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1. Supplementary information S1 (animation) Proposed role of syndecan-1 in the regulation of αVβ3 integrin- and VEGF-dependent angiogenesis. This interactive figure allows the user to navigate around the complex body of in vivo and in vitro data that relates to the functional interplay between αVβ3 integrin, vascular endothelial growth factor (VEGF) and VEGF receptor-2 (VEGFR2) in the regulation of pathological angiogenesis. The figure specifically highlights how potentially syndecan-1 may differentially regulate the αvβ3 integrin and VEGF–VEGFR signalling network (green boxes/arrow). The downstream effects of perturbing β3 integrin-dependent functions can be seen by clicking on the purple buttons to the left of the page, or on the β3 integrin functions in the top row of the figure. Users can return to the base model by clicking the red ‘Home’ button or by re-clicking on the disrupted β3 integrin function. The potential role of syndecan-1 in regulating these processes can be viewed by clicking the green button. References are included throughout the slide show. Perturbing αvβ3 integrin function in vivo can have remarkably different effects on pathological angiogenesis, depending on the nature of the perturbation and the cell type that is expressing the integrin (tumour (red) or host/endothelial (blue)). β3 integrin-deficient mice exhibit enhanced pathological angiogenesisas a consequence of increased VEGFR2 expression and signalling. Perturbation of tumour-cell αvβ3 integrin activation reduces pathological angiogenesis and tumour growth by suppressing VEGF secretion. Endothelial cell β3 integrin phosphorylation is required for VEGF-stimulated, VEGFR2-phosphorylation-dependent, pathological angiogenesis. Cross-activation of αVβ3 integrin and VEGFR2 is required for the formation of the αVβ3 integrin–VEGFR2 complex and endothelial cell migration. Inhibition of αVβ3 integrin engagement induces caspase-8-dependent apoptosis of angiogenic vessels and reduces tumour growth. In vitro studies suggest that syndecan-1, through the regulation of integrin activation and growth-factor presentation, could possibly modulate this β3 integrin–VEGF–VEGFR2 signalling network at various points. Start

2. The v3-integrin-VEGF/VEGFR Signalling Network Home b3 Activation b3 Signalling avb3 Engagement b3 orb3/b5 Expression Potential Syndecan-1 Function VEGF Secretion 3 Knock-out b3-VEGFR2 Complex Formation VEGFR2 Phosphorylation VEGF-VEGFR2 Engagement Caspase-8 Activation Normal Endothelial VEGFR2 Expression Inactive 3 Phospho-null 3 Endothelial Cell Migration Endothelial Cell Apoptosis v3 Antagonism Pathological Angiogenesis References Boxes/Arrows: The functional interplay between αvβ3-integrin, VEGF and VEGFR2 in the regulation of pathological angiogenesis; highlighting how potentially syndecan-1 may regulate this signalling network (green boxes/arrows) Functional regulation Syndecan-1-regulated Potentially Syndecan-1-regulated

3. Potential Syndecan-1 Function Soluble Syndecan-1 Ectodomain 1 Shedding Home 1 Syndecan-1 Potential Syndecan-1 Function 1 1 1 b3 Activation b3 Signalling avb3 Engagement b3 orb3/b5 Expression 3 Knock-out 2, 3 VEGF Secretion b3-VEGFR2 Complex Formation VEGFR2 Phosphorylation VEGF-VEGFR2 Engagement Caspase-8 Activation Normal Endothelial VEGFR2 Expression Inactive 3 2, 3 Phospho-null 3 Endothelial Cell Migration Endothelial Cell Apoptosis v3 Antagonism Pathological Angiogenesis References Boxes/Arrows: Functional regulation In vitro studies suggest that syndecan-1, through regulation of integrin activation and growth factor presentation, could possibly modulate the β3-integrin-VEGF/VEGFR2 signalling network Syndecan-1-regulated Potentially Syndecan-1-regulated

4. Integrin 3- or 3/5-Deficient Mice 3 or 3/5 -/- mice Home 4, 5 b3 Activation b3 Signalling avb3 Engagement b3 orb3/b5 Expression Potential Syndecan-1 Function VEGF Secretion 3 Knock-out b3-VEGFR2 Complex Formation VEGFR2 Phosphorylation VEGF-VEGFR2 Engagement Caspase-8 Activation Endothelial VEGFR2 Expression & Signalling Inactive 3 Phospho-null 3 Endothelial Cell Migration Endothelial Cell Apoptosis v3 Antagonism Pathological Angiogenesis References Boxes/Arrows: Functional regulation β3-integrin-deficient mice exhibit enhanced pathological angiogenesisas a consequence of increased VEGFR2 expression and signalling Syndecan-1-regulated Potentially Syndecan-1-regulated

5. Tumour Cell Expression of Inactivatable-3 Inactivatable 3 expression Home 6 b3 Activation b3 Signalling avb3 Engagement b3 orb3/b5 Expression Potential Syndecan-1 Function VEGF Secretion 3 Knock-out b3-VEGFR2 Complex Formation VEGFR2 Phosphorylation VEGF-VEGFR2 Engagement Caspase-8 Activation Normal Endothelial VEGFR2 Expression Inactive 3 Phospho-null 3 Endothelial Cell Migration Endothelial Cell Apoptosis v3 Antagonism Pathological Angiogenesis References Boxes/Arrows: Functional regulation Perturbation of tumour cell αvβ3-integrin activation reduces pathological angiogenesis and tumour growth by suppressing VEGF secretion Syndecan-1-regulated Potentially Syndecan-1-regulated

6. Phospho-null-3 Expression Phospho-incompetent 3 Home 7 b3 Activation b3 Signalling avb3 Engagement b3 orb3/b5 Expression Potential Syndecan-1 Function VEGF Secretion 8 7 3 Knock-out b3-VEGFR2 Complex Formation VEGFR2 Phosphorylation VEGF-VEGFR2 Engagement Caspase-8 Activation Normal Endothelial VEGFR2 Expression Inactive 3 Phospho-null 3 Endothelial Cell Migration Endothelial Cell Apoptosis v3 Antagonism Pathological Angiogenesis References Endothelial β3-integrin phosphorylation is required for VEGF-stimulated, VEGFR2-phosphorylation dependent, pathological angiogenesis. Cross-activation of αVβ3-integrin and VEGFR2 is required for the formation of the αVβ3-integrin–VEGFR2 complex and endothelial cell migration. Boxes/Arrows: Functional regulation Syndecan-1-regulated Potentially Syndecan-1-regulated

7. v3 Antagonism v3 antagonism Home 9, 10 b3 Activation b3 Signalling avb3 Engagement b3 orb3/b5 Expression Potential Syndecan-1 Function VEGF Secretion 3 Knock-out b3-VEGFR2 Complex Formation VEGFR2 Phosphorylation VEGF-VEGFR2 Engagement Caspase-8 Activation Normal Endothelial VEGFR2 Expression Inactive 3 Phospho-null 3 Endothelial Cell Migration Endothelial Cell Apoptosis v3 Antagonism Pathological Angiogenesis References Boxes/Arrows: Functional regulation Inhibition of αVβ3-integrin engagement induces caspase-8-dependent apoptosis of angiogenic vessels and reduces tumour growth Syndecan-1-regulated Potentially Syndecan-1-regulated

8. Reference List Home 1. Beauvais, D. M., Burbach, B. J. & Rapraeger, A. C. The syndecan-1 ectodomain regulates alphavbeta3 integrin activity in human mammary carcinoma cells. J Cell Biol167, 171-81 (2004). Fuster, M. M. et al. Genetic alteration of endothelial heparan sulfate selectively inhibits tumor angiogenesis. J Cell Biol177, 539-49 (2007). Subramanian, S. V., Fitzgerald, M. L. & Bernfield, M. Regulated shedding of syndecan-1 and -4 ectodomains by thrombin and growth factor receptor activation. J Biol Chem272, 14713-20 (1997). Reynolds, L. E. et al.Enhanced pathological angiogenesis in mice lacking beta3 integrin or beta3 and beta5 integrins. Nat Med8, 27-34 (2002). Reynolds, A. R. et al. Elevated Flk1 (vascular endothelial growth factor receptor 2) signaling mediates enhanced angiogenesis in beta3-integrin-deficient mice. Cancer Res64, 8643-50 (2004). De, S. et al. VEGF-integrin interplay controls tumor growth and vascularization. Proc Natl Acad Sci U S A102, 7589-94 (2005). 7. Mahabeleshwar, G. H., Feng, W., Phillips, D. R. & Byzova, T. V. Integrin signaling is critical for pathological angiogenesis. J Exp Med203, 2495-507 (2006). Mahabeleshwar, G. H., Feng, W., Reddy, K., Plow, E. F. & Byzova, T. V. Mechanisms of Integrin-Vascular Endothelial Growth Factor Receptor Cross-Activation in Angiogenesis. Circ Res (2007). Brooks, P. C. et al. Integrin alpha v beta 3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels. Cell79, 1157-64 (1994). 10. Stupack, D. G., Puente, X. S., Boutsaboualoy, S., Storgard, C. M. & Cheresh, D. A. Apoptosis of adherent cells by recruitment of caspase-8 to unligated integrins. J Cell Biol155, 459-70 (2001). Potential Syndecan-1 Function 3 Knock-out Inactive 3 Phospho-null 3 v3 Antagonism References