A systemic review on in vivo & in vitro models of angiogenesis & preliminary studies on CAM assay

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  • 1. A SYSTEMATIC REVIEWON IN VIVOANDIN VITROEXPERIMENTAL MODELS OFANGIOGENESIS& PRELIMINARY STUDIES ON CAMASSAYAProject report submittedinpartial fulfillment of therequirements fortheawardof theDegreeof Bachelorof PharmacyBy  ABHIJEET MIHIR(BPH/1019/2009)&NIRMAL TOPPO(BPH/1037/2009)UndertheguidanceofDr. S. P. PATTANAYAKAsst. ProfessorDEPARTMENT OF PHARMACEUTICALSCIENCESBIRLA INSTITUTE OF TECHNOLOGYMESRA - 835215, RANCHI
  • 2. CONTENTSS. NO. TITLES PAGE NO.1. INTRODUCTION 12. LITERATURE SURVEY 293. OBJECTIVE & PLAN OF WORK 354. RESEARCH METHODOLOGY 375. RESULTS & DISCUSSION 396. CONCLUSION 457. FUTURE SCOPE 468. REFERENCES 47
  • 3. ANGIOGENESISAngiogenesis means for the growth of new capillary blood vessels in the body is an important natural process in thebody used for healing and reproduction. The body controls angiogenesis by producing a precise balance of growth andinhibitory factors in healthy tissues. When this balance is disturbed, the result is either too much or too littleangiogenesis. Abnormal blood vessel growth either excessive or insufficient is now recognized as a “commondenominator” underlying many deadly and debilitating conditions including cancer, skin diseases, age relatedblindness, diabetic ulcers, cardiovascular disease, stroke and many others. Blood, carried in the vessels, deliversoxygen and nutrients to and removes waste products from the tissues. When new tissue is formed, blood vesselformation must occur as well. Thus, new tissue formed, for example, with the repair of wounds and the formation of theplacenta during pregnancy are normal examples of intense new blood vessel formation (angiogenesis). The list ofdiseases 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, whodescribed tumours as "hot and bloody. Angiogenesis is a multi-step process recruited for the formation of new bloodvessels is one of the crucial processes for the growth, survival, proliferation and metastasis of tumours. Under normalconditions angiogenesis is virtually essential for cell reproduction, development and wound healing, etc. The process ofneoangiogenesis involves endothelial cell proliferation, migration, and membrane degradation. The importance ofangiogenesis for the growth and survival of tumours is widely appreciated. Numerous studies are being focused on theunderstanding of angiogenesis and the antiangiogenic agents have received significant attention because of theirtherapeutic implications especially in extending the life expectancy of cancer patients. The recent studies in the field ofmolecular aspects of angiogenesis clearly. [2]Every organism is equipped with variety of enzymatic and non-enzymatic antioxidants for the stabilizationof free radicals. However, the increase in the concentration of free radicals leads to accumulation of oxidative stress: acause for cell dysfunctions and related degenerative diseases. [1][2]
  • 4. (Source: Carmeliet et al., 2003) [4]Figure.1.2: Formation of a vascular network. Endothelial progenitors differentiate to arterialand venous ECs, which assemble in a primitive capillary plexus. Vessels then sprout andbecome stabilized by SMCs, differentiating from their progenitors. HSCs contribute toangiogenesis directly and indirectly, by differentiating to leukocytes or platelets.
  • 5. 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(parentvessels).2. Growth factor-receptor binding activates signalling pathways within endothelial cells.3. Activated endothelial cells release proteolytic enzymes that dissolve the basement membrane surroundingparentvessels.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) dissolvethesurrounding 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 thevasculararchitecture.10. Blood flow begins in the mature stable vessel.These complex growth factor-receptor, cell-cell, and cell-matrix interactions characterize the
  • 6. (Source: Li e t al., 2004) [3]The Angiogenesis Cascade ofEvents
  • 7. Body Control Mechanismof Angiogenesis [5]Angiogenesis the growth of new blood vessels is an important natural process occurring in thebody both in health and in disease. Angiogenesis occurs in the healthy body for healing wounds and forrestoring blood flow to tissues after injury or insult. In females angiogenesis also occurs during the monthlyreproductive cycle (to rebuild the uterus lining, to mature the egg during ovulation) and during pregnancy (tobuild the placenta, the circulation between mother and foetus). The healthy body controls the formation ofnew blood vessels through a series of “on” and “off” switches.• The primary “on” switches are chemicals that stimulate bloodvessel formation.• The primary “off” switches are chemicals that inhibit blood vesselformation.When angiogenic growth factors (‘on’ switches) are created in greater amounts thanangiogenesis 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, thebody maintains a balance of angiogenesis regulators. In some disease states, the organs involved may losecontrol over angiogenesis. In these conditions, new blood vessels either grow too much or not enough.
  • 8. Angiogenesis Growth Factors[1]There are at least 20 different known angiogenic growth factors out of that five angiogenic growth factors arebeing tested in humans for growing new blood vessels to heal wounds and to restore blood flow to the heart, limbs andbrain. 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 factor1. 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)
  • 9. Angiogenesis Inhibitors[1]There are at least 28 known natural angiogenesis inhibitors found in the body. Other than this Angiogenesisinhibitors have also been discovered from natural sources in total more than 300 angiogenesis inhibitors have beendiscovered to date.sl.no. Known angiogenic inhibitors1. 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)
  • 10. TYPES OF ANGIOGENESIS[6][7][8]Tumours can grow to a size of approximately 1–2mm before their metabolic demands are restricted due to thediffusion limit of oxygen and nutrients. In order to grow beyond this size, the tumour switches to an angiogenicphenotype and attracts blood vessels from the surrounding stroma. This process is regulated by a variety of pro- andanti-angiogenic factors, and is a prerequisite for further outgrowth of the tumour. Next to sprouting angiogenesis, theprocess by which new vessels are formed from pre-existing vasculature, several other mechanisms ofneovascularization have been identified in tumours, including intussusceptive angiogenesis, the recruitment ofendothelial 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 metabolicwaste. Angiogenesis takes place in physiological situations, such as embryonic development, wound healing andreproduction. It also plays an important role in many pathologies, like diabetes, rheumatoid arthritis, cardiovascularischemic complications, and cancer. In cancer, sprouting angiogenesis is not only important in primary tumours, it isalso 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 cellspresent in pre- existing blood vessels.Second, the activated endothelial cells begin to release enzymes called proteases that degrade the basementmembrane in order to allow endothelial cells to escape from the original (parent) vessel walls. Theendothelial cells then proliferate into the surrounding matrix and form solid sprouts connectingneighbouring vessels. As sprouts extend toward the source of the angiogenic stimulus, endothelialcells migrate in tandem, using adhesion molecules, the equivalent of cellular grappling hooks, calledintegrin’s. These sprouts then form loops to become a full-fledged vessel lumen as cells migrate to thesite of angiogenesis.
  • 11. Intussusceptive angiogenesis: - A variant of angiogenesis, different from sprouting, is intussusceptiveangiogenesis. This process was first observed in postnatal remodelling of capillaries in the lung. In the third week of ratlife and during the first 2 years in humans, the volume of the lungs increases by more than 20 times. In thisdevelopmental process, a new concept of vessel formation was found where ngiopoietin vessels split in two newvessels by the formation of trans-vascular tissue pillar into the lumen of the vessel. Intussusceptive microvasculargrowth is a fast process that can take place within hours or even minutes, because it does not need proliferation ofendothelial 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 ashort 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 factorsand 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 andmyofibroblasts. These cells begin laying collagen fibres into the core to provide an extracellular matrix forgrowth 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 thenumber of capillaries without a corresponding increase in the number of endothelial cells. This is especially important inembryonic development as there are not enough resources to create a rich microvasculature with new cells every timea new vessel develops.In 1993, the first in vivo intussusceptive microvascular growth was demonstrated by video microscopy in achick chorioallantoic membrane. This process has now been detected in various organs, tissue repair processes andalso in tumour angiogenesis. Tissue pillars were detected in a colon carcinoma xenograft model. At the growing edgeboth sprouting and intussusceptive angiogenesis were observed, in the stabilised regions mostly intussusception was
  • 12. (Source: Hillen et al., 2007) [8]Figure 1.3: Different mechanisms of tumour vascularisation. This diagram represents the sixdifferent 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 theseprocesses, are indicated.
  • 13. ANGIOGENESIS BASEDDISEASES [12]In many serious diseases states the body loses control over angiogenesis and angiogenesis dependent diseases resultwhen 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 diseasedtissues, destroy normal tissues and in the case of cancer the new vessels allow tumour cells to escape into thecirculation and lodge in other organs (tumour metastases). Excessive angiogenesis occurs when diseased cellsproduce 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 (pathogensexpressangiogenic 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.
  • 14. Blood vessels: - Vascular malformations (Tie-2 mutation); DiGeorge syndrome (Low VEGF and neuropilin-1expression); 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 inAIDS patients.Eye: - Persistent hyperplastic vitreous syndrome (loss of Ang-2) or VEGF164; diabetic retinopathy; retinopathy ofprematurity;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. Inthese conditions blood vessel growth is inadequate and circulation is not properly restored leading to the risk of tissuedeath. 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 treatthese conditions.Diseases Characterized orCaused by Insufficient Angiogenesis orVessel Regression [4]Nervous system- Alzheimer disease – Vasoconstriction, micro vascular degeneration and cerebral angiopathydue to EC toxicity by amyloid-ß117.
  • 15. 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, butenhanced retinalneovascularization 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 bypathogens.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 andmaturation(SMC dropout) due to EC telomere shortening.Reproductive system- a. Pre-eclampsia – EC dysfunction resulting in organ failure, thrombosis and hypertension dueto 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 dueto 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 ofVEGF-driven angiogenesis, angiogenesis inhibitors prevent fracture healing.
  • 16. 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 appropriateassay. There are increasing numbers of angiogenesis assays being described both in vitro & in vivo . It has beenproved that it is necessary to use a combination of assays for identification of the cellular and molecular events inangiogenesis 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 typeinvolved in angiogenesis. The supporting cells (e.g. tumour cells, pericytes, smooth muscle cells and fibroblasts), theextracellular matrix produced by endothelial cells and their apposed mesenchymal cells, and the circulating blood withits cellular and humoral components are also involved. No in vitro assay exists currently to model/simulate this complexprocess. Whilst in vivo the components of the process are all present, disparate results and limitations also existdepending 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, physiologicallyrelevant. Here, we review the advantages and limitations of the principal assays in use, including those for theproliferation, migration and differentiation of endothelial cells in vitro , vessel outgrowth from organ cultures and in vivoassays such as sponge implantation, corneal, chamber, zebrafish, chick chorioallantoic membrane (CAM) and tumourangiogenesis models.IN-VIVOASSAYS [20][24]Matrigel Plug Assay: - This model is used for the evaluation of both angiogenic and anti-angiogenic agents. Themechanism involved in this model is injection of foreign substances in to the animal leads to the stimulation of theinflammatory cells including macrophages and neutrophils leads to the stimulation of angiogenesis. The mostly usedanimal model is mice. Matrigel is a gelatinous material derived from mouse tumour cells that is commonly used in vitroand in vivo as a substrate for cells. When pro- angiogenic and anti-angiogenic agents are also added to the matrigeland it is injected into the subcutaneous space of an animal, which forms the single solid gel plug will stimulate the new
  • 17. Sponge Implantation Method: - This model is used for the evaluation of angiogenesis and anti- angiogenicagents. 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 strengthenedwith sterile agarose and that is used for angiogenesis study. The animals are anaesthetized and an incision is given atmidline and gel piece is inserted in to subcutaneously. Animals are allowed to recover and at 14thday the animals aresacrificed and gel foams are harvested and quantification can be done for angiogenesis activity. Mostly used animalmodels 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 exogenousgrowth factors in the sponge. Corneal Angiogenesis Assay: - This is the “gold standard” method for the following the effect of defined substancesto 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 easilyidentified. Several corneal angiogenesis models in the rabbit eye have been described including direct intrasomalinjections of substances, chemical (or) thermal injury, intrasomal tumour implantation and sustained release sucralfateassay. Among these models the sustained release sucralfate assay is unique because it gives a predictable, persistentand aggressive neovascular response which is dependent on direct stimulation of blood vessels rather than on indirectstimulation by induction of inflammation. In this method a pocket is making in the cornea and the test substance whenintroduced into this pocket will stimulate the formation of new vessels from the peripheral limbal vasculature. Slowrelease materials such as ELVAX (ethylene vinyl acetate copolymer) or Hydron have been used for the introduce testsubstance into the corneal pocket. The sponge material to hold test cell suspensions or test substances to induceangiogenesis can also be used because the slow releasing formulations may cause toxic. The original method wasdeveloped 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 andfacilitate the Biomicroscopic grading of the neovascular response and the topical application of test drugs are the
  • 18. Sponge Granuloma Angiogenesis Assay: - This model is used for the evaluation of inflammatory angiogenesiswhich was described by Fajardo and colleagues. The sponge discs are prepared by cutting of sterile polyvinyl alcoholfoam sponges. A hole is cut into the disc centre to serve as a depot for administration of test substances and the backof the disc is close with the cotton plug. After adding the stimulants to the centre hole the sponge discs are coat with aninert 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 suturedto prevent disc dispersion. After 9-12 days the animals are sacrifice and the sponge discs are harvested. The discs arequantified for angiogenesis activity. Chick Chorioallantoic Membrane (CAM) Assay: - The cancer biologists, developmental biologists andophthalmologists have described the chick chorioallantoic membrane (CAM) as a model system for studyingdevelopment, cancer behaviour, properties of biomaterials, angiogenesis and photodynamic therapy. This assay is themost widely used assay for screening of angiogenesis activity. .This method is used for screening of both theangiogenesis and anti-angiogenesis substances. In this method the fertilized white leghorn chicken eggs on the secondday of incubation is collect and incubated at 370C and constant humidity. At the day of 3 small hole is drill at narrowend and the albumin is withdrawn. At the 7thday of incubation a small square window is open in the shell and testsubstances are implanted on the top of the membrane. The window was sealed and reincubated. Eggs are incubatedup 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 theentire membrane can be seen, rather than just a small portion through the shell window, multiple grafts can be placedon 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. Themechanism involved in this model is haemodynamic changes leads to the formation of new blood vessels i.e. whilelarge vessels with low flow tend to augmentation of blood flow which leads to the stimulation of vascular sprouting andmaintain the potency of the newly formed collateral vessels thereby providing blood flow to the ischemic tissue. So far
  • 19. overlying the middle portion of the hind limb. Then the proximal end of the femoral artery is ligating and distal portion ofsaphenous artery is ligating and artery and their side branches were dissected free. The femoral artery and attachedside branched are excised and overlying skin is then closed.Left Coronary Artery Ligation Model: - This model is mostly used for the myocardial ischemic studies andsubstance which have myocardial angiogenesis activity. The mechanism involved in this model is shear stress andstretch which leads to myocardium to up-regulate the adhesion molecules in the endothelium attraction of inflammatorycells and stimulation of endothelial cells to produce growth factors which causes angiogenesis. The rabbit is the mostlyused animal model for this study. In this model animals are anaesthetized. Under sterilization and artificial respirationthe left thoracotomy in the 5th‘intercostals’ space was done and heart is exposed. Then the left anterior descendingcoronary artery distal to its diagonal branch is ligating with a suture which produces myocardial ischemia, afterhaemodynamic 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-wellplate and polymerized for 30 min at 370. Then the endothelial cells are incubated in 1% FBS-containing growthmedium for 12 h respectively. Then they were trypsinized and resuspended in the same medium and dispersed ontothe matrigel. Then the cells were treated with the test substances. After 18h cord formation in each well is monitoredand 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 ofendothelial cell migration have for selecting biologically active assay for the evaluation of stimulants and inhibitors ofangiogenesis. This assay was carried out in a 48-well microchemotaxis chamber. The polycarbonate membrane with12-µm pore is coated with gelatine endothelial cells are resuspended in cell culture medium. The bottom chamber isloaded with endothelial cells and the membrane is laid over the cells. Invertation and incubation of the chamber iscarried out in sequence. After 2h incubation the upper wells are loaded with cell culture medium and test samples.
  • 20. 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 andcultured in medium at 370in 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 wellplates in the presence and absence of test samples and incubated for 48 h at 370.Then 10 ml of BrdU is added to eachwell and the cells are further incubated for 6 h at 370. Then the cells are fixed and incubated with anti-BrdU and thendetected by the substrate reaction. The reaction is stopped by the addition of 1 M H2SO4 and the absorbance ismeasured 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. Thecells from passages 4 to 7 are using for the angiogenesis study. Three dimensional collagen gels containingendothelial cells are prepared. After gelation at 370for 30 min the gels are overlaid with basal medium supplementedwith test substances at indicated concentrations. Gels are examined and the tube length is determined for each wellfollowed 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 matrixmetalloproteinase 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 gelatinaserapidly cleave it and it is easily incorporated into poly acryl amide gels. Test samples are diluted to a final proteinconcentration with distilled water and mixed with SDS sample buffer. The complexes are loaded onto the gel andelectrophoreses at 200 V for approximately 45 min at room temperature. After electrophoreses the gels are cut intopieces and one half of the gels are incubated for 18 h at 370analysed by densitography. Langendorff Isolated Heart model: - This method is example for the in-vitro coronary artery ligation model. Thiscan be performed by using the “isolated buffer perfused heart model”. The heart is isolated by performing tracheotomyand 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
  • 21. ORGAN CULTURE ASSAYS[20][24]Cultivation of Cardiac Myocytes in Agarose Medium: - This method is used for the angiogenic drugs which havethe action on cardiac myocytes. Cardiac myocytes are isolated from the left ventricular myocardium of the mice andplaced in culture medium. Heart explants are incubating for 7 days. Angiogenic Stimulants are added every other dayand 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 threeensionalcollagen gels with some modification of the method originally reported for the rat aorta. Thoracic aortas are removedtransferred to a culture dish containing ice cold serum free medium. The peri-aortic fibro adipose tissue is carefullymovedwithout damaging the aorta wall. After the aorta is cut into 1mm long rings and rinsing in minimum essential medium.useaortic rings are place in the middle of a 24-well plate. The rings are overloaded with matrigel and leave to polymerize1to 2 h at 370. The rings are exposed to hypoxia for 2 h followed by reoxygenation for 5-7days. The vessel sprouts areobserved and areas of sprouts are measured.Rat Blood Vessel Culture Assay: - The rat thoracic veins are isolated and fibro adipose tissue is removed. Thens arewashing with DMEM supplemented with 10% FBS. The veins are then cut into small fragments and cultured in fibrins whichare forming by addition of thrombin to the same medium containing fibrinogen in 12-wellplate. On the following day thesubstance in the same volume of medium is added to the fibrin gel in the wells. After 9th day tube formation and cell
  • 22. 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. Numerouschallenges exist in properly modelling each of the steps involved in angiogenesis both in vitro & in vivo . Rakesh Jainand colleagues delineated aspects of an ideal angiogenesis model. Among them they includea known release rate and spatial and temporal concentration distribution of angiogenic factors and inhibitors beingstudied 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 intoaccount 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 ofpathological 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 asinvestigating 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 awide variety of tumours in vitro and are beginning to be approved by the U.S. Food and Drug Administration for thetreatment of cancer.Endothelial cell differentiation—i.e., lumen or tube formation—can be studied in vitro both in two dimensions andin three dimensions. Endothelial cells cultured on plates coated with matrix proteins such as Matrigel (a matrix scaffoldthat incorporates extracellular matrix and basement membrane proteins), collagen, or fibrin can be induced to
  • 23. Matrigel Plug Assay: - Matrigel consist of purified basement membrane components (collagens, proteoglycans andlaminin) and, while it is liquid at temperatures just above 0 degrees, it forms a gel when it is warmed to 37 °C. Thus, thematerial can be cooled and then injected in the mice, where it will form a three dimensional gel, in which host bloodvessels can invade. Matrigel itself is a poor inducer of angiogenesis, but it can be mixed with angiogenic growthfactors and/or cells prior to injection leading to a controllable induction of blood vessel growth into the plug. Plugvessels are usually evaluated 7-21 days after implantation by gross examination / photography as well asimmunohistochemical staining (Akhtar et al, 2002). If the plug contains functional vessels, the blood (red) vessels canbe identified from the photograph.Also in this model, intravenous dye injection can be performed to evaluate vesselperfusion and leakiness.[26] Rat Aortic Ring Assay: - In this model, segments of rat aorta are placed in a matrix-containing environment withmonitoring of endothelial cell outgrowth. The outgrowth can be quantified by measuring the number and length ofmicrovessels growing out from the explant. This allows for the testing of pro- and anti- angiogenic substances as theyrelate to such outgrowth. The rat aortic model offers the benefit of culturing endothelial cells in the context of nativestromal cells and matrix to more closely mimic the in vivo environment. It also has the added benefit of havingquiescent cells at the start of the assay, which is the native characteristic of endothelial cells in vivo . The limitation ofthis assay, however, is that the aorta is most likely too large a vessel to accurately depict processes that are thought tobe initiated by smaller structures.  ‘‘Radial Invasion of Matrix by Aggregated Cells’’ Model of Vernon and Sage: - To study the angiogenic activity ofgrowth 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 : ECsprouting, lumen formation, and the formation of a branched network of tubes. Early passaged endothelial cells in adrop of medium with methylcellulose are suspended upside down in a parafilm-coated dish for 2 days and then, aftercareful removal of the medium, the resulting cell aggregate is embedded in a freshly prepared fibrin gel supported by awoven nylon mesh ring. The disks are then cultured in 24-well plates in assay medium containing the cytokine under
  • 24. receptors, reflecting the broader importance of cell-matrix interaction in angiogenesis. Studies have also shown that theinduction of capillary morphogenesis in vitro is dependent on the specific structure of the extracellular matrixcomponents. Langendorff Isolated Heart model: - This method is example for the in-vitro coronary artery ligation model. This can beperformed by using the “isolated buffer perfused heart model”. The heart is isolated by performing tracheotomy and thedominant branch of left circumflex coronary artery was sutured. Heart is excised and placed in iced buffer. Heart ishanging by using the aortic root on a Langendorff apparatus for retrograde non-recirculating buffer perfusion at aconstant pressure of 85 mm of Hg. Heart is continuously oxygenated with 95% O2 and 5% CO2. The perfusate iswarming 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, rabbitbyligating the vessels. The rabbit is mostly used animal model for this study. The reasons that most studies choose ther theexperimental animals are adequate cost, good management, easy maintenance and less complete formation ofls than thedog.Left Coronary Artery Ligation Model- This model is mostly used for the myocardial ischemic studies andce which havemyocardial 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.sedanimal models are mice and rat, the major disadvantage is implantation of the sponge materials is associated with non-
  • 25. Because the entire membrane can be seen, rather than just a small portion through the shell window, multiple graftscan 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 ispossible to gently cut a tiny pocket between the two layers, and in this pocket insert a pellet containing factors whichare to be investigated for their angiogenic or anti-angiogenic activities in vivo . Due to the transparent nature of thecornea and the strong red colour of perfused blood vessels, the angiogenic response can be followed kinetically bysimply taking photographs of the eye at different time points. This makes it possible to study the effects of angiogenicfactors, 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 thetechnical difficulty of implanting pellets into the mouse cornea.Zebrafish models of angiogenesis: - Zebrafish models have recently gained much attention as an angiogenesismodel 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 manytransgenic zebrafish strains which express fluorescent markers in particular cell types, organs or tissues, includingendothelial cells of the vasculature. By continuous observation of such transgenic embryos under the microscope, it ispossible to follow the dynamics of growing vessels during zebrafish development in real time. Such studies, haveyielded valuable insights into the process of vasculogenesis, which is the formation of the first embryonic vessels – theaorta, cardinal vein and thoracic duct – and on the origin of blood cells as well as the mechanism by which blood flow isinitiated.
  • 26. Literature Survey on AngiogenesisZetter, et al., (1998) [9]reported Angiogenesis, the recruitment of new blood vessels, is an essential component ofthe metastatic pathway. These vessels provide the principal route by which tumour cells exit the primary tumour siteand enter the circulation. For many tumours, the vascular density can provide a prognostic indicator of metastaticpotential, with the highly vascular primary tumours having a higher incidence of metastasis than poorly vasculartumours.  Ribatti, et al., (2002) [2]reported Angiogenesis is controlled by the net balance between molecules that havepositive and negative regulatory activity and this concept had led to the notion of the ‘‘angiogenic switch,’’ dependingon an increased production of one or more of the positive regulators of angiogenesis. Numerous inducers ofangiogenesis have been identified and this review offers a historical account of the relevant literature concerning thediscovery of the best-characterized angiogenic factors.  William, et al., (2003) [3]reported Angiogenesis, the growth of new blood vessels, is an important natural processrequired 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 treatingcrippling and life threatening conditions.  Yasufumi Sato (2003) [34]reported Angiogenesis is regulated by the balance of proangiogenic factors andangiogenesis inhibitors, and the imbalance of these regulators is the cause of pathological angiogenesis, includingtumor angiogenesis.  Tortora, et al., (2004) [11]reported the induction of neoangiogenesis is a critical step already present at the earlystages of tumour development and dissemination. The progressive identification of molecules playing a relevant role inneoangiogenesis 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
  • 27. Gregg L. Semenza (2007) [37]reported the concept of oxygen homeostasis will be presented as an organizingprinciple 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 cornealangiogenesis, 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 solidtumours. Anti-angiogenesis may arrest the tumour growth and keep check on cancer metastasis. Developingantiangiogenic agents have remained a significant hope in the mainstream of anticancer research. The free radicalimplications 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 tothe bedside and now represents an extra pillar of anticancer treatment. So far, anti-angiogenic therapy prolongssurvival in the order of months in some settings while failing to induce a survival benefit in others, in part because ofintrinsic refractoriness or evasive escape.  Prabhu, et al., (2011) [1]reported Angiogenesis means the growth of new capillary blood vessels in the body is animportant natural process used for healing and reproduction. The body controls angiogenesis by producing a precisebalance of growth and inhibitor factors in healthy tissues. When this balance is disturbed, the result is either too muchor too little angiogenesis.  Yoshiaki Kubota (2011) [36]reported Anti-angiogenic therapy is an anti-cancer strategy that targets the newvessels that grow to provide oxygen and nutrients to actively proliferating tumor cells. Most of the current anti-cancerreagents used in the clinical setting indiscriminately target all rapidly dividing cells, resulting in severe adverse effectssuch as immunosuppression, intestinal problems and hair loss. In comparison, anti-angiogenic reagents theoreticallyhave 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
  • 28. 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 vitromodels 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 ofendothelial 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 onthese 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 DiseasesJozsef, et al., (2001) [13]reported that discovery of the molecular mechanism of physiological vasculogenesis andpathological angiogenesis helped to recognize two class of diseases: one where therapeutic angiogenesis can repairthe tissue damages and the other one where inhibition of pathological angiogenesis can cure the disease or delay itsprogression (retinopathies, tumour progression, chronic inflammatory processes).Peter J. Polverini (2002) [39]reported the molecular mechanisms that regulate neovascularization continues toemerge, there is increasing hope that new discoveries will lead to newer therapies that target angiogenesis as areliable 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 generatedat a rapidly increasing pace, offering new therapeutic opportunities.Literature Survey on Current methods forassaying angiogenesis in vitro and in vivo[22]
  • 29. 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 inbehaviour of 7-day-old chick embryos.Staton, et al., (2004) [24]reported the advantages and limitations of the principal assays in use, including those forthe proliferation, migration and differentiation of endothelial cells in vitro, vessel outgrowth from organ cultures and invivo 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 promotersand potential promoters of angiogenesis. Full thickness rat skin autografts received a reproducible and uniform freezeinjury, before being applied to full thickness wounds, in order to delay revascularisation. Blood flow in the grafts wasmeasured 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. Thesemeasurements were corroborated by histological assessment of the graft vasculature, using a laminin stain to highlightvascular basement membrane. Freeze injury delayed but did not ultimately prevent full graft revascularisation (p -C0.01 for laser Doppler flowmetry and 133Xe clearance). Application of the angiogenic agent basic fibroblast growthfactor (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 thestimulation of a neovascular response. The purpose of this research was to develop such a model of angiogenesis inthe mouse cornea.Couffinhalet, et al., (1998) [30]reported development of a mouse model for angiogenesis particularly for hind limbischemia.[32]
  • 30. Leng, et al., (2004) [44]reported the use of chick chorioallantoic membrane (CAM) as a model system for the studyof 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 andStevens. According to Levine and Sleeman we reduced it to a hyperbolic equation and showed the existence ofcollapse in asymptotic behavior of the solution to a parabolic ODE system modeling tumour growth, Differential IntegralEquations. Also deal with the system in case the reduced equation is elliptic and show the existence of collapseanalogously. And application of the above result to another model proposed by Anderson and Chaplain arising fromtumour angiogenesis and show the existence of collapse. Further investigation of a contact point between these twomodels and a common property to them.Eming, et al., (2007) [43]reported mechanisms of angiogenesis would offer therapeutic options to amelioratedisorders 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 woundangiogenesis represents a paradigmatic model to study molecular mechanisms involved in the formation andremodeling of vascular structures.Ucuzian, et al., (2007) [25]reported clinically relevant models of angiogenesis in vitro that are crucial to theunderstanding 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 bloodvessel during the initiation of tumour-induced angiogenesis. The model represents an inceptive attempt to address thequestion of how unchecked sprouting of the parent vessel is avoided at the initiation of angiogenesis, based on theidea that feedback regulation processes play the dominant role. No chemical interaction between the proangiogenicand antiangiogenic factors is assumed. The model is based on corneal pocket experiments, and provides amathematical analysis of the initial spacing of angiogenic sprouts.Jensen, et al., (2012) [27]reported various Animal Models of Angiogenesis and Lymphangiogenesis with their
  • 31. OBJECTIVEAngiogenesis means the growth of new capillary blood vessels in the body, is an important natural processused for healing and reproduction. The body controls angiogenesis by producing a precise balance of growthand inhibitory factors in healthy tissues. When this balance is disturbed, the result is either too much or too littleangiogenesis. Excessive angiogenesis occurs in diseases such as cancer, diabetic blindness, age-relatedmacular 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 & invitro ) for the assay of angiogenesis process, which will foster the future researchers to work on a systematicpath 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 undereffects. Therapeutic angiogenesis aimed to stimulate new blood vessel growth with growth factors is beingdeveloped to treat these conditions. The chick embryo chorioallantoic membrane (CAM) is an extra embryonic membrane commonly used invivo 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
  • 32. 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 g allus 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.
  • 33. COLLECTION OF Gallus gallus EGGS (One Day)Fertilized White Leghorn chicken (Gallus g allus) eggs were collected. They must be hatched one day beforeincubation. They were placed in an incubator as soon as embryogenesis starts and are kept under constanthumidity 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 AnimalEthics committee. [44]REQUIREMENTSChemicals•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 PROTOCOLThe avian chorioallantoic membrane (CAM) is the outermost extraembryonic membrane lining the noncellulareggshell membrane. The CAM is formed by fusion of the splanchnic mesoderm of the allantois and the somaticmesoderm of the chorion. The fused CAM develops and covers the entire surface of the inner shell membrane of the
  • 34. sodium and chloride from the allantoic sac and calcium from the eggshell into the embryonic vasculature, and formspart of the wall of the allantoic sac, which collects excretory products. Because of low cost, the simplicity of the surgicalprocedure, and the possibility to continuously observe the test site without disrupting it, the CAM is a common methodfor studying biological processes such as transport, gas exchanges, tumour transplant experiments, toxicity, and morerecently angiogenesis. Typically, an opening is made in the shell to easily access and view the CAM. After havingapplied drugs, factors, or an implant to the CAM, the window is closed with a transparent tape or a glass slide, thusallowing easy viewing of the test site. [21]PROCEDUREEgg windowing: - Fertilized chicken eggs were incubated at 37°C with approximately 60% humidity. After 4 daysof 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 separatedfrom the top part of the shell, which allows for a small, 1.5-cm window to be cut in the shell of the egg, withoutdamaging the embryonic structures. The window was then sealed using a transparent tape and the egg was placedback in the incubator until day 7 of incubation. There CAM develops at the top as a flat membrane, reaching the edgeof the dish to provide a two-dimensional monolayer onto which grafts can be placed. Because the entire membranecan be seen, rather than just a small portion through the shell window, multiple grafts can be placed on each CAM andphotographs can be taken periodically to document vascular changes over time. [22]MICROSCOPICAL EVALUATION OF ANGIOGENESISModifications in the reported procedures of CAM which are discussed above, includes mounting of freshly isolatedCAM on glass slides using glycerine as mountant, was done. The mounted slides were then evaluated for variousparameters of angiogenesis. Evaluations of CAM were done for various CAM samples retrieved after completion ofprotocol under LEICA DME Microscope with Ex Digital Zoom 3.0 Software. Various photographs could be taken to
  • 35. RESULTSCAMASSAY:After 14 days of incubation, the eggs showed major vascularization in the window region. The blood vessels couldbe seen clearly for counting. The branching and secondary growth of the vessels could be observed at variousmagnification (10X, 40X, 100X) under microscope (LiecaDMEmicroscopewithExDigital Zoom3.0Software).(a) (b)(c) (d)
  • 36. (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 at15X x 40X, g) Observation at 10X x 40X, h) Observation at 10X x 10X.
  • 37. DISCUSSIONAngiogenesis is the physiological process through which new blood vessels form from pre-existing vessels. This isdistinct from vasculogenesis, which is the de no vo formation of endothelial cells from mesoderm cell precursors. Thefirst vessels in the developing embryo form through vasculogenesis, after which angiogenesis is responsible for most, ifnot all, blood vessel growth during development and in disease. Angiogenesis is a normal and vital process in growthand development, as well as in wound healing and in the formation of granulation tissue. However, it is also afundamental step in the transition of tumours from a benign state to a malignant one, leading to the use ofangiogenesis 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 growtheither excessive or insufficient is now recognized as a “common denominator” underlying many deadly and debilitatingconditions including cancer, skin diseases, age related blindness, diabetic ulcers, cardiovascular disease, stroke andmany others. Blood, carried in the vessels, delivers oxygen and nutrients to and removes waste products from thetissues. The purpose of this project was directed towards understanding these factors whose abnormal change maylead to excessive or too little angiogenesis and how these factors can be regulated so that normal angiogenesis can bemaintained again. In a broad term angiogenesis was studied to prevent the factor whose imbalance will lead toexcessive 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 ofpurified basement membrane components and it is liquid at temperatures just above 0 degrees, it forms a gel when it iswarmed to 37 °C. Matrigel itself is a poor inducer of angiogenesis, but it can be mixed with angiogenic growth factorsand/or cells prior to injection leading to a controllable induction of blood vessel growth into the plug. It can also be usedevaluate vessel perfusion and leakiness.Rat Aortic Ring consist of segments of rat aorta are placed in a matrix-containing environment with monitoring of
  • 38. It also has the added benefit of having quiescent cells at the start of the assay, which is the native characteristic ofendothelial cells in vivo. The limitation of this assay, however, is that the aorta is most likely too large a vessel toaccurately 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 activityof growth factors and their related mutants in vitro . This model recapitulates some of the basic steps of angiogenesisas it occurs in vivo : EC sprouting, lumen formation, and the formation of a branched network of tubes. This assayprovides a useful tool for determining the angiogenic potential in vitro of various growth factors with cells suspended infibrin gel.Langendorff Isolated Heart model is used for the in vitro coronary artery ligation model. Heart is hanging by usingthe aortic root on a Langendorff apparatus for retrograde non-recirculating buffer perfusion at a constant pressure of 85mm of Hg. Heart is continuously oxygenated with 95% O2 and 5% CO2. The perfusate is warming to 370. The perfusateis modified Krebs-hens let solution.In vivo models include Hind Limb Ischemia Model, Left Coronary Artery Ligation Model, Sponge ImplantationMethod, 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 usedanimal model for this study. The reasons that most studies choose the rabbit for the experimental animals areadequate 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 havemyocardial 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 usedanimal 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 exogenousgrowth 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
  • 39. Due to the transparent nature of the cornea and the strong red colour of perfused blood vessels, the angiogenicresponse can be followed kinetically by simply taking photographs of the eye at different time points. This makes itpossible to study the effects of angiogenic factors, either alone or in combination on different processes ofangiogenesis such as initial angiogenic expansion, vascular remodelling, maturation and stability in the same animalover 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 embryosdevelop outside of the uterus, which greatly facilitates imaging during development. Recently researchers have furtherexpanded the benefit of zebrafish-based model systems by generating many transgenic zebrafish strains whichexpress fluorescent markers in particular cell types, organs or tissues, including endothelial cells of the vasculature. Bycontinuous observation of such transgenic embryos under the microscope, it is possible to follow the dynamics ofgrowing vessels during zebrafish development in real time. Such studies, have yielded valuable insights into theprocess of vasculogenesis, which is the formation of the first embryonic vessels – the aorta, cardinal vein and thoracicduct – 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 (Gallusg allus) also exhibits more desirable properties for the testing of biomaterials over other CAM models, such as reptilesor even of other avian species. The vascular density of the CAM of a snapping turtle has been reported to besignificantly less than that of the chicken. Likewise, the CAM of a developing quail embryo has also been reported tohave a slightly lower vascular density. Maintaining an implant area well vascularized is often desirable and so greatervascular density is advantageous to the function and lifetime of the implant. A major advantage of the CAM model isthat 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 vascularnetwork and the vasodilation that invariably follows its manipulation may be hard to distinguish from the effects of thetest substance. Another limitation is nonspecific inflammatory reaction from the implant is that the histologic study ofCAM sections demonstrates the presence of perivascular inflammatory infiltrate together with any hyperplastic reaction
  • 40. Nonspecific inflammatory reactions are much less frequent when the implant is made very early in CAM developmentand the host’s immune system is relatively immature. Also one of the disadvantage of this model is that the testmaterials can be put into the system only for a limited amount of time. The chicken embryo will hatch after 21 days ofincubation. Because we need to window the eggs and wait for the full development of the CAM, the time for the implantis approximately 7–10 days. Although we showed this to be enough time for both the acute and chronic response of thetissue, 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.
  • 41. The present study on Angiogenesis and its various in vivo & in vitro models provide usthe conclusion that angiogenesis is a very important physiopathological phenomenon whichis responsible for various diseases like cancer, skin diseases, age related blindness, diabeticulcers, cardiovascular disease, stroke and many others. On the other hand it is also animportant physiological process through which new blood vessels form from pre-existingvessels.By studying various models of angiogenesis we concluded the advantages anddrawbacks of these models. From this information we can select a particular model suitedfor the process which we desire to perform in our laboratory conditions. Among this we alsofound that the chick CAM model allows for rapid, simple and low cost screening of tissuereactions to biomaterials. The CAM model is a true in vivo system that can be used as anintermediate step between a cell culture and a more complex mammalian model. The CAMmay also be used to verify the ability to inhibit the growth of capillaries by implanting tumoursonto the CAM and by comparing tumour growth and vascularization with or without theadministration of the anti-angiogenic substance.CAM is widely utilized as an in vivo system to study anti-angiogenesis. The rabbit corneapocket assay is used just as often as an in vivo system. CAM, however, offers the advantageof being relatively inexpensive and lends itself to large scale screening, while the very few
  • 42. From this piece of research, we came with a conclusion that, CAM can be used as anovel in vivo model for angiogenesis assay. A major advantage of the CAM model is that theegg window allows for visual inspection of the implant as well as easy application oftreatments (e.g., drugs, growth factors) to the test site. The capacity to image a growingembryo while simultaneously studying the developmental function of specific moleculesprovides invaluable information on embryogenesis.CAM assay can be done through in o vo method. The in o vo preparation is particularlyvaluable since it extends the period of time during which the developmental function of themolecule can be studied and it provides an easy, reproducible method for screening a batchof molecules. These advantages of CAM assay is not found in any other assaying techniquei.e. we cannot visualize the developmental function of molecule.These new techniques will prove very helpful in visualizing and understanding the role ofspecific molecules during embryonic morphogenesis, including blood vessel formation.
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