1. A SYSTEMATIC REVIEWON IN VIVOANDIN VITROEXPERIMENTAL MODELS OF
ANGIOGENESIS
& PRELIMINARY STUDIES ON CAMASSAY
AProject report submittedinpartial fulfillment of therequirements fortheawardof theDegree
of Bachelor
of Pharmacy
By
ABHIJEET MIHIR
(BPH/1019/2009)
&
NIRMAL TOPPO
(BPH/1037/2009)
Undertheguidanceof
Dr. S. P. PATTANAYAK
Asst. Professor
DEPARTMENT OF PHARMACEUTICAL
SCIENCES
BIRLA INSTITUTE OF TECHNOLOGY
MESRA - 835215, RANCHI

2. CONTENTS
S. NO. TITLES PAGE NO.
1. INTRODUCTION 1
2. LITERATURE SURVEY 29
3. OBJECTIVE & PLAN OF WORK 35
4. RESEARCH METHODOLOGY 37
5. RESULTS & DISCUSSION 39
6. CONCLUSION 45
7. FUTURE SCOPE 46
8. REFERENCES 47

4. ANGIOGENESIS
Angiogenesis means for the growth of new capillary blood vessels in the body is an important natural process in the
body used for healing and reproduction. The body controls angiogenesis by producing a precise balance of growth and
inhibitory factors in healthy tissues. When this balance is disturbed, the result is either too much or too little
angiogenesis. Abnormal blood vessel growth either excessive or insufficient is now recognized as a “common
denominator” underlying many deadly and debilitating conditions including cancer, skin diseases, age related
blindness, diabetic ulcers, cardiovascular disease, stroke and many others. Blood, carried in the vessels, delivers
oxygen and nutrients to and removes waste products from the tissues. When new tissue is formed, blood vessel
formation must occur as well. Thus, new tissue formed, for example, with the repair of wounds and the formation of the
placenta during pregnancy are normal examples of intense new blood vessel formation (angiogenesis). The list of
diseases that have angiogenesis as an underlying mechanism grows longer every year. [1]
The essential role of angiogenesis in tumour growth was first proposed in 1971 by Judah Folkman, who
described tumours as "hot and bloody. Angiogenesis is a multi-step process recruited for the formation of new blood
vessels is one of the crucial processes for the growth, survival, proliferation and metastasis of tumours. Under normal
conditions angiogenesis is virtually essential for cell reproduction, development and wound healing, etc. The process of
neoangiogenesis involves endothelial cell proliferation, migration, and membrane degradation. The importance of
angiogenesis for the growth and survival of tumours is widely appreciated. Numerous studies are being focused on the
understanding of angiogenesis and the antiangiogenic agents have received significant attention because of their
therapeutic implications especially in extending the life expectancy of cancer patients. The recent studies in the field of
molecular aspects of angiogenesis clearly. [2]
Every organism is equipped with variety of enzymatic and non-enzymatic antioxidants for the stabilization
of free radicals. However, the increase in the concentration of free radicals leads to accumulation of oxidative stress: a
cause for cell dysfunctions and related degenerative diseases. [1][2]

5. (Source: Carmeliet et al., 2003) [4]
Figure.1.2: Formation of a vascular network. Endothelial progenitors differentiate to arterial
and venous ECs, which assemble in a primitive capillary plexus. Vessels then sprout and
become stabilized by SMCs, differentiating from their progenitors. HSCs contribute to
angiogenesis directly and indirectly, by differentiating to leukocytes or platelets.

6. The Angiogenesis Cascade [3]
Angiogenesis occurs as an orderly cascade of molecular and cellular events in the wound bed:
1. Angiogenic growth factors bind to their receptors on the surface of endothelial cells in pre-existing venules
(parent
vessels).
2. Growth factor-receptor binding activates signalling pathways within endothelial cells.
3. Activated endothelial cells release proteolytic enzymes that dissolve the basement membrane surrounding
parent
vessels.
4. Endothelial cells proliferate and sprout outward through the basement membrane.
5. Endothelial cells migrate into the wound bed using cell surface adhesion Molecules known as integrins
(αvß3, αVß5,
and α5ß1).
6. At the advancing front of sprouting vessels, enzymes known as Matrix Metalloproteinases (MMPs) dissolve
the
surrounding tissue matrix.
7. Vascular sprouts form tubular channels which connect to form vascular loops.
8. Vascular loops differentiate into afferent (arterial) and efferent (venous) limbs.
9. New blood vessels mature by recruiting mural cells (smooth muscle cells and pericytes) to stabilize the
vascular
architecture.
10. Blood flow begins in the mature stable vessel.
These complex growth factor-receptor, cell-cell, and cell-matrix interactions characterize the

7. (Source: Li e t al., 2004) [3]
The Angiogenesis Cascade of
Events

8. Body Control Mechanismof Angiogenesis [5]
Angiogenesis the growth of new blood vessels is an important natural process occurring in the
body both in health and in disease. Angiogenesis occurs in the healthy body for healing wounds and for
restoring blood flow to tissues after injury or insult. In females angiogenesis also occurs during the monthly
reproductive cycle (to rebuild the uterus lining, to mature the egg during ovulation) and during pregnancy (to
build the placenta, the circulation between mother and foetus). The healthy body controls the formation of
new blood vessels through a series of “on” and “off” switches.
• The primary “on” switches are chemicals that stimulate blood
vessel formation.
• The primary “off” switches are chemicals that inhibit blood vessel
formation.
When angiogenic growth factors (‘on’ switches) are created in greater amounts than
angiogenesis inhibitors (‘off’ switches), the balance is tilted in favour of the growth of new blood vessels.
When inhibitors are present in greater amounts than stimulators, angiogenesis is stopped. In health, the
body maintains a balance of angiogenesis regulators. In some disease states, the organs involved may lose
control over angiogenesis. In these conditions, new blood vessels either grow too much or not enough.

9. Angiogenesis Growth Factors[1]
There are at least 20 different known angiogenic growth factors out of that five angiogenic growth factors are
being tested in humans for growing new blood vessels to heal wounds and to restore blood flow to the heart, limbs and
brain. Angiogenic gene therapy is also being developed as a method to deliver angiogenic growth factors to the heart,
limbs and wounds.Sl. No. Known angiogenic growth factor
1. Angiogenin
2. Angiopoietin-1
3. Del-1
4. Fibroblast growth factors: acidic (aGGF) and basic (bFGF)
5. Follistatin
6. Granulocyte colony-stimulating factor (G-CSF)
7 Hepatocyte growth factor (HGC)/ scatter factor (SF)
8. Interleukin-8 (IL-8)
9. Leptin
10. Midkine
11. Placental growth factor
12. Platelet-derived endothelial cell growth factor (PD-ECGF)
13. Platelet-derived growth factor –BB (PDGF-BB)
14. Pleiotrophin (PTN)
15. Progranulin
16. Proliferin
17. Transforming growth factor-alpha (TGF-alpha)
18. Transforming growth factor-beta (TGF-beta)
19. Tumor necrosis factor-alpha (TGF-alpha)
20. Vascular endothelial growth factor (VEGF)/vascular permeability factor (VPF)

10. Angiogenesis Inhibitors[1]
There are at least 28 known natural angiogenesis inhibitors found in the body. Other than this Angiogenesis
inhibitors have also been discovered from natural sources in total more than 300 angiogenesis inhibitors have been
discovered to date.sl.no. Known angiogenic inhibitors
1. Angioarrestin
2. Angiostatin (plasminogen fragment)
3. Antiangiogenic antithrombin III
4. Cartilage-derived inhibitor (CDI)
5. CD59 complement fragment
6. Endostatin (collagen XVIII fragment)
7. Fibronectin fragment
8. Gro-beta
9. Heparinases
10. Heparin hexasaccharide fragment
11. Human chorionic gonadotropin (hCG)
12. Interferon alpha/beta/gamma
13. Interferon inducible protein (IP-10)
14. Interleukin-12
15. Kringle 5 (plasminogen fragment)
16. Metalloproteinase inhibitor (TIMPs)
17. 2-methoxyestradiol
18. Placental ribonuclease inhibitor
19. Plasminogen activator inhibitor
20. Platelet factor-4(PF4)
21. Prolactin 16kD fragment
22. Proliferin-related protein (PRP)
23. Retinoids
24. Tetrahydrocortisol-s
25. Thrombospondin-1 (TSP-1)
26. Transforming growth factor –beta (TGF-b)
27. Vasculostatin
28. Vasostatin (calreticulin fragment)

11. TYPES OF ANGIOGENESIS[6][7][8]
Tumours can grow to a size of approximately 1–2mm before their metabolic demands are restricted due to the
diffusion limit of oxygen and nutrients. In order to grow beyond this size, the tumour switches to an angiogenic
phenotype and attracts blood vessels from the surrounding stroma. This process is regulated by a variety of pro- and
anti-angiogenic factors, and is a prerequisite for further outgrowth of the tumour. Next to sprouting angiogenesis, the
process by which new vessels are formed from pre-existing vasculature, several other mechanisms of
neovascularization have been identified in tumours, including intussusceptive angiogenesis, the recruitment of
endothelial progenitor cells, vessel cooption, vasculogenic mimicry and lymphangiogenesis.
Sprouting angiogenesis: - Sprouting angiogenesis is the growth of new capillary vessels out of pre-existing ones.
These blood vessels will provide expanding tissues and organs with oxygen and nutrients, and remove the metabolic
waste. Angiogenesis takes place in physiological situations, such as embryonic development, wound healing and
reproduction. It also plays an important role in many pathologies, like diabetes, rheumatoid arthritis, cardiovascular
ischemic complications, and cancer. In cancer, sprouting angiogenesis is not only important in primary tumours, it is
also involved in metastasis formation and further outgrowth of metastases. [6]
The process of sprouting angiogenesis involves several sequential steps.
First, biological signals known as angiogenic growth factors activate receptors present on endothelial cells
present in pre- existing blood vessels.
Second, the activated endothelial cells begin to release enzymes called proteases that degrade the basement
membrane in order to allow endothelial cells to escape from the original (parent) vessel walls. The
endothelial cells then proliferate into the surrounding matrix and form solid sprouts connecting
neighbouring vessels. As sprouts extend toward the source of the angiogenic stimulus, endothelial
cells migrate in tandem, using adhesion molecules, the equivalent of cellular grappling hooks, called
integrin’s. These sprouts then form loops to become a full-fledged vessel lumen as cells migrate to the
site of angiogenesis.

12. Intussusceptive angiogenesis: - A variant of angiogenesis, different from sprouting, is intussusceptive
angiogenesis. This process was first observed in postnatal remodelling of capillaries in the lung. In the third week of rat
life and during the first 2 years in humans, the volume of the lungs increases by more than 20 times. In this
developmental process, a new concept of vessel formation was found where ngiopoietin vessels split in two new
vessels by the formation of trans-vascular tissue pillar into the lumen of the vessel. Intussusceptive microvascular
growth is a fast process that can take place within hours or even minutes, because it does not need proliferation of
endothelial cells. In this process endothelial cells are remodelled by increasing in volume and becoming thinner.
Intussusception is believed to take place after vasculogenesis or angiogenesis to expand the capillary plexus, in a
short time and with a little amount of energy. Transmission electron microscopy revealed four Consecutive steps. [7][9]
First, the two opposing capillary walls establish a zone of contact.
Second, the endothelial cell junctions are reorganized and the vessel bilayer is perforated to allow growth factors
and cells to penetrate into the lumen.
Third, a core is formed between the two new vessels at the zone of contact that is filled with pericytes and
myofibroblasts. These cells begin laying collagen fibres into the core to provide an extracellular matrix for
growth of the vessel lumen.
Finally, the core is fleshed out with no alterations to the basic structure.
Intussusception is important because it is a reorganization of existing cells. It allows a vast increase in the
number of capillaries without a corresponding increase in the number of endothelial cells. This is especially important in
embryonic development as there are not enough resources to create a rich microvasculature with new cells every time
a new vessel develops.
In 1993, the first in vivo intussusceptive microvascular growth was demonstrated by video microscopy in a
chick chorioallantoic membrane. This process has now been detected in various organs, tissue repair processes and
also in tumour angiogenesis. Tissue pillars were detected in a colon carcinoma xenograft model. At the growing edge
both sprouting and intussusceptive angiogenesis were observed, in the stabilised regions mostly intussusception was

13. (Source: Hillen et al., 2007) [8]
Figure 1.3: Different mechanisms of tumour vascularisation. This diagram represents the six
different types of vascularisation observed in solid tumours, including sprouting angiogenesis,
intussusceptive angiogenesis, recruitment of endothelial progenitor cells, vessel co-option,
vasculogenic mimicry and lymph angiogenesis. The main key players involved in these
processes, are indicated.

14. ANGIOGENESIS BASEDDISEASES [12]
In many serious diseases states the body loses control over angiogenesis and angiogenesis dependent diseases result
when new blood vessels either grow excessively or insufficiently.
Excessive Angiogenesis[12]
Excessive Angiogenesis occurs in diseases such as cancer, diabetic blindness, age related macular degeneration,
rheumatoid arthritis, psoriasis and more than 70 other conditions. In these conditions new blood vessels feed diseased
tissues, destroy normal tissues and in the case of cancer the new vessels allow tumour cells to escape into the
circulation and lodge in other organs (tumour metastases). Excessive angiogenesis occurs when diseased cells
produce abnormal amounts of angiogenic growth factors overwhelming the effects of natural angiogenesis inhibitors.
Antiangiogenic therapies are aimed to halt new blood vessel growth there by used to treat these conditions.
Diseases Characterized orCaused by Abnormal orExcessive Angiogenesis [4]
Numerous organs: - Cancer (activation of oncogenes; loss of tumour suppressors); Infectious diseases (pathogens
express
angiogenic genes, induce Angiogenic programs or transform ECs);
Autoimmune disorders (Activation of mast cells and other leukocytes).
(a) (b)
(Source: http:// www.cancer.gov/cancertopics/understandingcancer/angiogenesis/ page3-assessed on-
15/03/2013) [46]
Figure 1.4: Tumour Angiogenesis.

15. Blood vessels: - Vascular malformations (Tie-2 mutation); DiGeorge syndrome (Low VEGF and neuropilin-1
expression); HHT
(mutations of endoglin or ALK-1); cavernous hemangioma; atherosclerosis;
transplant Arteriopathy.
Adipose tissue: - Obesity (angiogenesis induced by fatty diet; weight loss by Angiogenesis inhibitors).
Skin: - Psoriasis, warts, allergic dermatitis, scar keloids, pyogenic granulomas, blistering disease, Kaposi sarcoma in
AIDS patients.
Eye: - Persistent hyperplastic vitreous syndrome (loss of Ang-2) or VEGF164; diabetic retinopathy; retinopathy of
prematurity;
choroidal neovascularization (TIMP-3 mutation).
Lung: - Primary pulmonary hypertension (germline BMPR-2 mutation; somatic EC mutations); asthma; nasal polyps.
Intestines: - Inflammatory bowel and periodontal disease, ascites, peritoneal adhesions.
Reproductive system: - Endometriosis, uterine bleeding, ovarian cysts, ovarian Hyperstimulation.
Bone, joints: - Arthritis, synovitis, osteomyelitis, osteophyte formation.
Insufficient Angiogenesis[12]
Insufficient Angiogenesis occurs in diseases such as coronary artery disease, stroke and chronic wounds. In
these conditions blood vessel growth is inadequate and circulation is not properly restored leading to the risk of tissue
death. Insufficient angiogenesis occurs when tissues cannot produce adequate amounts of angiogenic growth factors.
Therapeutic angiogenesis is aimed to stimulate new blood vessel growth with growth factors is being developed to treat
these conditions.
Diseases Characterized orCaused by Insufficient Angiogenesis orVessel Regression [4]
Nervous system- Alzheimer disease – Vasoconstriction, micro vascular degeneration and cerebral angiopathy
due to EC toxicity by amyloid-ß117
.

16. Blood vessels- Atherosclerosis – Characterized by impaired collateral Vessel development.
Hypertension – Micro vessel rarefaction due to impaired vasodilation or angiogenesis.
Diabetes – Characterized by impaired collateral growth and angiogenesis in ischemic limbs, but
enhanced retinal
neovascularization secondary to pericyte dropout.
Restenosis – Impaired re-endothelialisation after arterial injury at old age.
Gastrointestinal- a. Gastric or oral ulcerations – Delayed healing due to production of angiogenesis inhibitors by
pathogens.
b. Crohn disease – Characterized by mucosal ischemia.
Skin- a. Hair loss – Retarded hair growth by angiogenesis inhibitors.
b. Skin purpura, telangiectasia and venous lake formation – Age- dependent reduction of vessel number and
maturation
(SMC dropout) due to EC telomere shortening.
Reproductive system- a. Pre-eclampsia – EC dysfunction resulting in organ failure, thrombosis and hypertension due
to deprivation of VEGF by soluble Flt-1.
b. Menorrhagia (uterine bleeding) – Fragility of SMC-poor vessels due to low Ang-1 Production.
Lung- a. Neonatal respiratory distress – Insufficient lung maturation and Surfactant production in premature mice due
to reduced HIF-2a and VEGF production.
b. Pulmonary fibrosis, Emphysema – Alveolar EC apoptosis upon VEGF inhibition.
Kidney- a. Nephropathy – Age-related vessel loss due to TSP-1 Production.
Bone- a. Osteoporosis, impaired bone fracture healing- Impaired bone formation due to age dependent decline of
VEGF-
driven angiogenesis, angiogenesis inhibitors prevent fracture healing.

17. CURRENT METHODS FORASSAYING ANGIOGENESIS IN VITRO& IN VIVO: - [20]
One of the most important technical challenges in such studies of angiogenesis is selection of the appropriate
assay. There are increasing numbers of angiogenesis assays being described both in vitro & in vivo . It has been
proved that it is necessary to use a combination of assays for identification of the cellular and molecular events in
angiogenesis and the full range of effects of a given test protein. Although the endothelial cell whose migration,
proliferation, differentiation and structural rearrangement is central to the angiogenic process, it is not the only cell type
involved in angiogenesis. The supporting cells (e.g. tumour cells, pericytes, smooth muscle cells and fibroblasts), the
extracellular matrix produced by endothelial cells and their apposed mesenchymal cells, and the circulating blood with
its cellular and humoral components are also involved. No in vitro assay exists currently to model/simulate this complex
process. Whilst in vivo the components of the process are all present, disparate results and limitations also exist
depending on specific microenvironments, organ sites, species used and manner of administration of test substances.
The ideal assay would be reliable, technically straightforward, easily quantifiable and, most importantly, physiologically
relevant. Here, we review the advantages and limitations of the principal assays in use, including those for the
proliferation, migration and differentiation of endothelial cells in vitro , vessel outgrowth from organ cultures and in vivo
assays such as sponge implantation, corneal, chamber, zebrafish, chick chorioallantoic membrane (CAM) and tumour
angiogenesis models.
IN-VIVOASSAYS [20][24]
Matrigel Plug Assay: - This model is used for the evaluation of both angiogenic and anti-angiogenic agents. 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 leads to the stimulation of angiogenesis. The mostly used
animal model is mice. Matrigel is a gelatinous material derived from mouse tumour cells that is commonly used in vitro
and in vivo as a substrate for cells. When pro- angiogenic and anti-angiogenic agents are also added to the matrigel
and it is injected into the subcutaneous space of an animal, which forms the single solid gel plug will stimulate the new

18. 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.
In this method the sponge can be prepared by using sterile absorbable gel foam. The gel foam is cut and strengthened
with sterile agarose and that is used for angiogenesis study. The animals are anaesthetized and an incision is given at
midline and gel piece is inserted in to subcutaneously. Animals are allowed to recover and at 14th
day the animals are
sacrificed and gel foams are harvested and quantification can be done for angiogenesis activity. Mostly used animal
models are mice and rat, the major disadvantage is implantation of the sponge materials is associated with non-
specific immune response which may cause a significant angiogenic response even in the absence of exogenous
growth factors in the sponge.
Corneal Angiogenesis Assay: - This is the “gold standard” method for the following the effect of defined substances
to promote neovascularization of the normally a vascular cornea. Naturally the eye does not contain any blood vessel.
So when applying test substances in the animal eye leads to the stimulation of angiogenesis which can be easily
identified. Several corneal angiogenesis models in the rabbit eye have been described including direct intrasomal
injections of substances, chemical (or) thermal injury, intrasomal tumour implantation and sustained release sucralfate
assay. Among these models the sustained release sucralfate assay is unique because it gives a predictable, persistent
and aggressive neovascular response which is dependent on direct stimulation of blood vessels rather than on indirect
stimulation by induction of inflammation. In this method a pocket is making in the cornea and the test substance when
introduced into this pocket will stimulate the formation of new vessels from the peripheral limbal vasculature. Slow
release materials such as ELVAX (ethylene vinyl acetate copolymer) or Hydron have been used for the introduce test
substance into the corneal pocket. The sponge material to hold test cell suspensions or test substances to induce
angiogenesis can also be used because the slow releasing formulations may cause toxic. The original method was
developed for rabbit eyes but now mostly used animal model is mice.
The advantages of this method is visibility, accessibility and avascularity of the cornea are highly advantageous and
facilitate the Biomicroscopic grading of the neovascular response and the topical application of test drugs are the

19. Sponge Granuloma Angiogenesis Assay: - This model is used for the evaluation of inflammatory angiogenesis
which was described by Fajardo and colleagues. The sponge discs are prepared by cutting of sterile polyvinyl alcohol
foam sponges. A hole is cut into the disc centre to serve as a depot for administration of test substances and the back
of the disc is close with the cotton plug. After adding the stimulants to the centre hole the sponge discs are coat with an
inert slow release ethylene vinyl acetate copolymer (ELVAX) and both the disc surfaces are sealed with filter paper.
The sterile discs are inserted into the subcutaneous layer at a site 2 cm distant from the incision which is then sutured
to prevent disc dispersion. After 9-12 days the animals are sacrifice and the sponge discs are harvested. The discs are
quantified for angiogenesis activity.
Chick Chorioallantoic Membrane (CAM) Assay: - The cancer biologists, developmental biologists and
ophthalmologists have described the chick chorioallantoic membrane (CAM) as a model system for studying
development, cancer behaviour, properties of biomaterials, angiogenesis and photodynamic therapy. This assay is the
most widely used assay for screening of angiogenesis activity. .This method is used for screening of both the
angiogenesis and anti-angiogenesis substances. In this method the fertilized white leghorn chicken eggs on the second
day of incubation is collect and incubated at 370
C and constant humidity. At the day of 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. There CAM develops at the top as a flat membrane,
reaching the edge of the dish to provide a two-dimensional monolayer onto which grafts can be placed. Because the
entire membrane can be seen, rather than just a small portion through the shell window, multiple grafts can be placed
on each CAM and photographs can be taken periodically to document vascular changes over time.[21][22][23]
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. So far

20. 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.
Left Coronary Artery Ligation Model: - This model is mostly used for the myocardial ischemic studies and
substance which have myocardial angiogenesis activity. The mechanism involved in this model is shear stress and
stretch which leads to myocardium to up-regulate the adhesion molecules in the endothelium attraction of inflammatory
cells and stimulation of endothelial cells to produce growth factors which causes angiogenesis. The rabbit is the mostly
used animal model for this study. In this model animals are anaesthetized. Under sterilization and artificial respiration
the left thoracotomy in the 5th
‘intercostals’ space was done and heart is exposed. Then the left anterior descending
coronary artery distal to its diagonal branch is ligating with a suture which produces myocardial ischemia, after
haemodynamic stability the pericardium and chest is closed.
IN-VITROASSAYS [20] [24]
Cell Cord Formation Assay: - In this method the growth factor reduced matrigel is pipette into a well of a 48-well
plate and polymerized for 30 min at 370
. Then the endothelial cells are incubated in 1% FBS-containing growth
medium for 12 h respectively. Then they were trypsinized and resuspended in the same medium and dispersed onto
the matrigel. Then the cells were treated with the test substances. After 18h cord formation in each well is monitored
and photographed using an inverted microscope. The tubular lengths of the cells are measured using software.
Cell Migration Assay: - A substantial number of published reports emphasize the predictable value that assays of
endothelial cell migration have for selecting biologically active assay for the evaluation of stimulants and inhibitors of
angiogenesis. This assay was carried out in a 48-well microchemotaxis chamber. The polycarbonate membrane with
12-µm pore is coated with gelatine endothelial cells are resuspended in cell culture medium. The bottom chamber is
loaded with endothelial cells and the membrane is laid over the cells. Invertation and incubation of the chamber is
carried out in sequence. After 2h incubation the upper wells are loaded with cell culture medium and test samples.

21. Cell Proliferation Assay: - The proliferation studies are based on cell counting, thymidine incorporation (or)
Immunohistochemical staining for proliferation (or) cell death. In this method the endothelial cells are isolated and
cultured in medium at 370
in a humidified atmosphere containing 5 % CO. Cell proliferation is determined using a 5-
bromo-2’-deoxyuridine (BrdU) colorimetric assay kit. Then the endothelial cells are seeded onto gelatine coated well
plates in the presence and absence of test samples and incubated for 48 h at 370.
Then 10 ml of BrdU is added to each
well and the cells are further incubated for 6 h at 370
. Then the cells are fixed and incubated with anti-BrdU and then
detected by the substrate reaction. The reaction is stopped by the addition of 1 M H2SO4 and the absorbance is
measured by using micro plate reader at 450 nm with 690 nm correction.
Tube Formation Assay: - The endothelial cells are isolated and cultured in medium in gelatine coated flasks. The
cells from passages 4 to 7 are using for the angiogenesis study. Three dimensional collagen gels containing
endothelial cells are prepared. After gelation at 370
for 30 min the gels are overlaid with basal medium supplemented
with test substances at indicated concentrations. Gels are examined and the tube length is determined for each well
followed by determination of each group by using software. All experiments are terminating at 48h.
Gelatine Zymography: - This assay can also be called as Matrix Metalloproteinase (MMP) assay. In this the matrix
metalloproteinase activities of the myocardial tissue is measured by using sodium dodecyl sulphate (SDS)
polyacrylamide gels. Gelatine is used as a substrate because connective tissue degrading enzymes such as gelatinase
rapidly cleave it and it is easily incorporated into poly acryl amide gels. Test samples are diluted to a final protein
concentration with distilled water and mixed with SDS sample buffer. The complexes are loaded onto the gel and
electrophoreses at 200 V for approximately 45 min at room temperature. After electrophoreses the gels are cut into
pieces and one half of the gels are incubated for 18 h at 370
analysed by densitography.
Langendorff Isolated Heart model: - This method is example for the in-vitro coronary artery ligation model. This
can be performed by using the “isolated buffer perfused heart model”. The heart is isolated by performing tracheotomy
and the dominant branch of left circumflex coronary artery was sutured. Heart is excised and placed in iced buffer.
Heart is hanging by using the aortic root on a Langendorff apparatus for retrograde non-recirculating buffer perfusion at

22. ORGAN CULTURE ASSAYS[20][24]
Cultivation of Cardiac Myocytes in Agarose Medium: - This method is used for the angiogenic drugs which have
the action on cardiac myocytes. Cardiac myocytes are isolated from the left ventricular myocardium of the mice and
placed in culture medium. Heart explants are incubating for 7 days. Angiogenic Stimulants are added every other day
and after 7 days endothelial sprouts are photographed and sprout formation is calculated.
The Aortic Ring Assay: - The angiogenesis can also evaluating by culturing rings of mouse aorta in three
ensional
collagen gels with some modification of the method originally reported for the rat aorta. Thoracic aortas are removed
transferred to a culture dish containing ice cold serum free medium. The peri-aortic fibro adipose tissue is carefully
moved
without damaging the aorta wall. After the aorta is cut into 1mm long rings and rinsing in minimum essential medium.
use
aortic rings are place in the middle of a 24-well plate. The rings are overloaded with matrigel and leave to polymerize
1
to 2 h at 370
. The rings are exposed to hypoxia for 2 h followed by reoxygenation for 5-7days. The vessel sprouts are
observed and areas of sprouts are measured.
Rat Blood Vessel Culture Assay: - The rat thoracic veins are isolated and fibro adipose tissue is removed. The
ns are
washing with DMEM supplemented with 10% FBS. The veins are then cut into small fragments and cultured in fibrin
s which
are forming by addition of thrombin to the same medium containing fibrinogen in 12-wellplate. On the following day the
substance in the same volume of medium is added to the fibrin gel in the wells. After 9th day tube formation and cell

23. IN VITROMODELS FORANGIOGENESIS[25][26]
In 1980, bovine capillary ECs were found to spontaneously form tubes when cultured in gelatine in vitro since then,
many in vitro models have been designed mimicking many of the basic steps of the in vivo process. Numerous
challenges exist in properly modelling each of the steps involved in angiogenesis both in vitro & in vivo . Rakesh Jain
and colleagues delineated aspects of an ideal angiogenesis model. Among them they include
a known release rate and spatial and temporal concentration distribution of angiogenic factors and inhibitors being
studied for forming dose-response curves;
(2) the assay should be able to quantify the structure of the new vasculature;
(3) the assay should be able to quantify the function of the new vasculature
(this includes EC migration rate, proliferation rate, canalization rate,
blood flow rate, and vascular permeability); and
(4) in vitro responses should be confirmed in vivo .
This final point is especially challenging as many models are carried out in two dimensions and may not take into
account the more complex three-dimensional arrangements involved in cell and extracellular environment interactions.
In vitro models of angiogenesis have many uses: the clinical testing of potential drug therapies; the modelling of
pathological conditions, such as intimal hyperplasia and intimal injury caused by interventions such as angioplasty;
study of the processes of endothelial cell differentiation, lumen formation, and vascular inoculation; as well as
investigating the molecular mechanisms associated with angiogenesis. Angiogenesis inhibitors such as bevacizumab,
erlotinib, and caplostatin, which interfere with growth factor production and function, have been shown to suppress a
wide variety of tumours in vitro and are beginning to be approved by the U.S. Food and Drug Administration for the
treatment of cancer.
Endothelial cell differentiation—i.e., lumen or tube formation—can be studied in vitro both in two dimensions and
in three dimensions. Endothelial cells cultured on plates coated with matrix proteins such as Matrigel (a matrix scaffold
that incorporates extracellular matrix and basement membrane proteins), collagen, or fibrin can be induced to

24. Matrigel Plug Assay: - Matrigel consist of purified basement membrane components (collagens, proteoglycans and
laminin) and, while it is liquid at temperatures just above 0 degrees, it forms a gel when it is warmed to 37 °C. Thus, the
material can be cooled and then injected in the mice, where it will form a three dimensional gel, in which host blood
vessels can invade. Matrigel itself is a poor inducer of angiogenesis, but it can be mixed with angiogenic growth
factors and/or cells prior to injection leading to a controllable induction of blood vessel growth into the plug. Plug
vessels are usually evaluated 7-21 days after implantation by gross examination / photography as well as
immunohistochemical staining (Akhtar et al, 2002). If the plug contains functional vessels, the blood (red) vessels can
be identified from the photograph.Also in this model, intravenous dye injection can be performed to evaluate vessel
perfusion and leakiness.[26]
Rat Aortic Ring Assay: - In this model, segments of rat aorta are placed in a matrix-containing environment with
monitoring of endothelial cell outgrowth. The outgrowth can be quantified by measuring the number and length of
microvessels growing out from the explant. This allows for the testing of pro- and anti- angiogenic substances as they
relate to such outgrowth. The rat aortic model offers the benefit of culturing endothelial cells in the context of native
stromal cells and matrix to more closely mimic the in vivo environment. It also has the added benefit of having
quiescent cells at the start of the assay, which is the native characteristic of endothelial cells in vivo . The limitation of
this assay, however, is that the aorta is most likely too large a vessel to accurately depict processes that are thought to
be initiated by smaller structures.
‘‘Radial Invasion of Matrix by Aggregated Cells’’ Model of Vernon and Sage: - To study the angiogenic activity of
growth factors and their related mutants in vitro , this model have been developed as a novel quantitative fibrin-based 3-
D angiogenesis model. This model recapitulates some of the basic steps of angiogenesis as it occurs in vivo : EC
sprouting, lumen formation, and the formation of a branched network of tubes. Early passaged endothelial cells in a
drop of medium with methylcellulose are suspended upside down in a parafilm-coated dish for 2 days and then, after
careful removal of the medium, the resulting cell aggregate is embedded in a freshly prepared fibrin gel supported by a
woven nylon mesh ring. The disks are then cultured in 24-well plates in assay medium containing the cytokine under

25. receptors, reflecting the broader importance of cell-matrix interaction in angiogenesis. Studies have also shown that the
induction of capillary morphogenesis in vitro is dependent on the specific structure of the extracellular matrix
components.
Langendorff Isolated Heart model: - This method is example for the in-vitro coronary artery ligation model. This can be
performed by using the “isolated buffer perfused heart model”. The heart is isolated by performing tracheotomy and the
dominant branch of left circumflex coronary artery was sutured. Heart is excised and placed in iced buffer. Heart is
hanging by using the aortic root on a Langendorff apparatus for retrograde non-recirculating buffer perfusion at a
constant pressure of 85 mm of Hg. Heart is continuously oxygenated with 95% O2 and 5% CO2. The perfusate is
warming to 370
. The perfusate is modified Krebs-hens let solution.
IN VIVOMODELS FORANGIOGENESIS [27][28][29][30][31][32]
Hind Limb Ischemia Model: - The animal model of persistent ischemia has been tried in a cat, canine, rabbit
by
ligating the vessels. The rabbit is mostly used animal model for this study. The reasons that most studies choose the
r the
experimental animals are adequate cost, good management, easy maintenance and less complete formation of
ls than the
dog.
Left Coronary Artery Ligation Model- This model is mostly used for the myocardial ischemic studies and
ce which have
myocardial angiogenesis activity. The rabbit is the mostly used animal model for this study.
Sponge Implantation Method- This model is used for the evaluation of angiogenesis and anti- angiogenic agents.
sed
animal models are mice and rat, the major disadvantage is implantation of the sponge materials is associated with non-

26. Because the entire membrane can be seen, rather than just a small portion through the shell window, multiple grafts
can be placed on each CAM and photographs can be taken periodically to document vascular changes over time.[21][22]
[23]
Cornea models: - The cornea is an avascular tissue consisting of two, thin, transparent layers in rodents. Thus it is
possible to gently cut a tiny pocket between the two layers, and in this pocket insert a pellet containing factors which
are to be investigated for their angiogenic or anti-angiogenic activities in vivo . Due to the transparent nature of the
cornea and the strong red colour of perfused blood vessels, the angiogenic response can be followed kinetically by
simply taking photographs of the eye at different time points. This makes it possible to study the effects of angiogenic
factors, either alone or in combination on different processes of angiogenesis such as initial angiogenic expansion,
vascular remodelling, maturation and stability in the same animal over time. The major limitation of the assay is the
technical difficulty of implanting pellets into the mouse cornea.
Zebrafish models of angiogenesis: - Zebrafish models have recently gained much attention as an angiogenesis
model system. Zebrafish embryos develop outside of the uterus, which greatly facilitates imaging during development.
Recently researchers have further expanded the benefit of zebrafish-based model systems by generating many
transgenic zebrafish strains which express fluorescent markers in particular cell types, organs or tissues, including
endothelial cells of the vasculature. By continuous observation of such transgenic embryos under the microscope, it is
possible to follow the dynamics of growing vessels during zebrafish development in real time. Such studies, have
yielded valuable insights into the process of vasculogenesis, which is the formation of the first embryonic vessels – the
aorta, cardinal vein and thoracic duct – and on the origin of blood cells as well as the mechanism by which blood flow is
initiated.

28. Literature Survey on Angiogenesis
Zetter, et al., (1998) [9]
reported Angiogenesis, the recruitment of new blood vessels, is an essential component of
the metastatic pathway. These vessels provide the principal route by which tumour cells exit the primary tumour site
and enter the circulation. For many tumours, the vascular density can provide a prognostic indicator of metastatic
potential, with the highly vascular primary tumours having a higher incidence of metastasis than poorly vascular
tumours.
Ribatti, et al., (2002) [2]
reported Angiogenesis is controlled by the net balance between molecules that have
positive and negative regulatory activity and this concept had led to the notion of the ‘‘angiogenic switch,’’ depending
on an increased production of one or more of the positive regulators of angiogenesis. Numerous inducers of
angiogenesis have been identified and this review offers a historical account of the relevant literature concerning the
discovery of the best-characterized angiogenic factors.
William, et al., (2003) [3]
reported Angiogenesis, the growth of new blood vessels, is an important natural process
required for healing wounds and for restoring blood flow to tissues after injury or insult. Angiogenesis therapies—
designed to “turn on” new capillary growth—are revolutionizing medicine by providing a unified approach for treating
crippling and life threatening conditions.
Yasufumi Sato (2003) [34]
reported Angiogenesis is regulated by the balance of proangiogenic factors and
angiogenesis inhibitors, and the imbalance of these regulators is the cause of pathological angiogenesis, including
tumor angiogenesis.
Tortora, et al., (2004) [11]
reported the induction of neoangiogenesis is a critical step already present at the early
stages of tumour development and dissemination. The progressive identification of molecules playing a relevant role in
neoangiogenesis has fostered the development of a wide variety of new selective agents.
Herr, et al., (2007) [6]
reported well-characterized angiogenic factors like vascular endothelial growth factor (VEGF)
or basic fibroblast growth factor (bFGF), some pregnancy-specific factors (e.g. human chorionic gonadotropin (hCG),
insulin-like growth factor-II (IGF-II) or alpha fetoprotein (AFP) were recently described to play a possible regulatory role

29. Gregg L. Semenza (2007) [37]
reported the concept of oxygen homeostasis will be presented as an organizing
principle for discussion of the phylogeny, ontogeny, physiology, and pathology of blood vessel formation and liomasing,
with a focus on molecular mechanisms and potential therapeutic applications.
Qazi, et al., (2009) [33]
reported the roles of both pro-angiogenic and anti-angiogenic molecular players in corneal
angiogenesis, proliferative diabetic retinopathy, exudative macular degeneration and retinopathy of prematurity,
highlighting novel targets that have emerged over the past decade.
Gacche, et al., (2010) [15]
reported Angiogenesis is a key process needed for the growth and survival of solid
tumours. Anti-angiogenesis may arrest the tumour growth and keep check on cancer metastasis. Developing
antiangiogenic agents have remained a significant hope in the mainstream of anticancer research. The free radical
implications in the initiation of cancers are well established.
Loges, et al., (2010) [35]
reported the concept of inhibiting tumor neovessels has taken the hurdle from the bench to
the bedside and now represents an extra pillar of anticancer treatment. So far, anti-angiogenic therapy prolongs
survival in the order of months in some settings while failing to induce a survival benefit in others, in part because of
intrinsic refractoriness or evasive escape.
Prabhu, et al., (2011) [1]
reported Angiogenesis means the growth of new capillary blood vessels in the body is an
important natural process used for healing and reproduction. The body controls angiogenesis by producing a precise
balance of growth and inhibitor factors in healthy tissues. When this balance is disturbed, the result is either too much
or too little angiogenesis.
Yoshiaki Kubota (2011) [36]
reported Anti-angiogenic therapy is an anti-cancer strategy that targets the new
vessels that grow to provide oxygen and nutrients to actively proliferating tumor cells. Most of the current anti-cancer
reagents used in the clinical setting indiscriminately target all rapidly dividing cells, resulting in severe adverse effects
such as immunosuppression, intestinal problems and hair loss. In comparison, anti-angiogenic reagents theoretically
have fewer side effects because, except in the uterine endometrium, neoangiogenesis rarely occurs in healthy adults.
Currently, the most established approach for limiting tumor angiogenesis is blockade of the vascular endothelial growth

30. Literature Survey on Types of Angiogenesis
Nakatsu, et al., (2003) [6]
reported Angiogenesis involves endothelial cell (EC) sprouting from the parent vessel,
followed by migration, proliferation, alignment, tube formation, and anastomosis to other vessels. Several in vitro
models have attempted to recreate this complex sequence of events with varying degrees of success.
Hillen, et al., (2007) [8]
reported the discovery of the contribution of intussusceptive angiogenesis, recruitment of
endothelial progenitor cells, vessel cooption, vasculogenic mimicry and lymphangiogenesis to tumour growth, anti-
tumour targeting strategies and concluded that future anti-vascular therapies might be most beneficial when based on
these strategies.
Makanya, et al., (2009) [7]
reported Primordial capillary plexuses expand through both Sprouting Angiogenesis
(SA) and Intussusceptive Angiogenesis (IA), but subsequent growth and remodeling are achieved through IA.
Literature Survey on Angiogenesis Based Diseases
Jozsef, et al., (2001) [13]
reported that discovery of the molecular mechanism of physiological vasculogenesis and
pathological angiogenesis helped to recognize two class of diseases: one where therapeutic angiogenesis can repair
the tissue damages and the other one where inhibition of pathological angiogenesis can cure the disease or delay its
progression (retinopathies, tumour progression, chronic inflammatory processes).
Peter J. Polverini (2002) [39]
reported the molecular mechanisms that regulate neovascularization continues to
emerge, there is increasing hope that new discoveries will lead to newer therapies that target angiogenesis as a
reliable option for disease therapy.
Carmeliet, et al., (2003) [4]
reported the formation of new blood vessels contributes to numerous malignant,
ischemic, inflammatory, infectious and immune disorders. Molecular insights into these processes are being generated
at a rapidly increasing pace, offering new therapeutic opportunities.
Literature Survey on Current methods forassaying angiogenesis in vitro and in vivo
[22]

31. Ribatti, et al., (2000) [21]
reported the fields of application of CAM in the study of anti-angiogenesis.
Hamamichi, et al., (2001) [23]
reported a shell-less culture system for video monitoring to observe change in
behaviour of 7-day-old chick embryos.
Staton, et al., (2004) [24]
reported the advantages and limitations of the principal assays in use, including those for
the proliferation, migration and differentiation of endothelial cells in vitro, vessel outgrowth from organ cultures and in
vivo assays.
Veeramani, et al., (2010) [20]
reported Advantages and Disadvantages of evaluating angiogenesis using in-vivo, in-
vitro and organ culture assay systems.
Literature Survey on In Vitro and In Vivo Models forAngiogenesis: -
Lees, et al., (1994) [40]
reported a new in viva model has been developed for the quantitative study of promoters
and potential promoters of angiogenesis. Full thickness rat skin autografts received a reproducible and uniform freeze
injury, before being applied to full thickness wounds, in order to delay revascularisation. Blood flow in the grafts was
measured during the healing period using noninvasive (laser Doppler llowmetry) and invasive (lJ3Xe clearance)
techniques. The increase in blood flow over a period of l&14 days was taken as an index of angiogenesis. These
measurements were corroborated by histological assessment of the graft vasculature, using a laminin stain to highlight
vascular basement membrane. Freeze injury delayed but did not ultimately prevent full graft revascularisation (p -C
0.01 for laser Doppler flowmetry and 133Xe clearance). Application of the angiogenic agent basic fibroblast growth
factor (bFGF), in slow release pellet form, stimulated angiogenesis in cryoinjured grafts in a dose-related fashion.
Kenyon, et al., (1996) [45]
reported the study of angiogenesis depends on reliable and reproducible models for the
stimulation of a neovascular response. The purpose of this research was to develop such a model of angiogenesis in
the mouse cornea.
Couffinhalet, et al., (1998) [30]
reported development of a mouse model for angiogenesis particularly for hind limb
ischemia.
[32]

32. Leng, et al., (2004) [44]
reported the use of chick chorioallantoic membrane (CAM) as a model system for the study
of the precision and safety of vitreoretinal microsurgical instruments and techniques.
Kubo, et al., (2007) [42]
reported a parabolic–ODE system modelling tumour growth proposed by Othmer and
Stevens. According to Levine and Sleeman we reduced it to a hyperbolic equation and showed the existence of
collapse in asymptotic behavior of the solution to a parabolic ODE system modeling tumour growth, Differential Integral
Equations. Also deal with the system in case the reduced equation is elliptic and show the existence of collapse
analogously. And application of the above result to another model proposed by Anderson and Chaplain arising from
tumour angiogenesis and show the existence of collapse. Further investigation of a contact point between these two
models and a common property to them.
Eming, et al., (2007) [43]
reported mechanisms of angiogenesis would offer therapeutic options to ameliorate
disorders that are currently leading causes of mortality and morbidity, including cardiovascular diseases, cancer,
chronic inflammatory disorders, diabetic retinopathy, excessive tissue defects, and chronic non-healing wounds.
Restoring blood flow to the site of injured tissue is a prerequisite for mounting a successful repair response, and wound
angiogenesis represents a paradigmatic model to study molecular mechanisms involved in the formation and
remodeling of vascular structures.
Ucuzian, et al., (2007) [25]
reported clinically relevant models of angiogenesis in vitro that are crucial to the
understanding of angiogenic processes and advances made in the development of these models.
Smith, et al., (2008) [41]
reported a simple mathematical model of the siting of capillary sprouts on an existing blood
vessel during the initiation of tumour-induced angiogenesis. The model represents an inceptive attempt to address the
question of how unchecked sprouting of the parent vessel is avoided at the initiation of angiogenesis, based on the
idea that feedback regulation processes play the dominant role. No chemical interaction between the proangiogenic
and antiangiogenic factors is assumed. The model is based on corneal pocket experiments, and provides a
mathematical analysis of the initial spacing of angiogenic sprouts.
Jensen, et al., (2012) [27]
reported various Animal Models of Angiogenesis and Lymphangiogenesis with their

34. OBJECTIVE
Angiogenesis means the growth of new capillary blood vessels in the body, is an important natural process
used for healing and reproduction. The body controls angiogenesis by producing a precise balance of growth
and inhibitory factors in healthy tissues. When this balance is disturbed, the result is either too much or too little
angiogenesis. Excessive angiogenesis occurs in diseases such as cancer, diabetic blindness, age-related
macular degeneration, rheumatoid arthritis, psoriasis, and more than 70 other conditions.
For this the chief objective of this systematic review is to find out the useful experimental tools (in vivo & in
vitro ) for the assay of angiogenesis process, which will foster the future researchers to work on a systematic
path to discover a potential anti-angiogenic target/ molecule by using these laboratory setups.
These anti-angiogenic therapies are aimed to halt new blood vessel growth by using angiogenesis inhibitors.
These can be easily discovered from natural sources to treat cancer and other diseases with vicinal under
effects.
Therapeutic angiogenesis aimed to stimulate new blood vessel growth with growth factors is being
developed to treat these conditions.
The chick embryo chorioallantoic membrane (CAM) is an extra embryonic membrane commonly used in
vivo to study both new vessel formation and its inhibition in response to tissues, cells, or soluble factors.
We have also set our objective to establish CAM as a novel in vivo model for assay of angiogenesis in our

35. PLAN OF WORK
Systematic Reviews on:
• Angiogenesis.
• Types of Angiogenesis.
• Angiogenesis Based Diseases.
• Current methods for assaying angiogenesis in vitro & in vivo .
• In Vitro & In Vivo Models for Angiogenesis
CAM Assay
• Collection of fresh Gallus g allus eggs(One day)
• 12 days Incubation of Eggs
• Egg Windowing
• Extraction of CAM
Result and Discussion
• Macroscopical Evaluations of the CAM Assay.
• Microscopical Evaluations of Angiogenesis in CAM on Various magnifications.
• Discussion of the various models of Angiogenesis and Observations of CAM Assay.
Conclusion of the Systematic Review.
Future Scope of Work.

37. COLLECTION OF Gallus gallus EGGS (One Day)
Fertilized White Leghorn chicken (Gallus g allus) eggs were collected. They must be hatched one day before
incubation. They were placed in an incubator as soon as embryogenesis starts and are kept under constant
humidity at 37°C.
During the period of incubation, eggs were monitored and rotated horizontally to maintain their normal growth.
Ethical Requirement
According to Ethical Guidelines, if the eggs are not 19 days old, there is no need for an approval from Animal
Ethics committee. [44]
REQUIREMENTS
Chemicals
•Dipotassium Hydrogen Phosphate
•Sodium Dihydrogen Phosphate
•Sodium Chloride
•Potassium Chloride
•Distilled Water
Instruments
•LEICA DME Microscope with Ex Digital Zoom 3.0 Software
•Digital HD Camera (SONY Cyber shot 14.1mega pixel)
INCUBATION PROTOCOL
The avian chorioallantoic membrane (CAM) is the outermost extraembryonic membrane lining the noncellular
eggshell membrane. The CAM is formed by fusion of the splanchnic mesoderm of the allantois and the somatic
mesoderm of the chorion. The fused CAM develops and covers the entire surface of the inner shell membrane of the

38. sodium and chloride from the allantoic sac and calcium from the eggshell into the embryonic vasculature, and forms
part of the wall of the allantoic sac, which collects excretory products. Because of low cost, the simplicity of the surgical
procedure, and the possibility to continuously observe the test site without disrupting it, the CAM is a common method
for studying biological processes such as transport, gas exchanges, tumour transplant experiments, toxicity, and more
recently angiogenesis. Typically, an opening is made in the shell to easily access and view the CAM. After having
applied drugs, factors, or an implant to the CAM, the window is closed with a transparent tape or a glass slide, thus
allowing easy viewing of the test site. [21]
PROCEDURE
Egg windowing: - Fertilized chicken eggs were incubated at 37°C with approximately 60% humidity. After 4 days
of incubation, the eggs were gently cleaned with a 70% ethanol solution. Using a 5-cc syringe and 18-gauge needle,
2.5 mL of albumen was extracted from the egg. By extracting the albumen, the CAM of the fertilized egg are separated
from the top part of the shell, which allows for a small, 1.5-cm window to be cut in the shell of the egg, without
damaging the embryonic structures. The window was then sealed using a transparent tape and the egg was placed
back in the incubator until day 7 of incubation. There CAM develops at the top as a flat membrane, reaching the edge
of the dish to provide a two-dimensional monolayer onto which grafts can be placed. Because the entire membrane
can be seen, rather than just a small portion through the shell window, multiple grafts can be placed on each CAM and
photographs can be taken periodically to document vascular changes over time. [22]
MICROSCOPICAL EVALUATION OF ANGIOGENESIS
Modifications in the reported procedures of CAM which are discussed above, includes mounting of freshly isolated
CAM on glass slides using glycerine as mountant, was done. The mounted slides were then evaluated for various
parameters of angiogenesis. Evaluations of CAM were done for various CAM samples retrieved after completion of
protocol under LEICA DME Microscope with Ex Digital Zoom 3.0 Software. Various photographs could be taken to

40. RESULTS
CAMASSAY:
After 14 days of incubation, the eggs showed major vascularization in the window region. The blood vessels could
be seen clearly for counting. The branching and secondary growth of the vessels could be observed at various
magnification (10X, 40X, 100X) under microscope (LiecaDMEmicroscopewithExDigital Zoom3.0Software).
(a) (b)
(c) (d)

41. (e) (f)
(g) (h)
Figure 5.1: Microscopical Observation of CAM: a) Egg Windowing, b) Egg Windowing, c)
Egg Windowing d) Observation at 10X x 15X, e) Observation at 10X x 15X, f) Observation at
15X x 40X, g) Observation at 10X x 40X, h) Observation at 10X x 10X.

42. DISCUSSION
Angiogenesis is the physiological process through which new blood vessels form from pre-existing vessels. This is
distinct from vasculogenesis, which is the de no vo formation of endothelial cells from mesoderm cell precursors. The
first vessels in the developing embryo form through vasculogenesis, after which angiogenesis is responsible for most, if
not all, blood vessel growth during development and in disease. Angiogenesis is a normal and vital process in growth
and development, as well as in wound healing and in the formation of granulation tissue. However, it is also a
fundamental step in the transition of tumours from a benign state to a malignant one, leading to the use of
angiogenesis inhibitors in the treatment of cancer.
The body controls angiogenesis by producing a precise balance of growth and inhibitory factors in healthy tissues.
When this balance is disturbed, the result is either too much or too little angiogenesis. Abnormal blood vessel growth
either excessive or insufficient is now recognized as a “common denominator” underlying many deadly and debilitating
conditions including cancer, skin diseases, age related blindness, diabetic ulcers, cardiovascular disease, stroke and
many others. Blood, carried in the vessels, delivers oxygen and nutrients to and removes waste products from the
tissues. The purpose of this project was directed towards understanding these factors whose abnormal change may
lead to excessive or too little angiogenesis and how these factors can be regulated so that normal angiogenesis can be
maintained again. In a broad term angiogenesis was studied to prevent the factor whose imbalance will lead to
excessive or insufficient angiogenesis i.e. to prevent the disease before occurring.
There are various In vitro Models like Matrigel, Rat Aortic Ring, ‘‘Radial Invasion of Matrix by Aggregated Cells’’
Model of Vernon and Sage and Langendorff Isolated Heart model, which are commonly used. Matrigel consist of
purified basement membrane components and it is liquid at temperatures just above 0 degrees, it forms a gel when it is
warmed to 37 °C. Matrigel itself is a poor inducer of angiogenesis, but it can be mixed with angiogenic growth factors
and/or cells prior to injection leading to a controllable induction of blood vessel growth into the plug. It can also be used
evaluate vessel perfusion and leakiness.
Rat Aortic Ring consist of segments of rat aorta are placed in a matrix-containing environment with monitoring of

43. It also has the added benefit of having quiescent cells at the start of the assay, which is the native characteristic of
endothelial cells in vivo. The limitation of this assay, however, is that the aorta is most likely too large a vessel to
accurately depict processes that are thought to be initiated by smaller structures.
Radial Invasion of Matrix by Aggregated Cells Model of Vernon and Sage is used to study the angiogenic activity
of growth factors and their related mutants in vitro . This model recapitulates some of the basic steps of angiogenesis
as it occurs in vivo : EC sprouting, lumen formation, and the formation of a branched network of tubes. This assay
provides a useful tool for determining the angiogenic potential in vitro of various growth factors with cells suspended in
fibrin gel.
Langendorff Isolated Heart model is used for the in vitro coronary artery ligation model. Heart is hanging by using
the aortic root on a Langendorff apparatus for retrograde non-recirculating buffer perfusion at a constant pressure of 85
mm of Hg. Heart is continuously oxygenated with 95% O2 and 5% CO2. The perfusate is warming to 370
. The perfusate
is modified Krebs-hens let solution.
In vivo models include Hind Limb Ischemia Model, Left Coronary Artery Ligation Model, Sponge Implantation
Method, Chick Chorioallantoic Membrane (CAM), Cornea models, Zebrafish models of angiogenesis.
Hind Limb Ischemia Model uses cat, canine, rabbit and rat by ligating the vessels. The rabbit is mostly used
animal model for this study. The reasons that most studies choose the rabbit for the experimental animals are
adequate cost, good management, easy maintenance and less complete formation of collaterals than the dog.
Left Coronary Artery Ligation Model is used for the myocardial ischemic studies and substance which have
myocardial angiogenesis activity. The rabbit is the mostly used animal model for this study.
Sponge Implantation Method is used for the evaluation of angiogenesis and anti- angiogenic agents. Mostly used
animal models are mice and rat, the major disadvantage is implantation of the sponge materials is associated with non-
specific immune response which may cause a significant angiogenic response even in the absence of exogenous
growth factors in the sponge.
The cornea is an avascular tissue consisting of two, thin, transparent layers in rodents. Thus it is possible to gently cut

44. Due to the transparent nature of the cornea and the strong red colour of perfused blood vessels, the angiogenic
response can be followed kinetically by simply taking photographs of the eye at different time points. This makes it
possible to study the effects of angiogenic factors, either alone or in combination on different processes of
angiogenesis such as initial angiogenic expansion, vascular remodelling, maturation and stability in the same animal
over time. The major limitation of the assay is the technical difficulty of implanting pellets into the mouse cornea.
Zebrafish models have recently gained much attention as an angiogenesis model system. Zebrafish embryos
develop outside of the uterus, which greatly facilitates imaging during development. Recently researchers have further
expanded the benefit of zebrafish-based model systems by generating many transgenic zebrafish strains which
express fluorescent markers in particular cell types, organs or tissues, including endothelial cells of the vasculature. By
continuous observation of such transgenic embryos under the microscope, it is possible to follow the dynamics of
growing vessels during zebrafish development in real time. Such studies, have yielded valuable insights into the
process of vasculogenesis, which is the formation of the first embryonic vessels – the aorta, cardinal vein and thoracic
duct – and on the origin of blood cells as well as the mechanism by which blood flow is initiated.
CAM Assay can be used to study the tissue response to Angiogenesis. The CAM of the domestic chicken (Gallus
g allus) also exhibits more desirable properties for the testing of biomaterials over other CAM models, such as reptiles
or even of other avian species. The vascular density of the CAM of a snapping turtle has been reported to be
significantly less than that of the chicken. Likewise, the CAM of a developing quail embryo has also been reported to
have a slightly lower vascular density. Maintaining an implant area well vascularized is often desirable and so greater
vascular density is advantageous to the function and lifetime of the implant. A major advantage of the CAM model is
that the egg window allows for visual inspection of the implant as well as easy application of treatments (e.g., drugs,
growth factors) to the test site. The major disadvantage of CAM is that it already contains a well- developed vascular
network and the vasodilation that invariably follows its manipulation may be hard to distinguish from the effects of the
test substance. Another limitation is nonspecific inflammatory reaction from the implant is that the histologic study of
CAM sections demonstrates the presence of perivascular inflammatory infiltrate together with any hyperplastic reaction

45. Nonspecific inflammatory reactions are much less frequent when the implant is made very early in CAM development
and the host’s immune system is relatively immature. Also one of the disadvantage of this model is that the test
materials can be put into the system only for a limited amount of time. The chicken embryo will hatch after 21 days of
incubation. Because we need to window the eggs and wait for the full development of the CAM, the time for the implant
is approximately 7–10 days. Although we showed this to be enough time for both the acute and chronic response of the
tissue, this model is not suitable for long-term studies when other factors (e.g., degradation, mineralization) play a role.
However, we believe that the advantages of this animal model clearly outweigh the above disadvantage.

47. The present study on Angiogenesis and its various in vivo & in vitro models provide us
the conclusion that angiogenesis is a very important physiopathological phenomenon which
is responsible for various diseases like cancer, skin diseases, age related blindness, diabetic
ulcers, cardiovascular disease, stroke and many others. On the other hand it is also an
important physiological process through which new blood vessels form from pre-existing
vessels.
By studying various models of angiogenesis we concluded the advantages and
drawbacks of these models. From this information we can select a particular model suited
for the process which we desire to perform in our laboratory conditions. Among this we also
found that the chick CAM model allows for rapid, simple and low cost screening of tissue
reactions to biomaterials. The CAM model is a true in vivo system that can be used as an
intermediate step between a cell culture and a more complex mammalian model. The CAM
may also be used to verify the ability to inhibit the growth of capillaries by implanting tumours
onto the CAM and by comparing tumour growth and vascularization with or without the
administration of the anti-angiogenic substance.
CAM is widely utilized as an in vivo system to study anti-angiogenesis. The rabbit cornea
pocket assay is used just as often as an in vivo system. CAM, however, offers the advantage
of being relatively inexpensive and lends itself to large scale screening, while the very few

49. From this piece of research, we came with a conclusion that, CAM can be used as a
novel in vivo model for angiogenesis assay. A major advantage of the CAM model is that the
egg window allows for visual inspection of the implant as well as easy application of
treatments (e.g., drugs, growth factors) to the test site. The capacity to image a growing
embryo while simultaneously studying the developmental function of specific molecules
provides invaluable information on embryogenesis.
CAM assay can be done through in o vo method. The in o vo preparation is particularly
valuable since it extends the period of time during which the developmental function of the
molecule can be studied and it provides an easy, reproducible method for screening a batch
of molecules. These advantages of CAM assay is not found in any other assaying technique
i.e. we cannot visualize the developmental function of molecule.
These new techniques will prove very helpful in visualizing and understanding the role of
specific molecules during embryonic morphogenesis, including blood vessel formation.

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