ANGIOGENESIS Endothelial Cell Migration Assay Endothelial Cell Tube Formation Assay

1. P02_Endothelial cells: Functional Analysis - Migration-Tube formation ANGIOGENESIS Endothelial Cell Migration Assay Endothelial Cell Tube Formation Assay

2. P02_Endothelial cells: Functional Analysis - Migration-Tube formation CONTENT • Angiogenesis: Definition & Overview • Angiogenesis: Assays Overview • Endothelial Cell Migration Assay (using FluoroBlokTM Insert system) • Endothelial Cell Tube Formation Assay

3. P02_Endothelial cells: Functional Analysis - Migration-Tube formation Angiogenesis: Definition • VASCULOGENESIS • embryonic formation and differentiation of the vascular system • formation of new blood vessels from angioblasts or progenitor stem cells (de-novo vessel synthesis) • ANGIOGENESIS • formation of new blood vessels (sprouting, branching etc.) from existing vessels

4. P02_Endothelial cells: Functional Analysis - Migration-Tube formation Angiogenesis: The Sprouting Process • TRIGGERS FOR SPROUTING • Infections • Tissue damage • Tumors • Hypoxia • SPROUTING SIGNALS • Mainly VEGF and HIF • SPROUTING EVENTS • Secretion of proteases (MMPs) • Digestion of basal lamina of vessels • Migration to signal source • Endothelial cell proliferation • Tube formation new vessels bring O2 and reduce sprouting trigger

5. Endothelial cells as target for drug screening DRUG e.g. Tyrosin kinase inhibitors TKI “mode of action” Inhibition of prolfieratio- Migration-Vessel formation? AR? 5

6. P02_Endothelial cells: Functional Analysis - Migration-Tube formation Angiogenesis: Activators and Inhibitors

7. P02_Endothelial cells: Functional Analysis - Migration-Tube formation Angiogenesis: The VEGF Pathway

8. P02_Endothelial cells: Functional Analysis - Migration-Tube formation Angiogenesis: VEGF as a Key Factor • Vascular Endothelial Cell Growth Factor (VEGF) • Secreted homodimeric growth factor • Expressed by a variety of vascularized tissue • Different isoforms. VEGF165 as key regulator of blood vessel growth • VEGF receptors • VEGF-R1, VEGF-R2, VEGF-R3 • VEGF-R2 as major mediator of EC proliferation, migration, angiogenesis • VEGF released from tumors • Potently induces angiogenesis in vivo • Induced vasodilation / vascular permeability / endothelial cell (EC) fenestration • ECs in new tumor vasculature display VEGF dependence • Promotes EC survival, proliferation and migration • Clinical angiogenic inhibitors • Bevacizumab (Avastin) (antibody against VEGF) • PTK787, ZD6474, SU6668, SU11248 (inhibits VEGF-R2)

9. P02_Endothelial cells: Functional Analysis - Migration-Tube formation Angiogenesis: Associated Diseases

10. P02_Endothelial cells: Functional Analysis - Migration-Tube formation Angiogenesis: Tumor Growth

11. P02_Endothelial cells: Functional Analysis - Migration-Tube formation Angiogenesis is sexy…

12. P02_Endothelial cells: Functional Analysis - Migration-Tube formation ANGIOGENESIS ASSAYS OVERVIEW

13. P02_Endothelial cells: Functional Analysis - Migration-Tube formation Organ Culture: Rat Aortic Ring Assay • slices of vessels are incubated on Matrigel • sprouting of vessels starting from the • endothelial layer of vessel

14. P02_Endothelial cells: Functional Analysis - Migration-Tube formation In Vivo Assays: CAM & Matrigel Plug Chick chorioallantoic membrane (CAM) assay: Angiogenic factors are administered Onto the allantoic membrane of the chicken Embryo and angiogenesis is analyzed Matrigel Plug Assay: A chilled mixture of cells/Matrigel HC is injected Subcutaneously. In the body, the plug will be broken down over Time (10-14 d) Invasion of vessels into plug starts at day 2-3

15. P02_Endothelial cells: Functional Analysis - Migration-Tube formation Angiogenesis: Endothelial Cells Commercially available endothelial cells come from many sources: HUVEC: Human Umbilical Vein Endothelial Cells* HMEC: Human dermal MicrovascularEndothelial Cells Capillary EC, easily accessible human material BAOEC: BovineAOrticEndothelialCells Bovine sources are cheap Different cell lines may respond differently in assays max. life cycle of EC is around 10-20 passages; thereafter, cells lose phenotype and die  We use passage 4

16. P02_Endothelial cells: Functional Analysis - Migration-Tube formation HUVEC: Material – Isolation-Characterization-long term stability

17. P02_Endothelial cells: Functional Analysis - Migration-Tube formation • Procedure: • Consent of the mother (+ Ethic commission) • Length of the cord 20-30 cm • Store immediately after birth in medium (RPMI 10% FCS + AB) • Subsequent Rrinsing with Heparin to avoid blood coagulation not possible • Transfer to the lab as outlined in PPP 01 • Storage at 4°C over night • Rinsing the vein using RPMI Medium • If blood is washed out: • Digest the inner endothelial cell monolayer by using one of these techniques • Control the absence of smooth muscle cells and fibroblast (prolonged digestion)

18. Isolation and propagation in specific medium • Microvascular EC • Tumor tissue • Foreskin • Using enzymatic digestion and selection by any technique • Bovine/swine aorta • Abrasion of the EC Monolayer Monolayer Cell Isolation - 2002

19. EC-quality control • Material: • Human EC derived from UC from healthy individuals • Enzymatic digestions as outlined • Cultivation in EGM-2 (Lonza) upt to 12th passage • Control by differentiation (vw-factor) and function • Freezing using PC-control device • Long term storage in Nitrogen • Thawing and quality control (WST, BrdU, Immunostaining vWF) • Upon release use in sreening/research HU 73 p6 Phasenkontrast HU 74 p5 vWF Immunfärbung HU 106 p7 Phasenkontrast 19

20. Quality control during serial passages • Cultivation of different batches up to passage 10 • Documentation of differentiation by Immunostaining • Quantifiation of vWF in % of total cells 20

21. P02_Endothelial cells: Functional Analysis - Migration-Tube formation Functional Analysis • Differentiation • Growth behavior • Migration • Tube (vessel) formation • Behavior under flow • 3D Models

22. HUVEC 73 Migrationsassay • Cells seede on 12 well MTP • Scratch after reaching confluence 4d • Microscopic control of EC migration into the “wound” Wound= Scratch control 2 h control 24 + 10µM /L Drug 24h 22

23. P02_Endothelial cells: Functional Analysis - Migration-Tube formation Cell migration (Scratch Assay) • Only horizontal • No Gradient of growth factors • Quantification by cell counting/imaging • Ready-to-use plates available ( expensive) Alternative: Boyden chamber assay – vertical from top to bottom

24. P02_Endothelial cells: Functional Analysis - Migration-Tube formation ENDOTHELIAL CELL MIGRATION ASSAY

25. P02_Endothelial cells: Functional Analysis - Migration-Tube formation In vitro Assays: EC migration and invasion

26. P02_Endothelial cells: Functional Analysis - Migration-Tube formation Migration / Invasion Assays: the hard & the smart way…

27. P02_Endothelial cells: Functional Analysis - Migration-Tube formation Principle Of Cell Migration Assay

28. P02_Endothelial cells: Functional Analysis - Migration-Tube formation Compatible Fluorophores for BD FluoroBlokTM Insert Systems

29. P02_Endothelial cells: Functional Analysis - Migration-Tube formation Endpoint or Real time kinetic Assay

30. P02_Endothelial cells: Functional Analysis - Migration-Tube formation Real-Time Analysis of HUVEC Migration

31. P02_Endothelial cells: Functional Analysis - Migration-Tube formation Correct coating may be critical for cell migration

32. P02_Endothelial cells: Functional Analysis - Migration-Tube formation Timecourse of Chemotaxis in Monocytic Cells • Response of Macrophage Chemoattractant Protein 1 (MCP-1)-induced chemotaxis in monocytic cells: • Calcein-prelabeled cells (top chamber) were incubated with 25 nM MCP-1 (bottom chamber) • Bottom fluorescence was measured at varying time points • Data on the graph are representative for a typical experiment • 20-25 min incubation is optimal

33. P02_Endothelial cells: Functional Analysis - Migration-Tube formation Importance of Optimizing Cell Seeding Density

34. P02_Endothelial cells: Functional Analysis - Migration-Tube formation ENDOTHELIAL CELL TUBE FORMATION ASSAY

35. P02_Endothelial cells: Functional Analysis - Migration-Tube formation EC Tube Formation: Assay Features • PRINCIPLE • ECs build up tube networks when cultured under appropriate conditions • labeled tube networks will be Calcein-stained and • tube length be measured using automated fluorescence microscopy • RAPID DATA • tube formation & assay data after 24 hours from seeding

36. P02_Endothelial cells: Functional Analysis - Migration-Tube formation EC tube formation assay EC are cultivated at desired cell density onto the top of high concentrated Matrigel (4-8 mg/ml Any drug/growth factors can be applied into the gel Monitoring of “branches” by eye or imaging software

37. P02_Endothelial cells: Functional Analysis - Migration-Tube formation EC Tube Formation: 96 well image acquisition

38. P02_Endothelial cells: Functional Analysis - Migration-Tube formation EC Tube Formation: Number of seeded cells is critical • cell lines differ in the number of cells that is optimal for tube formation • seeding cell density will result in cell growth as a layer rather than as tubes •  The optimal cell seeding number should be titrated cell-line specifically

39. P02_Endothelial cells: Functional Analysis - Migration-Tube formation EC Tube Formation: Data consistency of inter-day experiments

40. P02_Endothelial cells: Functional Analysis - Migration-Tube formation EC Tube Formation: Influence of Compounds II

41. IBIDI-Kammer 1 4 5 2 6 3 FLOW-orientiation 1 – 6 = imageposition P02_Endothelial cells: Functional Analysis - Migration-Tube formation Subcultivation into Flow-chamber: BdSMC, p11, 07.04.09Flow-Start: 08.04.09FLOW: 0,30 ml/minShear-Stress: 1,54 dyne/cm2Coating:uncoated Bild: 10.04.09 Control w/o flow (Start) 3 4 Smooth muscle cells under shear stress 5 6