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INTRODUCTION

In recent years the limitations of modern medicines being realized; especially in

management of many types of chronic diseases and the side effects of the drugs used for

the treatments are being evident. The tendency to use the ethnic and traditional medicines

is increased in all the parts of the world. All these systems may provide an integrated

medicinal approach to improve the human health. But the use of ethnic, traditional,

indigenous and Ayurvedic medicines can be acceptable even to modern therapist when

their animal tests based data that includes their effects, claims (tested), probable modes of

actions (which are worked out) will be provided. To ensure the use of these drugs the

consistency in the quality must be ensured. To check their quality control, bioassays

should be developed, as their chemical nature is hardly known in modern forms. This will

allow the drugs to be acceptable to any physician and the worldwide market will be

opened to these drugs.

Traditional medicines are referred as ‘Complementary’ or ‘Alternative’

medicines. At the website, http://www.who.int/mediacentre/factsheets/fs/134/en/ it has

been stated that popularity of the traditional medicines has been maintained in the

developing world and its use is rapidly spreading in industrialized countries. It has also

been mentioned at this site that there is extensive need of research of certain characters of

the drugs as well as of the efficacy and safety of several other practices and medicinal

plants. The web site also covers efforts of World Health Organization (WHO) in

promoting safe, effective and affordable traditional medicine. Keeping with the view of

WHO, many countries have integrated traditional medicines in to their health care

systems.

Natural products are a source of synthetic and traditional herbal medicine. They

have relatively fewer side effects and have been used clinically to treat various kinds of

diseases. The therapeutic efficacies of many indigenous plants for various diseases have

been described by traditional herbal medicine practitioners (Natrajan et al, 2003). Many

natural products are claimed to have many bioactive constituents and it will now be

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necessary to identify the chemical entities, which are responsible for their effects on

varied body systems including physiological units, cell biological units or any functional

unit of organism. Many herbs, their parts and products are known to contain different

bioactive potencies. Therefore, natural products have played a significant role in drug

discovery and development especially for agents against cancer and infectious diseases.

Natural compounds possess highly diverse and complex molecular structures compared

to small molecule synthetic drugs and often provide highly specific biological activities

likely derived from the rigidity and high number of chiral centers. Ethno- traditional use

of plant derived natural products has been a major source of discovery of potential

medicinal agents. The search for different compounds affecting the properties of ECs and

influencing the synthesis of vascular mediators is extensive and comprises different

strategies. There is a widespread belief that components of medicinal plants may protect

from diseases or inhibit their progression. The presence of various life-sustaining

constituents in plants has urged scientists to examine the plants for its angiogenic or

antiangiogenic properties.

Present project titled "Evaluation of influence of extracts of Pterocarpus

santalinus and Boerrhavia diffusa on angiogenesis by the chorioallantoic membrane

assay” is selected to reveal the mode of alterations in CAM vasculature with relevant

experimental work. For the study in vivo chick chorioallantoic membrane (CAM) assay

was used.

Objectives Of the study:

Present project is entitled "Evaluation of influence of extracts of Pterocarpus

santalinus and Boerrhavia diffusa on angiogenesis by the chorioallantoic membrane

assay" It was mainly aimed to analysis in detail the proangiogenic/antiangiogenic activity

of acetone, alcohol and benzene extracts of P. santalinus and B. diffusa plants in the

chick chorioallantoic membrane (CAM) in vivo. This study was also aimed to analyze the

effect of given plant extracts on development of CAM vasculature with area and diameter

of the CAM. In brief, the present study was mainly aimed to analyze the influence of P.

santalinus and B. diffusa extracts on angiogenesis and vasculogenesis. The results of

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which may form the first step in the drug development to treat angiogenesis based

diseases.

Significance of the present study:

The study of blood vessel formation began almost two decades ago in an attempt

to understand the role of vascularization in tumor growth. It has become an attractive

target for anticancer therapy due to its essential role for the progression of solid tumors.

The induction of new blood vessels provides tumors with a survival advantage. The

elucidation of the molecular mechanisms associated with tumor (pathologic) and

physiologic blood vessel formation has become the focus of an intense worldwide

research effort to develop treatment regimes for various pathologic states such as

cardiovascular disease and cancer (Carmeliet, 2000). Discerning these mechanisms may

lead to therapeutic options to improve or perhaps to cure these biologic disorders that are

now leading causes of morbidity and mortality in industrialized societies. It is now a major

field that includes the growth and regression of capillaries in embryological development,

physiological functions also. Angiogenesis depends upon complex interactions among

various classes of molecules, including adhesion molecules, proteases, structural proteins,

cell surface receptors, and growth factors. The early years of angiogenesis research were

dominated by intensive searches for precise growth factors that stimulate this process of

new blood vessel formation from pre-existing mature and quiescent vasculature. Both

angiogenic peptides and angiostatic steroids serve in an elaborate control system of

capillary growth, which in turn governs both growth and involution of tissues (Folkman,

1986a, 1986b). The discovery of angiogenic stimulators like bFGF and especially VEGF

in the mid-to-late 1980s were seminal events that significantly advanced the field. But

very soon afterward it became apparent that the angiogenic universe not only resolves

around the action of such stimulators but also depends on a large number of diverse

endogenous protein inhibitors.

A variety of antiangiogenic agents are currently in preclinical development; with

some of them now entering the clinical trials. However, the administration of

angiogenesis inhibitors usually causes cardiovascular complications including impaired

wound healing, bleeding, hypertension, proteinuria and thrombosis (Chen and Cleck,

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2009; Zangari et al, 2009; Higa and Abraham, 2009) due to their intrinsic cytotoxocity

against non-tumor associated endothelial cells (EC). In addition multiple signaling

pathways are involved in tumor angiogenesis inhibitors that affect a single pathway may

be insufficient and probably lead to resistance (Eikesdal and Kalluri, 2009). Thus,

protection and cure is obligatory to maintain the health. These problems highlight the

need for the development of multitarget agents with minimal side effects and toxicity.

For the reasons above in the present project two plants had been used to study the

bioactive potency from them 1. Pterocarpus santalinus. 2. Boerrhavia diffusa.

The bioactive compound analysis of these plants and their properties are reviewed

here in following pages to reveal the reasons why their efficacy had been studied on

angiogenesis a physiological process in animals.

Selection of Pterocarpus santalinus extracts:

Review of the properties of Pterocarpus santalinus:

Pterocarpus santalinus L.f. (Sanskrit: Raktachandan; Family: Fabaceae)

commonly called as Red sander is the evergreen tree found in the dry regions of south

India and in north India. It is an endangered plant species endemic to the state of

Andrapradesh in India (Pandey, 1980; Anuradha et al, 1999). It favors a dry rather than

rocky soil and a hot fairly dry climate (Pandey, 1980). It is described in Ayurveda for its

wide spectrum of medicinal properties. At present, P. santalinus is not only used as a

therapeutic by traditional medical practitioners but is also as health supplements readily

available in the commercial market. The diversity of highly desirable physiological effect

of P. santalinus has intrigued scientists for years. In general most of its actions have been

attributed to its various phytoconstituents.

The P. santalinus is renowned for its characteristic timber of luxury in Japan

(Reddy and Srivasuki, 1990). The coloring principle of red sanders is santalic acid or

santalin (C8H6O3). It is red, tasteless and odorless, crystalline powder, insoluble in ether,

with yellow color and in alcohol with blood red color. It likewise dissolves in alkalies and

acetic acid but not in essential oils. The heartwood of P. santalinus is used as astringent

tonic as external application for wounds, cuts and inflammation, in treating headache,

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skin diseases, fever, boils, scorpion sting and to improve sight (Jain, 1996; Chopra et al,

1956). Santalin, is a natural dye from red wood that is used as a coloring agent in

pharmaceutical preparation, foodstuffs. It is also used as a cooling agent in

pharmaceutical preparations. Fruit extract is used as astringent, diaphoretic, in

inflammation, headache, skin diseases, and bilious infections and chronic dysentery.

The P. santalinus contains a larger no. of such compounds as alkaloids, phenols,

saponins, glycosides, flavonoids, triterpenoids, sterols and tannins. Heartwood of P.

santalinus is known to possess isoflavone glycosides and triterpene. A new isoflavone

together with liquiritigenin and isoliquiritigenin has been isolated from the heartwood of

P. santalinus (Krishnaveni et al, 2000a; 2000b). Three new components viz

sesquiterpenes- pterocarptriol, isopterocarpolone and pterocarpodiolone together with β-

endesmol, pterocarpol and cryptomeridiol isolated from heartwood; while acetyloleanolic

acid, acetyloleanolic aldehyde and erythrodiol isolated from sapwood.

Recent research on the chemical nature of the red dyes isolated from Pterocarpus

santalinus found that it contains santalins A, B and C. Santalins A and B has some

similarities in structure with hematein. This is probably responsible for their staining

properties (Banerjee and Mukherjee, 1981). The structure of santalin B from wood

lupeol, epilupeol, lupenone, lup- (20)-ene, β-amyrone and sistesterol isolated

from barks and leaves. β-amyrine, stigmasterol, erythrodiol and Betulin also

isolated from bark and leaves. Structure and absolute configuration of

pterocarpol, a new triterpane lupenediol isolated from heartwood found to be

mixture of α-β-and γ isomers absolute configuration of pterocarpol. A small

amount of tannin is contained in red sanders.

The Phytoconstituents of P. santalinus have been employed for the treatment of

various disorders in the Ayurvedic herbal medicine. The fruit extract of P. santalinus is

mainly used as astringent, diaphoretic, in inflammation, headache, skin diseases, and

bilious infections and chronic dysentery (Anonymous, 1969). Anti-inflammatory activity

of savinin and lignan from P. santalinus is known to inhibit tumor necrosis factor-α

production and T-cell proliferation without displaying cytotoxicity (Cho et al, 2001).

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Kameswara (2001) and Manjunatha (2006) also reported a hepatoprotective

activity of aqueous suspension and ethanolic extract of stem bark of P. santalinus. These

investigators found that the ethanol extract of the stem bark of P. santalinus minimized

the toxic effects generated by the CCl4 in the liver. It has been also suggested that the

phytoconstituents like flavonoids, triterpenoids, saponins and alkaloids are known to

possess hepatoprotective activity. Phytoconstituents like flavonoids, triterpenoids are

known to promote wound healing process; mainly due to their astringent and

antimicrobial properties which are responsible for wound healing and increased rate of

epithelialization. (Ausprunk et al, 1974; Spanel-Borowski, 1989; Burton and Palmer,

1989; Dash and Murthy, 2011; Senthil et al, 2011).

Pharmacological study done by Biswas and Maity (2004) evaluated its toxicity as

well as wound-healing potential in animal studies and concluded that the P. santalinus

ointment is safe and effective in treating acute wounds in animal models. Kameswara

Rao et al. (2001) demonstrated that the ethanol extracts of bark extracts have

antihyperglycemic activity. Further experimental studies also evidenced that antioxidant

activity, acid inhibiting potential and the ability to maintain functional integrity; these

properties of P. santalinus plant help in the protectant against ibuprofen induced gastric

ulcers (Narayan, 2005). The recent study carried out by Narayan (2007) demonstrated

that the free radical scavenging capacity of P. santalinus help to prevent mitochondrial

dysfunctions and help in maintenance of lipid bilayer (Narayan, 2007). Anti-

inflammatory activity of savinin and lignan from P. santalinus is also known to inhibit

tumor necrosis factor-α production and T- cell proliferation without displaying

cytotoxocity (Chao et al, 2001). The people in the tribal groups of Western Ghats use

stem bark extract in treating diabetes, fever, snakebite, and jaundice and in wound

healing.

Reasons to select Pterocarpus santalinus for evaluation of its efficacy on

angiogenesis:

P. santalinus has a beneficial effect on wound healing in ethenic use. This

property has been studied on animal wound healing models (Rao et al. 2001, Biswas et al

2004). Because angiogenesis is an essential process in wound healing we hypothesized

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that P. santalinus extracts might contain potent angiogenic compounds. Besides the

above review of plant components and varied studies of the properties shows many

compounds with pro and anti angiogenic properties but its systemic or preliminary

analysis has not been done by using any of the in vitro or in vivo angiogenic model. Thus,

the present project was planned to evaluate the influence of acetone, alcohol and benzene

extracts of P. santalinus on angiogenesis by the chick chorioallantoic membrane (CAM)

assay for angiogenesis.

Selection of Boerrhavia diffusa extracts:

Review of the properties of Boerrhavia diffusa

Boerrhavia diffusa, commonly known as ‘punarnava’ (Family; Nictaginaceae) is

mainly a diffused perennial herbaceous creeping weed having spreading branches. The

plant was named in honor of Hermann Boerhaave, a famous Dutch physician of the 18th

century (Chopra, 1969). It is widely distributed in the tropical and temperate regions of

the world (Heywood, 1978). It is also indigenous to India; found throughout the warmer

parts of the country. It grows well on wetlands and in fields after the rainy season

(Chopra, 1969). The stem is prostrate, woody or succulent, cylindrical, often purplish,

hairy, and thickened at its nodes. The leaves are simple, thick, fleshy, and hairy, arranged

in unequal pairs, green and glabrous above and usually white underneath. The flowers are

minute, subcapitate, present 4-10 together in small bracteolate umbrellas, mainly red or

rose, but the white varieties are also known. The achene fruit is detachable, ovate,

oblong, pubescent, five-ribbed and glandular, anthocarpous and viscid on the ribs

(Thakur et al., 1989). The seeds germinate before the onset of the monsoon. The plant

grows profusely in the rainy season and mature seeds are formed in October-November.

It has a large root system bearing rootlets. The taproot is tuberous, cylindrical to narrowly

fusiform, conical or tapering, light yellow, brown or brownish gray. It is thick, fleshy and

very bitter in taste (Capasso et al, 2000).

B. diffusa is a plant, which has drawn the interest of many researchers in several

countries, either for its active principle or for the extremely important pharmacodynamic

or pharmatherapeutic properties. It exhibits a wide range of medicinal properties as per

Ayurvedic claims. The whole plant of B. diffusa has been employed for the treatment of

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various disorders like lumbar pain, myalgia, skin diseases, urinary infection, vesical

stone, anemia, dyspepsia, constipation, liver disorders, gastrointestinal disorders, and

heart diseases (Kirtikar and Basu, 1956; Chopra et al, 1996;Gaitonde et al, 1974).

In earlier studies of some groups it has shown to have laxative, diuretic,

antiurethritis, anticonvulsant, antifibrinolytic, antinematodal and antibacterial properties

(Chopra et al, 1923; 1956; Gaitonde et al, 1974; Nadkarni, 1976; Adesina, 1979; Jain and

Khanna, 1989; Vijayalakshmi et al, 1979; Olukoya et al, 1993). The plant has also been

screened for anti-inflammatory, antimicrobial, immunosuppressive, hepatoprotective,

antitumorogenic, antileprotic and antiasthmatic activities (Bhalla et al., 1968; Awasthi

and Menzel, 1986; Chakraborti and Handa, 1989; Mishra, 1980; Chandan et al, 1991;

Rawat et al., 1997). The root and leaves of punarnava is used in the form of juice and

decoction to treat anaemia, oedema, internal abscess, calculi, eye diseases, oedema during

pregnancy, haemoptysis, for inducing sleep, fever, rheumatic ailments, difficult labour,

vaginal pain and as rejuvinative (Chopra et al,1956; Adesina, 1979; Jain and Khanna,

1989; Olukoya et al, 1993).

Studies on its different extracts i.e. Hexane, chloroform and ethanol extracts of B.

diffusa had shown to block the activation of DNA binding of nuclear factor-KB and AP-

1, two major transcription factors centrally involved in expression of the IL-2 and IL-2R

gene, that are necessary for T cell activation and proliferation (Pandey et al, 2005;

Mehrotra et al, 2002). B. diffusa extracts were also able to attenuate the proliferation,

migration and differentiation of endothelial cells. Besides, B. diffusa plant showed much

higher inhibition of O2- production (Rachh et al, 2009). An aqueous extract of thinner

roots of B. diffusa at a dose of 2mg/kg exhibited the remarkable protection of various

enzymes such as serum glutanic-oxaloacetic transaminase, serum glutanic-

pyruvictransaminase, and bilirubin in serum against hepatic injury in rats (Rawat et al.,

1997). Due to the diuretic and anti-inflammatory activities, Punarnava is regarded

therapeutically highly efficacious for the treatment of renal inflammatory diseases and

common clinical problems such as nephritic syndrome, oedema, and as cites developing

at the early onset of the liver cirrhosis and chronic peritonitis. The root is used to treat

other renal ailments and cystitis, seminal weakness and blood pressure (Gaitonde et al.,

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1974) and as a diuretic (Singh et al., 1992; Anand, 1995). It is useful in the treatment of

nephritic syndrome disorders (Mudgal, 1975; Cruz, 1995). The flowers and seeds are

used as a contraceptive (Chopra et al., 1956). The extracts of B. diffusa have been shown

to inhibit the growth of many cancer cells.

It was also demonstrated that the drug decreased the albumin urea, increased the

serum protein and lowered serum cholesterol level (Ramabhimaiah et al., 1984). Singh

and Udupa (1972) reported that the dried root powder showed curative efficiency when

administered orally for one month to the children or adults suffering from the helminth

infection. The patients became worm-free within five days of the treatment. The drug,

singly or in combination with other drugs, was found to be efficient in liver disorders,

gastrointestinal disorders, heart diseases (hypertension, angina, cardiac failure, etc.),

respiratory tract infections, leucorrhoea, spermatorrhea, etc.

Further experimental studies also evidenced a beneficial activity of the Punarnava

root for the treatment of the jaundice (Singh and Pandey, 1980; Gopal and Shah, 1985).

The treatment with the watery extract from the root of B. diffusa induced leucocytosis

with predominant neutrophils, associated to the phagocytosis ability and it was

bactericidal to the neutrophils and the macrophages (Mungantiwar et al., 1997). The

recent study carried out by Pari et al. (2004) demonstrated that the leaves of B. diffusa

reduced the levels of glucose in the blood increasing the insulin release from the β cells

of pancreas. The watery extract of B. diffusa was proved to possess protective abilities to

the rodents suffering from the peritonitis induced by Escherichia coli (Hiruma-Lima et

al. 2000). It was evidenced that the leaves and root possessed antifibrinolitic and anti-

inflammatory activities (Hiruma-Lima, 2000). In the recent study, led by Mehrotra et al.

(2002) was reported that the ethanolic extract of B. diffusa showed a significant

immunosuppressive activity on human cells and on murine cells as well. Toxicological

studies conducted on B. diffusa demonstrated the absence of teratogenic and mutagenic

effects (Singh et al., 1991).

The whole plant analysis of B. diffusa is known to contain numerous

phytochemicals constituents that include flavonoids, alkaloids, triterpenoids, steroids,

lipids, lignins, tannins, phlobaphenes, ursolic acid, potassium nitrate, carbohydrates,

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proteins and glycoproteins (Agarwal and Dutt, 1936; Basu et al, 1947; Surange and

Pendse, 1972; Mishra and Tewari, 1971). Many rotenoids have been isolated from the

roots of the B. diffusa (Ahmed M. et al, 1990; Lami N. et al, 1991). Plant also includes a

series of boeravinone C, boeravinone D, boeravinone E and boeravinone F.

Punarnavoside, a phenolic glycoside, is reportedly present in roots (Jain and Khanna,

1989). C-methyl flavone also has been isolated from B. diffusa roots (Awasti et al,,

1985). Two known lignans viz., liriodendrin and syringaresinol mono-β-D-glycoside

have been isolated (Aftab et al., 1996, Lami N. et al., 1990). Presence of a purine

nucleoside hypoxanthine 9-L-arabinose (Ojewole JAO et al, 1985),

dihydroisofuroxanthone- borhavine (Ahmed and Yu, 1992), phytosterols have been

isolated from the plant (Kadota et al, 1987; 1989). It contains about 0.04 % of alkaloids

known as punarnavine and punernavoside, an antifibrinolytic agent. It also contains about

6 % of potassium nitrate, an oily substance, and ursolic acid. (Mishra and Tiwari, 1971),

punarnavoside (Jain and Khanna, 1989; Kokate et al, 2005).

The seeds of this plant

contain fatty acids and allantoin and the roots contain alkaloids (Aslam, 1996).

The stalk

of the plant has also been reported to contain Punarnavine (C17H22N2O mp 236–237°C)

(Agarwal and Dutt, 1936; Basu et al., 1947; Surange and Pendse, 1972), boerhavin,

boeravinone and boerhaavic acid (C10H81NO3 mp 108–109°C) (Kadota et al., 1989;

Lami N. et al, 1990).

Eupalitin-3-O-β-D-galactopyranoside isolated and purified from ethanolic leaf

extract (Pandey et al, 2005). Chopra et al. (1923) reported that the plant contained large

quantities of potassium nitrate, besides punarnavine. The herb and roots are rich in

proteins and fats. The herb contains 15 amino acids, including 6 essential amino acids,

while the root contains 14 amino acids, including 7 essential amino acids. Seth et al.

(1986) isolated a new antifibrinolytic compound ‘punarnavoside’ from the roots of B.

diffusa. Photochemical screening of the roots from garden-grown in vivo plants of B.

diffusa of different ages revealed that the maximum alkaloid content (2%) accumulated in

the roots of 3-year old mature plants. Analysis of the ash showed that it contained

potassium, magnesium, sodium, calcium, nitrate, phosphates, silica, and sulphates.

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Reasons to select Boerrhavia diffusa for evaluation of its efficacy on angiogenesis:

The drug, singly or in combination with other drugs, was found to be efficient in

liver disorders, gastrointestinal disorders, heart diseases (hypertension, angina, cardiac

failure etc.), respiratory tract infections, leucorrhoea, spermatorrhea, etc. (Sigh and

Udupa, 1972). Thus though plant has been screened for bioactivities against various

diseases; it has not been studied preliminarily or systematically against cardiovascular

diseases or related properties in vitro or in vivo animal models. Its angiogenic potential

remains to be studied. Our present studies as a part of our search for natural product-

based pro or antiangiogenic agents, included the influence of acetone, alcohol and

benzene extracts of B. diffusa on in vivo angiogenesis model of chicken chorioallantoic

membrane (CAM) in vivo. A systemic approach to detailed evaluations of quantitative

and histological analysis of the alterations in angiogenesis influenced by the extracts had

been studied in present project.

As it was decided to study the efficacy of plant extracts on angiogenesis for the

bioactivities in animal models for the claims of pro and anti angiogenic properties. The

available models were reviewed for their suitability with the project aims and objectives.

Following review includes the details.

Review of Models of angiogenesis study:

Many models to study angiogenesis have been developed so far. Some of the

models are as follows.

In vitro models:

In vitro models are based on the origin and passage number of endothelial cells,

the nature of the substrates (extracellular matrices), the angiogenic agents, and the levels

of endotoxins. These models used the cultures of the endothelial cells (EC) or fibroblasts

either derived from the walls of small capillaries or larger vessels. They also make use of

the cells derived from placenta or human umbilical veins. Considerable insight in the

molecular and cellular biology of angiogenesis has been obtained by in vitro studies

using endothelial cells, isolated from either capillaries or large vessels (Cockerill et al,

1995, Fan, 1997, Jain, 1997.). Most steps in the angiogenic cascade can be analyzed in

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vitro, including endothelial cell proliferation, migration and differentiation (Montesano,

1992).

a. Two dimensional model:

Two-dimensional models refer to those in which the planar organization of the

cells lies parallel to the surface of the culture plate. Reports state that CLS

formation could be observed spontaneously in long-term planar cultures (Feder et

al, 1983; Folkman and Haudenschild, 1980).

b. Three-Dimensional Models

Three-dimensional angiogenesis assays are based on the capacity of activated

endothelial cells to invade three-dimensional substrates. The matrix may consist

of collagen gels, plasma clot, purified fibrin, Matrigel, or a mixture of these

proteins with others. The culture medium may be added within the gel before

polymerization or on top of the gel.

In Vivo models:

Classical in vivo models of angiogenesis include the chick chorioallantoic

membrane, rabbit cornea assay, sponge implant models, matrigel plugs and conventional

tumor models (Cockerill, 1995; Fan, 1997; Jain, 1997; Ribatti and Vacca, 1999).

1. The rabbit cornea presents an in vivo avascular site. Therefore, any vessels

penetrating from the limbus into the corneal stroma can be identified as newly

formed. To induce an angiogenic response, slow release polymer pellets

containing an angiogenic substance (i.e. FGF-2 of VEGF) are implanted in

"pockets" created in the corneal stroma of a rabbit. This method is very reliable,

but technically more demanding and more expensive than the CAM assay, which

makes it not a practical screening assay.

2. Subcutaneous implantation of various artificial sponges (i.e. polyvinyl alcohol,

gelatin) in animals has been used frequently to study angiogenesis in vivo.

Compounds to be evaluated are either injected directly into the sponges or

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incorporated into ELVAX or hydron pellets, which are placed in the center of the

sponge. Neovascularization of the sponges is assessed either histologically,

morphometrically (vascular density), biochemically (hemoglobin content) or by

measuring the blood flow rate in the vasculature of the sponge using a radioactive

tracer (Hu et al., 1995). The differences in sponge materials, shape and size make

direct data comparison difficult. Moreover, implantation of these materials is

associated with non-specific immune responses, which may cause a significant

angiogenic response even in the absence of exogenous growth factors in the

sponge.

3. Matrigel is a matrix of a mouse basement membrane neoplasm known as

Engelbreth-Holm-Swarm murine sarcoma. It is a complex mixture of basement

membrane proteins including laminin, collagen type IV, heparan sulfate, fibrin

and growth factors, including EGF, TGF-b, PDGF and IGF-1. It was originally

developed to study endothelial cell differentiation in vitro. However, matrigel-

containing FGF-2 can be injected subcutaneously in mice. (Passaniti, et al., 1992)

Matrigel is liquid at 4°C but forms a solid gel at 37°C that traps the growth factor

to allow its slow release. After 10 days, the matrigel plugs are removed and

angiogenesis is quantified histologically or morphometrically in plug sections.

Matrigel is expensive but, unlike artificial sponges, it provides a more natural

environment to initiate an angiogenic response.

4. Numerous animal tumor models have been developed to test the anti-angiogenic

and anti-cancer activity of potential drugs. In many cases, tumor cells are

engrafted subcutaneously and tumor size is determined at regular time intervals.

5. Chick embryo chorioallantoic membrane is the most popular model to study

angiogenesis. Thus chorioallantoic membrane (CAM) assay is well established

and widely as a model to examine angiogenesis and anti-angiogensis effects

polylysine/ heparin stimulates angiogenesis in CAM (Pacini et al 2002). Similarly

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thymosin peptides are shown to promote angiogenesis (Koutrafouri et al, 2001).

Cigarette smoke has shown to inhibit growth and angiogenesis in the day 5th of

CMA (Melkonian et al 2002a). Protein C in activated form stimulates the

expression of angiogenic factors in human skin cells (Xue et al 2006). Effect of

resveratrol and platelet / fibrin acceleration of angiogenesis has been tested in

CAM (Mousa et al, 2005).

It is well verified model of angiogenesis. As the chicken embryo, develops

outside the mother, effects of external stresses on cardiovascular development can

be studied without interferences of maternal hormonal, metabolic, or

hemodynamic alterations. The most common causes of prenatal stress, namely

malnutrition and chronic hypoxia (as seen in placental insufficiency), can be

studied independently (Kempf et al, 1998; Ruijtenbeek et al, 2000; Xu and

Mortola 1989), and pharmacological or toxic substances are easily applicable

via

injections of compounds into the air cell (Carlo et al 2001). It is least costly,

easier to use and of limited ethical concerns than other in vivo models.

Ex-vivo model:

The rat aorta ring assay has gained a reputation of a “quasi in-vitro”

angiogenesis model because it mimics several aspects of “in vivo” angiogenesis animal

experiments, the latter being more expensive, requiring technical expertise and often

providing less reproducible data. In this system, aortic rings cultured in collagen gel give

rise to microvascular networks composed of branching endothelial channels. This

organotypic model can be used to study the angiogenic bioactivity of a large array of test

compounds, and provides important preliminary data about the angiogenesis mechanism

of the test factors. This methodology allows the study of angiogenic properties of

candidate molecules as an additional approach to utilize in conjunction or even replace

neomicrovasculature immunodetection, “in-vivo” and “in-vitro” models. It is not so

popular model.

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Selection of in vivo chick chorioallantoic membrane (CAM) assay:

In vivo animal model system used to study complex physiologic processes such as

angiogenesis or metastasis usually require weeks to months for occurrence of the end

point. This time constraint often limits the identification and characterization of

molecules that function in these processes. Numerous in vitro cell culture models attempt

to recapitulate distinct events in angiogenesis such as proliferation and migration of

endothelial cells and tube formation. However, few quantitative in vivo assays allow

analysis of and intervention in the whole process of angiogenesis. Classical assays for

studying angiogenesis in vivo include the hamster cheek pouch assay, the rabbit cornea

assay, matrigel plugs and conventional tumor models (Ribatti and Vacca, 1999). Several

new models have been recently introduced including subcutaneous implantation of

various three dimensional substrates including polyester sponge (Andrade et al, 1987),

polyvinyl-alcohol foam disk covered on both sides with a Millipore filter (Fajardo et al,

1988), and matrigel, a basement membrane rich extra cellular matrix (Passaniti et al,

1992).

As compared to these models CAM model in vivo was found suitable for the

project planned for following reasons.

1. It is easy to handle, fertilized eggs are available to work with.

2. In vivo it provides natural sterile environment for development.

3. The Morphogenesis has been studied in detail.

4. Organogenesis has been studied in detail. Points 2&3 had provided the suitability for

experimental designing.

5. It was possible to arrange drug schedule at the intervals when primary, secondary and

tertiary blood vessels originate.

6. Thus the experiments were designed so that both vasculogenesis, which is base of

angiogenesis, can be studied to get the differential action of extracts.

7. Teratogenic toxicity of extracts was also revealed.

Therefore it is popular model and is extensively used for angiogenic analysis and

other studies also.

Following review provides detail characters of CAM assay.

16

Chick embryo chorioallantoic membrane (CAM) assay:

Chick embryo chorioallantoic membrane (CAM) assay (Folkman and Shing, 1992;

Ribatti et al, 1996). Because of its suitability to assess angiogenesis, the development of

CAM and the factors that regulate angiogenesis in CAMs have been extensively studied

(Ribatti and Vacca, 1999).

Chicken eggs in the early phase of breeding are between in vitro and in vivo

system but may provide an immunodeficient, vascularized test environment (Kunz-Rappi

et al, 2001). The chorioallantoic membrane (CAM) of the chicken embryo is one of the

most important extra embryonic membranes, which serves as a gas exchange surface

(Romanoff, 1960). Its respiratory function is provided through an extensive capillary

network. (Billet et al, 1965). It is a highly vascularized membrane which lines inside the

surface of egg shell and is relatively thin and transparent. It facilitates oxygen, calcium

and nutrient transport to the embryo (Richardson and Singh, 2003; Tuan, 1987). The

chorioallantoic membrane (CAM) is formed on the fourth day of incubation by the fusion

of the mesodermal layer of the allantoic membrane with the mesodermal layer of chorion

(De fouw et al, 1989). At this stage, undifferentiated blood vessels are scattered in the

mesoderm of the CAM. They grow very rapidly until day 8; when some vessels

differentiate into capillaries and form a layer at the base of the ectoderm. After 10 days of

incubation it completely surrounds the embryo (Gilbert, 2003). At day 14, 6 days before

hatching, the capillary plexus is located at the surface of the ectoderm, adjacent to the

shell membrane (Ausprunk et al, 1974; 1977). The respiratory exchange in thee CAM

occurs by means of an extensive capillary plexus that develops initially adjacent to the

chorionic ectoderm and later interdigitates the ectodermal cells of the chorion (Ausprunk

et al, 1974; Burton and Palmer, 1989). As the capillary plexus develops, the spaces

within the plexus become subdivided. Initially and up to day 7, the major mechanism for

this subdivision is by spurting of pre-existing vessels; however after day 7,

intussusceptive angiogenesis becomes an important factor in subdividing the plexus

(Schlatter et al, 1997). During intussusceptive growth transcapillary pillars form and

partition the plexus (Patan et al, 1993; 1997; Djonov et al 2000). The CAM is attached

to the internal system of the shell membrane and provides a barrier between the watery

17

environment of the embryo and the air space. It is placenta like tissue consisting of three

distinct cellular layers: an ectodermal layer facing the egg cell, a sparsely populated

mesodermal layer, and an endodermal layer lining the allantois (Leeson and Leeson,

1963; Narbaitz, 1977; Packard and Packard, 1984). The structure allows the embryo to

harvest the calcium from the shell for bone development. It has also shown that vascular

CAM transports essential nutrients and gases to the graft, thereby facilitating

differentiation and cartilage formation in the limb. The CAM includes the chorioallantoic

fluid into which waste products are delivered. Its two-dimensional vascular structure can

be seen entirely with minimal preparation. This is one of the major reasons it has become

a popular assay tissue for putative angiogenic and antiangiogenic substances (Weiss et al,

1999). The CAM is a useful tool to studying angiogenesis because 1) it is a menable both

intravascular and topical administration of study agents.2) it is a relatively rapid assay

and 3) it can be adapted very easily to study angiogenesis dependent processes such as

tumor growth.

The chorioallantoic membrane (CAM) has been used as a reliable biomedical

assay system for many years. Exploitation of this assay enables a substantial reduction in

or substitution for subsequent animal experiments (Kunzi et al, 2001). It has been utilized

as an in vivo system to study angiogenesis, anti-angiogenesis and teratogenic effects of

individual compounds or complex plant extracts. The method is used for testing natural

compounds in small amounts for revealing various modes of action and the complex

mechanisms related to angiogenesis. A modified CAM assay allows for detection of

endothelial apoptosis induced by antiangiogenic substances (Gonzalez et al, 2003).

Angiogenesis involves coordinated signals to the adhesion, migration and survival

machinery within the target endothelial cells. Agents that interfere with any of these

processes may interfere and influence angiogenesis.

Many investigators have studied the histological and morphological changes

associated with the proliferation of new vessels and tumor neovascularization by direct

observation using CAM. (Folkman and Ingber, 1987; Melkonian et al, 2002b; Mostufa L.

et al., 1980; Pertruzzeli et al, 1993; Quigley and Armstrong, 1998; Folkman et al, 1983;

Illanens et al, 1999; Jacques et al, 1999; Lei et al, 2003; David et al, 2001).

18

However, the methods to quantify the extent of the angiogenesis or vascularization

in the chorioallantoic membrane are somewhat troublesome. They have been many

efforts developed at quantifying the number for example; one method is morphometric

measurement of microvessels of the chorioallantoic membrane by counting the number of

“vessel endpoints” with or without the assistance of a computerized image analysis

system (Neufeld et al, 1999). Later, a fractal analysis was applied on the method of

morphometric measurement, which was reported to be more accurate, reproducible, and

objective since the morphometric form of the vessel in the chorioallantoic membrane

poses hierarchical branching patterns (Kirchner et al, 1996).

Limitations of CAM assay:

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. The limitation of CAM assay is

represented by non-specific inflammatory reactions, which may develop as a result of

grafting and in turn induce a secondary vasoproliferative response eventually making it

difficult to quantify the primary response. In this connection, a study of histological

CAM section can help detecting the presence of a perivascular inflammatory infiltrate

together with a hyperplastic reaction, if any, of the chorionic epithelium (Ribatti et al,

1995). Another drawback is that polymers often do not adhere to the CAM surface.

Folkman has suggested hydrating the test substance with 5µl H2O on a sterile overslide

glass, which is then turned over and placed on the CAM. Saline solutions cannot be

employed because the hyperosmotic effect of crystal salts damages the chorion

epithelium and induces fibroblast proliferation. The substance must thus be used at

concentrations of pictograms to micrograms, as higher concentrations would cause this

hyperosmotic effect.

Many angiogenic factors have been investigated by using the CAM assay, they

include bFGF and VEGF. The results show that bFGF stimulates angiogenesis associated

with hyperplasia of chorion and fibroblast cell proliferation and its antibodies inhibit such

effects (Ribatti et al, 1997). In general, VEGF has the same effects as that of bFGF. In

addition, VEGF transfectants were found to be able to induce neovasculature with open

19

junctions and a fenestrated endothelium (Ribatti et al, 2001). This means that VEGF may

have a strong angiogenic potential in vivo in this model.

On the understanding of CAM assay and development specificities the design of

experiments i.e. drug treatment schedule was decided to get project’s aims and

objectives.

Following are some of the points explained.

Selection of developmental stages:

Selected hours to study of angiogenesis were 48, 55, 66, 72, 88 and 96 hrs. The

hours are according to development of CAM and vitelline veins of CAM.

a) Development 48 hrs:

At the end of second day area vasculosa is surrounded by sinus terminalis.

Meanwhile certain veins and arteries have extended from embryo in to the area

vasculosa. From the posterior end of the ductus venosus union of vessels passes outward

in to the area pellucida called as omphalomesentric veins. This vein in area vasculosa

gives extensions called right and left vitelline veins. Thus main right and left veins

originate at this stage.

b) Development at 55 hrs:

No allantois is formed and transitory vein develop toward the intestine.

c) Development 66 hrs:

Before the end of the third day one other new extra embryonic vessel starts to

appear the posterior vitelline vein.

d) Development at 72 hrs:

The vitelline arteries reach further out into the area vasculosa than during the

second day terminating near its border in network of capillaries, which empty into sinus

terminalis.

20

e) Development at 88 hrs:

It is a stage of rapid capillary development.

f) Development at 96 hrs:

By the end of the fourth day vitelline veins, such as anterior, posterior and lateral

vitelline veins are well developed and are more defined.

When treatments are applied, changes in the blood vessel formation can be

attributed to experimental procedures, hence it is possible to test a specific

molecule/herbal extract for its angiogenic or antiangiogenic properties by determining if

its presence causes distortion of and/or increase in the number of blood vessels, or it

causes oriented blood vessel growth in comparison with normal and control embryos.

Since effects can be observed on hatching, abnormalities can be studied.

Reasons to study CAM morphology, vasculogenesis along with angiogenesis:

Angiogenesis and vasculogenesis are accompanied with the development and

extension of CAM. CAM area influences healthy development of embryo. Extension of all

the types of blood vessels and capillaries occurs in the bed of CAM and its development

influences both vasculogenesis and angiogenesis influencing the length of vessels.

Vasculogenesis results in the formation of the major embryonic vessels, the dorsal aorta

and of the primary vascular plexus in the yolk sac. Adult blood vessels arise primarily

through angiogenesis; however, recent studies now support the contention that

vasculogenesis also contributes to the development of mature vascular networks (Asahara

et al, 1999). Angiogenesis is influenced by vasculogenesis. Some of the factors that

regulate the angiogenesis also regulate the angiogenesis, which are reviewed further. If

vasculogenesis is inhibited angiogenesis will also be inhibited. In chick CAM model it is

possible to distinguish these two inhibitory properties of bioactive compounds. Therefore

vasculogenesis study is also included in the work.

21

At the beginning of the experimental approach to evaluate the alteration process of

angiogenesis in chick must be known in details. Following is the review on angiogenesis

its regulation and related characters.

Review on Angiogenesis:

Need of Circulation and Networking:

Survival of the cell depends on a continuous supply of oxygen and nutrients

carried by the blood. Therefore every cell in the body must be sufficiently close to a

blood capillary to allow for efficient nutrient diffusion. A tissue cannot grow beyond

∼1mm in diameter before requiring new blood vessels to invade and nourish it (Rubanyi,

2000). An effective network of capillaries and larger blood vessels that nourish the cells

must surround them. For that the formation of blood vessels is required. The formation

and remodeling of the vascular system can be divided into two separate processes:

vasculogenesis and angiogenesis. These are the mechanisms of vascular network

formation, growth and remodeling in developing embryo (Patan, 2000). It is vital for

survival, growth and homeostasis in the vertebrate embryo.

To understand angiogenesis and its regulation; following review is presented.

Development of cardiovascular system:

The cardiovascular system is the first organ system formed during early embryonic

development in all vertebrates (Nguyen and D’Amore, 2001). It is composed of arteries,

resistance vessels, capillaries, venules and veins. Capillaries and post capillary venules are

tubes formed of a single layer of overlapping endothelial cells, which is surrounded

externally by the basal lamina, a 50-100 nm thick layer of fibrous proteins including

collagen and glycoproteins. Pericyte; isolated cells which can give rise to smooth muscle

cells during angiogenesis, adhere to the outside of the basal lamina, especially, in

postcapillary venules. The endothelial cells play a crucial role in controlling vascular

permeability, vasoconstriction, angiogenesis (growth of new blood vessels) and regulation

of coagulation.

22

Cells constructing blood vessels.

1.Endothelial cells and hematopoietic cells:

Endothelial cells (ECs) are the fundamental component of blood vessels. They are

not just the structural components of vessel walls but also take part in the regulation of

angiogenesis by secreting proangiogenic factors and proteases (Tiziana et al, 2003). The

first ECs that form in the gastrulating embryo originate from lateral and posterior

mesoderm (Murray, 1932). During gastrulation, EC differentiation occurs that leads to

formation of the mesoderm. During the primitive streak stage, groups of mesodermal cells

aggregate in the developing yolk sac, where they differentiate to EC and hematopoietic

cells (HC) of the extraembryonic blood islands. During their migration, the precursors

aggregate to clusters; termed hemangioblastic aggregates (Sabin, 1920). Cells at the

periphery of these aggregates differentiate into angioblast, the precursor of the blood

vessels, while cells in the interior become hematopoietic stem cells (HSCs), the precursor

of all the blood cells.

Endothelial cells (ECs) can initiate the angiogenic process; however,

periendothelial cell involvement is needed for vascular maturation.

2. Mural cells:

Mural cells are two types. (i) Smooth muscle cells: which is largely distributed

surrounding at arteries and veins. (ii) Pericytes: which are largely distributed surrounding

at capillaries and microvessels.

The mural cells strengthen immature vessel lumens by restraining endothelial

proliferation and migration. They provide growth factors such as VEGF for repair and

maintenance of the underlying endothelial cells (Tsurumi et al, 1997). These cells also

help stimulate production of the extracellular matrix (Carmeliet, 2000). Capillaries that

covered with pericytes survived exposure to hypoxia, in contrast to endothelial cells

without pericyte coverage (Benjamin et al, 1998). Disruption of endothelial- pericyte

associations resulted in endothelial cell death, excessive regression of vessels and

abnormal remodeling (Benjamin et al, 1998).

23

3. Extracellular Matrix (ECM):

The ECM provides contacts between endothelial cells and the surrounding tissues

and prevents vessels from collapsing. During angiogenesis, the basement membrane and

ECM are broken down proteolytically and the composition of the latter is altered

(remodeling). The remodeling of ECM is not just remove ECM; it also provides a

promigratory environment capable of supporting cell migration and survival. However,

the degree of remodeling of ECM must be tightly regulated. Insufficiency or excessive

breakdown of ECM does not favour angiogenesis (Bajou et al, 1998; Luttun et al, 2002).

Many ECM molecules, including laminin and fibronectin, promote endothelial cell

survival, growth, migration and tube formation. There are many proteolytic fragments of

ECM molecules being identified as antiangiogenic factors, for example, angiostatin

(derived from plasminogen), endostatin (from collagen XVIII) and tumstatin (from

collagen IV). Therefore, the ECM serves as a reservoir of angiogenic activators and

inhibitors.

4. Basement membrane:

Basement membrane is a thin sheet of ECM (50-100nm), which underlies the

endothelium of the blood vessel wall (Vracko and Strandness, 1967). Endothelial and

epithelial cells rest on basement membrane. The major components of basement

membrane include laminin, collagen IV, perlecan (a heparan- sulphate proteoglycan),

nidogen/ entactin and various growth factors and proteases (Paulsson, 1992). Dissolution

of basement membrane leads to a number of events. It liberates the endothelial cells to

migrate and proliferate, releases the sequestered growth factors, cytokines and proteases

(Vlodavsky et al, 1991) and exposes the cryptic domains that regulate angiogenesis and

lastly, leads to the detachment of the mural cells.

24

Formation of blood vessels:

Several distinct processes can contribute to new vessel formation in an organism

including vasculogenesis (formation of new vasculature from circulating vascular

precursor cells), angiogenesis (formation of new capillaries from pre-existing vessels) and

arteriogenesis (Formation of muscular arteries either de novo or from per-existing

collaterals). All of these processes are strictly controlled, both spatially and temporally,

under normal physiologic conditions.

Vasculogenesis:

Vasculogenesis refers to the differentiation of precursor cells (angioblasts) into

endothelial cells (ECs) and the de novo formation of a primitive vascular network is the

fundamental process by which blood vessels are formed (Drake, 2003). Successful

vasculogenesis is essential for normal embryo development and viability (Flamme et al,

1997). During vasculogenesis, blood vessels are created de novo from the lateral plate

mesoderm. In the first phase of vasculogenesis groups of splanchnic mesoderm cells are

specified to become hemangioblasts, the precursors of both the blood cells and the blood

vessels. In the second phase, the angioblasts multiply and differentiate into endothelial

cells, which form the lining of the blood vessels. In the third phase, the endothelial cells

form tubes and connect to form the primary capillary plexus, a network of capillaries

(Gilbert, 2003).

The yolk sac hematopoietic precursors mostly differentiate into primitive

erythrocytes, which are replaced, as development proceeds, by definitive hematopoietic

precursors generated in the embryo proper (Cumano et al., 2001, Dieterlen- Livre, 1975).

These definitive precursors are again observed to develop in close association with the

endothelium of the dorsal aorta (Jaffredo et al., 1998; Pardanaud et al, 1996).

Vasculogenesis results in the formation of the major embryonic vessels, the dorsal

aorta and of the primary vascular plexus in the yolk sac. Adult blood vessels arise

primarily through angiogenesis; however, recent studies now support the contention that

vasculogenesis also contributes to the development of mature vascular networks (Shi Q. et

al, 1998; Asahara et al, 1999).

25

Three growth factors may be responsible for initiating vasculogenesis. One of

these, basic fibroblast growth factor (bFGF) is required for the generation of

hemangioblasts from the splanchnic mesoderm. Vascular endothelial growth factor

(VEGF) is another protein appears to enable the differentiation of the angioblasts and the

multiplication to form endothelial tubes. The mesenchymal cells near the blood islands

secrete VEGF, and the hemangioblasts and angioblasts have receptors for VEGF. If mouse

embryos lack the genes encoding either VEGF or VEGFR-2 (Flk-1), yolk sac blood

islands fail to appear, and vasculogenesis fails to take place. Mice lacking genes for

VEGFR-1 have differentiated endothelial cells and blood islands, but these cells are not

organized into blood vessels (Carmeliet et al, 1996). Also, VEGF121 and VEGF165

regulate blood vessel diameter (Nakatsu et al, 2003). A third protein Angiopoietin-1 (Ang-

1) mediates the interaction between the endothelial cells and the pericytes- smooth muscle

like cells they recruit to cover them.

Angiogenesis

Angiogenesis is defined as the formation of new blood vessels from pre-existing

vascular network (Folkman and Shing, 1992). It is a complex multi-step process;

controlled by the balance between proangiogenic and angiogenic molecules (Folkman

and Kiagsburn, 1987; Poole and Ooffin, 1989; Risau and Lemmon, 1988, Yancopoulos et

al, 1998). The term, angiogenesis was first introduced by a British surgeon, Dr. John

Hunter, to describe blood vessels growing in reindeer antler in 1787.

a) The importance of angiogenesis

Numerous studies have demonstrated that angiogenesis is a fundamental step in

variety of pathophysiological conditions. It is a normal process in embryonic

development, wound healing, tissue remodeling, and regeneration. It also occurs in

female reproductive cycles, ovulation, placentation, menstruation and the corpus luteum

formation. Additionally, angiogenesis occurs in some circumstances such as tumor

progression and metastasis, diabetic retinopathy, rheumatoid arthritis, ischemic diseases,

macular degeneration, psoriasis, chronic inflammation including atherosclerosis,

(Folkman and Shing, 1992; Folkman 1995; Carmeliet 2003; Tonnesen et al, 2000; Arjan

26

and Grietje, 2000). Endometrial angiogenesis plays a role in endometrial remodelling

during the menstrual cycle and after conception during the implantation of the embryo

(Bacharach et al., 1992; Rogers and Gargett 1998; Smith 1998).

It is widely accepted that neovascularization is an absolute requirement for tumor

growth and vascular vessels are quite closely associated with tumor cell activation

(Ninomiya et al, 1991). Tumor cells as well as normal growing cells produce and secrete

several growth factors and signaling molecules involved in the control of angiogenesis

(Folkman and Kiagsburn, 1987). It is also a fundamental step in the transition of tumors

from a dormant state to a malignant state.

Many diseases are associated with imbalances in regulation of angiogenesis, in

which it is caused by either excessive or insufficient blood vessel formation (Ferrara et al,

2003; Carmeliet, 2003). Cancer, rheumatoid arthritis, psoriasis, and diabetic retinopathy

are the best known diseases caused by excessive or abnormal angiogenesis. On the other

hand, insufficient vessel growth or abnormal vessel regression causes stroke, Alzheimer

disease, amyotrophic lateral sclerosis, hypertension, osteoporosis, respiratory distress and

other disorders.

b) The process of angiogenesis

Angiogenesis occurs by the restructuring of existing vessels e.g. by splitting,

invagination or out pouching. In areas of the vascular bed, which were irreversibly

damaged or excised, the restoration of circulation is accomplished by an in growth of

new capillaries to reestablish a new vascular network (Schoefl, 1964). To make this

possible, endothelial cell (EC) in the vessels must separate from their original tissue and

migrate to the place where the new vessel is to be formed. The initial establishment of

blood vessels seems to be genetically predominated and in addition, epigenetic factors

such as metabolic, mechanical or hemodynamic play a significant role in the vessel

formation (Hudlioka, 1991).

The process of angiogenesis depends upon complex interactions among various

classes of molecules, including adhesion molecules, proteases, structural proteins, cell

surface receptors and angiogenic growth factors, whose molecular effectors must be

27

precisely regulated. In mature (non-growing) capillaries, the vessel wall is composed of

endothelial cells (EC), a basement membrane and a layer of cells called pericytes which

partially surround the epithelium, angiogenic factors bind to endothelial cell receptors

and initiate the sequence of angiogenesis. When the ECs are stimulated to grow, they

secrete proteases, which digest the basement membrane surrounding the vessel. The

junctions between ECs are altered; cell projections pass through the space created and the

newly formed sprout grows towards the source of the stimulus.

The process of angiogenesis occurs as an orderly series of events (Carmeliet,

2005). These steps include:

1. Diseased or injured tissues produce and release angiogenic growth factors

(proteins) that diffuse into the nearby tissues

2. The angiogenic growth factors bind to specific receptors located on the

endothelial cells (EC) of nearby preexisting blood vessels

3. Once growth factors bind to their receptors, the endothelial cells become

activated. Signals are sent from the cell's surface to the nucleus. The endothelial

cells’ machinery begins to produce new molecules including enzymes

4. Enzymes dissolve tiny holes in the sheath-like covering (basement membrane)

surrounding all existing blood vessels

5. The endothelial cells begin to divide (proliferate), and they migrate out through

the dissolved holes of the existing vessel towards the diseased tissue (tumor)

6. Specialized molecules called adhesion molecules, or integrins (avb3, avb5) serve

as grappling hooks to help pull the sprouting new blood vessel sprout forward

7. Additional enzymes (matrix metalloproteinases, or MMP) are produced to

dissolve the tissue in front of the sprouting vessel tip in order to accommodate it.

As the vessel extends, the tissue is remolded around the vessel

8. Sprouting endothelial cells roll up to form a blood vessel tube

9. Individual blood vessel tubes connect to form blood vessel loops that can circulate

blood

28

10. Finally, newly formed blood vessel tubes are stabilized by specialized muscle

cells (smooth muscle cells, pericytes) that provide structural support. Blood flow

then begins.

Fig1. Angiogenesis. (http://www.reishiscience.com).

c) Types of angiogenesis:

Depending on mechanism and application, angiogenesis are of four types.

1. In sprouting angiogenesis, biological signals activate receptors in endothelial cells

(ECs) present in pre-existing venular blood vessels. The activated ECs begin to release

proteases that degrade the basement membrane in order to allow ECs to escape from the

parent vessel walls. The EC then proliferate into the surrounding matrix and form solid

sprouts toward the source of the angiogenic stimulus. This sprouts then form loop to

become a full-fledged vessel lumen as cells migrate to the site of angiogenesis. It is

markedly different from splitting angiogenesis, however, because it forms entirely new

vessels as opposed to splitting existing vessel (Burri, 2004).

2. Intussusceptive angiogenesis (also called splitting angiogenesis) was first observed in

neonatal rats in which, the capillary wall extends into the lumen to split a single vessel in

two. Intussusception is important because is a reorganization of existing cells. It allows a

vast increase in the number of ECs. This is especially important in embryonic

29

development, as there are vast enough resources to create a rich microvasculature with

new cells every time a new vessel develops.

3. Therapeutic angiogenesis is the application of specific compounds, which may inhibit

or induce the creation of new blood vessels in the body in order to combat disease. The

presence of blood vessels where there should be none may affect the mechanical

properties of a tissue, increasing the likelihood of failure. The absence of blood vessels in

a repairing or otherwise metabolically active tissue may retard repair or some other

function. Several diseases (e.g. ischemic chronic wounds) are the result of failure or

insufficient blood vessel formation and may be treated by a local expansion of blood

vessels, thus bringing new nutrients to the site, facilitating repair. Other diseases, such as

age related macular degeneration might be created by a local expansion of blood vessels,

interfering with normal physiological processes.

4. The fourth one is mechanical angiogenesis. Mechanical stimulation is not well

characterized. There is a significant amount of controversy with regard to shear stress

acting on capillaries to cause angiogenesis; although current knowledge suggests that

increased muscle contraction may increase angiogenesis (Prior et al, 2004). This may be

due to an increase in the production of nitric oxide during exercise.

Arteriogenesis:

Besides vasculogenesis and angiogenesis a third mechanism of vessel formation

i.e. arteriogenesis might operate in the adult, which is responsible for the development of

angiographically visible collaterals in patients with advanced obstructive atherosclerotic

disease. This event is usually referred to the remodeling of pre-existing arteriole to form

major arteries. Once smooth muscle cells are mobilized to the sites of active

collateralization, they inundate the vessel and provide contractility for the developing

vasculature. It is now believed that signals regulating mural cell involvement in vascular

myogenesis are also implicated in arteriogenesis. Some of the stimuli that trigger this

process have been defined, for instance, shear stress and endothelial activation with

monocyte recruitment.

30

Mural cells develop specialized characteristics that provide or allow for the

maintenance of vascular tone. These contractile proteins and interstitial matrix

components include the intermediate filament desmin, MEF2C, elastin, fibrillin-2,

collagen and fibrillin-1. During pathological conditions involving inflammation. These

muscle cells may “de-differentiate” from a “contractile” to a “synthetic” phenotype (Li et

al, 1998; Carmeliet, 2000).

During arteriogenesis, vessels become inundated with pericytes and smooth

muscle cells, thus providing blood vessels with vasomotor tone that is essential for

adequate tissue perfusion (Risau, 1997).

Angiogenic regulators:

As described, angiogenesis is composed of a complex series of interdependent

events, controlled by a number of regulatory molecules called angiogenesis factors

(Iruela-Arispe and Dvorak, 1997). The angiogenic process results from a shift in the

balance of pro-angiogenic and antiangiogenic factors (Bergers and Benjamin 2003;

Hanahan and Folkman 1996). Cytokines and growth factors are the primary inducers.

The control of angiogenesis has been found to be altered in certain disease state

and in many cases the pathological damage associated with the disease is related to the

controlled angiogenesis. Knowledge of molecular mediators of angiogenesis is

fundamental in understanding the mechanisms that control its pathways and may

ultimately be useful in developing therapies for angiogenesis related diseases. Modulators

of angiogenesis are secreted by endothelial cells, tumor cells and by the surrounding

stroma.

31

Table.1. Angiogenesis stimulators and Inhibitors:

Angiogenesis Stimulators Angiogenesis inhibitors

Vascular endothelial growth factor (VEGF)/

vascular permeability factor (VPF)

Fibroblast growth factors: acidic (aFGF) and basic

(bFGF)

Placental growth factor (PIGF)

Angiopoietin-1 (Ang-1)

Platelet-derived growth factor-BB (PDGF-BB)

Hepatocyte growth factor (HGF) /scatter factor

(SF)

Tumor necrosis factor-alpha (TNF-alpha)

Epidermal growth facror (EGF)

Angiogenin

Follistatin

Granulocyte colony-stimulating factor (G-CSF)

Granulocyte/ macrophage-colony stimulating

factor (GM-CSF

Ephrins

Heparin

Interleukin-8 (IL-8)

Platelet-derived endothelial cell growth factor (PD-

ECGF)

Pleiotrophin (PTN)

Progranulin

Proliferin

Transforming growth factor-alpha (TGF-alpha)

Transforming growth factor-beta (TGF-beta)

Angiostatin (plasminogen fragment)

Endostatin(collagenXVIII fragment)

Interleukin-12

Interferon alpha/beta/gamma

Interferon inducible protein (IP-10)

Thrombospondin-1 (TSP-1)

Transforming growth factor-beta (TGF-

b)

Calreticulin

Vasculostatin

Vasostatin (calreticulin fragment)

Antiangiogenic antithrombin III

Cartilage-derived inhibitor (CDI)

CD59 complement fragment

Fibronectin fragment

Gro-beta

Heparinases

Heparin hexasaccharide fragment

Human chorionic gonadotropin (hCG)

Kringle 5 (plasminogen fragment)

Metalloproteinase inhibitors (TIMPs)

2-Methoxyestradiol

Placental ribonuclease inhibitor

Plasminogen activator inhibitor

Platelet factor-4 (PF4)

Prolactin 16kD fragment

Proliferin-related protein (PRP)

Retinoids

Tetrahydrocortisol-S

Angioarrestin

32

1. Angiogenic growth factors:

Growth factors are pivotal for the formation of functional blood vessels.

Therefore, modulating angiogenesis by targeting growth factors and their receptors is

extensively studied. Among more than 20 known angiogenic growth factors, Vascular

Endothelial Growth Factor (VEGF), Platelet-Derived Growth Factor (PDGF), Fibroblast

growth factors (aFGF, bFGF), and transforming growth factor-beta (TGF-β) are the most

common and well-studied ones.

VEGF Family:

VEGF, the most potent pro-angiogenic factor also known as vascular permeability

factor (VPF), is a heparin-binding glycoprotein specific for vascular endothelial cells

(Shinkaruk et al, 2003). It activates endothelial cell proliferation and increases the

expression of matrix metalloproteinases and plasminogen activators, which degrade the

extracellular matrix and thereby facilitates endothelial cell migration (Ferrara et al,

2003). VEGF is also a potent induces vasodilation and increases permeability of the

existing vessels by causing a loss of pericyte-endothelial integrity (Kerbel, 2008; Houck

et al, 1991). It is secreted from hypoxic, ischemic or malignant cells as a homodimer, and

is able to induce angiogenesis (Senger et al, 1983; Plouet et al, 1989; Houk et al, 1992).

Belonging to the vascular endothelium-specific growth factor super family, it consists of

five mammalian members: placental growth factor (PlGF) and VEGF-A, VEGF-B,

VEGF-C, VEGF-D. They act through interactions with endothelial specific-receptor

tyrosine kinases that have been shown definitively to play a role in the formation of the

embryonic vasculature (Shalaby et al, 1995). All these members have overlapping

abilities to interact with different receptors expressed mainly in the vascular endothelium

(Eriksson and Alitalo, 1999). VEGF-A is a potent growth factor for blood vessel

endothelial cells showing pleiotropic responses that induces endothelial cell proliferation,

cell migration, differentiation, tube formation and survival (Kim, 1993; Plate et al, 1992).

It also regulates apoptosis and plays an important role in the regulation of angiogenesis

(Ferrara 2004; Hoeben et al, 2004). It is also one of the most potent permeability factors,

so that VEGF-A is a common link of inflammation, permeability and angiogenesis. There

33

are two receptors for VEGF-A; VEGF-R1 (flt-1) and VEGF-R2 (KDR/flk-1). VEGF-A

induced endothelial proliferation and apoptosis can be regulated by changes in

endothelial expression levels of KDR and flt-1 (Hoeben et al, 2004). Regulators of

VEGF-A expression are the steroid hormones oestrogen and progesterone (Classen-

Linke et al, 2000; Hyder and Stancel 1999; Perrot-Applanat et al, 2000). Especially

VEGF-A mRNA expression by endometrial carcinoma cell lines and stromal cells were

found to be sensitive to steroidal stimulation (Charnock-Jones et al., 1993; Shifren et al.,

1996). Another stimulator of VEGF expression is hypoxia (Ferrara, 2004). VEGF-A

mRNA expression patterns are closely related to proliferation of blood vessels during the

developing embryo and wound healing. In the developing embryo cells within tissues

undergoing capillarization express VEGF-A mRNA. In most adult tissues, the level of

VEGF-A expression is low except in the kidney (Bowman’s capsule podocytes).

Expression of VEGF-A can be induced in macrophages, T-cells, astrocytes, osteoblasts,

smooth muscle cells, cardiomyocytes, skeletal muscle cells and keratinocytes. It is also

expressed in a variety of human tumors. VEGF-A, flt-1 and KDR proteins have been

detected in maternal decidual, epithelial and endothelial cells (Clark et al, 1996; Sharkey

et al., 1993; Sugino et al., 2002). VEGF expression is regulated by hypoxia, which occurs

during tumor expansion and ischemia (Minchenko et al, 1994).

Recent studies indicate that VEGF-B promotes angiogenesis through the

activation of protein kinase B (AKt/PKB) and endothelial nitric oxide synthase (eNOS)

relatively pathways (Silvestre et al, 2003). VEGF-C with related receptors VEGF-2 and

VEGF-3 (Flt-4) represents an apparently redundant pathway for postnatal angiogenesis.

VEGF-C was shown to stimulate NO release from ECs and to induce neovascularization

in a rabbit model of hindlimb ischemia (Witzenbichler et al, 1998). Evidence also

indicates a role for VEGF-C in pathological angiogenesis and lymphoangiogenesis

(Enholm et al, 1998; Ferrara and Alitalo, 1999). Placenta derived growth factor (PIGF),

which binds VEGF-1, enhances angiogenesis mainly under pathological conditions (Chen

et al, 2004). In concert with other growth/ differentiation factors, VEGF stimulation

results in basement membrane breakdown, migration and proliferation of endothelial

34

cells, and formation of functional blood carrying structures. Acutely, VEGF has

vasodilatory activity mediated by NO (Ware and Simons, 1999).

The Fibroblast growth factors (FGF) family:

Fibroblast growth factors (FGFs) are a family of heparin binding polypeptides

that consists of nine distinct members. (Burgess and Macaig, 1989). It has two pre-

dominant isoforms known as acidic FGF (aFGF) and basic FGF (bFGF), named after the

purification extraction (Slavin, 1995). They are presently called FGF-1 and FGF-2,

respectively. FGF-1 (acidic), FGF-2 (basic) and FGF-4 are potent angiogenic factors.

FGF-2 is highly proangiogenic factor, stimulates all major steps in the angiogenesis

cascade. It is ubiquitous and pleiotrophic growth factor and highly potent inducer of

DNA synthesis. As such it also plays an important role during embryonic development

and wound healing. It also reportedly contributes to cancer angiogenesis (Frank

Czubayko et al, 2003). It is present in the sub-endothelial basement membrane of blood

vessels in nearly all organs (Cordon-Cardo et al, 1990). During wound healing and tumor

growth it becomes active and upregulated. It interacts with endothelial cells through

binding to fibroblast growth factor receptor-1 (FGFR-1), a tyrosine kinase receptor and

exerts angiogenic activity in vivo and induces cell proliferation, migration and protease

production and chemotaxis in endothelial cells in vitro (Basilico and Moscatelli,1992;

Bikfavi et al, 1989). FGF receptors and low affinity, high capacity heparin sulfate

proteoglycan receptors (HSPGs) present on the cell surface and in the ECM (Johnson and

Williams, 1993; Rusnati and Presta, 1996).

It is produced by macrophages, endothelial cells and tumor cells and released in

the extracellular matrix, initiating angiogenesis. FGF-2 is associated with endothelial

ECM in vitro (Vlodasky et al, 1987; 1987b; Rogelji et al, 1989) and basement

membranes in vivo (Dimario et al, 1989; Hageman et al, 1991). It is involved in

endothelial cell proliferation and migration, and degradation of the extracellular matrix

(Itoh and Ornitz, 2004). In both mouse and human tumors, bFGF has been shown to be

involved in tumor growth and neovascularization (Presta et al, 2005). Newly synthesized

FGF-2 is stored in the ECM from where it is released to induce long-term stimulation of

35

target cells (Bashkin et al, 1989; Presta et al, 1989, Rogelji et al, 1989). VEGF and bFGF

act also an antiapoptotic factors for the newly formed blood vessels, since they induce

expression of antiapoptotic molecules, such as BC1-2, promoting endothelial cell survival

(Kim et al, 2001).

Placental growth factor (PlGF)

As the name implies, PlGF was found in the placenta and it shares biochemical

and functional features with VEGF and interacts with VEGFR-1. It is part of the VEGF

family. PlGF and VEGF-A have synergistic effects regarding angiogenesis, but PlGF-

induced vessels are more mature and stable than VEGF-induced vessels (Carmeliet et al,

2001). In contrast with VEGF, low oxygen tension results in reduced PlGF expression in

trophoblasts in vitro (Shore et al, 1997). PlGF is abundantly expressed in human

placenta, rising from the first-trimester to the late second-trimester and subsequently

declining from 30 weeks of gestation to delivery (Torry et al, 1998). PlGF mRNA is

expressed in villous and extra-villous trophoblast cells while the protein is detected in

vascular endothelium of term placental tissue (Clark et al, 1998). In addition, intense

staining for PlGF antigens is detected in decidual stromal cells (Khalig et al, 1996). The

receptor of PlGF, flt-1, is expressed on endothelial cells, perivascular smooth muscle

cells and (extravillous) trophoblast during pregnancy (Vuorela et al, 1997). PlGF may,

via flt-1, act as a regulator of decidual angiogenesis and an autocrine mediator of

trophoblast function (Khalig et al, 1996; Torry et al, 2004).

Angiopoietin family:

There are 3 members of the angiopoietin family of growth factors involved in

angiogenesis: Angiopoietin-1 (Ang-1), Angiopoietin-2 (Ang-2) and Angiopoietin-4

(Ang-4) (Davis et al., 1996; Suri et al, 1996). These growth factors all bind to the

endothelial tyrosine kinase receptor Tie-2 with equal affinity but the binding of each

ligand to this receptor results in widely different effects. When Ang-1 binds to Tie-2, it

activates the Tie-2 to increase endothelial cell migration and adhesion as well as

recruitment of pericytes and smooth muscle cells to stabilize vessels. It stimulates

36

interaction between ECs and pericytes (Suri et al., 1998; Chae et al 2000). It decreases

vascular permeability and plays a role in endothelial and vascular maturation after

VEGF-induced neovascularization (Geva-Jaffe 2000). Transgenic overexpression of

Ang-1 in mice results in the development of more complex vascular networks (Suri et al,

1998). Ang-1 activity involved in both physiological and pathological neovascularization

(Adams et al, 1999). Ang-2 is a functional antagonist of Ang-1 and is only expressed at

sites of vascular remodelling. When It binds to Tie-2, Tie-2 is inhibited therefore vessels

are de-stabilized by disruption of endothelial cells and perivascular cells that leads to

loosening of cell/cell interactions and allows access to angiogenic inducers like VEGF

(Maisonpierre et al, 1997). Ang-2 also increases the expression of matrix

metalloproteinase (MMP)-2, and acts with VEGF to promote angiogenesis with increase

in vascular permeability (Holash et al, 1999; Stratmann et al, 1998). But it induces

vascular regression in the absence of angiogenic signals (Asahara et al, 1998). Hypoxia

regulates both Ang-1 and Ang-2, i.e. regulates Ang-2 and destabilizes Ang-1 (Geva-Jaffe

2000). Ang-1 is widely expressed in the adult, whereas Ang-2 is restricted at sites of

active angiogenesis, like the uterus and placenta (Maisonpierre et al, 1997). In

endometrium, both Ang-1 and Ang-2 were detected in glands, stromal cells, and

endothelium (Hewett et al, 2002; Krikun et al, 2000). Tie-2 was mainly detected in

endothelium and glands and only small amounts were found in stromal cells (Hewett et

al, 2002; Krikun et al, 2000). Ang-1 and Ang-2 and TIE-2 are detected in human first-

trimester decidua; Tie-2 mainly in maternal endothelial cells, endo-vascular trophoblasts,

and (syn-) cytotrophoblasts and Ang-1 and -2 mainly in the latter. These findings suggest

an additional role for angiopoietins, besides their role in angiogenesis, in regulating

trophoblast behaviour in the development of uteroplacental circulation (Dunk et al, 2000;

Goldman-Wohl et al, 2000). The angiopoietins are regulated as gestation progresses:

Ang-2 is maximally present in the first-trimester and declines thereafter, whereas Ang-1

increases from first- to third-trimester. This suggests that Ang-2 is mainly involved in

first-trimester vasculogenesis and branching angiogenesis and Ang-1 in third-trimester

non-branching angiogenesis (Geva and Jafee, 2000).

37

Platelet derived growth factor (PDGF):

Platelet-derived growth factor (PDGF) and its receptor are not directly pro-

angiogenic. But all members of the PDGF family have strong angiogenic effect indirectly

stimulating proliferation of fibroblasts and vascular smooth muscle cells (Cao et al,

2002). PDGF-B and PDGFR- β is essential for recruitment of pericytes (supportive cells

to endothelium) and in maturation of the microvasculature (Lindahl et al, 1997). Lack of

PDGF leads to fragile neovasculature, a typical of pathological angiogenesis (Carmeliet,

2003). Combination of PDGF and VEGF results in the formation of more mature vessels

(Richardson et al, 2001). PDGF induces endothelial cell proliferation to sprout

development and subsequent tube formation, during which the plasticity of the

endothelial cell phenotype is an important feature of the angiogenic process. Recent

studies have emphasized the significance of tumor-derived PDGF-A (and potentially

PDGF-C) and PDGFR- α signaling in recruitment of the angiogenic stroma to produce

VEGF-A and other angiogenic factors (Dong et al, 2004). As FGF2 potently stimulates

EC proliferation but has almost no effect on chemotaxis and PDGF induces endothelial

cell migration but not proliferation, only when both systems become activated does

coordinated EC proliferation and migration occur, allowing for vessel growth (Yoshida et

al, 1996).

Transforming growth factor (TGF)

Transforming growth factor-alpha (TGF-a) is an angiogenic factor released by

tumor-associated macrophages and many tumor cells (Reppolee et al, 1988). TGF-a is

structurally and functionally related to epidermal growth factor (EGF) released by

macrophages and both induce endothelial cell DNA synthesis in vitro (Schreiber et al,

1986). TGF-a also stimulates capillary-like tube formation in collagen gels (Pepper et al,

1990; Sato et al, 1993). The ability of TGF-a to promote tube formation is dependent on

the release of substances like tissue-type plasminogen activator (tPA) and FGF by ECs

(Sato et al, 1993). TGF-a is more potent than EGF in promoting angiogenesis in the

hamster cheek pouch in vivo bioassay (Schreiber et al, 1986). In addition, TGF-a & EGF

are potent promoters of neoplastic cell growth in many carcinomas (Bicknell and Harris,

38

1991). TGF-ß, which is not structurally related to TGF-a, is synthesized by many normal

and neoplastic cells (Anzano et al, 1982; Derynck et al, 1985), TGF-ß stimulate

neovascularization in vivo when subcutaneously injected to mice (Roberts et al, 1986).

However, several studies utilizing two dimensional culture conditions found

microvascular endothelial cell proliferation to be inhibited by TGF-ß1, TGF-ß2 and TGF-

ß3 raising doubt as to whether TGF-ß functions as an angiogenic factor. It is possible that

TGF-ß promote angiogenesis by differentiating ECs by indirect stimulation of

angiogenesis (Pretoveera et al, 1994). Other studies also show that low concentrations of

TGF-ß1 enhance endothelial cell proliferation (Myoken et al, 1990) and in vitro

angiogenesis (Pepper et al, 1993), whereas high concentrations inhibit proliferation and

angiogenesis.

Hepatocyte growth factor / scatter factor (HGF/SF):

Hepatocyte growth factor/scatter factor (HGF/SF) is a mesenchyme-derived

cytokine that stimulates motility and invasiveness of epithelial and cancer cells. HGF/SF

is a potent angiogenic molecule in vivo and its angiogenic activity is mediated primarily

through direct actions on vascular endothelial cells. HGF activates its receptor c-met

expressed by ECs and hematopoietic stem cells, thereby resulting in stimulation of

angiogenesis. These include stimulation of cell migration, proliferation, protease

production, invasion, and organization into capillary-like tubes (Rosen et al, 1997).

Insulin-like growth factor I and interleukin 8:

Insulin-like growth factor I (IGF-I) is secreted by activated macrophages and it is

suggested to have a role in inflammatory angiogenesis (Filkins et al, 1980; Rom et al,

1988). IGF-I induces endothelial cell mitogenesis, migration and tube formation (Nakao-

Heyashi et al, 1992). Interleukin- 8 (IL-8) is produced by activated macrophages (Koch

et al, 1992), is mitogenic in vitro and stimulates angiogenesis in the rat corneal assay (Hu

et al, 1993). IL-6 induces VEGF transcription and regulates the VEGF promoter thereby

contributes to glioma angiogenesis (Rolhion et al, 2001). In contrast, IL-8 stimulates

angiogenesis through interaction with the C-X-C chemokine receptor 1(CXCR1),

39

CXCR2 and Duffy antigen receptor for cytokines and thereby can affect angiogenesis

independent of VEGF (Brat et al, 2004).

Tumor necrosis factor (TNF- α)

TNF-α is an inflammatory cytokine that induces tumor angiogenesis indirectly by

upregulating other angiogenic factors including VEGF (Maruno et al, 1997; Ryuto et al,

1996; Pusztai et al, 1994). TNF-α acts on endothelial cells to exert a procoagulant effect

(Van et al, 1990). Other in vitro and in vivo studies revealed that TNF-α stimulates

angiogenesis at low concentrations, whereas it inhibits angiogenesis at high

concentrations (Fajardo et al, 1992). The concentration of TNF-α is low in tissues in vivo

so that the overall effect of TNF-α is stimulatory to angiogenesis (Klagsbrun and

D’Amore, 1991). TNF-a stimulates angiogenesis in the rabbit cornea and in the CAM

(Leibovich et al, 1987). Hypoxia induces in vitro the release of TNF-a by macrophages in

vitro (Scannell et al, 1993).

Epidermal growth Factor (EGF):

Epidermal growth factor is a potent mitogenic factor for endothelial cells;

therefore binding to EGFR (ErbB-1, HER1) increases the proliferation of endothelial

cells (Tsai et al, 1997). Furthermore EGF can stimulate VEGF expression in gliomas,

which can act in an autocrine or paracrine manner (Ferrara et al, 2001). In tumors with

the mutant EGFRvIII, which is constitutively activated, VEGF expression is induced

through the Ras/MAPK and NF-κB pathways (Feldkamp et al, 1999a; Feldkamp et al,

1999b; Wu, 2004). Expression of EGFRvIII has been associated with faster rates of

double strand repair and increased radioresistance (Mukharjee et al, 2009).

Granulocyte / macrophage colony stimulating factor

Granulocyte-colony stimulating factor (G-CSF) and granulocyte/ macrophage-

colony stimulating factor (GM-CSF) are myeloid growth factors required for the survival,

growth, and differentiation of hemopoietic precursor cells. Both are produced in many

different tumour types (Fu et al, 1992) and are chemotactic for macrophages in vitro

40

(Mantovani et al, 1999; Wu et al, 1993). Recombinant G-CSF and GM-CSF stimulate the

migration and proliferation of human endothelial cells (Bussolino et al, 1989).

The Ephrins:

The Eph receptor tyrosine kinases comprise the largest known family of growth

factor receptors and use, the similarly numerous ephrins as their ligands. Ephrin-B2 and

Eph-B4 play an important role during initial distinction between arterial and venous

vessels. Interesting studies on mice lacking ephrin- B2 and Eph- B4 highlighted defects in

early angiogenic remodeling that are somewhat reminiscent of those seen in mice lacking

Ang-1or Tie-2 (Wang et al, 1998; Adams et al, 1999; Gerety et al, 1999). Moreover,

Ephrin-B2 and Eph-B4 display remarkably reciprocal distribution patterns during vascular

development with Ephrin-B2 marking the endothelium of primordial venous vessels.

Heparin:

Heparin induces endothelial cell proliferation and motility indirectly by increasing

the binding of FGFs to their endothelial receptors as well as by protecting FGFs from

inactivation (Folkman et al, 1988). Cell surface heparin-like molecules are also involved

in binding of VEGF to its high affinity receptor sites (Gitay-Goren et al, 1992). Released

FGF can be sequestered from its site of action by binding to heparan sulphate (HS) in the

ECM (Bashkin et al, 1989) to be kept for emergencies, such as wound repair and

neovascularization (Vlodavsky et al, 1991).

2. Antiangiogenic Factors:

Angiostatin:

Angiostatin is a specific inhibitor of endothelial cells proliferation and angiogenesis

in a number of primary and metastatic tumors. It is a 38-kDa-mouse plasminogen

fragment. Systemic injection of angiostatin has been shown to potently block

neovascularization and metastatic growth. Angiostatin inhibits ECM- enhanced and t-PA

catalyzed plasminogen activation (Stack et al, 1999). The inhibition of matrix enhanced

41

plasminogen activation leads to a reduced invasive activity, suggesting a crucial

involvement of angiostatin in cellular migration and invasion.

Endostatin:

Endostatin is a C-terminal fragment of collagen-XVIII, found in vessel walls and

basement membranes. It is a potent anti-angiogenic molecule that inhibits tumor growth

and also specifically inhibits endothelial cell proliferation and migration. It induces

endothelial cell apoptosis and cell cycle arrest in vitro (Hanai et al, 2002). Endostatin

significantly reduces endothelial as well as tumor cellular invasion into reconstituted

basement membrane in vitro (Kim et al, 2000).

Endostatin and angiostatin have been demonstrated to induce tumor regression and

tumor dormancy without drug resistance in several experimental models. Dormant tumors

secrete endostatin, angiostatin, thermospondins and tissue inhibitors of metalloproteinases

that prevents the tumors from increasing their size (Folkman, 2002).

Interleukin and Interferon:

Interferon (IFNs) have established antitumor and antiangiogenic actions. IFN-α

and retinoic acid have remarkably synergistic antiangiogenic effects able to inhibit both

the growth and the neovascularization of head an neck squamous cell carcinoma injected

into the mouth of nude mice (Lingen et al, 1998).

Interleukin-2 (IL-2) has a slight effect on angiogenesis in vivo in the rabbit cornea

model. IL-2 inhibits. IL-2 inhibits angiogenesis in the CAM in a dose dependent manner.

In vivo studies evidenced that mice treated with rat antibody raised against murine IFN-γ

results in neutralizing the antiangiogenic effect of murin interleukin-12 indicating that

IFN-γ is a mediator of the antiangiogenic effect of IL-2 (Majewasky et al, 1996).

Calreticulin:

Calreticulin is calcium binding molecule chaperone expressed primarily in the

lumen of the endoplasmic reticulum (ER). The 1-180 amino acids N-terminal domain of

42

Calreticulin is vasostatin, which inhibits endothelial cell proliferation, angiogenesis and

tumor growth.

The above review indicated many molecular categories that influence the

angiogenesis and vasculogenesis. Since we have used plant extracts to study their effects

on angiogenesis; the summary of angiogenesis influencing plant-originated compounds is

essential to compare the results.

Plant derived angiogenic and antiangiogenic compounds:

Numerous bioactive chemical compounds of plant origin may influence the

angiogenic activity. Some of the medicinal plants contain complex chemical cocktails

that act on multiple pathways that initiate and maintain angiogenesis. Plants with known

antiangiogenic activity are being used in cancer therapy (Tan et al, 2006). Various pro-

and antiangiogenic approaches have been recently tested for the compounds derived from

plants constitute the significant part of studies. It is reported that resveratrol isolated from

root of white hellebore (Veratrum grandiflorum) exhibits anticancer and antiangiogenic

properties (Mousa and Mousa, 2005). It has been shown to down-regulate the production

of several angiogenic cytokines, including VEGF and Interleukin-8 (IL-8). As

demonstrated by Brakenhielm et al, resveratrol can impede tumor growth by inhibition of

angiogenesis (Brakenhielm et al, 2001). Administration of Ginseng saponin and some

related triterpenoid compounds had anti- angiogenic activity in vitro or in vivo (Shibata et

al, 2001; Mochizuki et al, 1995). Administration of Ginko biloba extract was also found

to possess antiangiogenic properties. This is because of flavonoids (ginko- flavone

glycosides) and terpenoids (ginkgolides and biloblides), which inhibit angiogenesis by

down regulating VEGF. Experimental evidence suggests that administration of Green-tea

(Camellia sinesis) extracts significantly inhibit angiogenesis in mice. It may involve

inhibition of endothelial cell proliferation in response to stimulation with angiogenic

growth factors. Proanthocyanidin from edible berries inhibits VEGF expression induced

by tumor necrosis factor alpha (TNF-α). On the other hand, one study showed that,

proanthocyanidin can induce angiogenesis as part of normal tissue healing (Bagchi et al,

43

2000). In recent study Arbiser et al reported that curcumin and its derivatives

significantly inhibited basic fibroblast growth factor (bFGF) mediated corneal

neovascularization in the mouse (Arbiser et al, 1998). The predominant data referred to

compounds known as chemopreventive agents, which include resveratrol, catechins,

genistein, curcumin as well as others, such as diallyl sulfide, S-allyl cysteine, allicin,

lycopene, capsaicin, 6-gingerol, ellagic acid, urosoloic acid, silymarin, anethol and

eugenol (Aggarwal et al, 2004, Dorai et al, 2004). Those agents have been suggested to

suppress cancer cell proliferation, inhibit growth factor signaling pathways induce

apoptosis, as well as inhibit angiogenesis (Dorai et al, 2004).

Several other plant compounds and its derivatives like Isoliquiritin from licorice

root extract (Kobayashi et al, 1995), Shikonin from Lthospermum erythrorhizon (Hisa et

al, 1998), Viscum album coloratum extract (Yoon et al, 1995), ether fraction of water

soluble extracts of Populus nigra leaves (Glinkowska et al, 1997), Soybean

phytochemicals (Zhou et al, 1999), Chrysobalanus icaco methanol extract (Alves De

Paulo et al, 2000) an extract of the fern Polypodium leucotomos (Gonzalez et al, 2000),

sesquiterpene (torlin) from Torlis japonica (Kim et al, 2000) Cassia garrettiana

heartwood extract (Kimura et al, 2000), Agaricus blazei extract (Takaku et al, 2001),

Deoxypodophyllotoxin from Pulsatilla koreana (Kim et al, 2002) epigallacatechin

gallate from green tea (Jung and Ellis, 2001) have found to possess antiangiogenic

activity.

On the other hand, saponin from Ginseng Radix rubra (Morisaki et al, 1995),

asiaticoside isolated from Centella asiatica (Shukla et al, 1999), Ginko-biloba extracts

(Juarez et al, 2000), beta-sitosterol from Aloe vera (Choi et al, 2002) enhanced

angiogenesis in vivo. Wang et al (2004) screened 24 species of Chinese medicinal herbs

for their in vitro angiogenic and antiangiogenic activity on chick embryo chorioallantoic

membrane (CAM) model and cell proliferation of cultured bovine aortic endothelial cells

(BAECs). They reported that; the aqueous extracts of Epimedium sagittatum,

Trichosanthes kirilowii and Dalbergia odorifera showed the strong angiogenic activity

both in CAM and BAECs model; and the aqueous extracts of Berberis paraspecta,

Catharanthus roseus, Coptis chinesis, Taxus chinesis, Scutellaria basicalensis,

44

Polygonum cuspidatum and Scrophularia ningpoensis elicited significant inhibition of

angiogenesis.

As reasoned and discussed above the study was conducted on acetone, alcohol

and benzene extracts of P. santalinus and B. diffusa on CAM development,

vasculogenesis and angiogenesis. The parameters used evaluate the alterations are

1.Mortality and abnormalities studies to select the effective dose. 2. CAM diameter/area.

3.Number of Primary veins and area of their extension. 4. Number of secondary veins

and area of their extension. 5. Number of Tertiary veins and area of their extension. 6.

Histological analysis of CAM area.

The above data is presented in the thesis as different Chapters.

Thesis is divided into 5 Chapters with below stated plan.

Chapter I. Introduction:

Under this Chapter the problem is defined with the theoretical approach behind it.

Besides selection of animal model of angiogenesis doses P. santalinus and B. diffusa

extracts, experimental schedule and parameter studies are reasoned. Additionally review

of literature and reviews of relevant parameters and associated influences and processes

have been provided.

Chapter II. Materials and methods:

This chapter describes elaborately the materials and methods used in experiments;

where chick CAM model is described. The experimental schedule that includes CAM

exposure to Plant extracts was performed by window method. Experimental protocol is

provided in detail along with dose administration along with details of each of the

experimental group. The chicks CAM assay with associated quantitative, macroscopic

and histological evaluation in detail are provided.

Statistical methods used to valuate the observations have been provided.

45

Chapter III. Results and Discussion:

Results of the experimental studies conducted are presented under following

sections each section is provided with the discussion of the respective parameter.

Section1. Effect of P. santalinus extracts on chick CAM:

The alterations under experimental conditions are evaluated quantitatively,

macroscopically and histologically under following categories.

A) Mortality Studies and dose Selection

B) Influence of P. santalinus on CAM diameter and area and Vasculogenesis:

Evaluation of primary vitelline veins: Number and area covered.

C) Influence of P. santalinus on CAM Angiogenesis: (i) Evaluation of secondary

blood vessels: Number and area covered. (ii) Evaluation of tertiary blood vessels:

Number and area covered.

D) Macroscopic and histological analysis of chick CAM.

The alterations are related with the normal sprouting of blood vessels with cell

proliferation and angiogenic influence of P. santalinus extracts and discussed with the

relevant literature.

Section.2. Influence of Boerrhavia diffusa extracts on chick CAM.

The alterations under experimental conditions are evaluated quantitatively,

macroscopically and histologically under following categories.

A) Mortality Studies and dose Selection

B) Influence of B diffusa on CAM diameter, area and vasculogenesis: Evaluation

of primary vitelline veins: Number and area covered.

C) Influence of Boerrhavia diffusa on CAM Angiogenesis: (i) Evaluation of

secondary blood vessels: Number and area covered. (ii) Evaluation of tertiary

blood vessels: Number and area covered.

D) Macroscopic and histological analysis of chick CAM.

46

Chapter IV. General Discussion

Alterations in all the parameters studied are considered together to evaluate the

influence of P. santalinus and B. diffusa on in vivo CAM angiogenesis.

Chapter V. Concluding Remarks