1. Start Supplementary information S1 (animation) Proposed role of syndecan-1 in the regulation of α V β 3 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 α V β 3 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 α v β 3 integrin and VEGF–VEGFR signalling network (green boxes/arrow). The downstream effects of perturbing β 3 integrin-dependent functions can be seen by clicking on the purple buttons to the left of the page, or on the β 3 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 β 3 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 α v β 3 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)). β 3 integrin-deficient mice exhibit enhanced pathological angiogenesis as a consequence of increased VEGFR2 expression and signalling. Perturbation of tumour-cell α v β 3 integrin activation reduces pathological angiogenesis and tumour growth by suppressing VEGF secretion. Endothelial cell β 3 integrin phosphorylation is required for VEGF-stimulated, VEGFR2-phosphorylation-dependent, pathological angiogenesis. Cross-activation of α V β 3 integrin and VEGFR2 is required for the formation of the α V β 3 integrin–VEGFR2 complex and endothelial cell migration. Inhibition of α V β 3 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 β 3 integrin–VEGF–VEGFR2 signalling network at various points.

2. Pathological Angiogenesis Normal Endothelial VEGFR2 Expression 3 Activation VEGF Secretion Endothelial Cell Migration Endothelial Cell Apoptosis Caspase-8 Activation 3 -VEGFR2 Complex Formation VEGFR2 Phosphorylation 3 Signalling VEGF- VEGFR2 Engagement v 3 Engagement Potential Syndecan-1 Function 3 Knock-out v 3 Antagonism Inactive 3 Phospho-null 3 References 3 or 3 / 5 Expression Home Syndecan-1-regulated Functional regulation Boxes/Arrows: Potentially Syndecan-1-regulated The v 3 -integrin-VEGF/VEGFR Signalling Network The functional interplay between α v β 3 -integrin, VEGF and VEGFR2 in the regulation of pathological angiogenesis; highlighting how potentially syndecan-1 may regulate this signalling network (green boxes/arrows)

3. Pathological Angiogenesis 3 or 3 / 5 Expression Normal Endothelial VEGFR2 Expression 3 Activation VEGF Secretion Endothelial Cell Migration Endothelial Cell Apoptosis Caspase-8 Activation 3 Signalling VEGF- VEGFR2 Engagement v 3 Engagement Potential Syndecan-1 Function 3 Knock-out v 3 Antagonism Inactive 3 Phospho-null 3 References Potential Syndecan-1 Function Syndecan-1 Soluble Syndecan-1 Ectodomain Shedding Home Syndecan-1-regulated Functional regulation Boxes/Arrows: Potentially Syndecan-1-regulated 1 11 2, 3 1 1 In vitro studies suggest that syndecan-1, through regulation of integrin activation and growth factor presentation, could possibly modulate the β 3 -integrin-VEGF/VEGFR2 signalling network 3 -VEGFR2 Complex Formation VEGFR2 Phosphorylation

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

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

6. Pathological Angiogenesis Normal Endothelial VEGFR2 Expression 3 Activation VEGF Secretion Endothelial Cell Migration Endothelial Cell Apoptosis Caspase-8 Activation 3 Signalling VEGF- VEGFR2 Engagement v 3 Engagement Potential Syndecan-1 Function 3 Knock-out v 3 Antagonism Inactive 3 Phospho-null 3 References 3 or 3 / 5 Expression Phospho-null- 3 Expression Home Phospho- incompetent 3 Syndecan-1-regulated Functional regulation Boxes/Arrows: Potentially Syndecan-1-regulated 8 7 7 Endothelial β 3 -integrin phosphorylation is required for VEGF- stimulated, VEGFR2-phosphorylation dependent, pathological angiogenesis. Cross-activation of α V β 3 -integrin and VEGFR2 is required for the formation of the α V β 3 -integrin–VEGFR2 complex and endothelial cell migration. 3 -VEGFR2 Complex Formation VEGFR2 Phosphorylation

7. Pathological Angiogenesis Normal Endothelial VEGFR2 Expression 3 Activation VEGF Secretion Endothelial Cell Migration Endothelial Cell Apoptosis Caspase-8 Activation 3 Signalling VEGF- VEGFR2 Engagement v 3 Engagement Potential Syndecan-1 Function 3 Knock-out v 3 Antagonism Inactive 3 Phospho-null 3 References 3 or 3 / 5 Expression v 3 Antagonism Home v 3 antagonism Syndecan-1-regulated Functional regulation Boxes/Arrows: Potentially Syndecan-1-regulated 9, 10 Inhibition of α V β 3 -integrin engagement induces caspase-8- dependent apoptosis of angiogenic vessels and reduces tumour growth 3 -VEGFR2 Complex Formation VEGFR2 Phosphorylation

8. Potential Syndecan-1 Function 3 Knock-out v 3 Antagonism Inactive 3 Phospho-null 3 References 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 Biol 167, 171-81 (2004). 2.Fuster, M. M. et al. Genetic alteration of endothelial heparan sulfate selectively inhibits tumor angiogenesis. J Cell Biol 177, 539-49 (2007). 3.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 Chem 272, 14713-20 (1997). 4.Reynolds, L. E. et al. Enhanced pathological angiogenesis in mice lacking beta3 integrin or beta3 and beta5 integrins. Nat Med 8, 27-34 (2002). 5.Reynolds, A. R. et al. Elevated Flk1 (vascular endothelial growth factor receptor 2) signaling mediates enhanced angiogenesis in beta3-integrin-deficient mice. Cancer Res 64, 8643-50 (2004). 6.De, S. et al. VEGF-integrin interplay controls tumor growth and vascularization. Proc Natl Acad Sci U S A 102, 7589-94 (2005). 7. Mahabeleshwar, G. H., Feng, W., Phillips, D. R. & Byzova, T. V. Integrin signaling is critical for pathological angiogenesis. J Exp Med 203, 2495-507 (2006). 8.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). 9.Brooks, P. C. et al. Integrin alpha v beta 3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels. Cell 79, 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 Biol 155, 459-70 (2001).