NUTRACEUTICALS AS ANTI-ANGIOGENIC AGENTS : HOPES AND REALITY

JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2005, 56, Suppl 1, 51–69
www.jpp.krakow.pl
Review article
J. DULAK
NUTRACEUTICALS AS ANTI-ANGIOGENIC AGENTS:
HOPES AND REALITY
Department of Cell Biochemistry, Faculty of Biotechnology, Jagiellonian University,
Kraków, Poland
Angiogenesis, the formation of new blood vessels from preexisting vascular network
is a driving force of organ development in ontogeny, is necessary for ovulation and
hair growth, and is prerequisite for proper wound healing. It is also a critical
mechanism of numerous diseases, the most important of which are cancer and
atherosclerosis. Therefore, modulation of angiogenesis is considered as therapeutic
strategies of great importance for human health.
Numerous bioactive plant compounds, often referred to as nutraceuticals are recently
tested for the potential clinical applications. Among the most frequently studied are
resveratrol, a polyphenol present in red-wine and grape-seed, epigallocatechin-3-
gallate (EGCG) from green tea and curcumin from Curcuma longa. It is also possible
that components of other plants, including the constituents of local food diet may
find application for modulation of angiogenesis, provided that their effectiveness will
be confirmed in controlled, scientifically validated trials.
K e y w o r d s : angiogenesis, vascular endothelial growth factor, cancer, atherosclerosis,
polyphenols
Angiogenesis and diseases
Angiogenesis is the process in which the new blood vessels are formed from
pre-existing ones (for reviews see: 1-3). It is indispensable for embryonic
development as interruption of angiogenic events blocks the growth of the
embryo and results in early mortality. After birth angiogenesis plays both
adaptive role enabling the regeneration of the damaged body parts and is also
involved in numerous pathological changes. Understanding of the basic

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mechanisms of blood vessels formation is necessary for the establishment of the
effective therapeutic strategies for amelioration of diseases.
The major angiogenic regulator is vascular endothelial growth factor (VEGF),
named also VEGF-A, which is one of the several members of VEGF family (for
reviews see: 2,3). The most striking demonstration of its significance in blood
vessel formation is the early embryonic heterozygous lethality of knockout of
VEGF gene. Mice embryos devoid of only one functional allele of VEGF die at
day 9-11 of pregnancy and do not develop vasculature (4, 5).
In adult organisms physiological angiogenesis is limited and occurs during
regeneration of uterine epithelium in menstrual cycle, development of the ovum
and formation of corpus luteum and hair growth (3). It is also suggested that
spermatogenesis may be dependent on the activity of VEGF (6). Moreover, the
production of VEGF and other angiogenic mediators is prerequisite for the
reparative processes, such as healing of epidermal and internal wounds, including
bone fractures. Proper restoration of the continuity of the tissues requires the
coordinated actions of several pro-angiogenic mediators. Final establishment of
angiogenic homeostasis after effective repair of injured tissues is executed by
decrease in the synthesis and activity of those stimulants, but is also dependent on
the production of angiogenic inhibitors (Table 1).
Angiogenesis is also an important constituent of several pathological
processes. It is estimated that about a hundred of various diseases possess a
significant angiogenic component (Table 2). Among those diseases of particular
interest is angiogenesis in cancer and cardiovascular disorders.
Effect of plant derived compounds on tumor growth and angiogenesis
Food components can influence angiogenesis. On the one side this can
contribute to pathological changes. For example, ours (7,8) and others (9-11)
studies demonstrated that hypercholesterolemia enhances the synthesis of VEGF.
It is thus conceivable that long term changes in lipid blood constituents, which
may be the result of improper diet, can influence angiogenesis and add to the
pathogenesis of cardiovascular diseases. Whether diet compounds can be used to
beneficially modulate angiogenesis is not yet proven, although experimental data
suggest that this may be possible.
Various pro- and anti-angiogenic approaches have been recently tested for
the potential clinical application (Table 3). Among them the search for the
compounds derived from plants constitutes the significant part of studies,
which are supported by various funding bodies, including the European
Commission. Table 4 shows the examples of various components isolated from
different plants which demonstrated the anti-angiogenic activity. It has to be,
however, remembered that in a vast majority those are mostly preliminary
studies and more hard data are necessary to prove those activities. This can be
attained by careful and accurate characterization of the active chemical

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Table 1. Mediators stimulating or inhibiting formation of blood vessels (for a review see: 3).
Table 1. Mediators stimulating or inhibiting formation of blood vessels (for a review see: 3).
Stimulators
Inhibitors
Growth factors/cytokines :
angiopoietin-2 (in the absence of VEGF)
Vascular endothelial growth factors (VEGFA, VEGF-
B, VEGF-C, VEGF-D, VEGF-E)
Pigment epithelium derived factors
Placental growth factor (PlGF)
Fibroblasts growth factors (FGF-1, FGF-2, FGF-4)
tumor necrosis factor a (TNFa)
Angiopoietin -1 (Ang-1)
Angiopoietin-2 (Ang-2) (acting together with VEGF)
Platelet factor-4
Hepatocyte growth factor (HGF)
Platelet derived growth factor (PDGF)
Thrombospondin-1,-2 (TSP-1)
Transforming growth factor a, b (TGFa, b )
Insulin growth factor-1 (IGF- 1)
Inhbitors of proteases:
Tissue inhibitors of metalloproteinases (TIMP 1-4)
Ephrins and ephrin receptors
plasminogen activator inhibitor (PAI)


Proteases:
Maspin
Matrix Metalloproteinases (e.g. MMP-2, MMP-9)
Aminopeptidases (CD13/aminopeptidase N (APN)
Angiostatin
Urokinase-type plasminogen activator (uPA)
Endostatin
Interferon a, b, g (IFN a, b, g)
Transcription factors
IP-10
hypoxia-inducible factor-1 (HIF-1)

Cytokines/chemokines

IL-8

CXC

tumor necrosis factor a (TNFa)

Hormones & others
Kallikrein
Factor XIII
Angiogenin
Integrins
Tissue factor (TF)
inhibitor of DNA binding 1, 3 (Id1, Id3)
Enzymes:
Thymidine phosphorylase
Other angiogenic mediators
Nitric oxide
Hydrogen peroxide
Carbon monoxide
Prostaglandins
compounds, elucidation of the molecular mechanisms of their actions,
demonstration of the real efficacy by in vivo studies on proper animal models
of human diseases and finally by demonstration of their safety and
effectiveness in clinical trials. As all those studies require at least several years
of research, so far experiments with only few compounds have generated
sufficient amount of data which would allow their investigation in the clinical
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Table 2. Diseases in which angiogenesis is either enhanced or attenuated (after 3).
Table 2. Diseases in which angiogenesis is either enhanced or attenuated (after ref.3)
Diseases characterized by
Diseases characterized by excessive
abnormal/impaired angiogenesis
angiogenesis
Cardiovascular diseases:
Hemangiomas – tumors of the vessels
Atherosclerosis: heart and peripheral
ischemia
Cancer - growth and metastasis depend on
Diabetes: impaired collateral growth
angiogenesis
Restenosis: impaired reendothelialization

Psoriasis
Non-healing wounds:
gastric or oral ulcers
Obesity
Diabetic ulcers
Impaired bone fracture healing
Diabetic retinopathy
Crohn disease (mucosal ischemia)
Atherosclerosis (growth of the plaque)
Osteoporosis
Asthma
Nasal polyps
Hair loss
Skin pupura
Telangiectasia
Rheumatoid arthritis
Nephropathy
Inflammatory bowel disease
Neonatal respiratory distress
Endometriosis
Pulmonary fibrosis
Emphysema
Nervous system:
Alzheimer diseases
Amyotrophic lateral sclerosis
Diabetic nephropathy
studies. In this review the potential anti-angiogenic activities of three such
compounds are discussed.
Effect of plant polyphenols
As stated in a recent review (12), plants have a long history of use in the
treatment of cancer, though many of the claims for the efficacy of such treatments
should be viewed with some scepticism. Nevertheless, extensive research
suggests that regular consumption of certain fruits and vegetables can reduce the
risk of acquiring specific cancers (12). The effect seems to be related to the
chemicals present in this food. The predominant data referred to compounds
knowns as chemopreventive agents, which include resveratrol, catechins
genistein, curcumin, as well as others, such as diallyl sulfide, S-allyl cysteine,
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Table 3. Some possible ways of affecting angiogenesis
Table 3. Some possible ways of affecting angiogenesis
Enhancement
Mode
Inhibition
VEGF (VEGF-A, VEGF-C)
Protein factors
Anti-VEGF antibodies
FGFs
Soluble VEGF receptors
HGF
Angiogenic inhibitors (eg.
endostatin, angiostatin,
interferons)
Gene transfer of VEGF,
Gene therapy
Gene transfer of anti-
FGF-2, FGF-4, HGF
angiogenic genes (eg.
Other angiogenic mediators
endostatin)
Drugs/small molecular
Inhbitors of VEGF recepotors
Statins?
compounds
Thalidomide
HMG-CoA reductase
inhibitors (statins)?
COX-2 inhibitors
Bioactive food components
Resveratrol
Resveratrol/other
Curcumin
polyphenols
EGCG (green tea)
allicin, lycopene, capsaicin, 6-gingerol, ellagic acid, ursolic acid, silymarin,
anethol, and eugenol (13,14). Those agents have been suggested to suppress
cancer cell proliferation, inhibit growth factor signalling pathways, induce
apoptosis, as well as inhibit angiogenesis (14).
Effect of resveratrol on angiogenesis
One of the most widely investigated plant-derived bioactive compound is
resveratrol (trans-3,5,4'-trihydroxystilbene). It was first isolated in 1940 as a
constituent of the roots of white hellebore (Veratrum grandiflorum) (13).
Resveratrol has been found in various plants, including grapes, berries and peanuts.
Besides cardioprotective effects, resveratrol exhibits anticancer properties. Thus, it
suppressed proliferation of lymphoid and myeloid cancers, cancers of the breast,
prostate, stomach, colon, pancreas, and thyroid; melanoma; head and neck
squamous cell carcinoma; ovarian carcinoma; and cervical carcinoma (13).
Molecular mechanisms of inhibition of tumor cell proliferation have been partly
elucidated and involve suppression of several transcription factors, such as NF-?B,
AP-1 and Egr-1, downregulation of the expression of anti-apoptotic genes and
activation of caspases (13). As far as angiogenesis is concerned, resveratrol has
been shown to downregulate the production of several angiogenic cytokines,
including VEGF and interleukin-8 (IL-8) (for a review see: 15).
Protective effect of resveratrol in vascular cells is often ascribed to be related
to the scavenging of reactive oxygen species (ROS). Interestingly, ROS, such as
18

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Table 4. Bioactive herbal compounds and their effect on angiogenesis.

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hydrogen peroxide, appear also to be the crucial players in angiogenesis (16),
inducing the expression of angiogenic mediators, among them VEGF (17,18).
ROS are also generated by endothelial cells in response to stimulation with
growth factors, such as VEGF (19). Some data indicate that scavenging properties
of reseveratrol may contribute to its anti-angiogenic effects (20). In a recent study
Lin and co-workers (21) demonstrated that exposure of HUVECs to 1 to 2.5 µM
resveratrol significantly blocked VEGF-mediated cell migration and tube
formation but not cell proliferation. Resveratrol effectively abrogated VEGF-
mediated tyrosine phosphorylation of vascular endothelial (VE)-cadherin and its
complex partner, beta-catenin. VEGF stimulated an increase of peroxide which
has been shown to be involved in VE-cadherin phosphorylation and this was
strongly attenuated by resveratrol (21).
Importantly, those in vitro results have been corroborated by in vivo studies.
Brakenhielm et al. (22) demonstrated that resveratrol supplied in drinking water
suppresses the growth of new blood vessels in the chick chorioallantoic
membrane (CAM) assay and in the mice corneal neovascularization model,
directly inhibiting capillary endothelial cell growth. The effect was due to block
of both VEGF and bFGF-receptor mediated responses, involving the
phosphorylation of MAP kinases. In accordance with the role of angiogenesis in
tumor growth, oral administration of resveratrol inhibited the growth of murine
fibrosarcoma in mice, and significantly delayed wound healing, the process also
dependent on angiogenesis (22).
Mechanisms of the effect of red wine polyphenols on production of VEGF
have been studied in details by Oak et al. (23). Short-term and long-term exposure
of vascular smooth muscle cells to red wine polyphenolic compounds (RWPC)
inhibited VEGF mRNA expression and release of VEGF in response to platelet-
derived growth factor AB (PDGF-AB), transforming growth factor-beta1 (TGF-
?1) or thrombin. Short-term and long-term treatment of VSMCs with RWPCs
markedly reduced PDGF-AB-induced production of reactive oxygen species and
phosphorylation of p38 (23), again pointing to the role of ROS and downstream

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activation of p38 in induction of VEGF synthesis. By demonstration of such an
anti-angiogenic effect of red wine polyphenols the study add to the
atheroprotective effects of red wine and suggest that one of its mechanisms may
be related to the inhibition of atherosclerotic plaque growth by attenuation of the
synthesis of VEGF, which promotes the formation of vasa vasorum. Indeed, other
studies indicate that the growth of the atherosclerotic plaque is dependent on the
building of new vasculature. The size of the plaque correlates positively with the
extent of neovascularization (11) and other inhibitors of angiogenesis, such as
TNP-470 or endostatin can diminish the extent of atherosclerosis in apoE
knockout mice (24).
As demonstrated by Brakenhielm et al. (22) resveratrol can impede tumor
growth by inhibition of angiogenesis. Similarly, Tseng and co-workers have
shown that resveratrol at the dose 40 mg/kg/day suppressed the angiogenesis and
growth of tumour gliomas in vivo (25). The effect was dependent on the pro-
apoptotic effect of resveratrol on tumour cells and attenuation of VEGF
production. The authors also showed that resveratrol impaired the proliferation of
ECV304 cells, which they described as endothelial. However, it has to be stressed
that those cells are of tumour origin and recently it has been proven convincingly
that they are identical with bladder cancer cell line T24/83 (26).
Similar effects may be also exerted by resveratrol isolated from other sources,
as shown for compounds extracted from roots of Polygonum cuspidatum, which
prevented tumour growth and metastasis to lung and tumor-induced
neovascularization in Lewis lung carcinoma-bearing mice (27,28)
To complicate the picture, there is also some evidence that polyphenols may
exert pro-angiogenic effects. Accordingly, Sen and co-workers (17,29) have
shown that grape seed proanthycyanidin extract (GSPE) containing 5000 ppm
resveratrol potently upregulates oxidant and tumor necrosis factor-alpha (TNF?)
inducible VEGF expression in human keratinocytes. Their studies have
demonstrated that grape seed extract potentiate angiogenesis by enhancing the
oxidizing environment of the wound and in this way stimulating VEGF
production (29).
Such an effect can be related to stimulatory effect of tannins and
proanthocyanidins (condensed tannins) and other on wound healing. Thus, a
combination of grape seed proanthocyanidin extract and resveratrol facilitates
inducible VEGF expression, a key element supporting wound angiogenesis (17).
Effect of curcumin on angiogenesis
Curcumin is a small-molecular-weight compound isolated from the commonly
used spice turmeric. Curcumin down-regulates transcription factors NF-kappa B,
AP-1 and Egr-1, inhibits the expression of cyclooxygenase-2, lipooxygenase,
inducible nitric oxide synthase, matrix metalloproteinase-9, urokinase-type
plasminogen activator, TNF?, chemokines, cell surface adhesion molecules and

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cyclin D1; inhibits growth factor receptors (such as epidermal growth factor and
human epidermal growth factor receptor-2) activity and blocks the activity of c-
Jun N-terminal kinase, protein tyrosine kinases and protein serine/threonine
kinases (for references see: 30, 31) .
Effect of curcumin on the growth of cancers has also been investigated. Thus,
in animal models, curcumin and its derivatives were shown to inhibit the
progression of chemically induced colon and skin cancers (32). As far as
angiogenic events are considered, it has been recently demonstrated that blocking
of NF-?B and AP-1 by curcumin attenuated IL-8 expression in human breast
carcinoma cells but did not affect VEGF production (33).
On the other hand, curcumin was found to completely prevent the induction
of VEGF synthesis by microvascular endothelial cells stimulated with advanced
glycation end products (34). The effect might be mediated by downregulation
of NF-?B and AP-1 activity (34). Curcumin may also affect angiogenesis
dependent on other growth factors. 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 (32).
However, in that study curcumin had no effect on phorbol ester-stimulated
VEGF production (32).
Curcumin can also inhibit the activity of CD13/aminopeptidase N (APN), a
membrane-bound, zinc-dependent metalloproteinase that plays a key role in
tumour invasion and neovascularization (35). Accordingly, curcumin and other
known APN inhibitors strongly inhibited APN-positive tumour cell invasion and
bFGF-induced angiogenesis. Because curcumin did not affect the invasion of
APN-negative r cells, therefore it seems probable that the anti-invasive activity of
curcumin against tumours is attributable to the inhibition of APN (35).
A detailed analysis of gene expression affected by curcumin treatment was
performed by Kim and co-workers (36), who studied the effect of
demethoxycurcumin (DC), a structural analog of curcumin, isolated from
Curcuma aromatica, on genetic reprogramming in cultured human umbilical vein
endothelial cells (HUVECs) using cDNA microarray analysis. Of 1024 human
cancer-focused genes arrayed, 187 genes were up-regulated and 72 genes were
down-regulated at least 2-fold by DC. Interestingly, 9 angiogenesis-related genes
were down-regulated over 5-fold in response to DC. These data suggest that
change of MMP-9 gene expression is a major mediator for angiogenesis
inhibition by DC (36).
In accordance with its anti-angiogenic effect demonstrated in vitro, curcumin
inhibited also the growth of B16 melanoma cells in mice, attenuating
angiogenesis and NO and TNF? production (37). Finally, in one recent study,
Gururaj et al. (38) showed that curcumin, when injected intraperitoneally (i.p)
into mice, effectively decreased the formation of ascites fluid by 66% in Ehrlich
ascites tumor (EAT) bearing mice. The authors suggest that this effect can be due

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to anti-angiogenic action, as milimolar concentrations of curcumin inhibited
VEGF production by tumor cells (38).
However, the authors of discussed study (38) did not delineate that low
concentrations of curcumin (below 1 mM in vitro) in fact induced VEGF
expression, while the inhibitory effect was observed only at 1 mM concentration.
Meanwhile, curcumin appears to be very poorly absorbed from the intestine. In
phase I clinical trials in patients receiving orally very high, i.e. gram doses of
curcumin, the serum concentration of this compound was only at the nanomolar
range (39-41). This suggests that doses of curcumin required to attain level
sufficient to exert pharmacological activity are not feasible in humans and thus
curcumin may act only locally.
Green tea catechins
Tea is, besides water, the most widely consumed beverage worldwide. Of
several forms of this drink available, most studies examining the usefulness of tea
in prevention of cancer focused on green tea. This interest is based on
epidemiological evidence, which suggests that people who consume large
amounts of green tea have a lower risk of developing various cancers (42). Those
studies have been corroborated by experimental data from in vitro and in vivo
experiment on animal models
Green tea extracts contain (-)epigallocatechin gallate (EGCG), (-)-
epigallocatechin (EGC), (-)epicatechin gallate (ECG) and (-)epicatechin (EC)
(42). EGCG has been considered as a major acting constituent. In addition to
having pro-apoptotic activity on tumor cells, the compounds present in green tea
have been shown to inhibit tumor invasion and angiogenesis (Table 4).
Indeed, experimental evidence suggests that green tea consumption by mice
significantly inhibits angiogenesis (reviewed in 42). Mechanisms of anti-
angiogenic effects may involve inhibition of endothelial cell proliferation in
response to stimulation with angiogenic growth factors (43). This can be exerted by
inhibition of VEGF receptors and suppression of VE-cadherin and Akt
phosphorylation (44). Activation of certain transcription factors, such as AP-1, NF-
?B and Ets-1 is also blunted (45,46) and the production of metelalloproteinases
necessary for endothelial cell migration and invasion is attenuated (47-49). Finally,
EGCG can also inhibit the production of VEGF, bFGF and IL-8 (50-52). In human
colon cancer cells the attenuation of VEGF production was caused by EGCG-
mediated inhibition of Erk-1 and Erk-2 kinases (53).
However, a recent randomised phase II clinical trial in patients with prostate
cancer did not demonstrate any improvement in their conditions (54). Out of 42
patients who consumed large amounts of green tea, the level of prostate specific
antigen decreased only in one case. Further studies are ongoing (55) and their
results will be crucial in establishing the real effectiveness of green tea
consumption on tumor growth.

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