This document discusses in vitro and in vivo models for studying angiogenesis. It describes several in vitro models including cord formation assays, tube formation assays, cell migration assays, cell proliferation assays, and gelatin zymography that are used to study the molecular mechanisms and identify regulators of angiogenesis. Important considerations for developing angiogenesis studies include choosing an appropriate endothelial cell source and extracellular matrix. Several in vivo models are also described such as the rabbit corneal assay, sponge implantation models, matrigel plug assays, and the hind limb ischemia model. Both in vitro and in vivo techniques are important for advancing the understanding and treatment of diseases associated with angiogenesis like cancer.

1. In Vitro and in Vivo Models for
Studying Angiogenesis
Vijay Avin BR, Molecular Biomedicine Laboratory, Sahyadri Sceince
College, Shimoga, Karnataka, India

2. Growth of new blood vessels
Advanced cancers can secrete angiogenic
factors (VEGF, bFGF etc).

5. Basic research to elucidate molecular
mechanisms of angiogenesis:
Identify and characterize regulatory pathways that mediate various
steps of angiogenesis such as endothelial cell migration, invasion,
and tubulogenesis.
To develop treatments for cancer and other
diseases associated with angiogenesis:
Identification of compounds that inhibit or stimulate key steps in
the angiogenesis process.
Angiogenesis Research

6. Methods to Study Angiogenesis
In vitro Models
In Vivo Models

7. Important Considerations for
Developing
Angiogenesis Studies
 Choose appropriate endothelial cell source.
 Establish acceptable dynamic range to
measure stimulation and/or inhibition of
angiogenesis.
 Incorporate appropriate extracellular matrix
(ECM) protein(s) to facilitate cell functionality
and assay outcome.

8. Endothelial Cells are the most
important tool for in vitro
studies of Angiogenesis……

9. Tumor cell survival is
dependent on the health and
proliferation of endothelial
cells in surrounding blood
vessels………

10. Sources of Endothelial Cells
Large vessel
– aortic (e.g., HAEC)
– umbilical vein (e.g. HUVEC)
– pulmonary artery
Microvascular (e.g., HMVEC)
– brain
– lung
– dermis (e.g., HDMEC)
– myocardium

11. Human Umbilical Vein Endothelial Cells
HUVEC
Most commonly used EC type
for angiogenesis studies

12. ECM provides a physiological substrate that supports key
cellular functions:
• Structural organization of cells and tissue.
• Cell attachment, survival, and proliferation.
• Induction and maintenance of cell differentiation.
• Can influence signal transduction and regulation of
gene expression.
Examples: gelatin, fibronectin, vitronectin, laminin, collagen &
Matrigel
Extracellular Matrix

13. In vitro models for Angiogenesis
Cord Formation Assay
Tube Formation Assay
Cell Migration Assay
Cell Proliferation Assay
Gelatin Zymography

14. Cord Formation Assay
Endothelial cells are incubated in
growth factor containing matrigel
Then they were trypsinized and
resuspended in the same medium
and dispersed onto the matrigel.
Cord formation in each well is
monitored and photographed
using an inverted microscope.
Control
+ Inhibitor

15. The endothelial cells are isolated and cultured in medium in
gelatin coated flasks.
After gelation at 37C for 30 min the gels are overlaid with basal
medium supplemented with test substances at desired
concentrations.
Gels are examined and the tube length is determined for each
well followed by determination of each group by using software.
Tube Formation Assay

16. Cell Migration Assay
Polycarbonate + 12uM

17. Cell Proliferation Assay
Control
+ Inhibitor
The proliferation studies are based on
cell counting, thymidine incorporation
(or) Immunohistochemical staining for
proliferation (or) cell death.
Endothelial cells are isolated and
cultured in medium at 37C in a
humidified atmosphere containing 5 %
CO2. Cell proliferation is determined
using a 5-bromo-2'-deoxyuridine (BrdU)
colorimetric assay kit.

18. Gelatin Zymography
MMP
MMP
+
Inhibitor
This assay can also be called as Matrix
Metalloproteinase (MMP) assay.
Gelatin is used as a substrate and is
incorporated into poly acryl amide gels.
Samples are mixed with buffer loaded
onto the gel and electrophoreses.
After electrophoreses the gels are
incubated in activity buffer analyzed by
densitography

19. Invasion assay

20. Rabbit or rat corneal assay
Sponge implantation models
Matrigel plugs
Wound healing assay
In Vivo Models

21. Sponge Implantation Method
This model is used for the evaluation of angiogenesis and
anti- angiogenic agents.
The mechanism involved in this is stimulation of
inflammation by foreign substance leads to the angiogenesis.

22. Sponge implant models
Sterile absorbable gel foam is used as a sponge .
Incision is made at midline of the anaesthetized animal and
gel piece is inserted in to subcutaneously.
At 14th day the animals are sacrificed and gel foams are
harvested and quantification is done for angiogenesis activity.

23. Matrigel plug Assay
Used for the evaluation of both
angiogenic and anti-angiogenic
agents.
The mostly used animal model is
mice.
The mechanism involved in this
model is injection of foreign
substances in to the animal leads to
the stimulation of the inflammatory
cells including macrophages and
neutrophils that leads to the
stimulation of angiogenesis.
Matrigel is a gelatinous material
derived from mouse tumor cells

24. Matrigel plugs
Mice injected with VEGF supplemented Matrigel were left untreated or were
treated with the angiogenesis inhibitor which fully suppresses angiogenesis,
as visible macroscopically and microscopically.

25. Corneal Angiogenesis Assay
A pocket is created in the cornea where
the compound of interest is inserted.
 To induce an angiogenic response, a
variety of materials including sponges,
ethylene vinyl copolymer, or Hydron,
containing an angiogenic substance (i.e.
FGF-2 of VEGF) are implanted in
"pockets"
The vascular response can then be
monitored by direct observation using
either a microscope or can be quantified
by computer image analysis after
perfusion of the cornea.

27. Wound Healing Assay
Two circular holes of approximately 5 mm in diameter are
punched with a tissue puncher through the dorsal skin of
an anesthetized mouse.
Wound size, scar formation and re-epithelization of the
wounds should be recorded daily by photography and by
measuring the wound area with calipers.
Treatment can consist of pro-or anti-angiogenic
compounds, and their effects on angiogenesis is
determined post mortem after the regenerated tissue has
been excised, fixed and stained. Transgenic or knock-out
mice can be used, when available, to study the specific
effects of particular genes.
Inflammation, new tissue formation
and remodeling

28. Hind Limb Ischemia Model
This method is mostly used for the evaluation of angiogenesis
substances.
The mechanism involved in this model is haemodynamic changes
leads to the formation of new blood vessels i.e. while large vessels with low
flow tend to augmentation of blood flow which leads to the stimulation of
vascular sprouting and maintain the potency of the newly formed collateral
vessels thereby providing blood flow to the
ischemic tissue
the animals are anaesthetized and incision is making in the skin
overlying the middle portion of the hind limb. Then the proximal end of the
femoral artery is ligating and distal portion of saphenous artery is ligating
and artery and their side branches were dissected free. The femoral artery
and attached side branched are excised and overlying skin is then closed.

29. IN- OVA ASSAY
Pilot method for most of the angiogenesis
evaluation studies.

30. Fertilized chicken eggs on the second
day of incubation is and incubated at 37C
at constant humidity.
On day 3 small hole is drill at narrow
end and the albumin is withdrawn.
At the 7th day of incubation a small
square window is open in the shell and
test substances are implanted on the top
of the membrane.
The window was sealed and
reincubated. Eggs are incubated up to
appropriate incubation time and
angiogenesis is quantified

32. Xenograft model
 On day third window made
and removing 2-3 drops of
albumin
 Resealed and kept for
incubation
 On day 10 plastic rings
along with cells were placed
and kept for incubation
 On day 17th
the eggs were
opened and observed for
the formation of solid
tumor

33. Thank you