Download Microvascular Endothelial Cells

P02_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*
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
* Molecular Cell- and Tumour Biology * Summer 2013 * Naples*
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
* Molecular Cell- and Tumour Biology * Summer 2013 * Naples*
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
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 O2 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 prolfieratioMigration-Vessel formation?
AR?
* Molecular Cell- and Tumour Biology * Summer 2013 * Naples*
5
P02_Endothelial cells:
Functional Analysis - Migration-Tube formation
Angiogenesis: Activators and Inhibitors
* Molecular Cell- and Tumour Biology * Summer 2013 * Naples*
P02_Endothelial cells:
Functional Analysis - Migration-Tube formation
Angiogenesis: The VEGF Pathway
* Molecular Cell- and Tumour Biology * Summer 2013 * Naples*
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)
* Molecular Cell- and Tumour Biology * Summer 2013 * Naples*
P02_Endothelial cells:
Functional Analysis - Migration-Tube formation
Angiogenesis: Associated Diseases
* Molecular Cell- and Tumour Biology * Summer 2013 * Naples*
P02_Endothelial cells:
Functional Analysis - Migration-Tube formation
Angiogenesis: Tumor Growth
* Molecular Cell- and Tumour Biology * Summer 2013 * Naples*
P02_Endothelial cells:
Functional Analysis - Migration-Tube formation
Angiogenesis is sexy…
* Molecular Cell- and Tumour Biology * Summer 2013 * Naples*
P02_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 (Caco2)
* Molecular Cell- and Tumour Biology * Summer 2013 * Naples*
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
* Molecular Cell- and Tumour Biology * Summer 2013 * Naples*
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
* Molecular Cell- and Tumour Biology * Summer 2013 * Naples*
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 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*
P02_Endothelial cells:
Functional Analysis - Migration-Tube formation
HUVEC:
Material – Isolation-Characterization-long term stability
* Molecular Cell- and Tumour Biology * Summer 2013 * Naples*
16
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)
* 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 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
* Molecular Cell- and Tumour Biology * Summer 2013 * Naples*
19
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
HU 73-74-106
100
90
80
70
Angabe in %
60
alle positiven
Zellen
50
40
30
20
10
00
p3
p4
p6
p 10
* Molecular Cell- and Tumour Biology * Summer 2013 * Naples*
20
P02_Endothelial cells:
Functional Analysis - Migration-Tube formation
Functional Analysis
•
•
•
•
•
•
Differentiation
Growth behavior
Migration
Tube (vessel) formation
Behavior under flow
3D 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 4d
 Microscopic control of EC migration into the “wound”
control 2 h
control 24
+ 10µM /L Drug 24h
* Molecular Cell- and Tumour Biology * Summer 2013 * Naples*
22
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
* Molecular Cell- and Tumour Biology * Summer 2013 * Naples*
23
P02_Endothelial cells:
Functional Analysis - Migration-Tube formation
ENDOTHELIAL CELL
MIGRATION ASSAY
* Molecular Cell- and Tumour Biology * Summer 2013 * Naples*
P02_Endothelial cells:
Functional Analysis - Migration-Tube formation
In vitro Assays: EC migration and invasion
* Molecular Cell- and Tumour Biology * Summer 2013 * Naples*
P02_Endothelial cells:
Functional Analysis - Migration-Tube formation
Migration / Invasion Assays:
the hard & the smart way…
* Molecular Cell- and Tumour Biology * Summer 2013 * Naples*
P02_Endothelial cells:
Functional Analysis - Migration-Tube formation
Principle Of Cell Migration Assay
* Molecular Cell- and Tumour Biology * Summer 2013 * Naples*
P02_Endothelial cells:
Functional Analysis - Migration-Tube formation
Compatible Fluorophores for
BD FluoroBlokTM Insert Systems
* Molecular Cell- and Tumour Biology * Summer 2013 * Naples*
P02_Endothelial cells:
Functional Analysis - Migration-Tube formation
Endpoint or Real time kinetic Assay
* Molecular Cell- and Tumour Biology * Summer 2013 * Naples*
P02_Endothelial cells:
Functional Analysis - Migration-Tube formation
Real-Time Analysis of HUVEC Migration
* Molecular Cell- and Tumour Biology * Summer 2013 * Naples*
P02_Endothelial cells:
Functional Analysis - Migration-Tube formation
Correct coating may be critical for cell migration
* Molecular Cell- and Tumour Biology * Summer 2013 * Naples*
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
* Molecular Cell- and Tumour Biology * Summer 2013 * Naples*
P02_Endothelial cells:
Functional Analysis - Migration-Tube formation
Importance of Optimizing Cell Seeding Density
* Molecular Cell- and Tumour Biology * Summer 2013 * Naples*
P02_Endothelial cells:
Functional Analysis - Migration-Tube formation
ENDOTHELIAL CELL
TUBE FORMATION ASSAY
* Molecular Cell- and Tumour Biology * Summer 2013 * Naples*
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
* Molecular Cell- and Tumour Biology * Summer 2013 * Naples*
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
* Molecular Cell- and Tumour Biology * Summer 2013 * Naples*
P02_Endothelial cells:
Functional Analysis - Migration-Tube formation
EC Tube Formation: 96 well image acquisition
* Molecular Cell- and Tumour Biology * Summer 2013 * Naples*
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
* Molecular Cell- and Tumour Biology * Summer 2013 * Naples*
P02_Endothelial cells:
Functional Analysis - Migration-Tube formation
EC Tube Formation:
Data consistency of inter-day experiments
* Molecular Cell- and Tumour Biology * Summer 2013 * Naples*
P02_Endothelial cells:
Functional Analysis - Migration-Tube formation
EC Tube Formation:
Influence of Compounds II
* Molecular Cell- and Tumour Biology * Summer 2013 * Naples*
P02_Endothelial cells:
Functional Analysis - Migration-Tube formation
Subcultivation into Flow-chamber:
Flow-Start:
FLOW:
Shear-Stress:
Coating:
Bild:
BdSMC, p11, 07.04.09
08.04.09
0,30 ml/min
1,54 dyne/cm2
uncoated
10.04.09
IBIDI-Kammer
4
5
6
1
2
3
FLOW-orientiation
1 – 6 = imageposition
Control w/o flow (Start)
3
4
5
6
Smooth muscle cells under shear stress
* Molecular Cell- and Tumour Biology * Summer 2013 * Naples*