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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
2
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,
5
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).
6
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
7
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.,
9
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,
10
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.
11
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
12
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
13
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
14
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.
15
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