P 02Endothelial cells Functional Analysis MigrationTube formation ANGIOGENESIS

P 02_Endothelial cells: Functional Analysis - Migration-Tube formation ANGIOGENESIS Endothelial Cell Migration Assay Endothelial Cell Tube Formation Assay * Molecular Cell- and Tumour Biology * Summer 2013 * Naples*

P 02_Endothelial cells: Functional Analysis - Migration-Tube formation CONTENT • Angiogenesis: Definition & Overview • Angiogenesis: Assays Overview • Endothelial Cell Migration Assay (using Fluoro. Blok. TM Insert system) • Endothelial Cell Tube Formation Assay * Molecular Cell- and Tumour Biology * Summer 2013 * Naples*

P 02_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 * Molecular Cell- and Tumour Biology * Summer 2013 * Naples*

P 02_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 1. Secretion of proteases (MMPs) 2. Digestion of basal lamina of vessels 3. Migration to signal source 4. Endothelial cell proliferation 5. Tube formation new vessels bring O 2 and reduce sprouting trigger * Molecular Cell- and Tumour Biology * Summer 2013 * Naples*

Endothelial cells as target for drug screening DRUG e. g. Tyrosin kinase inhibitors TKI “mode of action” Inhibition of prolfieratio. Migration-Vessel formation? AR? * Molecular Cell- and Tumour Biology * Summer 2013 * Naples* 5

P 02_Endothelial cells: Functional Analysis - Migration-Tube formation Angiogenesis: Activators and Inhibitors * Molecular Cell- and Tumour Biology * Summer 2013 * Naples*

P 02_Endothelial cells: Functional Analysis - Migration-Tube formation Angiogenesis: The VEGF Pathway * Molecular Cell- and Tumour Biology * Summer 2013 * Naples*

P 02_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. VEGF 165 as key regulator of blood vessel growth VEGF receptors • VEGF-R 1, VEGF-R 2, VEGF-R 3 • VEGF-R 2 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) • PTK 787, ZD 6474, SU 6668, SU 11248 (inhibits VEGF-R 2) * Molecular Cell- and Tumour Biology * Summer 2013 * Naples*

P 02_Endothelial cells: Functional Analysis - Migration-Tube formation Angiogenesis: Associated Diseases * Molecular Cell- and Tumour Biology * Summer 2013 * Naples*

P 02_Endothelial cells: Functional Analysis - Migration-Tube formation Angiogenesis: Tumor Growth * Molecular Cell- and Tumour Biology * Summer 2013 * Naples*

P 02_Endothelial cells: Functional Analysis - Migration-Tube formation Angiogenesis is sexy… * Molecular Cell- and Tumour Biology * Summer 2013 * Naples*

P 02_Endothelial cells: Functional Analysis - Migration-Tube formation ANGIOGENESIS ASSAYS OVERVIEW In Vitro Organ Culture In Vivo EC migration Rat Aortic ring Chick chorioallantoic membrane (CAM) assay EC invasion Chick aortic arch Corneal angiogenesis assay EC tube formation Matrigel plug assay EC proliferation EC adhesion EC permeability (Caco 2) * Molecular Cell- and Tumour Biology * Summer 2013 * Naples*

P 02_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 * Molecular Cell- and Tumour Biology * Summer 2013 * Naples*

P 02_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 * Molecular Cell- and Tumour Biology * Summer 2013 * Naples*

P 02_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 Microvascular Endothelial Cells Capillary EC, easily accessible human material BAOEC: Bovine AOrtic Endothelial Cells 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 * Molecular Cell- and Tumour Biology * Summer 2013 * Naples*

P 02_Endothelial cells: Functional Analysis - Migration-Tube formation HUVEC: Material – Isolation-Characterization-long term stability * Molecular Cell- and Tumour Biology * Summer 2013 * Naples* 16

P 02_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) * Molecular Cell- and Tumour Biology * Summer 2013 * Naples* 17

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 Cell Isolation Monolayer * Molecular Cell- and Tumour Biology * Summer 2013 * Naples* 2002 - 18

EC-quality control Material: • Human EC derived from UC from healthy individuals • Enzymatic digestions as outlined • Cultivation in EGM-2 (Lonza) upt to 12 th passage • Control by differentiation (vw-factor) and function • Freezing using PC-control device • Long term storage in Nitrogen • Thawing and quality control (WST, Brd. U, Immunostaining v. WF) • Upon release use in sreening/research HU 73 p 6 Phasenkontrast HU 74 p 5 v. WF Immunfärbung * Molecular Cell- and Tumour Biology * Summer 2013 * Naples* HU 106 p 7 Phasenkontrast 19

Quality control during serial passages § Cultivation of different batches up to passage 10 § Documentation of differentiation by Immunostaining § Quantifiation of v. WF in % of total cells HU 73 -74 -106 100 90 80 Angabe in % 70 60 alle positiven Zellen 50 40 30 20 10 00 p 3 p 4 p 6 p 10 * Molecular Cell- and Tumour Biology * Summer 2013 * Naples* 20

P 02_Endothelial cells: Functional Analysis - Migration-Tube formation Functional Analysis • • • Differentiation Growth behavior Migration Tube (vessel) formation Behavior under flow 3 D Models * Molecular Cell- and Tumour Biology * Summer 2013 * Naples* 21

HUVEC 73 Migrationsassay Wound= Scratch § Cells seede on 12 well MTP § Scratch after reaching confluence 4 d § Microscopic control of EC migration into the “wound” control 2 h control 24 + 10µM /L Drug 24 h * Molecular Cell- and Tumour Biology * Summer 2013 * Naples* 22

P 02_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 * Molecular Cell- and Tumour Biology * Summer 2013 * Naples* 23

P 02_Endothelial cells: Functional Analysis - Migration-Tube formation ENDOTHELIAL CELL MIGRATION ASSAY * Molecular Cell- and Tumour Biology * Summer 2013 * Naples*

P 02_Endothelial cells: Functional Analysis - Migration-Tube formation In vitro Assays: EC migration and invasion * Molecular Cell- and Tumour Biology * Summer 2013 * Naples*

P 02_Endothelial cells: Functional Analysis - Migration-Tube formation Migration / Invasion Assays: the hard & the smart way… * Molecular Cell- and Tumour Biology * Summer 2013 * Naples*

P 02_Endothelial cells: Functional Analysis - Migration-Tube formation Principle Of Cell Migration Assay * Molecular Cell- and Tumour Biology * Summer 2013 * Naples*

P 02_Endothelial cells: Functional Analysis - Migration-Tube formation Compatible Fluorophores for BD Fluoro. Blok. TM Insert Systems * Molecular Cell- and Tumour Biology * Summer 2013 * Naples*

P 02_Endothelial cells: Functional Analysis - Migration-Tube formation Endpoint or Real time kinetic Assay * Molecular Cell- and Tumour Biology * Summer 2013 * Naples*

P 02_Endothelial cells: Functional Analysis - Migration-Tube formation Real-Time Analysis of HUVEC Migration * Molecular Cell- and Tumour Biology * Summer 2013 * Naples*

P 02_Endothelial cells: Functional Analysis - Migration-Tube formation Correct coating may be critical for cell migration * Molecular Cell- and Tumour Biology * Summer 2013 * Naples*

P 02_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 n. M 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 * Molecular Cell- and Tumour Biology * Summer 2013 * Naples*

P 02_Endothelial cells: Functional Analysis - Migration-Tube formation Importance of Optimizing Cell Seeding Density * Molecular Cell- and Tumour Biology * Summer 2013 * Naples*

P 02_Endothelial cells: Functional Analysis - Migration-Tube formation ENDOTHELIAL CELL TUBE FORMATION ASSAY * Molecular Cell- and Tumour Biology * Summer 2013 * Naples*

P 02_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 * Molecular Cell- and Tumour Biology * Summer 2013 * Naples*

P 02_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 * Molecular Cell- and Tumour Biology * Summer 2013 * Naples*

P 02_Endothelial cells: Functional Analysis - Migration-Tube formation EC Tube Formation: 96 well image acquisition * Molecular Cell- and Tumour Biology * Summer 2013 * Naples*

P 02_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 * Molecular Cell- and Tumour Biology * Summer 2013 * Naples*

P 02_Endothelial cells: Functional Analysis - Migration-Tube formation EC Tube Formation: Data consistency of inter-day experiments * Molecular Cell- and Tumour Biology * Summer 2013 * Naples*

P 02_Endothelial cells: Functional Analysis - Migration-Tube formation EC Tube Formation: Influence of Compounds II * Molecular Cell- and Tumour Biology * Summer 2013 * Naples*

P 02_Endothelial cells: Functional Analysis - Migration-Tube formation Subcultivation into Flow-chamber: Flow-Start: FLOW: Shear-Stress: Coating: Bild: Bd. SMC, p 11, 07. 04. 09 08. 04. 09 0, 30 ml/min 1, 54 dyne/cm 2 uncoated 10. 04. 09 IBIDI-Kammer 4 5 6 1 2 3 FLOW-orientiation Control w/o flow (Start) 1 – 6 = imageposition 3 4 5 6 Smooth muscle cells under shear stress * Molecular Cell- and Tumour Biology * Summer 2013 * Naples*