of active sequences and the evidence that these TSP-peptides are bona fide inhibitors of angiogenesis and tumor growth. 2. “DRUGGABLE” TSP-1 RECEPTORS AND LIGANDS TSP-1 interacts with a variety of receptors on the surface of endothelial cells, eliciting both anti- and pro-angiogenic responses. Therapeutic exploitation could therefore by achieved by TSP-1 mimetics in the first case, and by antagonists in the latter. Membrane Receptors CD36, the first TSP-1 membrane receptor identified [23, 24], is an 88 kDa glycoprotein expressed by platelets and many cell types, including microvascular but not large-vessel endothelial cells [25]. The TSP-1/CD36 interaction occurs between the conserved CLESH-1 domain in CD36 and the type I repeats in TSP-1 [26]. Homology searches indicate that the TSP-1 recognition motif of CD36 is found in several molecules, within and outside the CD36 family, including lysosomal integral membrane protein II (LIMPII) [27] and HIV envelope gp120 [28]. On the other hand, a large family of proteins - the TSR-superfamily - contains sequences similar to the type I repeats [29]. It is therefore plausible that the CLESH/TSR pattern of recognition extends beyond the CD36/TSP-1 system and is implicated in different molecular networks and biological processes. CD36 mediates the inhibitory effects of TSP-1 on FGF-2-induced endothelial cell migration, morphological organization [26, 30] and angiogenesis in the cornea assay [31]. By preventing free fatty acids uptake by CD36, TSP-1 causes inhibition of eNOS and therefore the NO-dependent response [32]. TSP-1 binding to CD36 induces endothelial cell apoptosis through the sequential activation of p59fyn, caspase-3 and p38 MAPK [31]. Apoptosis ultimately involves death receptors, the Fas/FasL (CD95/CD95L) or the TNFa/TNF-R1 systems, with possible differences in endothelial cell types and stimuli [33, 34]. Interestingly, the TSP-1/CD36 interaction affects the activity/expression of other receptors involved in angiogenesis. TSP-1, via CD36, down-modulates vascular endothelial growth factor (VEGF) receptor 2 (VEGFR-2) [30]. CD36 is also constitutively associated with ß1 integrin in endothelial cells [30], with interesting implications for the mechanisms of the anti-motility activity of TSP-1 and the possibility of cooperative antiangiogenic activity between integrin antagonists and CD36-binding sequences of TSP-1. CD47, also known as IAP (integrin-associated protein), was initially identified as the cell membrane receptor for the adhesive sequence 4N1 (KRFYVVMWKK) in the C-terminal G domain of TSP-1, mediating TSP-1-induced cell spreading [35, 36]. CD47 forms a signaling complex with vß3 and other integrins (reviewed in [21, 37]). The soluble 4N1 peptide reportedly inhibits tube formation, though not proliferation, of capillary endothelial cells, and FGF-2-induced neovascularization in the cornea assay [38]. The TSP-1/CD47 interaction is important in the inhibition of nitric oxide (NO) by TSP-1. Although both CD47 and CD36 contribute to NO regulation by TSP-1, only CD47 has proved necessary for this effect, since ligation of CD36 was unable to block NO signaling in cells lacking CD47 [32]. Regulation of NO signaling by TSP-1 has important consequences on the regulation not only of angiogenesis, but also of tumor blood flow, hence potentially on the activity of vasoactive and vascular targeting agents [39]. The N-terminal domain of TSP-1, mostly responsible for the pro-angiogenic activity of the molecule [17-20], binds heparin and related molecules, such as heparansulfate proteoglycans and sulfatides. The Nterminal domain of TSP-1 interacts with syndecan-4 to stimulate endothelial cell tubulogenesis and protect cells from apoptosis [40]. Syndecan-4 binds two TSP-
Inhibitors of Angiogenesis Based on Thrombospondin-1 Recent Advances in Angiogenesis and Antiangiogenesis, 2009 114 1 peptides bearing the consensus motifs for binding to glycosaminoglycans, Hep I (aa 17-35) and Hep II (aa 78-94). Hep II also comprises the binding sequence for 6 integrin, pointing to cooperation between the two ligands in the regulation of endothelial cell activity by TSP-1. The Hep I peptide in the heparin-binding domain of TSP-1 also signals through a receptor co-complex involving calreticulin and low-density lipoprotein receptor (LDLR)-related protein (LRP) to stimulate endothelial cell motility and focal adhesion disassembly through activation of the PI3K signaling pathway [41]. These molecules are endocytic receptors for TSP-1 [42]. The interaction of TSP-1 with VLDLR or LRP-1 and endocytosis of the TSP-VEGF complex contribute to the CD36-independent inhibition of VEGF activity by TSP-1 and have been proposed as the mechanism of the homeostatic activity of TSP in preserving the quiescence of normal endothelium [43, 44]. In addition, LRP-1 reportedly mediates TSP-2-dependent internalization of MMP-2 [45, 46]. TSP interacts with several integrins expressed by endothelial cells and involved in angiogenesis, including 3ß1, 4ß1, 5ß1, 6ß1, 9ß1, and vß3. Binding sites for ß1 integrins have been mapped in the N-terminal domain [19, 47, 48], the second and third type I repeats [49, 50] and the type II repeats (the latter reportedly not recognized by endothelial cells) [49]. Independently of whether the interaction between the intact TSP-1 and ß1 integrins elicited pro- or antiangiogenic functions in endothelial cells, preclinical studies indicate that small TSP-1 peptides containing the integrin binding site, as well as disintegrins or anti-integrin antibodies, can be used to block endothelial cell pro-angiogenic functions such as adhesion, proliferation, survival, wound healing, motility and angiogenesis in the chorioallantoic membrane (CAM) assay [18, 19, 51]. Interaction of TSP-1 or a peptide comprising the entire type I repeats with ß1 integrins inhibited VEGF-induced migration through a PI3k-dependent mechanism [50]. The integrin-recognition RGD sequence is present in the type III repeats of TSP-1, and interacts with vß3 and 5ß1 integrins [52]. Although cryptic in calcium loaded TSP-1, under certain conditions this sequence can mediate the pro-adhesive property of TSP-1 for endothelial cells [52]. The role of this sequence in angiogenesis still needs to be clarified. The usefulness of integrins as targets for antiangiogenic and antineoplastic therapies is still debated. Nonetheless, integrin antagonists have shown antiangiogenic and antineoplastic activity in preclinical studies, and some are currently undergoing clinical evaluation for cancer treatment (reviewed in [53, 54]). The use of integrin ligands for target-specific delivery of imaging or therapeutic agents is another possible means of exploiting integrin-binding sequences, including integrin recognizing TSP peptides. Soluble Ligands Consistent with its matricellular nature, TSP-1 binds and influences the activity/bioavailability of different mediators of angiogenesis, such as angiogenic factors, cytokines and proteases [2, 55, 56]. TSP-1 binds to angiogenic factors including FGF-2, VEGF, HGF, PDGF, and the viral protein tat [57-61]. We found that TSP-1 bound FGF-2 with high affinity - in the nanomolar range, similar to the affinity of the growth factor for heparin [58], and the binding site was located within the type III repeats [60]. Heparin prevented the TSP-1/FGF-2 interaction [58, 60], suggesting that negatively charged residues of TSP-1 bound the heparin-recognizing region of FGF-2. Indeed, TSP-1 prevented FGF-2 binding to heparan sulfate proteoglycans in the extracellular matrix and on the surface of endothelial cells, where they constitute the low-affinity receptors [59]. As a consequence, TSP-1 and the type III repeats prevent FGF-2 binding to cells and long-term internalization, and deplete the extracellular matrix of stored FGF-2, an important event in the regulation of FGF-2 location and bioavailability [58, 59]. TSP-1 also affects the interaction with the matrix of two other heparinbinding angiogenic factors, VEGF and HGF [57, 59] The FGF-2/TSP-1 interaction is modulated by calcium ions, since low calcium concentrations are required for binding [60], suggesting that, as for other active sites in the type III repeats, the FGF-2 binding site is not exposed in calcium-replete TSP-1, and its exposure/availability is regulated by environmental conditions. TSP-1 binds VEGF [44, 62, 63]. The VEGF-binding domain is conceivably located in the type I repeats of TSP-1, since the TSR in connective tissue growth factor (CTGF) binds specifically to VEGF165, preventing its binding to endothelial cells, and inhibiting VEGF-induced tube formation in vitro and angiogenesis in vivo in the Matrigel assay [63]. TSP-1 binding to VEGF inhibits VEGF association with the extracellular matrix [59]. TSP-1 has also been reported to influence VEGF internalization through LRP-1, acting as a regulator of ovarian angiogenesis and follicle development [44]. Tumors in mice that lack TSP-1 in the mammary gland have a high level of bioactive, receptorassociated VEGF [3]. Direct interaction of TSP-1 with the growth factor, or indirect mechanisms involving MMP-9 might contribute to the regulation of VEGF bioavailability by TSP-1. TSP-1 is an important regulator of protease activity, as it binds and inhibits the activity of matrix metalloproteinases (MMP), urokinase plasminogen activator, plasmin, neutrophil elastase and cathepsin G
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
Page 10 and 11: 2 Recent Advances in Angiogenesis a
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 32 and 33: Role of Thymidine Recent Advances i
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
Page 52: Index Recent Advances in Angiogenes

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