Cardiovascular Research 65 (2005) 550 – 563

www.elsevier.com/locate/cardiores

Review

The biology of vascular endothelial growth factors

Tuomas Tammela, Berndt Enholm, Kari Alitalo, Karri Paavonen*
Molecular/Cancer Biology Laboratory and Ludwig Institute for Cancer Research, Haartman Institute and Biomedicum Helsinki,
P.O. Box 63 (Haartmaninkatu 8) University of Helsinki and Helsinki University Central Hospital, Helsinki 00014, Finland

Received 30 September 2004; received in revised form 24 November 2004; accepted 2 December 2004
Time for primary review 18 days

Abstract

The discovery of the vascular endothelial growth factor (VEGF) family members VEGF, VEGF-B, placental growth factor (PlGF),
VEGF-C and VEGF-D and their receptors VEGFR-1, -2 and -3 has provided tools for studying the vascular system in development as
well as in diseases ranging from ischemic heart disease to cancer. VEGF has been established as the prime angiogenic molecule during
development, adult physiology and pathology. PlGF may primarily mediate arteriogenesis, the formation of collateral arteries from

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preexisting arterioles, with potential future therapeutic use in for example occlusive atherosclerotic disease. VEGF-C and VEGF-D are
primarily lymphangiogenic factors, but they can also induce angiogenesis in some conditions. While many studies have addressed the role
of angiogenesis and the blood vasculature in human physiology, the lymphatic vascular system has until recently attracted very little
attention. In this review, we will discuss recent advances in angiogenesis research and provide an overview of the molecular players
involved in lymphangiogenesis.
D 2004 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.

Keywords: Angiogenesis; Lymphangiogenesis; Growth factors; Endothelial receptors; VEGF

1. Introduction recently attracted very little attention. The vascular endo-

Many human diseases are characterized by disorders of
the vasculature. In ischemic heart disease or peripheral
artery disease, insufficient blood vasculature leads to tissue
ischemia. In cancer, tumor angiogenesis contributes to
tumor growth and metastasis. Out of the many players in
the angiogenesis field, the vascular endothelial growth
factors are by far the best characterized and several VEGFs
have already entered clinical use in a variety of human
conditions. While many studies have addressed the role of
angiogenesis and the blood vasculature in human physiol-
ogy and pathology, the lymphatic vascular system has until
* Corresponding author. Molecular/Cancer Biology Laboratory, Bio-
medicum Helsinki, P.O. Box 63 (Haartmaninkatu 8), 00014 University of
Helsinki, Finland. Tel.: +358 9 1912 5537; fax: +358 9 1912 5510.
E-mail address: karri.paavonen@helsinki.fi (K. Paavonen).
0008-6363/$ - see front matter D 2004 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.cardiores.2004.12.002
thelial growth factor (VEGF) family members VEGF,
VEGF-B, placental growth factor (PlGF), VEGF-C and
VEGF-D bind their cognate receptors VEGFR-1, VEGFR-2
and VEGFR-3 found on the vascular endothelium. VEGF is
the prime hypoxia inducible angiogenic factor. VEGF
inhibition has been validated as clinical cancer therapy.
Furthermore, several studies have reported beneficial effects
of VEGF when used as a proangiogenic therapy in the
setting of tissue ischemia. PlGF may primarily mediate
arteriogenesis, the formation of collateral arteries from
preexisting arterioles, and thus it has potential as a
therapeutic in for example occlusive atherosclerotic disease.
VEGF-C and VEGF-D are primarily lymphangiogenic
factors, inducing the growth of lymphatic vessels during
development as well as in the adult. Inhibition of VEGF-C
and VEGF-D signal transduction may prove crucial in the
inhibition of lymphatic metastasis in cancer and stimulation
of this pathway may be effective in the treatment of
lymphedema.
 T. Tammela et al. / Cardiovascular Research 65 (2005) 550–563
2. VEGF
VEGF binds VEGFR-1 and VEGFR-2 as well as
neuropilin-1 (Nrp-1) and Nrp-2; the latter are receptors
for semaphorins, molecules involved in axonal guidance
during neuronal development [1,2]. VEGF induces pro-
liferation, sprouting, migration and tube formation of
endothelial cells (ECs) [3]. VEGF is also a potent survival
factor for ECs during physiological and tumor angio-
genesis and it has been shown to induce the expression of
antiapoptotic proteins in the ECs [4,5]. VEGF was
originally described as a permeability factor, as it increases
permeability of the endothelium through the formation of
intercellular gaps, vesico-vascular organelles, vacuoles and
fenestrations [6]. VEGF also causes vasodilatation through
the induction of the endothelial nitric oxide synthase
(eNOS) and the subsequent increase in nitric oxide
production [7,8].
Although VEGF acts mostly on ECs, it has been shown
to also bind VEGF receptors on hematopoietic stem cells
(HSCs), monocytes, osteoblasts and neurons [3]. Besides
angiogenesis, VEGF induces HSC mobilization from the
bone marrow, monocyte chemoattraction, osteoblast-medi-
ated bone formation and neuronal protection [3,9].
Furthermore, VEGF stimulates inflammatory cell recruit-
ment and promotes the expression of proteases implicated
in pericellular matrix degradation in angiogenesis [10–12].
Many cytokines including platelet-derived growth factor,
epidermal growth factor, basic fibroblast growth factor and
transforming growth factors induce VEGF expression in
cells [13].
At least six VEGF isoforms of variable amino acid
number are produced through alternative splicing:
VEGF121, VEGF145, VEGF165, VEGF183, VEGF189 and
VEGF206 (Table 1) [3]. VEGF121, VEGF165 and VEGF189
are the major forms secreted by most cell types [14].
After secretion, VEGF121 may diffuse relatively freely in
tissues, while approximately half of the secreted VEGF165
binds to cell surface heparan sulfate proteoglycans
(HSPGs). VEGF189 remains almost completely seques-
tered by HSPGs in the extracellular matrix making
HSPGs a reservoir of VEGF that can be mobilized via
proteolysis [3].
Table 1
A table showing the chromosomal localization, major mRNA transcript and major protein sizes of the VEGF receptors
Gene Sequence homology Chromosomal
localization
VEGF 6p23.1
VEGF-B 45% homology with VEGF-A 11q13
VEGF-C 30% homology with VEGF-A165 4q34
VEGF-D 61% homology with VEGF-C; Xp22.31
31% with VEGF-A165
PlGF 42% homology with VEGF-A 14q24
* Denotes splice variants that bind to heparan sulfate proteoglycans (HSPGs).
551
VEGF is first expressed mainly in the anterior portion of
mouse embryos and it directs the migration of VEGFR-1
and VEGFR-2 positive cells in embryonic tissues [15]. In
general, VEGF expression is stronger at sites of active
vasculogenesis and angiogenesis in embryos [16]. Homo-
zygous VEGF knockout mice die at E8-E9 from defects in
blood island formation, EC development and vascular
formation [13]. The levels of VEGF protein during develop-
ment appear critical as mice lacking even a single VEGF
allele die at E11-E12, displaying defects in early vascular
development [13]. The different biological functions of
VEGF isoforms were illustrated by studies on isoform-
specific VEGF knockout mice. Mice expressing only
VEGF120 (homologue of human VEGF121) die soon after
birth and those that survive succumb to ischemic cardiomy-
opathy and multiorgan failure [17]. Mice expressing only
VEGF188 (human VEGF189) display impaired arteriolar
development and approximately half die at birth [18]. Mice
expressing only VEGF164 (human VEGF165) are viable and
healthy [18]. These studies underline the importance of
VEGF165 as the principal effector of VEGF action, with
intermediate diffusion and matrix-binding properties.
VEGF is strongly induced in hypoxic conditions via
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hypoxia inducible factor (HIF) regulated elements of the
VEGF gene [19]. Constitutive degradation of hypoxia
inducible factor (HIF)-1a is blocked in hypoxia because
of the oxygen requirement of HIF prolyl hydroxylases,
followed by stabilization of HIF-1a and its heterodime-
rization with HIF-1h, also called the aryl hydrocarbon
nuclear translocator (ARNT). These complexes then bind
hypoxia-responsive elements (HREs) in the promoters of
hypoxia inducible genes and initiate transcription of a set
of more than a hundred genes, including genes involved
in glucose transport, glycolysis, and angiogenesis [19,20].
Interestingly, Bartonella henselae, the causative agent of
cat-scratch fever, can induce hypoxia via an intracellular
oxygen consumption mechanism, leading to VEGF
induction and an angiomatous tumor [21]. Examples of
other hypoxia-regulated genes include cyclooxygenase-2
(COX-2), MMP-2, VEGF and VEGFR-1 [19]. Deletion of
a HRE from the mouse VEGF gene promoter results in
progressive motoneuron degeneration, presumably due to
insufficient vascular perfusion of nervous tissue and
Splice Major mRNA Major protein
variants transcript size (kb) size (kDa)
121,145, 165*, 3.7, 4.5 21
183*, 189*, 206*
167*, 186 1.4 21, 30
– 2.4 20–21
– 2.2 20–21
131, 152*, 219 1.7, 1.2 38, 30
552 T. Tammela et al. / Cardiovascular Research 65 (2005) 550–563
impaired motoneuron survival via loss of VEGF induction
[22].
The skin has been widely used as a model for studying
VEGF action in vivo; for example, transgenic mice over-
expressing VEGF in the skin have abundant cutaneous
angiogenesis and an inflammatory skin condition resembling
psoriasis [23]. Overexpression of VEGF in mouse skin also
accelerates experimental tumor growth [24]. In contrast, mice
with a targeted deletion of VEGF in the epidermis exhibit
delayed wound healing, while chemically induced skin
papillomas developed less frequently in these animals [25].
VEGF blocking monoclonal antibodies or VEGF receptor
inhibition reduce the growth of experimental tumors in mice
and humans [13,26,27]. In humans, VEGF is expressed in
practically all solid tumors studied as well as in some
hematological malignancies [3]. In fact, correlations have
been found between the level of VEGF expression, disease
progression and survival [28].
The effects of VEGF on the lymphatic vasculature
have also been recently studied. Adenoviral overexpres-
sion of the murine VEGF164 in the skin induced formation
of giant lymphatic vessels [29], while another study
employing the human VEGF165 isoform reported only
dilatation of cutaneous lymphatics (Fig. 3A) [30]. How-
ever, VEGF did not induce lymphangiogenesis in a
number of other tissue types [31–34]. The lymphangio-
genic effects of VEGF may be linked to the recruitment
of inflammatory cells, such as macrophages, which
express VEGFR-1 and secrete lymphangiogenic factors
[35–37]. At least in midgestation mouse embryos, VEGF-
C but not VEGF had the capacity to induce migration of
endothelial cells committed to the lymphatic endothelial
lineage [38].
3. Placental growth factor
PlGF is predominantly expressed in the placenta, heart
and lungs [39]. PlGF homodimers bind VEGFR-1 and
Nrp-1, while PlGF heterodimerization with VEGF may
also occur. Activation of VEGFR-1 by either PlGF or
VEGF induced different gene expression profiles and
phosphorylation of distinct tyrosine residues in the tyrosine
kinase (TK) domain of VEGFR-1 [40], while combined
administration of these factors enhanced VEGF driven
angiogenesis [41]. This is probably due to the fact that
PlGF/VEGF heterodimers bind VEGFR-2 and also
VEGFR-1/VEGFR-2 heterodimers in vitro [42]. Of the
three reported human isoforms of PlGF (PlGF-1, -2 and
-3), only PlGF-2 binds HSPGs [42]. PlGF knockout mice
do not have an apparent phenotype. However, these mice
recover poorly from experimental myocardial infarction
and exhibit impaired collateral formation in response to
hind limb ischemia [43]. Overexpression of PlGF in the
skin of transgenic mice results in a hypervascular
phenotype with increased inflammatory and permeability
responses [44,45], while local administration of PlGF with
recombinant adenoviruses or as a recombinant protein
induces the formation of mature, leakage-resistant vessels
in a macrophage-dependent manner [46–48].
4. VEGF-B
VEGF-B (also called VEGF-related factor/VRF) is a
ligand for VEGFR-1 and Nrp-1, and it can form heterodimers
with VEGF (Fig. 1) [49]. In humans, VEGF-B is expressed as
two different isoforms, VEGF-B167 and VEGF-B186 [50].
VEGF-B167 is nonglycosylated, binds HSPGs and is mostly
sequestered in the extracellular matrix while VEGF-B186 is
O-glycosylated, and freely diffusible. In vivo, VEGF-B167 is
the predominant form and is abundantly expressed in brown
fat, in the myocardium and skeletal muscle (Fig. 2) [49].
The precise role of VEGF-B in vivo is not known. As
VEGF-B is highly expressed in striated muscle, myocardium
and brown fat [51,52], its function may be linked to high
cellular energy metabolism. Interestingly, at least in one
genetic background mice deficient in VEGF-B had smaller
hearts and impaired recovery after experimental myocardial
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infarctions suggesting that the regeneration of coronary
collaterals through arteriogenesis could at least in part be
dependent on VEGF-B [53]. In another genetic background, a
prolonged PQ interval was observed in the electrocardio-
grams of VEGF-B gene targeted mice [54]. Furthermore, one
report has claimed that VEGF-B knockout mice fail to
develop pulmonary hypertension in response to hypoxia [55].
Conversely, overexpression of VEGF-B in the lung protected
rats from hypoxic pulmonary hypertension in a manner
comparable to VEGF [56]. VEGF-B knockout mice also
display reduced angiogenic responses in collagen-induced
arthritis, suggesting a role for VEGF-B in inflammatory
angiogenesis [57]. Correspondingly, VEGF-B may have
subtle angiogenic effects, as overexpression of VEGF-B in
vivo by injection of recombinant plasmids in a mouse model
of hind limb ischemia induced angiogenesis [58].
5. VEGF-C
Where VEGF-A and VEGF-B isoforms are formed
through alternative splicing, different forms of VEGF-C
and VEGF-D are the result of proteolytic processing.
VEGF-C is produced as a precursor protein, which is
activated by intracellular secretory proprotein convertases
furin, PC5 and PC7 [59,60]. The secreted, VEGFR-3
binding 31/29 kD subunits of VEGF-C are bound together
by disulphide bonds, but the factor is further proteolyzed
in the extracellular environment by plasmin and other
proteases to generate a 21 kD non-disulfide-linked
homodimeric protein with high affinity for both VEGFR-
2 and VEGFR-3. The mature form of VEGF-C induces
mitogenesis, migration and survival of ECs [61]. During
 T. Tammela et al. / Cardiovascular Research 65 (2005) 550–563
Fig. 1. The VEGF family ligands and their receptors. VEGF, VEGF-B and PlGF bind VEGFR-1 and VEGFR-2 on the blood vascular endothelium. VEGF-C
and VEGF-D primarily bind VEGFR-3 on lymphatic endothelium.
development, VEGF-C is expressed along with its receptor
VEGFR-3 predominantly in regions where lymphatic
vessels develop [38,62]. The expression then decreases in
most tissues, remaining high in the lymph nodes [63].
VEGF-C induces selective lymphangiogenesis without
accompanying angiogenesis as demonstrated by early
experiments in the chick chorioallantoic membrane assay
and in transgenic mice overexpressing VEGF-C in the skin
[64,65]. Adenoviral VEGF-C gene transduction has been
shown to induce growth of functional lymphatic vessels in
several different animal models (Fig. 3C) [30,66]. In vivo,
VEGFR-3 activation is sufficient to induce lymphangio-
genesis whereas the increase in blood vascular perme-
ability induced by VEGF-C and VEGF-D is mediated by
VEGFR-2 [30,67].
Mice lacking both VEGF-C alleles fail to develop
lymphatic vessels and succumb to tissue edema at E15.5–
VEGF165
PIGF
VEGF-B
VEGF-C
VEGF-D
40 30 20 10 0 suggesting that prostanoids could induce VEGF-C driven
Percentage of residue substitution
Fig. 2. A dendrogram showing the relationships VEGF family members
estimated by the amino acid residue substitution analysis.
553
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E17.5 [38]. Loss of one VEGF-C allele results in
lymphedema characterized by hypoplasia of the cutaneous
lymphatic vessels indicating that the VEGF-C protein
concentration is critical for the development of the
lymphatic vasculature [38]. However, VEGF-C is not
essential for the development of blood vessels unlike its
receptors VEGFR-2 and VEGFR-3. Sprouting of endothe-
lial cells committed to the lymphatic endothelial lineage in
VEGF-C gene targeted mice could be rescued by applica-
tion of recombinant VEGF-C and to a lesser degree by
VEGF-D, but not with VEGF. This indicates that develop-
ment of the lymphatic vessels is dependent on VEGFR-3
and VEGF-C in a nonredundant fashion [38].
VEGF-C mRNA transcription was induced in ECs in
response to proinflammatory cytokines [68]. The regulation
of VEGF-C mRNA transcription by these cytokines
indicates that VEGF-C could regulate lymphatic vessel
function during inflammation, reflecting the role of the
lymphatic vasculature in the control of immune function and
leukocyte trafficking. The promoter of the VEGF-C gene
has been shown to contain putative NF-nB binding sites that
could mediate the activation of VEGF-C mRNA tran-
scription by proinflammatory cytokines [69]. A recent
report demonstrated that prostaglandin E2 mediated activa-
tion of COX-2 increased VEGF-C mRNA transcription
lymphangiogenesis [70]. Recent reports have shown
that inflammatory reactions in human kidney transplants
undergoing rejection are accompanied by abundant lym-
phangiogenesis and that VEGFR-3 signaling contributes to
554 T. Tammela et al. / Cardiovascular Research 65 (2005) 550–563

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Fig. 3. Blood and lymphatic vascular effects of various adenovirally expressed human VEGFs in the mouse skin two weeks after treatment. (A) VEGF165
induces a prominent angiogenic response (blood vessels stained with antibodies for PECAM-1/CD31, green), accompanied by enlargement of lymphatic
vessels (stained with antibodies for the lymphatic marker LYVE-1, red). (B) VEGF-B167 does not induce either angiogenesis or lymphangiogenesis. (C) VEGF-
C is a powerful inducer of lymphangiogenesis, while its angiogenic activity is weak. (D) The AdLacZ control corresponds to nonstimulated, normal skin.

reactive lymphadenitis [71]. In a rabbit cornea model of
inflammatory angiogenesis and lymphangiogenesis, selec-
tive depletion of macrophages blocked lymphangiogenesis
demonstrating that inflammatory cells can mediate the
formation of lymphatic vessels [37]. The angiogenic and
lymphangiogenic responses were blocked by a VEGF
inhibitor. The recruited macrophages expressed abundant
amounts of VEGF-C and VEGF-D, suggesting that the
lymphangiogenic effect of VEGF is indirect and most likely
mediated by inflammatory cytokines that induce the
expression of lymphangiogenic factors in the inflammatory
infiltrate. In this regard, it is interesting to note that COX-2
up-regulated VEGF-C and promoted lymphangiogenesis in
human lung adenocarcinoma [70]. VEGF-C is also highly
expressed in arthritic joint synovium in patients with
rheumatoid arthritis in the absence of lymphangiogenesis
per se, suggesting that the lymphangiogenic response is
insufficient in this disorder or that the induced VEGF-C
participates in other functions [72].
In experimental models, tumor cells overexpressing
VEGF-C or VEGF-D induce peritumoral and in some cases
stromal lymphangiogenesis and tumor cell invasion into
lymphatic vessels [61,73,74]. Furthermore, overexpression
of VEGF-C in pancreatic islet cell tumors of transgenic mice
induced lymphangiogenesis and promoted lymph node
metastasis [75]. Interestingly, neutralization of VEGF-C
and VEGF-D in an experimental tumor model by systemic
overexpression of a soluble VEGFR-3 fused to the Fc domain
of immunoglobulin g chain (VEGFR-3-Ig) inhibited lym-
phangiogenesis and lymph node metastasis [76,77]. Peritu-
moral lymphatics were recently found to originate from the
preexisting lymphatic vasculature while circulating endothe-
lial progenitor cells did not contribute to the process [78].
Although enlarged lymphatic vessels at the tumor edge have
been reported in human cancers, functional intratumoral
lymphatic vessels have not been reported [79]. Interestingly,
several clinical studies of cancer patients have shown a
positive correlation between VEGF-C expression and lym-
phatic invasion, lymphatic metastasis and patient survival
[80]. However, although breast cancers frequently metasta-
size to the lymph nodes, at least one study did not detect
tumor lymphangiogenesis in breast cancer [81].
 T. Tammela et al. / Cardiovascular Research 65 (2005) 550–563
6. VEGF-D
Like VEGF-C, the human VEGF-D is processed in its N-
terminal and C-terminal ends; the mature form binds to and
activates VEGFR-2 and VEGFR-3, is mitogenic for EC and
angiogenic as well as lymphangiogenic in vivo [61]. Mouse
VEGF-D binds only VEGFR-3, indicating a different role
for VEGF-D in mice [82]. VEGF-D is present in most
human tissues, most abundantly in the lung and skin during
embryogenesis [83]. In experimental tumors VEGF-D
increases lymphatic vessel growth and lymphatic metastasis
[84]. VEGF-D is expressed by melanoma cells and has been
proposed to have a role in tumor angiogenesis and
lymphangiogenesis in this tumor [85]. VEGF-D, along with
VEGF-C, has recently been shown to be involved in
lymphatic metastasis in experimental intrathoracic lung
cancer [86]. In humans, VEGF-D has been shown to be of
prognostic value for lymphatic vessel invasion and also
survival in certain human cancers [80]. The fully processed
form of VEGF-D is able to induce strong angiogenesis in
addition to lymphangiogenesis in the rabbit hind limb
muscle [32,87].
7. VEGFR-1
VEGFR-1 (fms-like tyrosine kinase, Flt-1) is composed
of seven extracellular immunoglobulin (Ig) homology
domains, a single transmembrane region and an intra-
cellular tyrosine kinase (TK) domain that is interrupted by
a kinase-insert domain (Fig. 1, Table 2) [88]. VEGFR-1
binds VEGF, VEGF-B and PlGF with high affinity.
VEGFR-1 by itself transmits only weak mitogenic signals
in ECs [3], but it can heterodimerize with VEGFR-2
forming a complex with stronger signaling properties than
VEGFR-1 or VEGFR-2 homodimers [43,89] (Table 2).
VEGFR-1 is expressed in ECs as well as osteoblasts,
monocytes/macrophages, pericytes, placental trophoblasts,
renal mesangial cells and also in some hematopoietic stem
cells [3,90]. VEGFR-1 expression is up-regulated during
angiogenesis and also by hypoxia, unlike that of VEGFR-2
and VEGFR-3 [91,92].
During development, VEGFR-1 is first expressed in
angioblasts and in the endothelium, although less strongly
than VEGFR-2; VEGFR-1 expression subsides during later
embryonic development [93–95]. VEGFR-1 gene targeted
Table 2
A table showing the chromosomal localization, major mRNA transcripts
and major protein sizes of VEGF receptors
Gene Chromosomal Major mRNA Major protein
localization transcript size (kb) size (kDa)
VEGFR-1 13q12-q13 7.5, 8.0 180
VEGFR-2 4q11-q12 5.8, 7.0 230
VEGFR-3 5q33-qter 4.5, 5.8 195
555
mice die at E8.5 due to disorganization of blood vessels
and overgrowth of endothelial cells [3]. Although VEGFR-
1 gene targeted mice do not survive, mice lacking only the
intracellular kinase (TK) domain are normal except for
slightly impaired angiogenesis during pathologic condi-
tions [96,97]. Activation of the VEGFR-1 TK domain is a
requisite for monocyte migration [98]. Moreover, VEGFR-
1 mediated angiogenesis as well as arteriogenesis have
been shown to be dependent on monocytes [47,48], and
angiogenesis during experimental tumor growth is at least
partially inhibited by anti-VEGFR-1 antibodies [47].
VEGFR-1 signaling is also involved in the recruitment
and survival of bone marrow derived progenitor cells
[99,100].
A naturally occurring soluble form of VEGFR-1
(sVEGFR-1), consisting of the extracellular part of
VEGFR-1, that is able to inhibit VEGF action has been
described in humans and mice [3]. sVEGFR-1 has recently
been linked to the development of gestational toxemia and
preeclampsia [101]. Women with preeclampsia have ele-
vated circulating sVEGFR-1 levels and decreased circulat-
ing levels of PlGF and VEGF possibly leading to EC
dysfunction in the maternal and placental vasculatures
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[102].
8. VEGFR-2
The overall structure of VEGFR-2 (kinase-insert
domain receptor, KDR/fetal liver kinase, Flk-1) is similar
to that of VEGFR-1 (Table 2) [103,104]. VEGFR-2 binds
VEGF, VEGF-C and VEGF-D. Although the binding
affinity of VEGF towards VEGFR-2 is lower than that
for VEGFR-1, selective activation of VEGFR-1 and
VEGFR-2 has shown that VEGFR-2 is the primary
receptor transmitting VEGF signals in endothelial cells
[105–107]. The expression of VEGFR-2 is autoregulated:
VEGF, VEGF-C and VEGF-D up-regulate the expression
of VEGFR-2 [84,108]. In addition to the ECs, VEGFR-2 is
also expressed on neuronal cells, osteoblasts, pancreatic
duct cells, retinal progenitor cells, megakaryocytes and
hematopoietic stem cells [3,109]. During development
VEGFR-2 is expressed by the primitive endoderm,
embryonic angioblasts and in the blood islands as well
as in angiogenic vessels [93]. VEGFR-2 gene targeted
mice die at E8.5-E9.5 due to lack of development of the
blood islands, embryonic vasculature and hematopoietic
cells [110,111]. VEGFR-2 expression is down-regulated in
the adult blood vascular ECs, and is again up-regulated in
the endothelium of angiogenic blood vessels [92]. On the
other hand, sequestration of VEGF results in down-
regulation of VEGFR-2 and in apoptotic death of some
capillary endothelial cells in vivo [112]. Moreover,
VEGFR-2 may be associated with integrin-dependent
migration of ECs, as it forms a complex with integrin
aVh3 upon binding VEGF [113,114]. An interaction
556 T. Tammela et al. / Cardiovascular Research 65 (2005) 550–563
between VEGFR-2 and VE-cadherin, a cell–cell adhesion
molecule has also been described [115].
9. VEGFR-3
VEGFR-3 (fms-like tyrosine kinase 4, Flt4) has only six
Ig-homology domains as the fifth Ig-homology domain is
proteolytically cleaved soon after biosynthesis and the
resulting polypeptide chains remain linked via a disulfide
bond (Table 2) [116,117]. In humans, alternative splicing
of the VEGFR-3 gene generates two isoforms of VEGFR-
3 that differ in their C-termini [118]. VEGFR-3 binds
VEGF-C and VEGF-D [119,120]. VEGFR-3 is present on
all endothelia during development but in the adult it
becomes restricted to lymphatic ECs and certain fenes-
trated blood vascular ECs [121,122]. VEGFR-3 is up-
regulated on blood vascular ECs in pathologic conditions
such as in vascular tumors and in the periphery of solid
tumors [92,123]. Interestingly, EC contact with SMCs has
been shown to down-regulate VEGFR-3 in ECs [124],
suggesting that VEGFR-3 signaling is important in nascent
blood vessels, and it becomes redundant as the vessels
mature. Indeed, VEGFR-3 gene targeted mice exhibit a
dramatic blood vascular phenotype, with embryonic lethal-
ity at E9.5 from defective remodeling of the primary
vascular plexus and disturbed hematopoiesis [125,126].
Transgenic mice overexpressing a soluble VEGFR-3-Ig
fusion protein in the skin lack dermal lymphatic vessels
and have hypoplastic deeper lymphatic vessels, although
the phenotype is less pronounced as the mice age [127].
Missense mutations in VEGFR-3 have been linked to
hereditary lymphedema in humans and also to a similar
condition in a mouse model of lymphedema [128–130].
More recently, VEGFR-3 has also been shown to modulate
adaptive immunity in an experimental model of corneal
transplantation [131].
10. Neuropilins
The neuropilins, Nrp-1 and Nrp-2, have roles in
immunology and neuronal development but they are also
involved in angiogenesis [132,133]. Neuropilins bind class
3 semaphorins, which are secreted molecules that mediate
repulsive signals during neuronal axon guidance [1,2,134].
Nrp-1 also binds VEGF, VEGF-B and PlGF while Nrp-2
binds VEGF, VEGF-C and PlGF [132]. Nrp-1 acts as a
co-receptor enhancing VEGF-VEGFR-2 interactions,
forming complexes with VEGFR-1 and augmenting tumor
angiogenesis in vivo [135]. Overexpression of Nrp-1 in
chimeric mice leads to excessive formation of capillaries
and blood vessels and hemorrhages in addition to cardiac
malformations [136]. In chick embryos, endothelial Nrp-1
expression is mostly restricted to arteries, whereas Nrp-2
primarily marks veins, indicating a role in mediating the
arterial/venous identity of ECs [137]. Nrp-2 is expressed
also on lymphatic ECs, and mutated Nrp-2 induces
abnormalities in the formation of small lymphatic vessels
and lymphatic capillaries in mice [138]. Nrp-1 gene
targeted mice die at E13 from vascular defects such as
insufficient development of yolk sac vascular networks,
deficient neural vascularization and transposition of large
vessels [139,140]. It is thought that Nrp-1 is required for
cardiovascular development because it regulates VEGF165
levels [139]. While Nrp-2 gene targeted mice have normal
blood vasculature, the compound Nrp-1/Nrp-2 knockout
mouse has a phenotype resembling that of the VEGF and
VEGFR-2 gene targeted mice [141].
11. Disorders of the vascular system linked to the
VEGFs
11.1. Therapeutic angiogenesis/arteriogenesis in ischemic
heart disease and peripheral vascular disease
Angiogenic growth factors may be useful for attempts
to increase collateral vessel formation in ischemic heart
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disease (IHD) or critical limb ischemia/claudication or in
the treatment of diabetic neuropathy of the lower
extremities [142]. Reduction of blood flow in thromboem-
bolic disease leads to collateral formation via arterio-
genesis, a process during which preexisting arterioles
enlarge, leading to a 10- to 20-fold increase in blood flow
[143]. Arteriogenic pathways such as the PlGF-VEGFR-1
pathway recruit monocytes and act directly on ECs and
SMCs by inducing the growth of these cell populations.
Recently, PlGF was reported to induce formation of vascular
collaterals in experimental models of myocardial infarction
and lower limb ischemia in mice by amplification of VEGF
signaling [40,43,47]. Thus, VEGFR-1 agonists are poten-
tially useful in restoration of blood perfusion in settings of
tissue ischemia.
Human trials utilizing proangiogenic therapy have
arrived at some successful endpoints [142]. Attempts at
treating inoperable IHD have been made with gene transfer
of VEGF by adenovirus and naked plasmid injections
[144,145]. Preliminary reports targeting ischemic skeletal
muscle suggested that in patients with critical limb
ischemia/claudication, injection of VEGF plasmid may
induce the growth of functional collateral vessels [146]. In
the recent regional angiogenesis with VEGF (RAVE)
randomized trial patients with claudication were treated
with adenoviral delivery of VEGF121 but the researchers
failed to see clinical benefits [147]. However, VEGF
driven angiogenesis in progressive ischemic disease may
not be sufficient to restore the vascular perfusion because
the formed vessels are not sufficiently functional, and even
angiomas may develop [148]. Because of the permeability
side effects of VEGF, it has been suggested that a
combined approach with several growth factors such as
 T. Tammela et al. / Cardiovascular Research 65 (2005) 550–563
VEGF and angiopoietins, platelet-derived growth factors,
or the more arteriogenic PlGF may lead to better results
[42].
11.2. The role of VEGF family growth factors in athero-
sclerotic plaque formation
Atherosclerotic plaque progression is associated with
inflammatory angiogenesis through increased secretion of
angiogenic growth factors such as VEGF and basic
fibroblast growth factor (bFGF) [149]. Plaque angiogenesis
results in atherosclerotic plaque growth, increased plaque
instability and with the increase in the number of the
adventitial vasa vasora, an increased risk of intraplaque
hemorrhage [150]. These factors contribute to the growing
instability of the atherosclerotic plaque.
It is well known that plaque progression is associated
with inflammatory cell recruitment and deposition of
oxidized low-density lipoprotein (OxLDL). OxLDL
increases VEGF production and VEGF secretion by
inflammatory cells in vitro [151]. The number of VEGF
positive cells has been shown to correlate with the degree of
atherosclerotic plaque vascularization [152]. Experimental
atherosclerosis can be induced with VEGF and experimental
atherosclerotic changes can be reduced with antiangiogenic
therapy [153,154]. In a model of experimental cardiac
allografts, VEGF was found to mediate the progression of
arteriosclerosis [155]. However, recent results have sug-
gested that the angiopoietins, another family of angiogenic
molecules that bind and activate Tie2 tyrosine kinase
receptors on ECs, could actually prevent some of the
inflammatory changes in the vessel wall [156].
11.3. The role of endothelial progenitor cells in vascular
pathology
The blood circulation has been suggested to contain
endothelial progenitor cells (EPCs) that incorporate into
newly formed vessels at sites of active angiogenesis [157].
The EPCs are recruited to sites of angiogenesis where they
differentiate in situ into mature endothelial cells much like
during the embryonic vasculogenesis. EPCs express a
variety of endothelial cell surface markers including platelet
endothelial cell adhesion molecule-1, von Willebrand factor
(vWF) and VEGF receptors. EPCs are mobilized from the
bone marrow by cytokines such as macrophage chemo-
attractant protein-1 (MCP-1), transforming growth factor-
beta (TGF-h), VEGF and PlGF [157]. Skeletal muscle has
also been suggested to contain cells with stem cell proper-
ties that can contribute to angiogenesis, raising the
possibility that tissues can contain resident angioblasts
[158]. EPCs have been reported to incorporate into forming
blood vessels during physiological angiogenesis such as
during the female reproductive cycle, during pathologic
angiogenesis in experimental solid tumors or in the limb
muscles or the myocardium during ischemia [157]. How-
557
ever, the capacity of bone marrow derived EPCs to
incorporate into the vessel wall and transdifferentiate into
mature ECs in vivo has also been questioned [159–161].
11.4. Tumor angiogenesis
Blood vessels in tumors display several distinct features
that have implications for tumor biology and treatment.
Blood vessels in tumors have a chaotic structure and are
leaky [162]. As a consequence, blood flow in the tumor is
sluggish and the interstitial fluid pressure is high [162]. The
sluggish blood flow results in hypoxic regions and
perpetuates VEGF production while high interstitial fluid
pressure within the tumor hampers delivery of therapeutic
agents [162]. Since tumor growth is angiogenesis depend-
ent, therapeutic targeting of the tumor vasculature is an
attractive alternative or adjunct to conventional therapy. ECs
in tumors express a number of molecules that are unique to
blood vessels undergoing angiogenesis, such as VEGFR-2
and integrin aVh3 that can be targeted for therapeutic
purposes. Furthermore, work by Ruoslahti et al. has
demonstrated that angiogenic blood vessel endothelium
expresses certain peptides/integrins on their cell surface and
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these markers may serve as bzip codesQ for targeting
antiangiogenic molecules to tumor vessel endothelium
[163].
An important step in antiangiogenic cancer therapy was
recently taken when the anti-VEGF blocking antibody
bevacizumab (Genentech, USA) showed remarkable
results in the treatment of metastatic colorectal cancer,
and it has recently been approved by the FDA for
treatment of metastatic colorectal cancer [27,164]. This
may only mark the beginning of antiangiogenic therapy
for tumors, as at present bevacizumab is being evaluated
in Phase III trials of metastatic breast cancer, nonsmall
cell lung cancer, pancreatic cancer and renal cell
carcinoma (see www.clinicaltrials.gov).
11.5. Some other human diseases involving angiogenesis
Excessive ocular neovascularization contributes to visual
loss in retinopathy of prematurity and diabetic retinopathy
as well as in age-related macular degeneration. Retinal
hypoxia and excessive secretion of angiogenic factors like
VEGF leads to inappropriate retinal neovascularization and
hemorrhages [165]. Experimental retinal neovascularization
can be inhibited by antiangiogenic therapy [166–168].
Phase III clinical trials are underway to treat wet age-related
macular degeneration with ranibizumab, an antibody frag-
ment to VEGF (Genentech USA). An aptamer inhibitor
specific for the VEGF165 isoform (pegaptanib, EyeTech,
USA) has also entered phase II–III clinical trials with similar
indications [169].
Psoriasis is a chronic inflammatory skin disorder
characterized by dermal angiogenesis and overexpression
of angiogenic factors such as VEGF [170]. In fact, trans-
558 T. Tammela et al. / Cardiovascular Research 65 (2005) 550–563
genic mice with sustained overexpression of murine VEGF
in the skin have many of the classical features of psoriasis,
and treatment with VEGF Trap, a VEGF antagonist
(Regeneron, USA), was shown to reverse this phenotype
[23]. High VEGF levels are also involved in the pathologic
angiogenesis observed in endometriosis and possibly also
other gynecological disorders such as uterine bleeding
disorders [171].
11.6. Lymphedemas
In lymphedema, lymphatic drainage is impaired and
protein-rich fluid accumulates in the subcutaneous tissue
leading to fibrosis, impaired immune responses and fatty
degeneration of the connective tissue. Lymphedemas are
classified as primary, congenital lymphedemas and secon-
dary or acquired lymphedemas. Secondary lymphedema is
usually caused by filariasis or by iatrogenic trauma such
as radiation therapy, surgery or infection [172]. Primary
lymphedemas develop at birth or during adolescence.
Congenital hereditary lymphedema, or Milroy disease, is
characterized by hypoplastic superficial lymphatic vessels,
while the lymphatics in Meige disease, or late-onset
congenital lymphedema, appear enlarged and are dysfunc-
tional [173]. The genetic defects underlying several
primary lymphedemas have recently been characterized.
In some patients with Milroy disease, missense mutations
found in the TK domain of VEGFR-3 inhibit the function
of this receptor with resulting lymphedema [129]. In
lymphedema distichiasis, mutations in the FOXC2 gene
have been identified that lead to abnormal mural cell
coating of lymphatic vessels and lack of lymphatic valves
[174,175]. Other human diseases involving lymphedema
with putative, as yet uncharacterized molecular mecha-
nisms include Turner syndrome and cholestasis-lymphe-
dema.
11.7. Tumor lymphangiogenesis
The intravasation of tumor cells into lymphatic vessels is
one of the first steps in lymphatic metastasis. Lymphangio-
genesis around solid tumors could promote lymphatic
metastasis by providing a larger target for the intravasation
of tumor cells [80]. Although enlarged lymphatic vessels at
the tumor edge have occasionally been reported in human
cancers, functional intratumoral lymphatic vessels have not
been reported [79,176]. One explanation could be that the
high interstitial fluid pressure in tumors compresses
lymphatic vessels. According to another hypothesis, com-
pressed lymphatic vessels seen in the tumor stroma did not
arise by lymphangiogenesis but are preexisting lymphatic
vessels that were engulfed by the expanding tumor [177].
Although lymphatic metastasis is not necessarily dependent
on lymphangiogenesis [176], tumor lymphangiogenesis has
been reported to be a prognostic factor in cutaneous
melanoma and squamous cell cancers of the head and neck
[178–180]. Several reports have documented a correlation
between expression levels of the lymphangiogenic factors
VEGF-C and VEGF-D and lymphatic metastasis in cancer
patients [80]. Thus the blocking of VEGF-C and VGEF-D
signals with for example a soluble VEGFR-3-Ig fusion
protein could be an attractive approach for inhibition of
lymph node metastasis in human cancer patients, as already
indicated by the results from preclinical studies [76,77].
12. Conclusion
The vascular endothelial growth factors and their recep-
tors play a paramount role in the development of the vascular
system, via vasculogenic and angiogenic mechanisms, as
well as in the formation of the lymphatic vascular system.
Later in life, these molecules are required for processes
involving tissue repair, such as wound healing and the cyclic
reconstitution of the female endometrium. Aberrant angio-
genesis is a key mechanism in the pathophysiology of, e.g.,
atherosclerosis as well as tumor growth, while both angio-
genesis and lymphangiogenesis may contribute to tumor
metastasis. Controlling these processes with targeted molec-
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ular therapies is therefore at the heart of research directed at
disease treatment. While proangiogenic therapies could be
employed in the treatment of various ischemic diseases,
generation of new lymphatic vessels could help patients with
lymphedema. Meanwhile, a renaissance seems to be unrav-
eling around the VEGF growth factors, as novel functions for
them continue to be discovered outside the scope of vascular
biology.
Acknowledgements
The authors wish to thank Dr. Marika K7rkk7inen for
helping with the graphics. The authors have been supported
by the Human Frontier Science Program, EU (Lymphangio-
genomics LSHG-CT-2004-503573 and AngioNet QLG1-
CT-2001-01172), the Novo Nordisk Foundation, the Finnish
Academy of Sciences, the Farmos Research Foundation, the
Ida Montin Foundation, the Paulo Foundation, the Bio-
medicum Helsinki Foundation and the Finnish Medical
Foundation.
References
[1] Kolodkin AL, Levengood DV, Rowe EG, Tai YT, Giger RJ, Ginty
DD. Neuropilin is a semaphorin III receptor. Cell 1997;90:753 – 62.
[2] Chen H, Chedotal A, He Z, Goodman CS, Tessier-Lavigne M.
Neuropilin-2, a novel member of the neuropilin family, is a high
affinity receptor for the semaphorins Sema E and Sema IV but not
Sema III. Neuron 1997;19:547 – 59.
[3] Ferrara N, Gerber H-P, LeCouter J. The biology of VEGF and its
receptors. Nat Med 2003;9:669 – 76.
[4] Benjamin LE, Keshet E. Conditional switching of vascular endo-
thelial growth factor (VEGF) expression in tumors: induction of
 T. Tammela et al. / Cardiovascular Research 65 (2005) 550–563
endothelial cell shedding and regression of hemangioblastoma-like
vessels by VEGF withdrawal. Proc Natl Acad Sci U S A
1997;94:8761 – 6.
[5] Gerber H-P, Dixit V, Ferrara N. Vascular endothelial growth factor
induces expression of the antiapoptotic proteins Bcl-2 and A1 in
vascular endothelial cells. J Biol Chem 1998;273:13313 – 6.
[6] Bates DO, Hillman NJ, Williams B, Neal CR, Pocock TM.
Regulation of microvascular permeability by vascular endothelial
growth factors. J Anat 2002;200:581 – 97.
[7] Hood JD, Meininger CJ, Ziche M, Granger HJ. VEGF upregulates
ecNOS message, protein, and NO production in human endothelial
cells. Am J Physiol 1998;274:H1054 – 8.
[8] Kroll J, Waltenberger J. A novel function of VEGF receptor-2
(KDR): rapid release of nitric oxide in response to VEGF-A
stimulation in endothelial cells. Biochem Biophys Res Commun
1999;265:636 – 99.
[9] Storkebaum E, Lambrechts D, Carmeliet P. VEGF: once regarded as
a specific angiogenic factor, now implicated in neuroprotection.
BioEssays 2004;26:943 – 54.
[10] Pepper MS, Ferrara N, Orci L, Montesano R. Vascular endothelial
growth factor (VEGF) induces plasminogen activators and plasmi-
nogen activator inhibitor-1 in microvascular endothelial cells.
Biochem Biophys Res Commun 1991;181:902 – 6.
[11] Unemori EN, Ferrara N, Bauer EA, Amento EP. Vascular endothelial
growth factor induces interstitial collagenase expression in human
endothelial cells. J Cell Physiol 1992;153:557 – 62.
[12] Mandriota SJ, Seghezzi G, Vassalli J-D, Ferrara N, Wasi S, Mazzieri
R, et al. Vascular endothelial growth factor increases urokinase
receptor expression in vascular endothelial cells. J Biol Chem
1995;270:9709 – 16.
[13] Ferrara N. Vascular endothelial growth factor: basic science and
clinical progress. Endocr Rev 2004;25:581 – 611.
[14] Robinson CJ, Stringer SE. The splice variants of vascular
endothelial growth factor (VEGF) and their receptors. J Cell Sci
2001;114:853 – 65.
[15] Hiratsuka S, Kataoka Y, Nakao K, Nakamura K, Morikawa S,
Tanaka S, et al. VEGF-A is involved in guidance of VEGF-receptor-
positive cells to the anterior portion of early embryos. Mol Cell Biol
2005;25:355 – 63.
[16] Weinstein BM. What guides early embryonic blood vessel forma-
tion? Dev Dyn 1999;215:2 – 11.
[17] Carmeliet P, Ng YS, Nuyens D, Theilmeier G, Brusselmans K,
Cornelissen I, et al. Impaired myocardial angiogenesis and ischemic
cardiomyopathy in mice lacking the vascular endothelial growth factor
isoforms VEGF164 and VEGF188. Nat Med 1999;5:495 – 502.
[18] Stalmans I, Ng YS, Rohan R, Fruttiger M, Bouche A, Yuce A, et al.
Arteriolar and venular patterning in retinas of mice selectively
expressing VEGF isoforms. J Clin Invest 2002;109:327 – 36.
[19] Pugh CW, Ratcliffe PJ. Regulation of angiogenesis by hypoxia: role
of the HIF system. Nat Med 2003;9:677 – 84.
[20] Luttun A, Brusselmans K, Fukao H, Tjwa M, Ueshima S, Herbert
JM, et al. Loss of placental growth factor protects mice against
vascular permeability in pathological conditions. Biochem Biophys
Res Commun 2002;295:428 – 34.
[21] Kempf VAJ, Lebiedziejewski M, Alitalo K, W7lzlein J-H, Ehehalt U,
Fiebig J, et al. Activation of HIF-1 in bacillary angiomatosis: evidence
for a role of HIF-1 in bacterial infections. Circ Res 2005;3:623 – 32.
[22] Oosthuyse B, Moons L, Storkebaum E, Beck H, Nuyens D,
Brusselmans K, et al. Deletion of the hypoxia-response element in
the vascular endothelial growth factor promoter causes motor neuron
degeneration. Nat Genet 2001;28:131 – 8.
[23] Xia YP, Li B, Hylton D, Detmar M, Yancopoulos GD, Rudge JS.
Transgenic delivery of VEGF to the mouse skin leads to an
inflammatory condition resembling human psoriasis. Blood 2003;
102:161 – 8.
[24] Larcher F, Murillas R, Bolontrade M, Conti CJ, Jorcano JL. VEGF/
VPF overexpression in skin of transgenic mice induces angiogenesis,
559
vascular hyperpermeability and accelerated tumor development.
Oncogene 1998;17:303 – 11.
[25] Rossiter H, Barresi C, Pammer J, Rendl M, Haigh J, Wagner EF, et
al. Loss of vascular endothelial growth factor a activity in murine
epidermal keratinocytes delays wound healing and inhibits tumor
formation. Cancer Res 2004;64:3508 – 16.
[26] Sepp-Lorenzino L, Rands E, Mao X, Connolly B, Shipman J,
Antanavage J, et al. A novel orally bioavailable inhibitor of kinase
insert domain-containing receptor induces antiangiogenic effects and
prevents tumor growth in vivo. Cancer Res 2004;64:751 – 6.
[27] Kabbinavar F, Hurwitz HI, Fehrenbacher L, Meropol NJ,
Novotny WF, Lieberman G, et al. Phase II, randomized trial
comparing bevacizumab plus fluorouracil (FU)/leucovorin (LV)
with FU/LV alone in patients with metastatic colorectal cancer. J
Clin Oncol 2003;21:60 – 5 [comment].
[28] Ferrara N. Role of vascular endothelial growth factor in physio-
logical and pathological conditions: therapeutic implications. Semin
Oncol 2002;29:10 – 4.
[29] Nagy JA, Vasile E, Feng D, Sundberg C, Brown LF, Detmar MJ,
et al. Vascular permeability factor/vascular endothelial growth
factor induces lymphangiogenesis as well as angiogenesis. J Exp
Med 2002;196:1497 – 506.
[30] Saaristo A, Veikkola T, Enholm B, Hytonen M, Arola J, Pajusola
K, et al. Adenoviral VEGF-C overexpression induces blood vessel
enlargement, tortuosity, and leakiness but no sprouting angio-
genesis in the skin or mucous membranes. FASEB J 2002;16:
1041 – 9.
Downloaded from by guest on December 22, 2014
[31] Kubo H, Cao R, Brakenhielm E, Makinen T, Cao Y, Alitalo K.
Blockade of vascular endothelial growth factor receptor-3 signaling
inhibits fibroblast growth factor-2-induced lymphangiogenesis in
mouse cornea. Proc Natl Acad Sci U S A 2002;99:8868 – 73.
[32] Rissanen TT, Markkanen JE, Gruchala M, Heikura T, Puranen A,
Kettunen MI, et al. VEGF-D is the strongest angiogenic and
lymphangiogenic effector among VEGFs delivered into skeletal
muscle via adenoviruses. Circ Res 2003;92:1098 – 106.
[33] Cao R, Eriksson A, Kubo H, Alitalo K, Cao Y, Thyberg J.
Comparative evaluation of FGF-2-, VEGF-A-, and VEGF-C-induced
angiogenesis, lymphangiogenesis, vascular fenestrations, and per-
meability. Circ Res 2004;94:664 – 70.
[34] Lee CG, Link H, Baluk P, Homer RJ, Chapoval S, Bhandari V, et al.
Vascular endothelial growth factor (VEGF) induces remodeling and
enhances T(H)2-mediated sensitization and inflammation in the lung.
Nat Med 2004;10:1095 – 103.
[35] Clauss M, Weich H, Breier G, Knies U, Rockl W, Waltenberger J,
et al. The vascular endothelial growth factor receptor Flt-1 mediates
biological activities. Implications for a functional role of placenta
growth factor in monocyte activation and chemotaxis. J Biol Chem
1996;271:17629 – 34.
[36] Rafii S, Avecilla S, Shmelkov S, Shido K, Tejada R, Moore MA,
et al. Angiogenic factors reconstitute hematopoiesis by recruiting
stem cells from bone marrow microenvironment. Ann N Y Acad
Sci 2003;996:49 – 60.
[37] Cursiefen C, Chen L, Borges LP, Jackson D, Cao J, Radziejewski C,
et al. VEGF-A stimulates lymphangiogenesis and hemangiogenesis
in inflammatory neovascularization via macrophage recruitment.
J Clin Invest 2004;113:1040 – 50.
[38] Karkkainen MJ, Haiko P, Sainio K, Partanen J, Taipale J, Petrova
TV, et al. Vascular endothelial growth factor C is required for
sprouting of the first lymphatic vessels from embryonic veins. Nat
Immunol 2004;5:74 – 80.
[39] Persico MG, Vincenti V, DiPalma T. Structure, expression and
receptor-binding properties of placenta growth factor (PlGF). In: L.
Claesson-Welsh editor. Vascular growth factors and angiogenesis.
Springer-Verlag, Berlin: Springer-Verlag, MG Persico, V Vincenti,
DiPalma T. 1999, p. 31-40.
[40] Autiero M, Waltenberger J, Communi D, Kranz A, Moons L,
Lambrechts D, et al. Role of PlGF in the intra- and intermolecular
560 T. Tammela et al. / Cardiovascular Research 65 (2005) 550–563
cross talk between the VEGF receptors Flt1 and Flk1. Nat Med
2003;9:936 – 43.
[41] Park JE, Chen HH, Winer J, Houck KA, Ferrara N. Placenta growth
factor. Potentiation of vascular endothelial growth factor bioactivity,
in vitro and in vivo, and high affinity binding to Flt-1 but not to Flk-
1/KDR. J Biol Chem 1994;269:25646 – 54.
[42] Nagy JA, Dvorak AM, Dvorak HF. VEGF-A(164/165) and PlGF:
roles in angiogenesis and arteriogenesis. Trends Cardiovasc Med
2003;13:169 – 75.
[43] Carmeliet P, Moons L, Luttun A, Vincenti V, Compernolle V, De Mol
M, et al. Synergism between vascular endothelial growth factor and
placental growth factor contributes to angiogenesis and plasma
extravasation in pathological conditions. Nat Med 2001;7:575 – 83.
[44] Odorisio T, Schietroma C, Zaccaria ML, Cianfarani F, Tiveron C,
Tatangelo L, et al. Mice overexpressing placenta growth factor
exhibit increased vascularization and vessel permeability. J Cell Sci
2002;115:2559 – 67.
[45] Oura H, Bertoncini J, Velasco P, Brown LF, Carmeliet P, Detmar M.
A critical role of placental growth factor in the induction of
inflammation and edema formation. Blood 2003;101:560 – 7.
[46] Carmeliet P. VEGF gene therapy: stimulating angiogenesis or
angioma-genesis? Nat Med 2001;6:1102 – 3.
[47] Luttun A, Tjwa M, Moons L, Wu Y, Angelillo-Scherrer A, Liao F,
et al. Revascularization of ischemic tissues by PlGF treatment, and
inhibition of tumor angiogenesis, arthritis and atherosclerosis by
anti-Flt1. Nat Med 2002;8:831 – 40.
[48] Pipp F, Heil M, Issbrucker K, Ziegelhoeffer T, Martin S, van den
Heuvel J, et al. VEGFR-1-selective VEGF homologue PlGF is
arteriogenic: evidence for a monocyte-mediated mechanism. Circ
Res 2003;92:378 – 85.
[49] Olofsson B, Jeltsch M, Eriksson U, Alitalo K. Current biology of
VEGF-B and VEGF-C. Curr Opin Biotechnol 1999;10:528 – 35.
[50] Olofsson B, Pajusola K, von Euler G, Chilov D, Alitalo K, Eriksson
U. Genomic organization of the mouse and human genes for vascular
endothelial growth factor B (VEGF-B) and characterization of a
second splice isoform. J Biol Chem 1996;271:19130 – 317.
[51] Enholm B, Paavonen K, Ristimaki A, Kumar V, Gunji Y, Klefstrom
J, et al. Comparison of VEGF, VEGF-B, VEGF-C and Ang-1 mRNA
regulation by serum, growth factors, oncoproteins and hypoxia.
Oncogene 1997;14:2475 – 83.
[52] Salven P, Lymboussaki A, Heikkila P, Jaaskela-Saari H, Enholm B,
Aase K, et al. Vascular endothelial growth factors VEGF-B and
VEGF-C are expressed in human tumors. Am J Pathol 1998;153:
103 – 38.
[53] Bellomo D, Headrick JP, Silins GU, Paterson CA, Thomas PS,
Gartside M, et al. Mice lacking the vascular endothelial growth
factor-B gene (Vegfb) have smaller hearts, dysfunctional coronary
vasculature, and impaired recovery from cardiac ischemia. Circ Res
2000;86:E29 – 35 [Online].
[54] Aase K, von Euler G, Li X, Ponten A, Thoren P, Cao R, et al.
Vascular endothelial growth factor-B-deficient mice display an atrial
conduction defect. Circulation 2001;104:358 – 64.
[55] Wanstall JC, Gambino A, Jeffery TK, Cahill MM, Bellomo D,
Hayward NK, et al. Vascular endothelial growth factor-B-deficient
mice show impaired development of hypoxic pulmonary hyper-
tension. Cardiovasc Res 2002;55:361 – 8.
[56] Louzier V, Raffestin B, Leroux A, Branellec D, Caillaud JM, Levame
M, et al. Role of VEGF-B in the lung during development of chronic
hypoxic pulmonary hypertension. Am J Physiol, Lung Cell Mol
Physiol 2003;284:L926 – 37.
[57] Mould AW, Tonks ID, Cahill MM, Pettit AR, Thomas R, Hayward
NK, et al. Vegfb gene knockout mice display reduced pathology and
synovial angiogenesis in both antigen-induced and collagen-induced
models of arthritis. Arthritis Rheum 2003;48:2660 – 9.
[58] Silvestre JS, Tamarat R, Ebrahimian TG, Le-Roux A, Clergue M,
Emmanuel F, et al. Vascular endothelial growth factor-B promotes in
vivo angiogenesis. Circ Res 2003;93:114 – 23.
[59] Joukov V, Sorsa T, Kumar V, Jeltsch M, Claesson-Welsh L, Cao Y, et
al. Proteolytic processing regulates receptor specificity and activity
of VEGF-C. EMBO J 1997;16:3898 – 911.
[60] Siegfried G, Basak A, Cromlish JA, Benjannet S, Marcinkiewicz J,
Chretien M, et al. The secretory proprotein convertases furin, PC5,
and PC7 activate VEGF-C to induce tumorigenesis. J Clin Invest
2003;111:1723 – 32.
[61] Saharinen P, Tammela T, Karkkainen MJ, Alitalo K. Lymphatic
vasculature: development, molecular regulation and role in tumor
metastasis and inflammation. Trends Immunol 2004;25:387 – 95.
[62] Kukk E, Lymboussaki A, Taira S, Kaipainen A, Jeltsch M, Joukov V,
et al. VEGF-C receptor binding and pattern of expression with
VEGFR-3 suggests a role in lymphatic vascular development.
Development 1996;122:3829 – 37.
[63] Lymboussaki A, Olofsson B, Eriksson U, Alitalo K. Vascular
endothelial growth factor (VEGF) and VEGF-C show overlapping
binding sites in embryonic endothelia and distinct sites in differ-
entiated adult endothelia. Circ Res 1999;85:992 – 9.
[64] Oh SJ, Jeltsch MM, Birkenhager R, McCarthy JE, Weich HA, Christ
B, et al. VEGF and VEGF-C: specific induction of angiogenesis and
lymphangiogenesis in the differentiated avian chorioallantoic mem-
brane. Dev Biol 1997;188:96 – 109.
[65] Jeltsch M, Kaipainen A, Joukov V, Meng X, Lakso M, Rauvala H,
et al. Hyperplasia of lymphatic vessels in VEGF-C transgenic mice.
Science 1997;276:1423 – 5.
[66] Enholm B, Karpanen T, Jeltsch M, Kubo H, Stenback F, Prevo R,
et al. Adenoviral expression of VEGF-C induces lymphangio-
Downloaded from by guest on December 22, 2014
genesis in the skin. Circ Res 2001;88:623 – 9.
[67] Veikkola T, Jussila L, Makinen T, Karpanen T, Jeltsch M, Petrova
TV, et al. Signalling via vascular endothelial growth factor receptor-3
is sufficient for lymphangiogenesis in transgenic mice. EMBO J
2001;20:1223 – 31.
[68] Ristimaki A, Narko K, Enholm B, Joukov V, Alitalo K. Proin-
flammatory cytokines regulate expression of the lymphatic endothe-
lial mitogen vascular endothelial growth factor-C. J Biol Chem
1998;273:8413 – 8.
[69] Chilov D, Kukk E, Taira S, Jeltsch MM, Kaukonen J, Palotie A, et al.
Genomic organization of human and mouse genes for vascular
endothelial growth factor C. J Biol Chem 1997;272:25176 – 83.
[70] Su JL, Shih JY, Yen ML, Jeng YM, Chang CC, Hsieh CY, et al.
Cyclooxygenase-2 induces EP1- and HER-2/Neu-dependent vascu-
lar endothelial growth factor-C up-regulation: a novel mechanism of
lymphangiogenesis in lung adenocarcinoma. Cancer Res 2004;64:
554 – 64.
[71] Kerjaschki D, Regele HM, Moosberger I, Nagy-Bojarski K,
Watschinger B, Soleiman A, et al. Lymphatic neoangiogenesis in
human kidney transplants is associated with immunologically active
lymphocytic infiltrates. J Am Soc Nephrol 2004;15:603 – 12.
[72] Paavonen K, Mandelin J, Partanen T, Jussila L, Li T-F, Alitalo K,
et al. Vascular endothelial growth factors -C and -D and their
VEGFR-2 and -3 receptors in blood and lymphatic vessels in
healthy and rheumatoid synovium. J Rheumatol 2002;29:39 – 45.
[73] Skobe M, Hawighorst T, Jackson DG, Prevo R, Janes L, Velasco P,
et al. Induction of tumor lymphangiogenesis by VEGF-C promotes
breast cancer metastasis. Nat Med 2001;7:192 – 8.
[74] Karpanen T, Alitalo K. Lymphatic vessels as targets of tumor
therapy? J Exp Med 2001;194:F37 – 42.
[75] Mandriota SJ, Jussila L, Jeltsch M, Compagni A, Baetens D,
Prevo R, et al. Vascular endothelial growth factor-C-mediated
lymphangiogenesis promotes tumour metastasis. EMBO J
2001;20:672 – 82.
[76] Karpanen T, Egeblad M, Karkkainen MJ, Kubo H, Yla-Herttuala S,
Jaattela M, et al. Vascular endothelial growth factor C promotes
tumor lymphangiogenesis and intralymphatic tumor growth. Cancer
Res 2001;61:1786 – 90.
[77] He Y, Kozaki K, Karpanen T, Koshikawa K, Yla-Herttuala S,
Takahashi T, et al. Suppression of tumor lymphangiogenesis and
 T. Tammela et al. / Cardiovascular Research 65 (2005) 550–563
lymph node metastasis by blocking vascular endothelial growth
factor receptor 3 signaling. J Natl Cancer Inst 2002;94:819 – 25.
[78] He Y, Rajantie I, Ilmonen M, Makinen T, Karkkainen MJ, Haiko P,
et al. Preexisting lymphatic endothelium but not endothelial
progenitor cells are essential for tumor lymphangiogenesis and
lymphatic metastasis. Cancer Res 2004;64:3737 – 40.
[79] Reis-Filho JS, Schmitt FC. Lymphangiogenesis in tumors: what do
we know? Microsc Res Tech 2003;60:171 – 80.
[80] He Y, Karpanen T, Alitalo K. Role of lymphangiogenic factors in
tumor metastasis. Biochim Biophys Acta 2004;1654:3 – 12.
[81] Vleugel MM, Bos R, van der Groep P, Greijer AE, Shvarts A, Stel
HV, et al. Lack of lymphangiogenesis during breast carcinogenesis.
J Clin Pathol 2004;57:746 – 51.
[82] Baldwin ME, Catimel B, Nice EC, Roufail S, Hall NE, Stenvers KL,
et al. The specificity of receptor binding by vascular endothelial
growth factor-d is different in mouse and man. J Biol Chem
2001;276:19166 – 71.
[83] Farnebo F, Piehl F, Lagercrantz J. Restricted expression pattern of
vegf-d in the adult and fetal mouse: high expression in the embyonic
lung. Biochem Biophys Res Commun 1999;257:891 – 4.
[84] Stacker SA, Caesar C, Baldwin ME, Thornton GE, Williams RA,
Prevo R, et al. VEGF-D promotes the metastatic spread of tumor
cells via the lymphatics. Nat Med 2001;7:186 – 91.
[85] Achen MG, Williams RA, Minekus MP, Thornton GE, Stenvers K,
Rogers PA, et al. Localization of vascular endothelial growth factor-
D in malignant melanoma suggests a role in tumour angiogenesis.
J Pathol 2001;193:147 – 54.
[86] Ishii H, Yazawa T, Sato H, Suzuki T, Ikeda M, Hayashi Y, et al.
Enhancement of pleural dissemination and lymph node metastasis of
intrathoracic lung cancer cells by vascular endothelial growth factors
(VEGFs). Lung Cancer 2004;45:325 – 37.
[87] Byzova TV, Goldman CK, Jankau J, Chen J, Cabrera G, Achen MG,
et al. Adenovirus encoding vascular endothelial growth factor-D
induces tissue-specific vascular patterns in vivo. Blood 2002;
99:4434 – 42.
[88] Shibuya M, Yamaguchi S, Yamane A, Ikeda T, Tojo A, Matsushime
H, et al. Nucleotide sequence and expression of a novel human
receptor type tyrosine kinase gene (flt) closely related to the fms
family. Oncogene 1990;5:519 – 24.
[89] Huang K, Andersson C, Roomans GM, Ito N, Claesson-Welsh L.
Signaling properties of VEGF receptor-1 and -2 homo- and
heterodimers. Int J Biochem Cell Biol 2001;33:315 – 24.
[90] Zachary I, Gliki G. Signaling transduction mechanisms mediating
biological actions of the vascular endothelial growth factor family.
Cardiovasc Res 2001;49:568 – 81.
[91] Gerber HP, Conorelli F, Park J, Ferrara N. Differential transcriptional
regulation of the two vascular endothelial growth factor receptor
genes. Flt-1, but not Flk-1/KDR, is up-regulated by hypoxia. J Biol
Chem 1997;272:23659 – 67.
[92] Partanen TA, Alitalo K, Miettinen M. Lack of lymphatic vascular
specificity of vascular endothelial growth factor receptor 3 in 185
vascular tumors. Cancer 1999;86:2406 – 12.
[93] Kaipainen A, Korhonen J, Pajusola K, Aprelikova O, Persico MG,
Terman BI, et al. The related FLT4, FLT1, and KDR receptor
tyrosine kinases show distinct expression patterns in human fetal
endothelial cells. J Exp Med 1993;178:2077 – 88.
[94] Peters KG, De Vries C, Williams LT. Vascular endothelial growth
factor receptor expression during embryogenesis and tissue repair
suggests a role in endothelial differentiation and blood vessel growth.
Proc Natl Acad Sci U S A 1993;90:8915 – 9.
[95] Fong GH, Rossant J, Gertsenstein M, Breitman ML. Role of the Flt-1
receptor tyrosine kinase in regulating the assembly of vascular
endothelium. Nature 1995;376:66 – 70.
[96] Hiratsuka S, Minowa O, Kuno J, Noda T, Shibuya M. Flt-1 lacking
the tyrosine kinase domain is sufficient for normal development
and angiogenesis in mice. Proc Natl Acad Sci U S A 1998;
4:9349 – 54.
561
[97] Hiratsuka S, Maru Y, Okada A, Seiki M, Noda T, Shibuya M.
Involvement of Flt-1 tyrosine kinase (vascular endothelial growth
factor receptor-1) in pathological angiogenesis. Cancer Res 2001;
61:1207 – 13.
[98] Barleon B, Sozzani S, Zhou D, Weich HA, Mantovani A, Marme D.
Migration of human monocytes in response to vascular endothelial
growth factor (VEGF) is mediated via the VEGF receptor flt-1.
Blood 1996;87:3336 – 43.
[99] Carmeliet P, Luttun A. The emerging role of the bone marrow-
derived stem cells in (therapeutic) angiogenesis. Thromb Haemost
2001;86:289 – 97.
[100] Hattori K, Heissig B, Wu Y, Dias S, Tejada R, Ferris B, et al.
Placental growth factor reconstitutes hematopoiesis by recruiting
VEGFR1(+) stem cells from bone-marrow microenvironment. Nat
Med 2002;8:841 – 9.
[101] Maynard SE, Min JY, Merchan J, Lim KH, Li J, Mondal S, et al.
Excess placental soluble fms-like tyrosine kinase 1 (sFlt1) may
contribute to endothelial dysfunction, hypertension, and proteinuria
in preeclampsia. See editorial. J Clin Invest 2003;111:649 – 58.
[102] Levine RJ, Maynard SE, Qian C, Lim KH, England LJ, Yu KF, et al.
Circulating angiogenic factors and the risk of preeclampsia. New
Engl J Med 2004;350:672 – 83.
[103] Matthews W, Jordan CT, Gavin M, Jenkins NA, Copeland NG,
Lemischka IR. A receptor tyrosine kinase cDNA isolated from a
population of enriched primitive hematopoetic cells and exhibiting
close genetic linkage to c-kit. Proc Natl Acad Sci U S A
1991;88:9026 – 30.
Downloaded from by guest on December 22, 2014
[104] Terman BI, Carrion ME, Kovacs E, Rasmussen BA, Eddy RL,
Shows TB. Identification of a new endothelial cell growth factor
receptor tyrosine kinase. Oncogene 1991;6:1677 – 83.
[105] Meyer M, Clauss M, Lepple-Wienhues A, Waltenberger J, Augustin
M, Ziche M, et al. A novel vascular endothelial growth factor
encoded by Orf virus, VEGF-E, mediates angiogenesis via signaling
through VEGFR-2 (KDR) but not VEGFR-1 (Flt-1) receptor tyrosine
kinases. EMBO J 1999;18:363 – 74.
[106] Wise LM, Veikkola T, Mercer AA, Savory LJ, Fleming SB, Caesar
C, et al. The vascular endothelial growth factor (VEGF)-like protein
from orf virus NZ2 binds to VEGFR2 and neuropilin-1. Proc Natl
Acad Sci U S A 1999;96:3071 – 6.
[107] Gille H, Kowalski J, Li B, LeCouter J, Moffat B, Zioncheck TF, et al.
Analysis of biological effects and signaling properties of Flt-1
(VEGFR-1) and KDR (VEGFR-2). A reassessment using novel
receptor-specific vascular endothelial growth factor mutants. J Biol
Chem 2001;276:3222 – 30.
[108] Shima DT, Deutsch U, D’Amore PA. Hypoxic induction of vascular
endothelial growth factor (VEGF) in human epithelial cells is
mediated by increases in mRNA stability. FEBS Lett 1995;
370:203 – 8.
[109] Matsumoto T, Claesson-Welsh L. VEGF receptor signal trans-
duction. Science’s STKE 2001;112:1 – 17.
[110] Shalaby F, Rossant J, Yamaguchi TP, Gertsenstein M, Wu XF,
Breitman ML, et al. Failure of blood-island formation and vasculo-
genesis in Flk-1-deficient mice. Nature 1995;376:62 – 6.
[111] Shalaby F, Ho J, Stanford WL, Fischer KD, Schuh AC, Schwartz L,
et al. A requirement for Flk1 in primitive and definitive hematopoi-
esis and vasculogenesis. Cell 1997;89:981 – 90.
[112] Baffert F, Thurston G, Rochon-Duck M, Le T, Brekken R, McDonald
DM. Age-related changes in vascular endothelial growth factor
dependency and angiopoietin-1-induced plasticity of adult blood
vessels. Circ Res 2004;94:984 – 92.
[113] Soldi R, Mitola S, Strasly M, Defilippi P, Tarone G, Bussolino F.
Role of avh3 in the activation of vascular endothelial growth factor
receptor-2. EMBO J 1999;18:882 – 92.
[114] Hutchings H, Ortega N, Plouet J. Extracellular matrix-bound
vascular endothelial growth factor promotes endothelial cell adhe-
sion, migration and survival through integrin ligation. FASEB J
2003;17:1520 – 2.
562 T. Tammela et al. / Cardiovascular Research 65 (2005) 550–563
[115] Carmeliet P, Lampugnani MG, Moons L, Breviario F, Comper-
nolle V, Bono F, et al. Targeted deficiency or cytosolic truncation
of the VE-cadherin gene in mice impairs VEGF-mediated
endothelial survival and angiogenesis. Cell 1999;98:147 – 57.
[116] Pajusola K, Aprelikova O, Pelicci G, Weich H, Claesson-Welsh L,
Alitalo K. Signalling properties of FLT4, a proteolytically processed
receptor tyrosine kinase related to two VEGF receptors. Oncogene
1994;9:3545 – 55.
[117] Lee J, Gray A, Yuan J, Louth S-M, Avraham H, Wood W. Vascular
endothelial growth factor-related protein: a ligand and specific
activator of the tyrosine kinase receptor Flt4. Proc Natl Acad Sci
U S A 1996;93:1988 – 92.
[118] Hughes DC. Alternative splicing of the human VEGFGR-3/FLT4
gene as a consequence of an integrated human endogenous
retrovirus. J Mol Evol 2001;53:77 – 9.
[119] Joukov V, Pajusola K, Kaipainen A, Chilov D, Lahtinen I, Kukk E,
et al. A novel vascular endothelial growth factor, VEGF-C, is a
ligand for the Flt4 (VEGFR-3) and Kdr (VEGFR-2) receptor
tyrosine kinases. EMBO J 1996;15:290 – 8.
[120] Achen MG, Jeltsch M, Kukk E, Makinen T, Vitali A, Wilks AF, et al.
Vascular endothelial growth factor D (VEGF-D) is a ligand for the
tyrosine kinases VEGF receptor 2 (Flk1) and VEGF receptor 3
(Flt4). Proc Natl Acad Sci U S A 1998;95:548 – 53.
[121] Kaipainen A, Korhonen J, Mustonen T, van Hinsbergh VM, Fang
G-H, Dumont D, et al. Expression of the fms-like tyrosine kinase
FLT4 gene becomes restricted to endothelium of lymphatic vessels
during development. Proc Natl Acad Sci U S A 1995;92:3566 – 70.
[122] Partanen T, Arola J, Saaristo A, Jussila L, Ora A, Miettinen M, et al.
VEGF-C and VEGF-D expression in neuroendocrine cells and their
receptor, VEGFR-3, in fenestrated blood vessels in human tissues.
FASEB J 2000;14:2087 – 96.
[123] Skobe M, Hamberg LM, Hawighorst T, Shirner M, Wolf GL, Alitalo
K, et al. Concurrent induction of lymphangiogenesis, angiogenesis
and macrophage recruitment by VEGF-C in melanoma. Am J Pathol
2001;159:893 – 903.
[124] Veikkola T, Lohela M, Ikenberg K, Makinen T, Korff T, Saaristo
A,et al. Intrinsic versus microenvironmental regulation of lym-
phatic endothelial cell phenotype and function. FASEB J
2003;17:2006 – 13.
[125] Dumont DJ, Jussila L, Taipale J, Mustonen T, Pajusola K, Breitman
M, et al. Cardiovascular failure in mouse embryos deficient in VEGF
receptor-3. Science 1998;282:946 – 9.
[126] Hamada K, Oike Y, Takakura N, Ito Y, Jussila L, Dumont DJ, et al.
VEGF-C signaling pathways through VEGFR-2 and VEGFR-3 in
vasculoangiogenesis and hematopoiesis. Blood 2000;96:3793 – 800.
[127] Makinen T, Jussila L, Veikkola T, Karpanen T, Kettunen MI,
Pulkkanen KJ, et al. Inhibition of lymphangiogenesis with resulting
lymphedema in transgenic mice expressing soluble VEGF receptor-
3. Nat Med 2001;7:199 – 205.
[128] Irrthum A, Karkkainen MJ, Devriendt K, Alitalo K, Vikkula M.
Congenital hereditary lymphedema caused by a mutation that
inactivates VEGFR3 tyrosine kinase. Am J Hum Genet 2000;67:
295 – 301.
[129] Karkkainen MJ, Ferrell RE, Lawrence EC, Kimak MA, Levinson
MA, McTigue MA, et al. Missense mutations interfere with VEGFR-
3 signalling in primary lymphoedema. Nat Genet 2000;25:153 – 9.
[130] Karkkainen MJ, Saaristo A, Jussila L, Karila KA, Lawrence EC,
Pajusola K, et al. A model for gene therapy of human hereditary
lymphedema. Proc Natl Acad Sci U S A 2001;98:12677 – 82.
[131] Chen L, Hamrah P, Cursiefen C, Zhang Q, Pytowski B, Streilein JW,
et al. Vascular endothelial growth factor receptor-3 mediates
induction of corneal alloimmunity. Nat Med 2004;10:13 – 5.
[132] Klagsbrun M, Takashima S, Mamluk R. The role of neuropilin in
vascular and tumor biology. Adv Exp Med Biol 2002;515:33 – 48.
[133] Bagri A, Tessier-Lavigne M. Neuropilins as semaphorin receptors: in
vivo functions in neuronal cell migration and axon guidance. Adv
Exp Med Biol 2002;515:13 – 31.
[134] Kawasaki T, Bekku Y, Suto F, Kitsukawa T, Taniguchi M, Nagatsu I,
et al. Requirement of neuropilin 1-mediated Sema3A signals in
patterning of the sympathetic nervous system. Development
2002;129:671 – 80.
[135] M. Klagsbrun, J. Folkman, Angiogenesis. In: Sporn, M.B. Roberts,
A.B. Peptide growth factors and their receptors. Springer-Verlag,
New York: Springer-Verlag, M. Klagsbrun, Folkman J; 1991:549-86.
[136] Kitsukawa T, Shimono A, Kawakami A, Kondoh H, Hajime F. Over
expression of a membrane protein, neuropilin, in chimeric mice
causes anomalities in the cardiovascular system, nervous system and
limbs. Development 1995;121:4309 – 18.
[137] Herzog Y, Kalcheim C, Kahane N, Reshef R, Neufeld G. Differential
expression of neuropilin-1 and neuropilin-2 in arteries and veins.
Mech Dev 2001;109:115 – 9.
[138] Yuan L, Moyon D, Pardanaud L, Breant C, Karkkainen MJ, Alitalo
K, et al. Abnormal lymphatic vessel development in neuropilin 2
mutant mice. Development 2002;129:4797 – 806.
[139] Kawasaki T, Kitsukawa T, Bekku Y, Matsuda Y, Sanbo M, Yagi T,
et al. A requirement for neuropilin-1 in embryonic vessel
formation. Development 1999;126:4895 – 902.
[140] Gu C, Rodriguez ER, Reimert DV, Shu T, Fritzsch B, Richards LJ,
et al. Neuropilin-1 conveys semaphorin and VEGF signaling during
neural and cardiovascular development. Dev Cell 2003;5:45 – 57.
[141] Takashima S, Kitakaze M, Asakura M, Asanuma H, Sanada S,
Tashiro F, et al. Targeting of both mouse neuropilin-1 and neuropilin-
2 genes severely impairs developmental yolk sac and embryonic
angiogenesis. Proc Natl Acad Sci U S A 2002;99:3657 – 62.
Downloaded from by guest on December 22, 2014
[142] Yla-Herttuala S, Alitalo K. Gene transfer as a tool to induce
therapeutic vascular growth. Nat Med 2003;9:694 – 701.
[143] Scholz D, Cai WJ, Schaper W. Arteriogenesis, a new concept of
vascular adaptation in occlusive disease. Angiogenesis 2001;4:
247 – 57.
[144] Rosengart TK, Patel SR, Crystal RG. Therapeutic angiogenesis:
protein and gene therapy delivery strategies. J Cardiovasc Risk
1999;6:29 – 40.
[145] Freedman SB, Vale P, Kalka C, Kearney M, Pieczek A, Symes J,
et al. Plasma vascular endothelial growth factor (VEGF) levels
after intramuscular and intramyocardial gene transfer of VEGF-1
plasmid DNA. Hum Gene Ther 2002;13:1595 – 603.
[146] Baumgartner I, Isner JM. Gene therapy for peripheral vascular
disease. Israel Med Assoc J 2000;2:27 – 32.
[147] Rajagopalan S, Mohler ERr, Lederman RJ, Mendelsohn FO,
Saucedo JF, Goldman CK, et al. Regional angiogenesis with vascular
endothelial growth factor in peripheral arterial disease: a phase II
randomized, double-blind, controlled study of adenoviral delivery of
vascular endothelial growth factor 121 in patients with disabling
intermittent claudication. Circulation 2003;108:1933 – 8.
[148] Carmeliet P. VEGF gene therapy: stimulating angiogenesis or
angioma-genesis? Nat Med 2000;6:1102 – 3.
[149] Sueishi K, Yonemitsu Y, Nakagawa K, Kaneda Y, Kumamoto M,
Nakashima Y. Atherosclerosis and angiogenesis. Its pathophysio-
logical significance in humans as well as in an animal model induced
by the gene transfer of vascular endothelial growth factor. Ann N Y
Acad Sci 1997;811:311 – 24.
[150] Hayden MR, Tyagi SC. Vasa vasorum in plaque angiogenesis,
metabolic syndrome, type 2 diabetes mellitus, and atheroscleropathy:
a malignant transformation. Cardiovasc Diabetol 2004;3:1.
[151] Salomonsson L, Pettersson S, Englund MC, Wiklund O, Ohlsson
BG. Post-transcriptional regulation of VEGF expression by
oxidised LDL in human macrophages. Eur J Clin Invest 2002;32:
767 – 74.
[152] Nakagawa K, Chen YX, Ishibashi H, Yonemitsu Y, Murata T, Hata
Y, et al. Angiogenesis and its regulation: roles of vascular endothelial
cell growth factor. Semin Thromb Hemost 2000;26:61 – 6.
[153] Celletti FL, Waugh JM, Amabile PG, Brendolan A, Hilfiker PR,
Dake MD. Vascular endothelial growth factor enhances atheroscler-
otic plaque progression. Nat Med 2001;7:425 – 9.
 T. Tammela et al. / Cardiovascular Research 65 (2005) 550–563
[154] Moulton KS, Vakili K, Zurakowski D, Soliman M, Butterfield C,
Sylvin E, et al. Inhibition of plaque neovascularization reduces
macrophage accumulation and progression of advanced atheroscle-
rosis. Proc Natl Acad Sci U S A 2003;100:4736 – 41.
[155] Lemstrom KB, Krebs R, Nykanen AI, Tikkanen JM, Sihvola RK,
Aaltola EM, et al. Vascular endothelial growth factor enhances
cardiac allograft arteriosclerosis. Circulation 2002;105:2524 – 30.
[156] Cho CH, Kammerer RA, Lee HJ, Yasunaga K, Kim KT, Choi HH,
et al. Designed angiopoietin-1 variant, COMP-Ang1, protects
against radiation-induced endothelial cell apoptosis. Proc Natl
Acad Sci U S A 2004;101:5553 – 8.
[157] Rafii S, Lyden D. Therapeutic stem and progenitor cell trans-
plantation for organ vascularization and regeneration. Nat Med
2003;9:702 – 12.
[158] Majka SM, Jackson KA, Kienstra KA, Majesky MW, Goodell MA,
Hirschi KK. Distinct progenitor populations in skeletal muscle are
bone marrow derived and exhibit different cell fates during vascular
regeneration. J Clin Invest 2003;111:71 – 9.
[159] Wagers AJ, Sherwood RI, Christensen JL, Weissman IL. Little
evidence for developmental plasticity of adult hematopoietic stem
cells. Science 2002;297:2256 – 9.
[160] Ziegelhoeffer T, Fernandez B, Kostin S, Heil M, Voswinckel R,
Helisch A, et al. Bone marrow-derived cells do not incorporate into
the adult growing vasculature. Circ Res 2004;94:230 – 8.
[161] Rajantie I, Ilmonen M, Alminaite A, Ozerden U, Alitalo K, Salven P.
Adult bone marrow-derived cells recruited during angiogenesis
comprise precursors for periendothelial vascular mural cells. Blood
2004;104:2084 – 6.
[162] Jain RK. Molecular regulation of vessel maturation. Nat Med
2003;9:685 – 93.
[163] Ruoslahti E. Vascular zip codes in angiogenesis and metastasis.
Biochem Soc Trans 2004;32:397 – 402.
[164] Yang JC, Haworth L, Sherry RM, Hwu P, Schwartzentruber DJ,
Topalian SL, et al. A randomized trial of bevacizumab, an anti-
vascular endothelial growth factor antibody, for metastatic renal
cancer. New Engl J Med 2003;349:427 – 34.
[165] Bainbridge JW, Mistry AR, Thrasher AJ, Ali RR. Gene therapy for
ocular angiogenesis. Clin Sci 2003;104:561 – 75.
[166] Aiello LP, Pierce EA, Foley ED, Takagi H, Chen H, Riddle L, et al.
Suppression of retinal neovascularization in vivo by inhibition of
vascular endothelial growth factor (VEGF) using soluble VEGF-
receptor chimeric proteins. Proc Natl Acad Sci U S A 1995;92:
10457 – 61.
[167] Robinson GS, Pierce EA, Rook SL, Foley E, Webb R, Smith LES.
Oligodeoxynucleotides inhibit retinal neovascularization in a murine
563
model of proliferative retinopathy. Proc Natl Acad Sci U S A 1996;
93:4851 – 6.
[168] Ozaki H, Seo MS, Ozaki K, Yamada H, Yamada E, Okamoto N,
et al. Blockade of vascular endothelial cell growth factor receptor
signaling is sufficient to completely prevent retinal neovasculari-
zation. Am J Pathol 2000;156:697 – 707.
[169] Ishida S, Usui T, Yamashiro K, Kaji Y, Amano S, Ogura Y, et al.
VEGF164-mediated inflammation is required for pathological, but
not physiological, ischemia-induced retinal neovascularization. J Exp
Med 2003;198:483 – 9.
[170] Detmar M. Evidence for vascular endothelial growth factor
(VEGF) as a modifier gene in psoriasis. J Invest Dermatol
2004;122:xiv – v.
[171] Reynolds LP, Grazul-Bilska AT, Redmer DA. Angiogenesis in the
female reproductive organs: pathological implications. Int J Exp
Pathol 2003;83:151 – 63.
[172] Rockson SG. Lymphedema. Am J Med 2001;110:288 – 95.
[173] Northup KA, Witte MH, Witte CL. Syndromic classification of
hereditary lymphedema. Lymphology 2003;36:162 – 89.
[174] Fang J, Dagenais SL, Erickson RP, Arlt MF, Glynn MW, Gorski JL,
et al. Mutations in FOXC2 (MFH-1), a forkhead family transcription
factor, are responsible for the hereditary lymphedema-distichiasis
syndrome. Am J Hum Genet 2000;67:1382 – 8.
[175] Petrova TV, Karpanen T, Norrmen C, Mellor R, Tamakoshi T,
Finegold D, et al. Defective valves and abnormal mural cell
recruitment underlie lymphatic vascular failure in lymphedema
distichiasis. Nat Med 2004;10:974 – 81.
Downloaded from by guest on December 22, 2014
[176] Padera TP, Kadambi A, di Tomaso E, Carreira CM, Brown EB,
Boucher Y, et al. Lymphatic metastasis in the absence of functional
intratumor lymphatics. Science 2002;296:1883 – 6.
[177] Achen MG, Williams RA, Baldwin ME, Lai P, Roufail S, Alitalo K,
et al. The angiogenic and lymphangiogenic factor vascular endothe-
lial growth factor-D exhibits a paracrine mode of action in cancer.
Growth Factors 2002;20:99 – 107.
[178] Maula SM, Luukkaa M, Grenman R, Jackson D, Jalkanen S,
Ristamaki R. Intratumoral lymphatics are essential for the metastatic
spread and prognosis in squamous cell carcinomas of the head and
neck region. Cancer Res 2003;63:1920 – 6.
[179] Dadras SS, Paul T, Bertoncini J, Brown LF, Muzikansky A, Jackson
DG, et al. Tumor lymphangiogenesis: a novel prognostic indicator
for cutaneous melanoma metastasis and survival. Am J Pathol
2003;162:1951 – 60.
[180] Straume O, Jackson DG, Akslen LA. Independent prognostic impact
of lymphatic vessel density and presence of low-grade lymphangio-
genesis in cutaneous melanoma. Clin Cancer Res 2003;9:250 – 6.