Abstract. This review focuses on vascular endothelial growthfactors (VEGF), a group of structurally-related proteins thatserve as key growth factors for tumor-associated angiogenesis.Pathways induced by VEGF proteins that regulate biologicalfunctions of key cell types involved in tumor angiogenesis,including vascular endothelial cells, pericytes and tumor cells,are discussed. Strategies that are currently being developed andtested based on the emerging definitions of the roles of themultiple cell types involved in tumor vessel development, theirselective production of VEGF-related proteins and other pro-angiogenic growth factors, their expression of associatedreceptors as well as identification of signal transductionpathways involved in VEGF-induced tumor survival andtumor-associated angiogenesis will be reviewed.

Neovascularization is the development of blood vessels and

is a fundamental and highly regulated process necessary for

appropriate embryonic development (1). In adult mammals,

this activity occurs only during normal physiological

processes such as the female reproductive cycle (2) and

wound healing (3). While tumors that are relatively small

(< 200 Ìm) use existing vasculature as a source of oxygen

and nutrients, in a process termed vessel co-option (4),

tumors that are beyond this size have increased nutritional

requirements and exist within a hypoxic, metabolically-

deficient microenvironment. Studies over the last four

decades have demonstrated that actively growing tumors

have an absolute requirement for the development of new

blood vessels, which support increases in tumor mass, their

survival and provide an avenue for tumor invasion and

metastasis. Without this process, defined as tumor-

associated angiogenesis (5), apoptotic signal transduction

pathways are triggered, leading to tumor dormancy and

regression.

Growth Factors in Tumor-associated Angiogenesis

Developing tumors are in a prevascular state and exist in a

state of equilibrium between proliferation and apoptosis.

As tumor cells proliferate and their layers accumulate, the

distance between malignant cells within the expanding

tumor mass and the nearest blood vessel increases. When

tumors reach a size of 1-2 mm, tumor cells located at a

distance greater than 200 Ìm from the nearest blood vessel

become hypoxic and are deficient in critical nutrients (6).

Without initiation of tumor-associated angiogenesis, tumor

growth is restricted (7) and tumors become dormant (8). To

allow for tumor expansion, survival and metastasis, tumors

undergo what has been defined as the "angiogenic switch",

which results from a shift in the balance of anti-angiogenic

growth factors that inhibit the formation of new vasculature

to favor the production of pro-angiogenic growth factors

such as VEGF which induce tumor-associated angiogenesis

(9,10) (Figure 1A). Tumor-associated angiogenesis is a

complex process that involves the coordinated activity of a

number of different cell types within the tumor

microenvironment, including tumor cells and stromal cell

populations of vascular endothelial cells and pericytes (11).

While the literature is replete with studies that define the

role of tumor cells during tumor-associated angiogenesis,

the role of vascular endothelial cells is currently being

elucidated. In contrast, the role of pericytes during this

525

Correspondence to: F.M. Robertson, Department of Molecular

Virology, Immunology, and Medical Genetics, 2184 Graves Hall,

333 West 10th Avenue, Columbus, OH 43210, U.S.A. Tel: (614)

499-1833, Fax: (614) 292-0643, e-mail: robertson.48@osu.edu

Key Words: Akt/Protein kinase B, anti-angiogenic drugs,

inositidylphosphate-3-kinase (IP3-K), pericytes, platelet-derived

growth factor beta, (PDGF‚), platelet-derived growth factor

receptor beta, (PDGFR-‚), review, splice variant, angiogenesis,

vascular endothelial cells, vascular endothelial growth factor

(VEGF), vascular targeting.

in vivo 18: 525-542 (2004)

Review

Vascular Endothelial Growth Factor as aSurvival Factor in Tumor-associated Angiogenesis

NESRINE I. AFFARA and FREDIKA M. ROBERTSON

Department of Molecular Virology, Immunology, and Medical Genetics, The Ohio State University, 333 West 10th Avenue, Columbus, OH 43210, U.S.A.

0258-851X/2004 $2.00+.40

process has only recently been recognized and remains to

be completely defined. Figure 1B provides a schematic

representation of tumor-associated angiogenesis and the

role of these individual cell types in this process. Following

the angiogenic switch, tumors acquire an angiogenic

phenotype, produce pro-angiogenic growth factors such as

VEGF and express the associated VEGF receptors, which

confers the ability to respond to these proteins in an

autocrine manner.

VEGF production is stimulated following activation or

mutation of oncogenes such as Bcr-Abl, ras and erbB-

2/HER-2/neu (12-14). In addition, the hypoxic tumor

microenvironment and activation of survival pathways work

synergistically with oncogenes to increase VEGF production

(15,16). As a pro-angiogenic growth factor, VEGF activates

vascular endothelial cells within the capillaries adjacent to

expanding tumors to express specific VEGF receptors which

allow these cells to respond to VEGF. Once activated by

VEGF, vascular endothelial cells secrete proteolytic enzymes

that degrade basement membranes and the extracellular

matrix surrounding the pre-existing blood vessel. Digestion

of these elements allows the vascular endothelial cells to

detach from pericytes that reside beneath the vascular

endothelial cells. Following their detachment, vascular

endothelial cells proliferate and migrate toward the source

of pro-angiogenic growth factor production by invasion

in vivo 18: 525-542 (2004)

526

Figure 1. Schematic of tumor-associated angiogenesis. A) "The angiogenic switch" results from production of inducers of angiogenesis which are pro-angiogenic growth factors with a simultaneous down-regulation of inhibitors of angiogenesis. B) Tumor cells and infiltrating inflammatory cells, includingmast cells, neutrophils and macrophages, produce VEGF splice variants that activate vascular endothelial cells lining adjacent blood vessels. Theendothelial cells then detach from the underlying pericytes and extracellular matrix, proliferate, migrate and then organize and differentiate into capillarytubes which provide oxygen and nutrients to expanding tumors.

through spaces created by enzyme degradation (Figure 1B).

These recruited vascular endothelial cells then secrete

extracellular matrix proteins which provide the building

blocks for the development of new basement membranes

that support the tumor vessels (17,18). In addition, vascular

endothelial cells secrete VEGF and an additional protein,

platelet-derived growth factor beta (PDGF‚), which induce

migration of pericytes into the area of active tumor vessel

formation (19).

The vascular endothelial cells then change from an

actively proliferating population of cells to subsequently

undergo differentiation and organization into hollow tubes

that connect the expanding tumor mass to nearby vascular

beds. Direct interactions between pericytes and vascular

endothelial cells stimulate differentiation of pericytes into

vascular smooth muscle cells that stabilize and maintain the

newly-formed tumor vasculature (20). Blood flow is then

initiated which inhibits the rate of apoptosis within tumors,

providing both a source of oxygen and nutrients and a route

for tumor invasion and metastasis to distant organ sites.

VEGF also acts as a chemotactic factor which results in

recruitment and activation of non-vascular cells, including

inflammatory mast cells, macrophages and neutrophils (Figure

1B) (21-23). Recruited inflammatory cells produce pro-

angiogenic products as well as synthesize matrix metallo-

proteinases (24-29), which has led to these cells being defined

as co-conspirators in tumor-associated angiogenesis (26).

Other cell types have also been identified as playing a role in

tumor-associated angiogenesis such as bone marrow-derived

stromal cells, which respond to pro-angiogenic growth factors,

migrate from the bone marrow to the tumor site and

differentiate into capillary-like structures (30). Additionally,

in a unique process defined as vasculogenic mimicry, tumor

cells within aggressively growing malignancies such as

melanomas trans-differentiate into cells with phenotypic and

functional characteristics of vascular endothelial cells (31).

These cells form a vascular network rich in extracellular

matrix proteins without the need to activate endothelial cells

that are usually required for angiogenesis. It is clear that

further investigation will reveal additional cell types that

contribute to tumor vessel formation as well as elucidate

previously undefined contributions of cells that are known to

play a role in tumor-associated angiogenesis.

Early studies demonstrated the presence of tumor

angiogenesis factors (TAF), which were initially defined as

soluble factors that diffuse into the tumor microenvironment

(32). Although the first pro-angiogenic growth factor

identified was basic fibroblast growth factor (bFGF, FGF-2)

(33), this protein is now believed to be a general angiogenic

growth factor that is not critical for angiogenesis, based on

studies demonstrating that the loss of bFGF does not result

in any significant phenotypic alteration in the vasculature

during embryonic development (34).

The second angiogenic growth factor was identified based

on its ability to induce vascular permeability in guinea pig

skin and was named vascular permeability factor (VPF) (35).

VPF was later found to be similar to proteins purified from

pituitary folliculostellate cells that stimulated proliferation

of cultured endothelial cells and was therefore defined as

vascular endothelial growth factor-A (VEGF-A) (36-38).

Studies using knock-out mice demonstrated that the loss of

only one VEGF-A allele resulted in early embryonic

lethality, indicating that VEGF-A was critical to angiogenesis

during development (39,40). Studies have revealed that

VEGF-A is part of a family of VEGF-related molecules

including VEGF-B (41), VEGF-C (42,43), VEGF-D (44),

VEGF-E (45,46) and placental-derived growth factor (PlGF)

(47). For the purpose of this review, VEGF-A will be

designated as VEGF.

VEGF is recognized as a pivotal growth factor in tumor-

associated angiogenesis (48). Since it can activate and

recruit vascular endothelial cells to undergo all of the

necessary activities required to form new capillary beds,

VEGF has been defined as a complete angiogenic growth

factor (49). VEGF stimulates vasodilation and increases

permeability of pre-existing capillaries, leading to leakage

of plasma proteins (50,51) and deposition of an

extravascular fibrin gel around developing vasculature. This

provides a matrix to support the subsequent proliferation

and migration of vascular endothelial cells (52). As an

additional critical component of its activity as a complete

angiogenic growth factor, VEGF activates signal

transduction pathways that regulate survival of tumor cells

(53-55), vascular endothelial cells (56-58) and pericytes (59),

which exist in the tumor microenvironment that is hypoxic

and deprived of nutrients. The pivotal nature of VEGF to

tumor-associated angiogenesis is demonstrated by the

detection of its presence in all tumors thus far examined and

by the correlation between production of VEGF with tumor

stage and tumor progression (60,61).

In addition to bFGF and VEGF-related proteins, other

pro-angiogenic growth factor families have been identified

which have distinct activities in tumor-associated angiogenesis

and act in concert with VEGF. Angiopoietin-1 (Ang-1)

(62,63), Ang-2 (64), Ang-3 and Ang-4 (65) are members of

the Angiopoietin family of proteins which bind to receptors

within the tyrosine kinase immunoglobulin-like and

epidermal growth factor homology domains (Tie) receptor

family that are expressed by tumor cells, vascular endothelial

cells and pericytes. During embryonic development, ligand

binding of Ang-1 to the Tie-2 receptor tyrosine kinase

increases adherence of vascular endothelial cells to pericytes,

resulting in stabilization of newly-formed blood vessels (66).

In addition, Ang-1 regulates vascular endothelial cell survival,

sprouting and organization into tubules (67). Ang-1 can also

inhibit VEGF-induced permeability and reduce inflammation

Affara and Robertson: VEGF-induced Survival in Tumor Angiogenesis

527

(68). Despite the fact that each of the ligands bind to the

same receptor, Ang-2 and Ang-3 inhibit the Tie-2 tyrosine

phosphorylation induced by Ang-1, which counteracts the

stabilizing effects of Ang-1 (5). Molecular profiling studies

have demonstrated that VEGF up-regulates Ang-2 during

tumor-associated angiogenesis, resulting in destabilization of

existing vasculature, loss of pericytes and subsequent

enhancement of tumor vessel formation (69,70). The co-

operative activities of VEGF and Ang-2 may, in part, account

for the morphologically distinct tortuous blood vessels

formed during tumor-associated angiogenesis and may also

be responsible for the less stable, leaky vessels that develop

adjacent to expanding tumors (71). Although the members of

the Angiopoietin family and their associated Tie-2 receptor

in vivo 18: 525-542 (2004)

528

Figure 2. Structure and functions of human and murine VEGF exons. A) The human VEGF gene gives rise to 7 distinct VEGF splice variants, includingVEGF121, VEGF145, VEGF165, VEGF165b, VEGF183, VEGF189 and VEGF206. VEGF165b, which has inhibitory activity, lacks exons 6 and 8 andcontains a novel 6 amino acid sequence SLTRKD, designated as exon 9. B) The mouse VEGF gene gives rise to splice variants that are all one aminoacid shorter than the corresponding human splice variants. Mouse splice variants include VEGF120, VEGF144, VEGF164 and VEGF188. The uniquemouse splice variant VEGF205* lacks exons 7 and 8 and contains exon 6, which includes a 61 bp extension of exon 6a, designated as exon 6’, whichencodes a unique 7 amino acid carboxyl-terminal tail, YVGAAAV, that is significantly different from the carboxyl-terminal tail of any VEGF proteinidentified to date.

have opposing roles in angiogenesis, it is clear that the

balance in production of these growth factors plays a key role

in regulating tumor vessel formation (72). Disruption of this

delicate balance in relative amounts of Ang-1 or Ang-2 has

been observed to result in significant defects in tumor-

associated angiogenesis (73). Recent evidence suggests that

VEGF, Tie-2, Ang-1 and Ang-2 are simultaneously expressed

during tumor growth, yet are differentially-expressed spatially

within the tumor microenvironment as well as temporally-

expressed during tumor vessel development (71). It is clear

that further studies, which continue to define the roles of the

members of this family of proteins, will provide insight into

their use as therapeutics and as targets for therapeutics, not

only in VEGF-induced tumor-associated angiogenesis, but

also in disorders associated with enhanced inflammatory

responses.

In addition to the ability of tumor cells and vascular

endothelial cells to respond to VEGF and the Angiopoietins

during tumor-associated angiogenesis, pericytes also

respond to pro-angiogenic growth factors during this

process (74). Pericytes produce VEGF (75) as well as

respond to it by expression of VEGF receptors (76),

resulting in enhanced recruitment of pericytes to the local

tumor microenvironment and stabilization of newly-

developed tumor vessels. In addition, vascular endothelial

cells produce PDGF‚. Pericytes, as well as their precursors

Affara and Robertson: VEGF-induced Survival in Tumor Angiogenesis

529

Figure 3. VEGF acts as a survival factor through IP3-K/Akt. IP3-K is activated by binding of VEGF to VEGFR-2. IP3-K then catalyzes the productionof phosphatidylinositol 3,4,5-triphosphate (PI-3,4,5,-P3), which binds to the pleckstrin homology domain (PH) of Akt and PDK-1. Akt is then translocatedfrom the cytoplasm to the plasma membrane and undergoes conformational changes. Akt can then be phosphorylated at Thr308 by PDK1 and at Ser473by ILK. Activated Akt then promotes VEGF-induced endothelial cell survival by phosphorylating and inhibiting the pro-apoptotic proteins of the Forkheadtranscription factors and by increasing the expression of the anti-apoptotic FLICE-inhibitory protein (FLIP). VEGF also promotes increased cell massby activation of mTOR/S6K. Activated Akt also induces cell cycle entry by inactivating glycogen synthase kinase-3 beta (GSK-3‚), which results instabilization of cyclin D1 and by inhibiting p21Cip1/WAF1. LY292004 is a specific inhibitor of IP3-K, while Rapamycin is a selective inhibitor of mTOR.

derived from mesenchymal cells, respond to PDGF‚ viaexpression of the associated receptor tyrosine kinase,

platelet derived growth factor receptor beta (PDGFR-‚).

Further studies are required to more completely define the

interactions of pro-angiogenic growth factors with each of

the cell types involved in tumor-associated angiogenesis.

Structure and Function of Human and MurineVEGF Alternative Splice Variants

VEGF is encoded for by a single gene organized into 8

exons in mice and rats and 9 exons in humans (Figure 2).

Alternative splicing generates distinct peptides named for

the number of amino acids contained within them (77-80).

In humans, the most abundantly produced VEGF splice

variants are VEGF121, VEGF165 and VEGF189 (78) (Figure

2A). The less abundantly produced human VEGF splice

variants identified to date are VEGF145 (80), VEGF183 (81),

and VEGF206 (79). Although the majority of human VEGF

splice variants are pro-angiogenic, an anti-angiogenic splice

variant VEGF165b was found to be down-regulated in renal

tumors, and the loss of VEGF165b coincided with induction

of the angiogenic switch in this tissue (82). VEGF165b lacks

exon 8, which is replaced by exon 9, resulting in the

production of a protein with the same length as VEGF165

but with a carboxyl-terminal tail composed of 6 unique

amino acids, SLTRKD (Figure 2A). A very recent study

reported that VEGF165b is present in differentiated renal

podocytes but was not present in this cell type undergoing

active proliferation, suggesting that alternative splicing of

VEGF pre-mRNA is an integral part of regulating

neovascularization (83). Further studies may reveal the

existence of other human VEGF splice variants with

functional activities that vary from those described to date.

Elucidation of the activities of the distinct VEGF splice

variants may provide insight into their utility as both

therapeutics and therapeutic targets.

In contrast to human VEGF splice variants, murine

VEGF variants are one amino acid shorter than the

analogous human splice variants, but in most cases the

mouse VEGF proteins possess biological activities similar

to human VEGF splice variants (Figure 2B). The murine

VEGF splice variants identified to date include VEGF120,

VEGF144, VEGF164 and VEGF188 (84). In addition to these

murine proteins, our laboratory identified, sequenced and

characterized the activities of a novel murine splice variant

designated as VEGF205* (Genbank Accession AY120866)

(Figure 2B). This VEGF splice variant was first identified

by detection of its mRNA and protein only in mouse skin

papillomas and squamous cell carcinomas, which was

completely undetectable in normal skin (85). VEGF205*

contains exons 1-6 and lacks exons 7 and 8. In place of these

exons is a 61 bp extension to exon 6a, defined as exon 6’

(Figure 2B). Due to the presence of a stop codon in exon

6’, translation of murine VEGF205* mRNA results in the

production of a truncated 145 amino acid protein containing

a carboxyl-terminal tail consisting of 7 unique amino acids,

YVGAAAV. The exon structure of murine VEGF205* and

the resultant VEGF splice variant protein are significantly

different from any other human or mouse VEGF splice

in vivo 18: 525-542 (2004)

530

Figure 4. VEGF205* stimulates phospho-Akt-1-Ser473 which is inhibited by LY294002. A) Western blots of anti-phospho-Akt-1-Ser-473, Akt-1 and ‚-actin. B) Densitometric analysis of Western blots shown in A. Data are shown as the ratio of phospho-Akt-1-Ser473 to Akt-1 and are expressed as mean± S.E., N=3. *, p<0.02.

Affara and Robertson: VEGF-induced Survival in Tumor Angiogenesis

531

Figure 5. Strategies to target cells involved in tumor-associated angiogenesis. A) Targeting tumor cells and tumor-associated endothelial cells. Avastinìis a humanized anti-VEGF monoclonal antibody (MAb VEGF) that binds and neutralizes VEGF. VEGFR-2 chimeric receptors are composed of theextracellular VEGF-binding domain of VEGFR-2 and the intracellular domain of the apoptotic receptor Fas. Following binding of VEGF, apoptosis ofboth tumor cells and vascular endothelial cells is induced. B) Targeting vascular endothelial cells and pericytes. Blockade of Tie-2 and PDGFR-‚ receptortyrosine kinases using Gleevecì/Imatinib, SU6668, or SU11248 targets pericytes and tumor endothelial cells. SU5416 and Avastinì block VEGFR-2. C)Targeting tumor cells, vascular endothelial cells and pericytes. LY294002 can inhibit survival of tumor cells, vascular endothelial cells and pericytes.

in vivo 18: 525-542 (2004)

532

variant described to date, including human VEGF206

(Figure 2 A and B). The size, amino acid sequence of the

carboxyl-terminal tail and biological activities of VEGF205*

are unique.

The biological activity of VEGF splice variants occurs

following receptor-ligand binding and subsequent

transduction of specific signals. Identification of VEGF

receptor families has been crucial to understanding the

functions of the individual VEGF splice variants. Thus far,

there have been two distinct VEGF receptor families

identified, which include the VEGF receptor tyrosine kinase

family and the Neuropilin family. The VEGF receptor

tyrosine kinase family includes VEGF Receptor-1

(VEGFR-1), which was first isolated and characterized as

Fms-like tyrosine kinase-1 (Flt-1) (86-88), VEGFR-2, also

defined as Fetal Liver Kinase 1/Kinase-insert- Domain-

containing Receptor (Flk-1/KDR) (89,90) and VEGFR-3,

first defined as Flt-4 (91).

Despite their structural homology and the ability of

VEGFR-1 and VEGFR-2 to bind VEGF and induce

receptor autophosphorylation at specific tyrosine residues,

these receptors have distinct expression patterns within

different organs, which may overlap, and they also activate

specific signal transduction pathways (92). The differential

ability of VEGF receptor-ligand binding to induce selective

biological activities, including increased vascular permeability

as well as proliferation, migration, differentiation and

organization of vascular endothelial cells into vascular tubules

during tumor-associated angiogenesis, is due to phosphorylation

of specific VEGFR-associated tyrosine residues (93). For

example, following binding of VEGF to VEGFR-2, vascular

endothelial cells undergo proliferation, which is due to

phosphorylation of tyrosine residue 1059, while migration of

these cells is stimulated following phosphorylation of tyrosine

residue 951 (94). Conversely, binding of VEGF to VEGFR-1

inhibits VEGFR-2-mediated endothelial cell proliferation, but

not migration (95). VEGFR-1 can also act as a decoy

receptor that sequesters VEGF, preventing it from binding

to VEGFR-2 (96,97). In contrast to the ability of VEGF

splice variants to differentially bind VEGFR-1 and VEGFR-

2, VEGFR-3 binds only to VEGF-C and VEGF-D, and this

VEGF receptor is primarily involved in, and is critical to,

appropriate development of lymphatic vessels (98). In

addition, VEGFR-3 is expressed on a variety of tumor cells

of different origins and may be useful as a marker of early

stages of tumor progression (42,99). Although further studies

are required to completely define the roles of these VEGF

receptors in tumor-associated angiogenesis, there are a

number of therapeutic approaches that are based on the

interactions of VEGF and VEGFR-2 currently being

developed that target tumor vessels (100-102).

The Neuropilins are a second family of VEGF receptors

whose members identified thus far include neuropilin-1 (NP-1)

(103) and neuropilin-2 (NP-2) (104). NP-1 and NP-2 belong to

a family of collapsins/semaphorins receptors first described as

neuronal axon guidance factors present in developing

embryos (105,106). These receptors are now known to be

expressed by tumor cells, vascular endothelial cells and

pericytes (103,104). Both NP-1 and NP-2 are alternatively

spliced (107) and contain short intracellular domains which,

at least in part, confer the biological activities of these

receptors (108). NP-1 and NP-2 bind VEGF165 as well as

the heparin-binding form of PlGF, PlGF-2 (109) and

VEGF-B (110). NP-1 acts as a co-receptor for VEGFR-2

(103), resulting in enhanced binding affinity of VEGF165 to

VEGFR-2 and effectively increasing the local concentration

of VEGFR-2 at the cell surface (111,112). Only NP-2 can

bind VEGF145 (104). In contrast, neither NP-1 nor NP-2

binds VEGF121, a VEGF splice variant which lacks the

ability to bind heparin and heparan sulfate proteoglycans

(103,104). These observations have prompted speculation

that one of the primary functions of NP-1 and NP-2 is to act

as receptors for specific VEGF splice variants. Taken

together with a recent report that blocking NP-1 effectively

inhibits angiogenesis in murine retinal neovascularization

(113), these studies suggest that the Neuropilin receptors

may serve as targets for the development of VEGF splice

variant-specific anti-angiogenic therapies. Further studies are

necessary to determine whether there are other members of

this receptor family, to fully elucidate the role of these

receptors in tumor-associated angiogenesis and to determine

if these receptors can be used to target this process.

Production of multiple VEGF splice variants allows this

one group of proteins to regulate specific functions of tumor

cells, vascular endothelial cells and pericytes, resulting in

precise regulation of tumor-associated angiogenesis

(49,55,58,59). In part, the distinct biological activities of

VEGF splice variants are determined by the exons present

within the splice variant pre-mRNA (Figure 2 A and B).

Exon 1 encodes for the signal sequence common to all

VEGF splice variants. The presence of Exon 1 indicates that

each of the splice variant proteins can be secreted. Exon 2

encodes for a specific N-terminal sequence which is also

common to all known human and rodent VEGF splice

variants. Exon 3 is also contained within all known VEGF

splice variants and encodes for VEGF dimerization sites

and for binding to VEGFR-1 (86-88). Exon 4 encodes for

binding of VEGF alternative splice variants to VEGFR-2

(89,90). Exon 5 encodes for a plasmin cleavage site, which

allows production of smaller VEGF peptide fragments

through proteolytic cleavage (114,115). Exon 6 encodes for

binding of VEGF splice variants to heparin and heparan

sulfate proteoglycans on the surface of cells (116). Exon 6

is either not present, as occurs with murine VEGF120 and

VEGF164 (Figure 2B), and human VEGF165b (Figure 2A),

or is significantly altered by loss of part of exon 6 designated

as exon 6b, which occurs in murine VEGF188 and VEGF144

(Figure 2B). Exon 6 is also altered by extension of 6a,

designated as exon 6’, which occurs in VEGF205* (Figure

2B). Exon 7 encodes for an additional heparin- binding

domain (116) and it is also lost in a number of VEGF splice

variants including murine VEGF120, VEGF144 and

VEGF205*. The presence or absence of exons 6 and 7

determines the ability of the specific VEGF splice variants

to either freely diffuse within the tumor microenvironment

or to bind heparin and heparan sulfate proteoglycans,

therefore remaining sequestered and acting as slow release

molecules. For example, VEGF121 lacks the charged basic

amino acids encoded by exons 6 and 7 and, therefore, is a

soluble growth factor that does not bind heparin (114)

(Figure 2A). In contrast, VEGF189 contains exons 6 and 7,

and has a high affinity for binding heparin, resulting in

sequestration of this splice variant by the extracellular

matrix and heparan sulfate proteoglycans on the cell surface

(115). Other VEGF splice variants that contain either exon

6 or exon 7 but not both possess only one of the two

heparin-binding domains (Figure 2B). VEGF splice variants

with this exon configuration, such as VEGF164, can both

freely diffuse in the area surrounding the tumor as well as

remain bound to the cell surface and extracellular matrix.

The number of VEGF splice variants which have deletions,

insertions, or alterations in either exon 6 and/or exon 7

suggests that production of VEGF splice variants that

differentially bind heparin or freely diffuse is tightly

regulated during angiogenesis. For example, the exon

structure of the unique murine VEGF205* splice variant

predicts that it is actively secreted based on the presence of

exon 1, that it can dimerize and bind both VEGFR-1 and

VEGFR-2 based on the presence of exons 3 and 4.

Additionally, VEGF205* is predicted to bind heparin and

heparan sulfate proteoglycans based on the presence of

exon 6, resulting in its retention within the local tumor

microenvironment. Since VEGF205* contains an extension

of exon 6a, designated as 6’, that encodes for a protein with

a novel carboxyl-terminus tail, this VEGF splice variant is

expected to have activities that differ from other VEGF

splice variants. We have defined one such activity of

VEGF205*, which is stimulation of survival pathways of

vascular endothelial cells.

VEGF-induced Signal Transduction Pathways

VEGF splice variants act as survival factors through the

inositidylphosphate-3-kinase (IP3-K)/Akt signal transduction

pathway (57,58,117). Akt (also known as Protein kinase B) is

a serine/threonine kinase with homology to both protein

kinase C (PKC) and cyclic-AMP-dependent protein kinase A

(PKA) (118-120). As shown in Figure 3, Akt is a core

component of the IP3-K signaling pathway (121,122) and is

composed of an amino-terminal pleckstrin homology (PH)

domain that binds phospholipids (123), a kinase domain, and

a proline-rich carboxyl-terminal regulatory domain. In

response to growth factors including VEGF splice variants,

IP3-K is recruited to the inner surface of the plasma

membrane resulting in the generation of the membrane-

bound lipid phosphatidylinositol 3,4,5-triphosphate (PIP3)

(57,124,125) (Figure 3). Following binding of PIP3 to the PH

domain, Akt is translocated from the cytoplasm to the inner

surface of the plasma membrane (126). This results in

conformational changes rendering Akt accessible to

phosphorylation at two amino acid residues, threonine-308

(Thr-308), which lies within the Akt kinase domain and

serine-473 (Ser-473), which is located within the Akt

regulatory domain (127). Simultaneous phosphorylation of

Akt at Thr-308 and Ser-473 (128) is carried out by two

distinct kinases: 3-phosphoinositide-dependent protein

kinase-1 (PDK-1), which phosphorylates Thr-308 (129) and

integrin-linked kinase-1 (ILK-1), which phosphorylates Ser-

473 (130-132) (Figure 3).

The majority of studies that have examined VEGF as a

survival factor have used human VEGF165 splice variant

(57,117). Activation of IP3-K/Akt by VEGF165 occurs

through its selective association with VEGFR-2 (133,134).

VEGF activates Akt, resulting in protection of cells against

apoptosis by inducing the expression of anti-apoptotic

proteins, including FLICE-inhibitory protein (FLIP), an

inhibitor of FLICE/caspase 8. Inhibition of FLICE protects

vascular endothelial cells from apoptosis induced by Fas-

mediated caspase 8 activation (135). VEGF also stimulates

phosphorylation and subsequent inactivation of pro-

apoptotic proteins within the family of Forkhead

transcription factors, which occurs through an IP3-K/Akt-

dependent pathway (Figure 3) (136).

Although the majority of studies to date have focused on

the role of Akt as a survival factor, Akt can also act as a

multi-functional protein kinase which regulates both cell

proliferation and increases in cell mass (Figure 3) (137). Akt

phosphorylates and consequently blocks the activity of

glycogen synthase kinase-3 beta (GSK-3‚), thereby

preventing degradation of cyclin D1, which is a protein

required for progression of cells from the G1-phase of the

cell cycle into the S-phase (Figure 3) (138). In addition, Akt

regulates cell proliferation by phosphorylating the cell cycle

inhibitory protein, p21Cip1/WAF1, which inhibits interactions

between p21Cip1/WAF1 and proliferating cell nuclear antigen

(PCNA), thereby reversing repression of DNA replication

(139). In addition to its ability to regulate cell proliferation,

Akt can control cell mass by directly activating the

mammalian target of rapamycin (mTOR), also known as

FK506-binding protein-rapamycin-associated protein,

FRAP1 (137,140,141). mTOR was identified through

characterization of the activity of Rapamycin (Sirolimus;

Affara and Robertson: VEGF-induced Survival in Tumor Angiogenesis

533

Rapamune), a macrolide antibiotic isolated from a strain of

Streptomyces hygroscopicus contained in a soil sample

collected on Easter Island, Rapa Nui. mTOR phosphorylates

and activates ribosomal protein S6 kinase (S6K), which

stimulates translation of mRNAs that regulate cell mass and

regulate the cell-cycle such as cyclin D1 (142,143). In

addition, activated Akt can directly phosphorylate S6 kinase.

Taken together, these studies demonstrate that VEGF can

induce signal transduction pathways associated with survival,

proliferation and expansion in cell mass (144) (Figure 3).

Our laboratory has found that the IP3-K/Akt pathway is

activated by specific VEGF splice variants, suggesting that

these variants can selectively block apoptosis, allowing

proliferation of cells involved in tumor-associated

angiogenesis, which leads to increased tumor mass. As

shown in Figure 4, the novel murine VEGF splice variant

VEGF205* (50 ng/ml), but not VEGF120, led to a 3.5-fold

increase (p<0.02) in Akt activation in human vascular

endothelial cells. This response was measured by the

quantitation of phosphorylation levels of Akt at Serine-473

residue by means of a phospho-specific antibody, which was

inhibited by the specific IP3-K inhibitor, LY294002 (145).

These results demonstrate that recombinant VEGF205*

selectively stimulates the IP3-K signal transduction pathway

in vascular endothelial cells. Activation of the IP3-K/Akt

pathway by distinct VEGF splice variants may lead to

multiple biological effects which are dependent upon the

cell types that respond to VEGF through expression of

specific receptors, as well as on the stage of tumor-

associated angiogenesis during which specific VEGF splice

variants activate Akt.

Targeting Tumor-associated Angiogenesis UsingVEGF-directed Strategies

The recognition that tumors undergoing active expansion

are dependent upon tumor-associated angiogenesis has

brought significant advances in anti-tumor therapy. Instead

of using conventional chemotherapeutics, which target all

cells undergoing active proliferation, including bone marrow

and the gastrointestinal tract, approaches that selectively

target tumor vessels are now being developed. Tumors are

genetically unstable and accumulate mutations as they

progress, invade and metastasize. In contrast, vascular

endothelial cells and pericytes within the stromal

compartment are genetically stable. Since agents that block

tumor-associated angiogenesis are primarily targeted at cell

populations within the stromal compartment, they bypass

the development of drug resistance by tumor cells that

commonly occurs with administration of conventional

chemotherapeutic drugs (146,147). Furthermore,

endothelial cells lining the inner surface of tumor vessels are

readily accessible to systemically delivered anti-angiogenic

drugs, which is in contrast to standard anti-tumor

chemotherapies given intravenously that do not reach all of

the tumor cells within a large tumor mass (148). The

approaches that are now being explored to block tumor-

associated angiogenesis are relatively new and, although

some show promise, approval for widespread common

clinical use has not yet been obtained.

Since both tumor cells and vascular endothelial cells

respond to VEGF, the first approach to block tumor growth

would be to simultaneously target both of these cell types

(Figure 5A). One such approach under clinical evaluation,

which shows promise as an adjunct to conventional

chemotherapeutic regimens, is recombinant humanized

monoclonal anti-VEGF165 antibody (rhuMAb VEGF),

Avastinì (Bevacizumab; Genentech, San Francisco, CA,

USA) (102). Clinical trials are currently ongoing using

Avastinì in patients with metastatic colorectal carcinoma

in combination with a conventional chemotherapy regimen

of 5-Fluorouracil/Leucovorin (149,150). There are also

other clinical trials now ongoing including phase III clinical

trials using Avastinì in combination with cytotoxic

chemotherapies for treatment of metastatic breast cancer,

lung cancer and renal cell cancer, and phase II clinical trials

using Avastinì as an adjunct to conventional treatment

regimens in a variety of other malignancies (149,151,152).

Widespread use of Avastinì awaits approval by the U.S.

Food and Drug Administration, which is predicted to occur

within 2004 (http://www.gene.com/gene/pipeline/status/

oncology/avastin.

While Avastinì was initially developed to target tumor-

associated vascular endothelial cells, the successful results

of clinical trials using this treatment approach as well as our

continued understanding of the role of pericytes in tumor-

associated angiogenesis suggest that Avastinì may inhibit

pericyte functions during this process (Figure 5B), and may

also inhibit VEGF-induced activation of survival pathways

in tumor cells (Figure 5A). Additional targeting strategies

currently being developed include the use of small

interfering RNA (SiRNA) (153,154) and anti-sense therapy

(155,156) to knock-down VEGF gene expression, which

suppresses VEGF synthesis and reduces tumor growth.

Since both tumor cells and vascular endothelial cells

express VEGFR-2, development of chimeric receptors

consisting of the extracellular VEGF-binding domain of

VEGFR-2 combined with the intracellular domain of a

death-triggering molecule would reverse VEGF-induced

survival of both of these cell populations (Figure 5A). For

example, VEGF-induced apoptosis of vascular endothelial

cells was triggered following transfection of these cells with

a chimeric receptor composed of the extracellular VEGF-

binding domain of VEGFR-2, and the intracellular domain

of the apoptotic receptor Fas (157). Another therapeutic

strategy is the use of nuclease-resistant ribozymes directed

in vivo 18: 525-542 (2004)

534

against VEGFR-2, which degrades mRNA encoding for that

receptor. One such anti-VEGFR-2 ribozyme therapeutic is

Angiozyme (Ribozyme, Boulder, CO, USA), which is

currently being tested in phase II clinical trials (158).

An alternative anti-angiogenic strategy, that has been

defined as antiangiogenic scheduling or metrononomic

scheduling of chemotherapy, is based on administration of

cytotoxic drugs at lower doses and for longer times than is

conventionally used (159). This approach is based on the

observation that apoptosis of vascular endothelial cells

preceded apoptosis of tumor cells following standard

cytotoxic chemotherapy (160). However, during the

treatment-free interval that is required for recovery of bone

marrow when administering standard chemotherapy at the

maximum tolerated dose, vascular endothelial cells involved

in tumor-associated angiogenesis resumed rapid proliferation,

thus supporting tumor regrowth. In addition tumor cells can

acquire drug resistance during this treatment-free interval. To

overcome tumor recurrence, cytotoxic drugs such as

cyclophosphamide combined with the anti-angiogenic agent

TNP-470 were administered at lower doses and at regular

intervals. This anti-angiogenic scheduling provided maximum

and continuous exposure of tumor-associated vascular

endothelial cells to an anti-angiogenic drug and low doses of

a cytotoxic anti-tumor drug (159). This scheduling strategy

resulted in sustained tumor regression due to loss of tumor

vessels, which occurred prior to acquisition of resistance to

cytotoxic chemotherapeutic drugs by tumor cells (159-161).

Tumor vessels development occurs due to the coordinated

activity of vascular endothelial cells and pericytes. One

current strategy in anti-angiogenic therapy is to target both

of these cell types that comprise capillary sprouts formed

during tumor-associated angiogenesis (162-164) (Figure 5B).

In contrast to vascular endothelial cells in quiescent and

mature vasculature, during active tumor-associated

angiogenesis these cells secrete PDGF‚, whose receptor,

PDGFR-‚ is selectively expressed on the surface of

pericytes. In contrast, neither PDGF‚ nor PDGFR-‚ are

produced or expressed by tumor cells. Therefore, targeting

pericytes by inhibiting the PDGFR-‚ receptor tyrosine

kinase with simultaneous inhibition of the VEGFR-2

receptor tyrosine kinase expressed by both vascular

endothelial cells and pericytes would block activation of

VEGF-induced survival pathways resulting in apoptosis of

these cells. Ultimately, this strategy would result in

diminished tumor-associated angiogenesis leading to tumor

regression (Figure 5B). Use of inhibitors of PDGFR-‚ and

VEGFR-2 is a newly-described therapeutic approach that

was recently defined as a multiplex receptor-targeting

strategy (162,165). Regression of tumors was observed

following treatment with either SU6668 (Sugen/Pharmacia

& Upjohn, Bridgewater, NJ, USA), or Gleevecì (STI571/

Imatinib; Novartis Pharmaceuticals, East Hanover, NJ,

USA) (166), which inhibit the PDGFR-‚ receptor tyrosine

kinase expressed by pericytes, in combination with SU5416

(Sugen), which inhibits VEGFR-2 receptor tyrosine kinase

that is expressed by both vascular endothelial cells and

pericytes, as well as inhibits production of matrix- degrading

enzymes produced during tumor vessel formation (162,165).

This combination of inhibitors disrupted pericyte-endothelial

associations within tumors to a greater extent than either of

the single agents alone. Using a similar strategy, inhibition

of the Tie-2 receptor tyrosine kinase was observed to

significantly decrease tumor-associated angiogenesis and

diminish tumor growth (167,168). Since Tie-2 and VEGFR-

2 share homology in their ATP binding kinase domains

(169), compounds that simultaneously target homologous

regions contained within both of these receptors may hold

promise as effective anti-angiogenic therapies. For example,

nakijiquinone C-derived analogues are being developed that

simultaneously target the ATP binding kinase domains of

these two receptors (169). Taken together, these studies

clearly indicate that combinatorial strategies that take

advantage of our understanding of the complex nature of

tumor-associated angiogenesis and target the multiple cell

populations, receptors, growth factors and signal

transduction pathways involved in this process will be

significantly more effective than the use of single agents.

The last strategy to be discussed for targeting VEGF-

induced tumor-associated angiogenesis involves targeting

pathways that are shared by all three cell types involved in

tumor vessel formation (Figure 5C). Until tumor angio-

genesis is completed and the activated vascular endothelial

cells organize into functional new blood vessels, all of these

cells including tumor cells, vascular endothelial cells and

pericytes are within a hypoxic and nutritionally-deprived

milieu and are at risk of undergoing apoptosis. Since

VEGF-induced IP3-K/Akt signaling is a survival pathway

common to all of these cell types, targeting this pathway

may represent a successful therapeutic strategy to

simultaneously induce apoptosis of each of these cell types

(Figure 5C). Recent pre-clinical data support the anti-

angiogenic properties of LY294002 (145), which was

observed to inhibit survival of vascular endothelial cells by

inducing the p53 tumor suppressor gene and stimulating

expression of p21Cip1/WAF1 (170). Alternatives to LY294002

that also target the IP3-K/Akt signaling pathway include

agents that inhibit the serine/threonine kinase mTOR

(Figure 3), such as Rapamycin and its ester derivative, cell

cycle inhibitor-779 (CCI-779; Wyeth, Madison, NJ, USA).

These agents have been shown to have both anti-tumor and

anti-angiogenic activities (171,172).

The ability of tumor cells to trans-differentiate into cells

that have capabilities of vascular endothelial cells increases

the complexity of development of therapeutic strategies to

target the tumor vasculature. Recent studies demonstrated

Affara and Robertson: VEGF-induced Survival in Tumor Angiogenesis

535

that the anti-angiogenic inhibitor Endostatin, which is a

20 kD proteolytic fragment of collagen type XVIII, failed

to inhibit tumor-derived vascular networks that arose from

vasculogenic mimicry (31). However, the identification of

IP3-K/Akt as an essential regulatory survival pathway

common to all of the cell types now recognized to be

involved in tumor-associated angiogenesis holds promise

as a means of targeting this process, regardless of the

specific cell population involved in formation of tumor

vessels (Figure 5C) (173).

While the use of agents that target Akt-associated

survival and proliferation pathways hold promise, systemic

treatment with Akt inhibitors can have severe side-effects

including cardiotoxicity (174) and toxicity to the nervous

system (175). To overcome such limitations, new drug-

delivery strategies include conjugating the IP3-

K/Akt/mTOR pathway inhibitors, LY294002 and

Rapamycin, to peptides that home to tumor vessels (Figure

5C). This drug-delivery strategy takes advantage of the fact

that tumor-associated vascular endothelial cells express

specific surface markers, defined as molecular addresses,

that are absent in mature, quiescent blood vessels (176-178).

For example, tumor-associated vascular endothelial cells

express integrins such as ·V‚3 and ·V‚5, which enable their

attachment to extracellular matrix proteins (177,179). A

technique based on in vivo phage display technology can be

used to deliver therapeutic agents to tumor-associated blood

vessels (Figure 5C). This uses small synthetic peptides

displayed within the protein coat of M13 filamentous

bacteriophage, which act as homing ligands that direct anti-

angiogenic agents, cytotoxic drugs and/or pro-apoptotic

molecules to the integrins expressed only on tumor-

associated vascular endothelial cells (180). In contrast to the

multiple studies that have defined proteins present on

vascular endothelial cells during tumor vessel formation,

peptides that are differentially-expressed by tumor-

associated pericytes are only now being identified (181).

Following elucidation of the molecular addresses present on

cells involved in tumor vessel development and stabilization,

other therapeutic opportunities will be available to

differentially target tumor-associated pericytes by combining

this directed drug-delivery strategy with agents such as

inhibitors of the VEGFR-2 and PDGFR-‚ receptor tyrosine

kinases, which include SU5416, SU6668, SU11248 (182) and

Gleevecì/Imatinib (Figure 5B).

Development of improved strategies for treatment of

tumors is dependent upon our continued understanding of

the complexity of tumor-associated angiogenesis. Successful

therapeutic approaches will probably target pathways that

are common to the cell populations known to be involved

in this process, which will be used in combination with

enhanced drug-delivery methods. This has the potential to

significantly improve the efficacy of anti-tumor therapy by

targeting tumor-associated vasculature without affecting the

angiogenesis that occurs under normal conditions such as

wound healing. These approaches will also limit the toxicity

and drug resistance that are commonly observed using

conventional treatment protocols.

Conclusion

Development of new blood vessels is required for tumor

survival and expansion of the tumor mass. It is clear that we

are on the verge of defining the cell types involved in tumor-

associated angiogenesis. In addition, the growth factors and

their associated receptors expressed by specific cell types

involved in this process, that work in concert with VEGF

splice variants to precisely co-regulate the temporal

sequence of events critical to tumor-associated angiogenesis,

are only now being defined and their function elucidated.

As this field emerges, our improved understanding of the

role of VEGF proteins in tumor-associated angiogenesis will

continue to provide opportunities to enhance the selectivity

and specificity of anti-angiogenic and anti-tumor

therapeutics.

Acknowledgements

The authors acknowledge Dr. Robert M. Snapka (The Ohio State

University, Columbus, Ohio, USA) for critical reading of the

manuscript and helpful discussion.

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Received February 2, 2004Accepted May 14, 2004

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