1. SYNTHETIC EXTRACELLULAR MATRICES FOR PROMOTING ANGIOGENESIS Eduardo A. Silva Harvard University, Cambridge, MA, USA and Faculdade de Engenharia da Universidade do Porto, Porto, Portugal December 2004 Porto, Portugal

2. MOTIVATION Cardiovascular Diseases: - Principal cause of mortality in western countries Current Approaches to treat Ischemic Heart Diseases: Pharmacologic Invasive Surgical Therapeutic Angiogenesis

3. ISCHEMIA Carmeliet & Jain Nature, 2000 Ischemia - i sch- is restriction, hemia or haemia is blood

4. ANGIOGENESIS Sprouting of new blood vessels from pre-existent vessels

5. VASCULAR ENDOTHELIAL GROWTH FACTOR VEGF Is a key regulator of blood vessels formation and function Endothelial Cells are the primary target of VEGF 45 kDa homodimer Four isoforms obtained by alternative splicing: VEGF 121, VEGF 165, VEGF 189, VEGF 206 Muller et al., Structure, 5:1325-1338 (1997) Fairbrother et al., Structure, 6:637-648 (1998)

6. AIM I: Investigate VEGF signaling in cardiac cells under hypoxia. AIM I

7. VEGF SECRETION UNDER HYPOXIA

8. TISSUE ENGINEERING STRATEGY Localized, sustained delivery from bioactive polymeric systems Constant local concentration Protection from degradation Minimal side effects

9. AIM II Development of an injectable polymeric system for controlled and defined delivery of angiogenic molecules VEGF incorporated in an injectable polymer

10. POLYMERIC SYSTEM Alginate O OH CO 2 - O O OH - O 2 C O HO CO 2 - OH O HO CO 2 - OH O O O O CO 2 - O OH GGMMG Ca 2+  -L- Guluronic acid  -D- Mannuronic acid Gelation process Hydrogel

11. MODEL SYSTEM Distinct approach used to incorporate VEGF Simple mixing VEGF and alginate Pre-incorporation of VEGF in polymeric microspheres and immobilization in the alginate ++ Ca 2+ Angiogenic moleculesAlginate ++ Microspheres Polymeric system Bioactive molecules

12. ALGINATE MODIFICATIONS Combination of binary molecular weight distribution and partial oxidation Oxidation with NaIO 4  Irradiation (5 Mrad) CHO

13. Solution Properties

14. Gel Properties

15. VEGF RELEASE

16. BIOACTIVITY OF VEGF RELEASED

17. ANGIOGENESIS

18. PDGF RELEASE

19. VEGF/PDGF RELEASE

20. In vivo evaluation of VEGF releasing materials Model: Ischemic hind limb of Apo E null (Apo E -/- ) LDPI image

21. In vivo evaluation of VEGF releasing materials Blank VEGF& PDGF VEGF Week 2Week 4Week 6

22. In vivo evaluation of VEGF releasing materials

23. CONCLUSIONS Sustainable and controlled angiogenic molecules delivery can be achieved with binary alginate Blood vessel formation and perfusion can be significantly improved by using binary alginate as local dual delivery vehicle

24. FUTURE WORK VEGF 121 AND VEGF 165 PLAY DISTINCT ROLES IN THE STIMULATION OF COLLATERAL VESSEL AND BLOOD FLOW RE-ESTABLISHMENT IN AN ISCHEMIC HEART VEGF 121µm VEGF 165 - +

25. ACKNOWLEDGMENTS Professor David J. Mooney Professor Mário Barbosa Department of Radiology Division of Cardiovascular Diseases Department of Internal Medicine University of Michigan Paul M. Grossman Sanjay Rajagopalan Qinghua Sun Mooney Lab 2nd PGDB FCT - Fundação para a Ciência e a Tecnologia IGC/FCG - Fundação Calouste Gulbenkian NIH - National Institutes of Health Harvard University/University of Michigan & Universidade do Porto

26. Thinking about Curia 2005

27. A - Alginate; B - New tissue; C - Blood Vessel; D - Inflammatory cells