Vascular endothelial growth factor (VEGF) signaling in tumor progression.

Abstract

Vascular endothelial cells are ordinarily quiescent in adult humans and divide less than once per decade. When tumors reach a size of about 0.2-2.0mm in diameter, they become hypoxic and limited in size in the absence of angiogenesis. There are about 30 endogenous pro-angiogenic factors and about 30 endogenous anti-angiogenic factors. In order to increase in size, tumors undergo an angiogenic switch where the action of pro-angiogenic factors predominates, resulting in angiogenesis and tumor progression. One mechanism for driving angiogenesis results from the increased production of vascular endothelial growth factor (VEGF) following up-regulation of the hypoxia-inducible transcription factor. The human VEGF family consists of VEGF (VEGF-A), VEGF-B, VEGF-C, VEGF-D, and placental growth factor (PlGF). The VEGF family of receptors consists of three protein-tyrosine kinases and two non-protein kinase receptors (neuropilin-1 and -2). Owing to the importance of angiogenesis in tumor progression, inhibition of VEGF signaling represents an attractive cancer treatment.

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Critical Reviews in Oncology/Hematology 62 (2007) 179–213
Vascular endothelial growth factor (VEGF)
signaling in tumor progression
Robert Roskoski Jr.∗
Blue Ridge Institute for Medical Research, 3754 Brevard Road, Suite 116A, Box 19, Horse Shoe, NC 28742, USA
Accepted 29 January 2007
Contents
1.Vasculogenesis and angiogenesis.......................................................................................
1.1.Definitions ....................................................................................................
1.2.Physiological and non-physiological angiogenesis.................................................................
1.3. Activators and inhibitors of angiogenesis .........................................................................
1.4.Sprouting and non-sprouting angiogenesis........................................................................
1.5. Tumor vessel morphology.......................................................................................
The vascular endothelial growth factor (VEGF) family ...................................................................
Properties and expression of the VEGF family ..........................................................................
3.1.VEGF-A ......................................................................................................
3.2.VEGF-B ......................................................................................................
3.3.VEGF-C ......................................................................................................
3.4.VEGF-D ......................................................................................................
3.5.Placental growth factor (PlGF) ..................................................................................
3.6.VEGF-E ......................................................................................................
VEGF receptors......................................................................................................
4.1. VEGFR1 (Flt-1) ...............................................................................................
4.2.VEGFR2 (Flk-1/KDR) .........................................................................................
4.3. VEGFR3 (Flt-4) ...............................................................................................
4.4.Neuropilin-1 and -2 ............................................................................................
4.4.1.Properties and expression ...............................................................................
4.4.2. Tumor progression .....................................................................................
4.5.Essential nature of the VEGF receptors...........................................................................
Proteolysis of VEGF isoforms and release from heparan sulfate proteoglycans..............................................
5.1.VEGF isoforms................................................................................................
5.2. Plasminogen activators, plasmin, and matrix metalloproteases ......................................................
5.3. VEGF isoform proteolysis by plasmin............................................................................
5.4. VEGF isoform proteolysis by urokinase type of plasminogen activator ..............................................
5.5.VEGF isoform proteolysis by matrix metalloproteases .............................................................
5.6. Differential stimulation of VEGF isoform action by heparin ........................................................
Phenotypes of mice expressing specific VEGF isoforms..................................................................
Regulation of VEGF gene expression by oxygen, growth factors, and oncogenes............................................
7.1. Hypoxia-inducible transcription factor (HIF) family ...............................................................
7.2.HIF-1? prolyl hydroxylation and proteosomal degradation .........................................................
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∗Tel.: +1 828 891 5637; fax: +1 828 890 8130.
E-mail address: rrj@brimr.org.
1040-8428/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.critrevonc.2007.01.006

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7.3.
7.4.
7.5.
7.6.
VEGF and tumor progression..........................................................................................
8.1.Tumor growth and angiogenesis .................................................................................
8.2. VEGF expression in tumors.....................................................................................
Inhibition of VEGF family signaling ...................................................................................
9.1.Anti-VEGF antibodies..........................................................................................
9.2.VEGF traps (genetically engineered VEGF-binding proteins).......................................................
9.3.VEGF receptor protein-tyrosine kinase inhibitors..................................................................
Tumor metastasis, the pre-metastatic niche, and VEGFR1 ...............................................................
VEGF and vascular endothelial cell survival ...........................................................................
Epilogue ...........................................................................................................
Reviewer............................................................................................................
Acknowledgements...................................................................................................
References ..........................................................................................................
HIF-1? asparaginyl hydroxylation and transcription ...............................................................
Responses to hypoxia...........................................................................................
Growth factors and hormones ...................................................................................
Oncogenes ....................................................................................................
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Abstract
Vascularendothelialcellsareordinarilyquiescentinadulthumansanddividelessthanonceperdecade.Whentumorsreachasizeofabout
0.2–2.0mmindiameter,theybecomehypoxicandlimitedinsizeintheabsenceofangiogenesis.Thereareabout30endogenouspro-angiogenic
factorsandabout30endogenousanti-angiogenicfactors.Inordertoincreaseinsize,tumorsundergoanangiogenicswitchwheretheactionof
pro-angiogenic factors predominates, resulting in angiogenesis and tumor progression. One mechanism for driving angiogenesis results from
the increased production of vascular endothelial growth factor (VEGF) following up-regulation of the hypoxia-inducible transcription factor.
The human VEGF family consists of VEGF (VEGF-A), VEGF-B, VEGF-C, VEGF-D, and placental growth factor (PlGF). The VEGF family
of receptors consists of three protein-tyrosine kinases and two non-protein kinase receptors (neuropilin-1 and -2). Owing to the importance
of angiogenesis in tumor progression, inhibition of VEGF signaling represents an attractive cancer treatment.
© 2007 Elsevier Ireland Ltd. All rights reserved.
Keywords: Angiogenesis; Hypoxia; Neuropilin; Proteolysis; Receptor protein-tyrosine kinase; Vasculogenesis
1. Vasculogenesis and angiogenesis
1.1. Definitions
The intricately branched circulatory network of vascular
endothelial and supporting cells is essential for transport-
ing oxygen, nutrients, and signaling molecules to and the
removal of carbon dioxide and metabolic end products
from cells, tissues, and organs [1]. Neovascularization, or
new blood vessel formation, is divided into two com-
ponents: vasculogenesis and angiogenesis. Embryonic or
classical vasculogenesis is the process of new blood ves-
sel formation from hemangioblasts that differentiate into
blood cells and mature endothelial cells [2]. In the embryo
and yolk sac, early blood vessels develop by aggrega-
tion of angioblasts into a primitive network of simple
endothelial tubes [3]. As primitive vessels are remodeled
into a functioning circulatory system, they undergo local-
ized proliferation and regression, as well as branching
and migration. In contrast, angiogenesis is the process of
new blood vessel formation from pre-existing vascular net-
works by capillary sprouting. During this process, mature
endothelial cells divide and are incorporated into new capil-
laries. Vascular endothelial growth factor (VEGF) signaling
is required for the full execution of vasculogenesis and
angiogenesis.
Many observations associated with tissue ischemia and
tumor formation are consistent with the concept that vascu-
logenesis also occurs during postnatal vessel development
[4]. Asahara et al. were the first to describe the existence
of endothelial progenitor cells in adult human blood that
can differentiate into endothelial cells [5]. These progenitor
cells normally reside in the bone marrow but may become
mobilized into the circulation by cytokine or angiogenic
growthfactorsignals[6].Duringadultvasculogenesis,mobi-
lizedprogenitorcellspromotevesselformationbyintegrating
into vessels and by supplying growth factors. Bone-marrow-
derived endothelial progenitor cells may be recruited to sites
of infarction, ischemia, or tissue trauma where they differ-
entiate into mature endothelial cells and combine with other
cells to form new vessels. These findings suggest that vascu-
logenesis and angiogenesis might constitute complementary
mechanismsforpostnatalneovascularization.Notallstudies,
however,supporttheconceptofadultvasculogenesis[7],and

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181
additional work will be required to sort out the inconsisten-
cies.
1.2. Physiological and non-physiological angiogenesis
Adult human vascular endothelial cells constitute an esti-
mated 1kg of tissue and line the vessels of every organ [1].
These endothelial cells correspond to an estimated surface
area of 1000m2, about the size of a tennis court [8]. In
adult humans, most endothelial cells are quiescent; only 1
in every 10,000 endothelial cells is in the cell division cycle
at any one time [9]. However, there is an increased rate of
endothelialcellmitosisandangiogenesisduringwoundheal-
ingandtissuerepair,duringovariancorpusluteumformation,
and during placental development establishing pregnancy
[10].Inhibitionofangiogenesisrepresentsapotentialtherapy
for disorders with non-physiological angiogenesis includ-
ing neovascular age-related macular degeneration of the eye,
diabetic retinopathy, endometriosis, psoriasis, rheumatoid
arthritis, and tumor growth and metastasis [10]. Decipher-
ing the mechanisms of developmental, physiological, and
aberrant angiogenesis has assumed considerable biomedical
importance during the past 35 years.
1.3. Activators and inhibitors of angiogenesis
Angiogenesis, which is regulated by both endogenous
activators and inhibitors, is under stringent control [9]. There
are about 30 known endogenous pro-angiogenic factors, sev-
eral of which are listed in Table 1. Three families of receptor
protein-tyrosine kinases play pivotal roles in vasculogenesis
and angiogenesis. The VEGF/VEGFR (vascular endothelial
growthfactor/VEGFreceptor)familyisthemoststudiedreg-
ulator of vascular development, and it is the central focus
of this review. The angiopoietin/Tie system controls vessel
maturation and quiescence [11] while the eph/Ephrin system
controls positional guidance cues and arterio-venous asym-
metry [12]. Acidic and basic fibroblast growth factors also
play important and well-studied roles in angiogenesis [13].
There are about 30 endogenous anti-angiogenic factors;
several of these are listed in Table 2. The most studied neg-
ative regulators include angiostatin [15], endostatin [16],
and thrombospondin [17]. Under most physiological con-
ditions in mature animals, the action of negative regulators
predominates and angiogenesis is quiescent. Under certain
pathological conditions, for example, during tumor progres-
sion, the vasculature undergoes the so-called angiogenic
switch, the action of positive regulators predominates, and
angiogenesisisactive[9].Inthecontextofthisreview,tumor
progressionrepresentstheprocessoftumorgrowthoccurring
in conjunction with new blood vessel formation.
1.4. Sprouting and non-sprouting angiogenesis
Angiogenesis in tumors and elsewhere is an intricate
process that involves interactions between regulatory and
Table 1
Selected endogenous pro-angiogenic factors
FactorMW (kDa)a
Swiss prot
accession no.
Acidic fibroblast growth
factor (aFGF, FGF1)b
Angiogeninb
Angiopoietin-1
Angiopoietin-2
Basic fibroblast growth factor
(bFGF, FGF2)b
Ephrin-A1
Ephrin-B1
Ephrin-B2
Epidermal growth factor
(EGF)b
Granulocyte
colony-stimulating factor
(GCSF)
Macrophage-granulocyte
colony-stimulating factor
(GM-CSF)
Hepatic growth factor (HGF,
scatter factor)b
Interleukin-8 (Il-8, CXCL8)b
Leptin
Placental growth factor
(PlGF)b
Platelet-derived endothelial
growth factor (PD-EGF)b
Platelet-derived growth
factor-A (PDGF-A)b
Platelet-derived growth
factor-B (PDGF-B)b
Transforming growth factor-?
(TGF-?)b
Transforming growth factor-?
(TGF-?)b
Tumor necrosis factor
(TNF-?)b
Vascular endothelial growth
factor (VEGF-A)b
VEGF-Bb
VEGF-Cb
VEGF-Db
17.5P05230
16.6
57.5
56.9
17.3
P03950
Q15389
O15123
P09038
23.8
38.0
36.9
134
P20827
P98172
P52799
P01133
16.3 P09919
16.3 P04141
83.1 P14210
11.1
18.6
24.8
P10145
P41159
P49763
50.0 P19971
24.0 P04085
27.3P01127
17.0 P01135
44.3P01137
25.6 P01375
27.0P15692
21.6
46.9
40.4
P49765
P49767
O43915
aMolecular weight (MW) corresponding to the unprocessed human pre-
cursor.
bCommonly found in human tumors.
effector molecules. Pepper divided classical angiogenesis
into a phase of sprouting and a phase of resolution [18]. The
phase of sprouting consists of six components: (i) increased
vascularpermeabilityandextravascularfibrindeposition,(ii)
vessel wall disassembly, (iii) basement membrane degrada-
tion, (iv) cell migration and extracellular matrix invasion, (v)
endothelialcellproliferation,and(vi)capillarylumenforma-
tion. The phase of resolution consists of five components: (i)
inhibition of endothelial cell proliferation, (ii) cessation of
cell migration, (iii) basement membrane reconstitution, (iv)
junctional complex maturation, and (v) vessel wall assembly
including recruitment and differentiation of smooth muscle
cells and pericytes, both of which are mural cells (mural,
wall).

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Table 2
Selected endogenous anti-angiogenic factorsa
InhibitorDescriptionMW (kDa)b
Swiss prot
accession no.
(A) Derived from the extracellular matrix
Anastellin
Arresten
Canstatin
Chondromodulin-1
EFC-XV
Endorepellin
Fragment of fibronectin
Fragment of type IV collagen ?1 chain
Fragment of type IV collagen ?2 chain
Secreted cartilage glycoprotein
Endostatin-like fragment from type XV collagen
Fragment of perlecan, a basement membrane-specific
heparan-sulfate-proteoglycan core protein
Fragment of collagen type XVIII (residues 1334–1516)
Fibulins 1–5 are secreted extracellular matrix and
basement membrane proteins
263
161
168
37.1
142
469
P02751
P02462
P08572
O75829
P39059
P98160
Endostatin
Fibulin fragments
154
≈77
P39060
P23142, P98095,
Q12805, O95967,
Q9UBX5
P07996, P35442
Thrombospondin-1 and -2Extracellular matrix glycoproteins that are proteolyzed
to produce anti-angiogenic proteins; Tsp-1 was the first
recognized naturally occurring angiogenesis inhibitor
Fragment of type IV collagen ?3 chain
129
Tumstatin162Q01955
(B) Non-matrix derived factors
Angiostatin
Antithrombin III (cleaved)
Hemopexin-like domain (PEX)
Interferon-?, -?, -?
Fragment of plasminogen (residues 98–465)
Fragment of antithrombin III
Fragment of MMP-2
Cytokines
90.6
52.6
73.9
≈22
P00747
P01008
P08253
P01574, P01574,
P01579
P01584, P05112,
P29459, Q14116
Interleukin-1, -4, -12, -18Cytokines
≈17
2-Methoxyestradiol
Pigment epithelium-derived factor (PEDF)
Plasminogen kringle-5
Platelet factor-4
Prolactin fragments
Prothrombin kringle-2
Semaphorin-3F
Soluble VEGFR1
TIMP-2
Troponin I
TrpRS
Vasostatin
Endogenous estrogen metabolite
Growth factor
Fragment of angiostatin/plasminogen
Released by platelets
8- and 16-kDa fragments of prolactin
Fragment of prothrombin
VEGF family antagonist
Fragment of VEGFR1
Tissue inhibitor of metalloprotease-2
Inhibitory subunit of muscle troponin
Fragment of tryptophanyl-tRNA synthetase
Fragment of calreticulin
46.3
90.6
10.8
25.9
70.0
88.4
151
24.4
21.2
53.2
48.1
P36955
P00747
P02776
P01236
P00734
Q13275
P17948
P16035
P48788
P23381
P27797
aAdapted from ref. [14].
bMolecular weight (MW) corresponding to the unprocessed human precursor.
Besides classical angiogenesis, various forms of non-
sprouting angiogenesis have been described in tumors [19].
These include intussusceptive vascular growth, co-option,
formationofmosaicvessels,andvasculogenicmimicry.Dur-
ing intussusceptive vascular growth, a column of interstitial
cells is inserted into the lumen of a pre-existing vessel,
thereby dividing the lumen and yielding two vessels [20].
The column is invaded by fibroblasts and pericytes and accu-
mulates extracellular matrix proteins. This process does not
require the immediate proliferation of endothelial cells but
rather the rearrangement and remodeling of existing ones.
The advantage of this mechanism of growth over sprouting
is that blood vessels are generated in a metabolically eco-
nomicprocessbecauseextensivecellproliferation,basement
membrane degradation, and invasion of the surrounding tis-
sue are not required. By yet another mechanism, developing
tumors can surround vessels in the tissue or organ of origin
andincorporate,orco-opt,thesevessels[21].Co-optionmay
be important when tumors arise in or metastasize to vascular
organs such as the lung or brain.
Tumor cells, along with endothelial cells, may together
form the luminal surface of capillaries thus generating a
mosaic vessel [22]. Chang et al. found that about 15% of
vessels in human colon carcinoma implants (xenografts) in
athymic hairless, or nude, mice and in biopsies of human
colon carcinomas were mosaic channels lined with both
endothelial and tumor cells [22].
In vasculogenic mimicry, first described in ocular
melanoma, vascular channels develop that are extracellular-
matrix-rich tubular networks [23]. These tubular networks or
channels lack endothelial cells but contain circulating red
blood cells. Vasculogenic mimicry has been described in
breast, lung, ovarian, and prostate carcinoma and in rhab-
domyosarcoma [24]. However, Auguste et al. point out that
someinvestigatorsdisagreewiththeconceptofvasculogenic
mimicry [19].

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Table 3
VEGF receptor ligands and VEGF family isoforms that bind to heparan sulfate proteoglycansa
VEGFR1VEGFR2VEGFR3Neuropilin-1Neuropilin-2Syndecanb
VEGF
VEGF-B
VEGF (110–165)c
VEGF-C
Pro- and mature VEGF-C
Pro- and mature VEGF-D
VEGF-165
PlGF-152
VEGF (145–165)c
PlGF-152
VEGF (145–206)c
PlGF-152, PlGF-224
PlGFVEGF-D
VEGF-E
Pro-VEGF-Cd
Pro-VEGF-Dd
Semaphorin-3C
Semaphorin-3F
Semaphorin-3A
VEGF-B
VEGF-E
Pro- and mature VEGF-Cd
Pro-VEGF-Dd
Semaphorin-3C
Semaphorin-3F
VEGF-B167
aFrom ref. [30] unless otherwise noted.
bOne form of heparan sulfate proteoglycan.
cIsoforms within the range of the specified number of amino acids.
dFrom ref. [31].
Sprouting angiogenesis, which is the most studied
mechanism of new blood vessel formation, occurs under
physiologicalandnon-physiologicalconditionsasdoesintus-
susceptive vascular growth [19]. It is uncertain whether
co-option, mosaic vessels, or vasculogenic mimicry play a
role in physiological vasculogenesis or angiogenesis. The
nature of the factors that determine which combinations of
these angiogenic processes occur during tumor progression
is unknown.
1.5. Tumor vessel morphology
Tumor vessels exhibit abnormal morphology while nor-
mal vessels are organized in a hierarchical fashion with
arterioles, capillaries, and venules that are readily distin-
guishable [2,25]. Pioneering work by Algire and Chalkley
using tumors grown in a transparent chamber in rats in
vivo demonstrated that capillaries in rapidly growing tumors
are about five times the diameter of those in normal tissue
[26]. These capillaries rarely differentiated into arterioles
or venules. Moreover, three-dimensional microscopy of
tumor vascular casts revealed an absence of normal arteri-
ole, capillary, and venule structure with arteriolar–venular
shunts, frequent blind endings, and incomplete and abnor-
mal endothelial cell lining [27]. Tumor vessels often develop
into disorganized bundles containing numerous vascular
sprouts while exhibiting irregular vessel lumen diameters
[28]. Owing to the abnormal organization and structure of
tumor vessels, blood flow in tumors is chaotic [27].
2. The vascular endothelial growth factor (VEGF)
family
The VEGF family plays an integral role in angiogen-
esis, lymphangiogenesis, and vasculogenesis. The human
VEGFfamilyconsistsoffivemembers:VEGF(orVEGF-A),
VEGF-B, VEGF-C, VEGF-D, and placental growth factor
(PlGF) [10,29]. Each of these proteins contains a signal
sequencethatiscleavedduringbiosynthesis.Moreover,alter-
native splicing of their corresponding pre-mRNAs generates
multiple isoforms of VEGF, VEGF-B, and PlGF. There are
three receptor protein-tyrosine kinases for the VEGF fam-
ily of ligands (VEGFR1, VEGFR2, and VEGFR3) and two
non-enzymatic receptors (neuropilin-1 and -2). Moreover,
several of the VEGF family ligands bind to heparan sul-
fate proteoglycans that are found on the plasma membrane
and in the extracellular matrix. See Table 3 for a list of the
VEGF receptor ligands and the VEGF isoforms that bind to
the extracellular matrix heparan sulfate proteoglycans.
VEGF binding sites were identified on vascular endothe-
lial cells [32,33] corresponding to VEGFR1 (Flt-1) [34]
and VEGFR2 (Flk-1/KDR) [35–37]. This distribution on
endothelial cells accounts for the selectivity and speci-
ficity of action of VEGF. VEGFR3 (Flt-4) [38], which is
in the same receptor family, binds VEGF-C and VEGF-D.
Each of these receptors is a type V (five) protein-tyrosine
kinasethatconsistsofanextracellularcomponentcontaining
seven immunoglobulin-like domains, a single transmem-
brane segment, a juxtamembrane segment, an intracellular
protein-tyrosine kinase domain that contains a kinase insert
of 70–100 amino acid residues, and a carboxyterminal tail
(see ref. [39] for a description of types I–IX receptor protein-
tyrosine kinases). The three VEGF receptors are related to
the platelet-derived growth factor receptors (? and ?), the
fibroblast growth factor receptors (1–4), the stem cell factor
receptor (Kit), the Flt ligand receptor (Flt-3), and the colony-
stimulating factor-1 receptor (CSF-1R), all of which contain
extracellular immunoglobulin domains and a kinase insert
[39,40].
3. Properties and expression of the VEGF family
3.1. VEGF-A
ThediscoveryofVEGF(VEGF-A)representstheconver-
gence of work by several groups beginning in the 1980s. In
1983, Senger et al. isolated and partially purified a protein
from ascites fluid and from conditioned medium induced by