A SYSTEMATIC REVIEW ON IN VIVO AND IN VITRO EXPERIMENTAL MODELS OF ANGIOGENESIS & PRELIMINARY STUDIES ON CAM ASSAY

A Project report submitted in partial fulfillment of the requirements for the award of the Degree of Bachelor of Pharmacy By  ABHIJEET MIHIR (BPH/1019/2009) & NIRMAL TOPPO (BPH/1037/2009)

Under the guidance of Dr. S. P. PATTANAYAK Asst. Professor

DEPARTMENT OF PHARMACEUTICAL SCIENCES

BIRLA INSTITUTE OF TECHNOLOGYMESRA - 835215, RANCHI

2013

CONTENTS

S.

NO.

TITLES PAGE

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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

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]

(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.

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 angiogenesis process, regardless of the inciting stimuli or its location in the body.

(Source: Li et al., 2004) [3]

The Angiogenesis Cascade of Events

Body Control Mechanism of 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.

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. Angiogenin2. Angiopoietin-13. Del-14. Fibroblast growth factors: acidic (aGGF) and basic (bFGF)5. Follistatin6. Granulocyte colony-stimulating factor (G-CSF)7 Hepatocyte growth factor (HGC)/ scatter factor (SF)8. Interleukin-8 (IL-8)9. Leptin10. Midkine11. Placental growth factor12. Platelet-derived endothelial cell growth factor (PD-ECGF)13. Platelet-derived growth factor –BB (PDGF-BB)14. Pleiotrophin (PTN)15. Progranulin16. Proliferin17. 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)

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. Angioarrestin2. Angiostatin (plasminogen fragment)3. Antiangiogenic antithrombin III4. Cartilage-derived inhibitor (CDI)5. CD59 complement fragment6. Endostatin (collagen XVIII fragment)7. Fibronectin fragment8. Gro-beta9. Heparinases10. Heparin hexasaccharide fragment11. Human chorionic gonadotropin (hCG)12. Interferon alpha/beta/gamma13. Interferon inducible protein (IP-10)14. Interleukin-1215. Kringle 5 (plasminogen fragment)16. Metalloproteinase inhibitor (TIMPs)17. 2-methoxyestradiol18. Placental ribonuclease inhibitor19. Plasminogen activator inhibitor20. Platelet factor-4(PF4)21. Prolactin 16kD fragment22. Proliferin-related protein (PRP)23. Retinoids24. Tetrahydrocortisol-s25. Thrombospondin-1 (TSP-1)26. Transforming growth factor –beta (TGF-b)27. Vasculostatin28. Vasostatin (calreticulin fragment)

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.

Sprouting occurs at a rate of several millimetres per day, and enables new vessels to grow across gaps in the vasculature. It is markedly different from splitting angiogenesis, however, because it forms entirely new vessels as opposed to splitting existing vessels. [8]

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 detected. Patan et al. also hypothesised that intravascular blood flow patterns or changes in shear stress are parameters that regulate pillar formation. In mammary tumours of c-neu transgenic mice, smaller tumour regions exhibited numerous sprouts, while in larger tumours regions frequently pillar- and mesh formations were observed.[8]

(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.

ANGIOGENESIS BASED DISEASES [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 or Caused by Abnormal or Excessive 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.

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 or Caused by Insufficient Angiogenesis or Vessel Regression [4]

Nervous system- Alzheimer disease – Vasoconstriction, micro vascular degeneration and cerebral angiopathy due to EC toxicity by amyloid-ß117. Amyotrophic lateral sclerosis & Diabetic neuropathy - Impaired perfusion and neuroprotection, Causing motor neuron or axon degeneration due to insufficient VEGF production. Stroke – Correlation of survival with angiogenesis in brain; stroke due to arteriopathy.

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.

The 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 (ischemic diseases, arteriosclerosis etc.) and the other one where inhibition of pathological angiogenesis can cure the disease or delay its progression (retinopathies, tumour progression, chronic inflammatory processes). Though there are an exponentially growing number of new synthetic molecules characterized mostly by antiangiogenic properties, the discovery of the large battery of natural pro- and anti-angiogenic factors suggest that this may provide a more physiological approach to treat these diseases in the near future.[19]

CURRENT METHODS FOR ASSAYING 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-VIVO ASSAYS [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 blood vessels invade the matrigel. This is the basis of an assay referred to as the “Matrigel plug” assay. The matrigel can be harvested and the new vessel formation in the plug can be assessed. The difficulty with the traditional matrigel plug assay is that the matrigel disperses easily in the subcutaneous tissue and does not form a tight solid mass.

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 advantages of this method. The disadvantage of this method is it needs technically more demanding and more expensive than the CAM assay which makes it is not a potential screening assay.

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 370C 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 the animal model of persistent ischemia has been tried in a 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, in this method 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.

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-VITRO ASSAYS [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. Then the chamber is reincubated for 2 h and membrane filter is fixed and staining Diff-Quick reagent. The number of cells that migrated through the filter is counted under a microscope, the advantage of this assay is it is reproducible in practice to very format screening for angiogenesis modifiers.

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 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.

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 dimensional collagen gels with some modification of the method originally reported for the rat aorta. Thoracic aortas are removed and transferred to a culture dish containing ice cold serum free medium. The peri-aortic fibro adipose tissue is carefully removed without damaging the aorta wall. After the aorta is cut into 1mm long rings and rinsing in minimum essential medium. Mouse aortic rings are place in the middle of a 24-well plate. The rings are overloaded with matrigel and leave to polymerize for 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 veins are washing with DMEM supplemented with 10% FBS. The veins are then cut into small fragments and cultured in fibrin gels which are forming by addition of thrombin to the same medium containing fibrinogen in 12-wellplate. On the following day the test substance in the same volume of medium is added to the fibrin gel in the wells. After 9th day tube formation and cell growth are examined using a microscope and graded.  Chick Aortic Arch Assay: - This method is the modification method of the rat aortic ring assay. It is rapid method and it takes 1-3 days with serum-free medium. The chick aortic arch assay can be performed by incubating chick aortic arch ring in culture medium contain test substances. The aortic arches are isolated from 12-14 chick embryos and cut into 1mm rings and cultured in well plate containing matrigel. Average sprouting is measured.

IN VITRO MODELS FOR ANGIOGENESIS[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 differentiate and lend themselves to a common model of in vitro angiogenesis.  

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 investigation. The disks are photographed digitally, and capillary sprout length is quantified. The disks are then fixed and sectioned for light and transmission electron microscopy. This assay provides a useful tool for determining the angiogenic potential in vitro of various growth factors with cells suspended in fibrin gel. Previous studies have demonstrated the importance of extracellular matrix component interactions with cells, growth factors and their related

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 VIVO MODELS FOR ANGIOGENESIS [27][28][29][30][31][32]

  Hind Limb Ischemia Model: - The animal model of persistent ischemia has been tried in a 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- This model is mostly 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- This model 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.   Chick Chorioallantoic Membrane (CAM): - 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 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]

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.

Literature Survey on AngiogenesisZetter, 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 in Placental vascular development. 

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 factor (VEGF) pathway.   Bikfalvi, et al., (2011) [38] reported that antiangiogenesis treatment in the experimental gliomas model drives expression of critical genes which relate to disease aggressiveness in glioblastoma patients. A molecular mechanism in tumour cells that allows the switch from an angiogenic to invasive programme.

Literature Survey on Types of AngiogenesisNakatsu, 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 for assaying angiogenesis in vitro and in vivoWingerter, et al., (1998) [22] reported an assay of angiogenesis for soluble factors in the quail

CAM that supports perturbation of the entire vascular tree. This experimental approach, when combined with fractal morphometry, is sensitive to subtle changes in vascular branching pattern and density.

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 for Angiogenesis: -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.

Carmelietet, et al., (1998) [32] reported Mouse models of angiogenesis, arterial stenosis, atherosclerosis and haemostasis.

Goldbrunner, et al., (2000) [31] reported Models for assessment of angiogenesis in gliomas i.e., antiangiogenic treatment of experimental brain tumours.

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 advantages and disadvantages.

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 laboratory for the future scope of the researchers working in the field of angiogenesis.

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 AngiogenesisCAM Assay• Collection of fresh Gallus gallus eggs(One day)• 12 days Incubation of Eggs• Egg Windowing• Extraction of CAMResult 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.

COLLECTION OF Gallus gallus EGGS (One Day)Fertilized White Leghorn chicken (Gallus gallus) 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 RequirementAccording 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 WaterInstruments•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 chicken by day 12 of incubation. The chick will normally hatch at day 21. The CAM serves as a support for the extraembryonic respiratory capillaries, actively transports

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]

PROCEDUREEgg 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 check Vascularization, Blood flow, No. of Blood Vesses/ Branches of Per Selected Area, Blood Vessel Density, Blood Vessel Diameter, Shape Irregularities, Permeablity.

RESULTS CAM ASSAY:

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 (Lieca DME microscope with Ex Digital Zoom 3.0 Software).

(a) (b)

(c) (d)

(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.

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 novo 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 endothelial cell outgrowth. 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 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 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 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 gallus) 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 of the chorionic epithelium.

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.

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 restrictions to its use are essentially due to nonspecific inflammatory reactions and to the presence of pre-existing vessels which make it difficult to determine the true extent of anti-angiogenesis.

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 ovo method. The in ovo 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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