Monographs
`
`1097M
`
` Genes & Cancer
`2(12) 1097 –1105
`© The Author(s) 2011
`Reprints and permission:
`sagepub.com/journalsPermissions.nav
`DOI: 10.1177/1947601911423031
`http://ganc.sagepub.com
`
`Vascular Endothelial Growth Factor (VEGF)
`and Its Receptor (VEGFR) Signaling
`in Angiogenesis: A Crucial Target for
`Anti- and Pro-Angiogenic Therapies
`
`Masabumi Shibuya
`
`Abstract
`The vascular endothelial growth factor (VEGF) and its receptor (VEGFR) have been shown to play major roles not only in physiological but also in most
`pathological angiogenesis, such as cancer. VEGF belongs to the PDGF supergene family characterized by 8 conserved cysteines and functions as a
`homodimer structure. VEGF-A regulates angiogenesis and vascular permeability by activating 2 receptors, VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk1
`in mice). On the other hand, VEGF-C/VEGF-D and their receptor, VEGFR-3 (Flt-4), mainly regulate lymphangiogenesis. The VEGF family includes other
`interesting variants, one of which is the virally encoded VEGF-E and another is specifically expressed in the venom of the habu snake (Trimeresurus
`flavoviridis). VEGFRs are distantly related to the PDGFR family; however, they are unique with respect to their structure and signaling system. Unlike
`members of the PDGFR family that strongly stimulate the PI3K-Akt pathway toward cell proliferation, VEGFR-2, the major signal transducer for
`angiogenesis, preferentially utilizes the PLCγ-PKC-MAPK pathway for signaling. The VEGF-VEGFR system is an important target for anti-angiogenic
`therapy in cancer and is also an attractive system for pro-angiogenic therapy in the treatment of neuronal degeneration and ischemic diseases.
`
`Keywords: VEGF, VEGF receptor, tumor angiogenesis, anti-angiogenic therapy, neuronal degeneration, pro-angiogenic therapy
`
`Introduction
`Angiogenesis, the formation and main-
`tenance of blood vessel structures, is
`essential for the physiological functions
`of tissues and is important for the pro-
`gression of diseases such as cancer and
`inflammation.1,2 In recent decades, a
`variety of signaling molecules, such as
`VEGF-VEGFRs, ephrin-Eph receptors,
`angiopoietin-Tie, and the Delta-Notch
`system, have been identified as playing
`important roles in angiogenesis. Among
`these, vascular endothelial growth fac-
`tors (VEGFs) and receptors (VEGFRs)
`regulate both vasculogenesis, the devel-
`opment of blood vessels from precursor
`cells during early embryogenesis, and
`angiogenesis, the formation of blood
`vessels from pre-existing vessels at a
`later stage3 (Fig. 1). The VEGF family
`of genes contains at least 7 members,
`including
`the viral genome–derived
`VEGF-E, whereas the VEGFR family of
`genes has 3 to 4 members depending on
`the vertebrate species.4,5 VEGF-A and
`its receptors VEGFR-1 and VEGFR-2
`play major roles in physiological as well
`
`as pathological angiogenesis, including
`tumor angiogenesis. VEGF-C/D and
`their receptor VEGFR-3 can regulate
`angiogenesis at early embryogenesis but
`mostly function as critical regulators of
`lymphangiogenesis.6
`VEGF-A has a variety of functions,
`including pro-angiogenic activity, vas-
`cular permeability activity, and the stim-
`ulation of cell migration in macrophage
`lineage and endothelial cells. Recently,
`anti–VEGF-VEGFR drugs such as an
`anti–VEGF-A neutralizing antibody and
`multikinase inhibitors have been devel-
`oped and widely used for the treatment
`of major solid tumors.7,8 The clinical
`efficacy of these medicines has been
`well evaluated; however, none of them
`provide a complete cure for cancer
`patients. The molecular basis of the
`refractoriness in some tumors and the
`acquisition of resistance to these medi-
`cines should be extensively studied to
`develop more efficient anti-angiogenic
`therapies.
`On the other hand, VEGFs have pro-
`angiogenic potential for the maintenance
`
`of various tissues at physiological levels
`and for the formation of new blood ves-
`sels to overcome ischemic diseases. The
`utility of VEGF family members in pro-
`angiogenic medicine, together with the
`possible side effects, should be charac-
`terized
`in more detail for clinical
`applications.
`
`Structure and Function
`of the VEGF Family
`VEGF, also known as VEGF-A, is a pro-
`tein with vascular permeability activity
`that was originally purified from a fluid
`secreted by a tumor.9 A few years later,
`a protein with angiogenic activity was
`independently purified and named
`
`Jobu University, Isesaki, Japan
`Department of Molecular Oncology, Tokyo Medical
`and Dental University, Tokyo, Japan
`University of Tokyo, Tokyo, Japan
`
`Corresponding Author:
`Masabumi Shibuya, MD, PhD, Department of Molecular
`Oncology, Tokyo Medical and Dental University, 1-5-45
`Yushima, Bunkyo-ku, Tokyo 113-8519, Japan
`Email: shibuya@ims.u-tokyo.ac.jp
`
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`

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`1098
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`M Monographs
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`Regulation of :
`Tip cell formation,
`Placental circulation,
`Corneal
`.
`Corneal avascularity
`
`Trophoblast(sFlt-1
`synthesis)
`
`VEGF-E
`
`VEGF-A
`
`VEGF-C
`VEGF-D
`
`PlGF
`VEGF-B
`
`sFlt-1
`
`maternal
`circulation
`
`fetal
`circulation
`
`Placenta
`
`VEGFR-1
`(Flt-1)
`
`VEGFR-2
`(KDR/Flk-1)
`
`VEGFR-3
`(Flt-4)
`
`Angiogenesis Lymphangiogenesis
`
`Figure 1. The VEGF and VEGFR system. VEGF-A and its receptors, VEGFR-1 and VEGFR-2, play
`a major role in vasculogenesis and angiogenesis. In addition, sFlt-1, a soluble form of VEGFR-1, is
`expressed in various cells such as trophoblasts and negatively regulates angiogenesis.
`
`VEGF.10 Molecular cloning, however,
`revealed that these 2 proteins were identi-
`cal and encoded by a single gene.3 The
`VEGF family includes VEGF-A, VEGF-
`B, VEGF-C, VEGF-D, PlGF (placental
`growth factor), VEGF-E (Orf-VEGF),
`and Trimeresurus flavoviridis svVEGF.
`With the exception of the latter 2 mem-
`bers, 5 genes of the VEGF family exist in
`mammalian genomes, including humans.
`Essentially, all the VEGFs have 8 con-
`served cysteine residues at fixed posi-
`tions, which are very similar to the PDGF
`family such as M-CSF (CSF-1), SCF
`(stem cell factor), and Flt3L (Flt3 ligand).
`Among the 8 cysteines, 6 residues form 3
`S-S intramolecular bonds and generate 3
`loop structures.11 The remaining 2 cyste-
`ines form 2 S-S intermolecular bonds,
`contributing to the stable homodimer
`structure of VEGF. We have shown that a
`structure combined with loop 1 and loop
`3 in VEGF-A and VEGF-E is essential
`for
`the binding and activation of
`VEGFR-2.12
`
`VEGF-A
`Through alternative splicing, the VEGF-
`A protein contains subtypes, such as
`peptides of 121, 165, 189, and 206
`amino acids in humans.3 Except for
`
`VEGF-A
`, the other peptides have a
`121
`basic stretch near the carboxyl terminus.
`The basic stretch of VEGF-A
` has
`165
`a weak affinity for acidic materials
`such as heparin/heparan sulfate and to
`neuropilin-1, a membrane protein involved
`in neuronal cell regulation and a corecep-
`tor for VEGF-A. The basic stretch of
`VEGF-A
` has a strong binding affinity
`189
`to heparin/heparan sulfate, and thus, most
`of the VEGF-A
` molecules appear to be
`189
`localized on the cell surface or in the
`extracellular matrix.
`The VEGF-A gene is unique in terms
`of its haploid insufficiency: even if only a
`single copy of the VEGF-A gene is defi-
`cient
`(heterozygotic VEGF-A gene
`knockout mice; VEGF-A+/– mice), the
`mutant embryo dies at early embryogen-
`esis (E10-E11) due to immature forma-
`tion and dysfunction of the circulatory
`system.3 This indicates that the local con-
`centration of VEGF-A in tissue is tightly
`regulated in embryogenesis, and half the
`level of the VEGF-A protein is insuffi-
`cient to complete the formation of the
`closed circulatory system in the body.
`Among subtypes of VEGF-A, VEGF-
` is most important both quantita-
`A
`165
`tively and qualitatively. Maes et al.
`reported that VEGF-A
` is essential and
`165
`
`Genes & Cancer / vol 2 no 12 (2011)
`
`for angiogenesis because
`sufficient
` transgenic mice in a VEGF-
`VEGF-A
`164
`A–null genetic background are alive and
`essentially healthy.13 More
`recently,
`another subtype of VEGF-A, VEGF-
`, was reported in humans.14 VEGF-
`A
`xxxb
`A
` activates the receptor much more
`xxxb
`weakly than the normal VEGF-A, sug-
`gesting that VEGF-A
` could be a phys-
`xxxb
`iological competitor against VEGF-A.
`VEGF-A binds to and activates both
`VEGFR-1 and VEGFR-2, promoting
`angiogenesis, vascular permeability,
`cell migration, and gene expression.5
`In addition, Lee et al. showed that an
`autocrine loop of VEGF-A and its recep-
`tor system exist within vascular endo-
`thelial cells, contributing to endothelial
`functions.15
`
`PlGF and VEGF-B
`These molecules bind to and activate
`only VEGFR-1. As will be described
`later, VEGFR-1 has the ability to bind
`tightly to its ligands but has a weak tyro-
`sine kinase activity, generating signals
`weaker than VEGFR-2. Both PlGF–/–
`and VEGF-B–/– mice are alive at birth
`with no significant defects related to
`angiogenesis, suggesting that these genes
`are dispensable at embryogenesis. How-
`ever, under pathological conditions, syn-
`ergism between PlGF and VEGF-A has
`been shown to contribute to angiogene-
`sis.16 VEGF-B–/– mice have the pheno-
`type of an atrial conduction defect.17 In
`addition, VEGF-B was recently reported
`to protect against the degeneration of sen-
`sory neurons.18 These results indicate
`that, although PlGF and VEGF-B are not
`essential at embryogenesis, they have a
`variety of functions under pathological or
`stressed conditions.
`
`VEGF-C and VEGF-D
`These 2 members of the VEGF family
`are produced as premature forms and are
`cleaved by proteases such as furin in
`both the amino- and carboxyl-terminal
`portions.19 After processing, these mol-
`ecules develop a higher affinity for
`VEGFR-3, which is expressed on lym-
`phatic endothelial cells and stimulates
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`

`VEGF and VEGFR signaling in angiogenesis / Shibuya
`
`Monographs
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`M
`
`1099
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`VEGF-A165 PlGF/VEGF-B VEGF-E T.f. svVEGF
`
`
`VEGFR 1 VEGFR 2 R 1 R 2VEGFR-1 VEGFR-2 R-1 R-2
`
`(Flt-1)
`(KDR/Flk-1)
`
`
`
`
`
`R 1 R 2R-1 R-2
`
`
`
`R 1 R 2R-1 R-2
`
`VEGFVEGF
`
`activity
`
`VPF
`activity
`
`+++ +/-
`
`+++ +
`
`+++
`+++
`
`+
`+
`
`+
`+
`(++ in acute phase)
`
`+++
`+++
`
`Figure 2. Unique activation of VEGFRs by VEGF-E and Trimeresurus flavoviridis svVEGF. VEGF-E
`encoded in the Orf viral genome binds to and activates only VEGFR-2, inducing well-organized
`blood vessels. T. flavoviridis svVEGF stimulates vascular permeability by activation of VEGFRs in
`a specific manner.
`
`the receptor for lymphangiogenesis. In
`addition, these proteins have a weak
`affinity for VEGFR-2, activating angio-
`genesis to some extent. VEGF-C is
`expressed during embryogenesis, whereas
`VEGF-D is expressed after birth during
`adult stages. This difference in gene
`expression is thought to be a major cause
`for lethality in VEGF-C–/– mice but not
`in VEGF-D–/– mice. VEGF-C–/– mice
`show severe accumulation of fluid in tis-
`sues due to poor development of lymph
`vessels.6
`
`VEGF-E, an Angiogenic Protein
`Encoded in the Pro-Angiogenic
`Orf Virus Genome
`The Orf virus, a parapoxvirus infecting
`sheep, goats, and sometimes humans, is
`known to induce angiogenesis at sites of
`infection on the skin. In 1994, Lyttle et al.
`found that a gene in the viral genome
`encodes a protein distantly related to
`VEGF/PDGF.20 The amino acid identity
`of this gene product in the NZ7 strain of
`the Orf virus shows a limited (25%)
`degree of identity to human VEGF-A
`and 19% to human PDGF. This low
`homology suggested that NZ7-derived
`proteins bind weakly to VEGFR and/or
`PDGFR. However, to our surprise, we
`
`(designated
`this protein
`that
`found
`VEGF-E
`) tightly binds to and acti-
`NZ7
`vates VEGFR-2, but not other VEGFRs
`(VEGFR-1, VEGFR-3) nor PDGFR21
`(Fig. 2). Therefore, VEGF-E is unique in
`terms of its specificity to VEGFR-2.
`None of the angiogenic factors encoded
`in the human genome has been reported
`to exhibit such VEGFR-2 specificity. The
`products of other Orf virus clones, such
`as NZ2 and D1701 strains, also show
`essentially the same specificity.22,23
`Because the human genome does not
`contain the original VEGF-E gene, and
`because the genomic structure of VEGF-E
`in the Orf virus genome suggests an
`insertion of a DNA sequence carrying
`the VEGF-E gene into the viral sequence,
`the original VEGF-E might be present
`in the genome of some vertebrates other
`than mammals. Infection of an animal
`by the precursor Orf virus might have
`led to the incorporation of the VEGF-E
`gene into the viral genome, maintaining
`the gene as a pro-angiogenic factor to
`facilitate viral production in the infected
`dermal tissues. Such a capture of a bio-
`logically active gene into a viral genome
`is well known to have occurred in the
`cases of viral oncogene–containing ret-
`roviruses such as Rous sarcoma virus.24
`
`T. flavoviridis svVEGF, a VEGF-Like
`Molecule Secreted in Snake Venom
`Snake venom contains a variety of
`molecules that attack target animals
`both directly (as toxins) and indirectly
`(as toxin-promoting materials). From
`T. flavoviridis (habu) snake venom,
`Takahashi et al. purified a protein bear-
`ing weak angiogenic activity and strong
`vascular permeability activity.25 Interest-
`ingly, this protein, named T. flavoviridis
`svVEGF, binds tightly to VEGFR-1 and
`weakly to VEGFR-2 (Fig. 2). Further-
`more, the T. flavoviridis svVEGF pro-
`tein is synthesized only in the venom
`tissue and secreted into the venom fluid,
`which appears to be the first case of a
`VEGF family protein being secreted
`from tissues of the body. This may sug-
`gest that the biological function of this
`protein is not for increasing the vascular
`permeability within the snake body itself
`but rather promoting vascular permea-
`bility in the local tissues of targeted ani-
`mals. Because T. flavoviridis svVEGF
`itself is not toxic to cultured mammalian
`cells, the purpose of this vascular per-
`meability activity is considered to be
`linked to the circulation of real toxins
`into the target animals and promotion of
`their efficacy.
`
`Structure of VEGFRs
`VEGFRs are typical tyrosine kinase recep-
`tors (TKRs) carrying an extracellular
`domain for ligand binding, a transmem-
`brane domain, and a cytoplasmic domain,
`including a tyrosine kinase domain4 (Fig.
`1). The overall structure of VEGFRs is
`similar to that of the PDGFR family mem-
`bers; however, these 2 receptor families
`have clear differences: the PDGFR extra-
`cellular domain contains 5 immunoglobu-
`lin (Ig)–like domains, whereas VEGFRs
`bear 7 Ig-like domains. Both TKRs share a
`tyrosine kinase domain with a long kinase
`insert (KI) of 60 to 70 amino acids; how-
`ever, the amino acid sequences in the KI of
`these 2 TKRs are very different from each
`other. The KIs in PDGFR family members
`contain 1 or 2 Tyr(Y)-x-x-Met(M) motifs
`as autophosphorylation sites, and these
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`
`motifs have been shown to be strong bind-
`ing sites for the SH2 domain of the p85
`subunit in the PI3-kinase complex and to
`activate the PI3K pathway. These auto-
`phosphorylation sites were demonstrated
`to be crucial for the cell growth signal
`mediated by PDGFR and for the cell trans-
`formation signal mediated by v-Fms, an
`activated form of M-CSFR (a member of
`the PDGFR family). However, none of the
`VEGFRs contain this Y-x-x-M motif in
`their KI region or in the carboxyl-terminal
`region, indicating that the downstream sig-
`naling from VEGFRs may be different
`from that of the PDGFR family.5
`
`Signaling of VEGFRs
`activates
`VEGF-A binds
`to
`and
`VEGFR-1 (Flt-1) and VEGFR-2 (KDR/
`Flk-1 in mice). VEGFR-1 has a high
`affinity for VEGF-A (Kd = 1~10 pM),
`which is one order higher than that of
`VEGFR-2, whereas its tyrosine kinase
`activity is approximately 10-fold weaker
`than that of VEGFR-2.26 The major pro-
`angiogenic signal is generated from the
`ligand-activated VEGFR-2. Within the
`KI or carboxyl-terminal region, TKRs
`have tyrosine autophosphorylation sites,
`which are important for the downstream
`signal. Unlike most of the TKRs that
`activate the Ras pathway or PI3K path-
`way, we found that the PLCγ-PKC-
`MAPK pathway is highly activated in
`VEGF-bound VEGFR-2 and used as a
`crucial signal for endothelial prolifera-
`tion. An SH2 domain of PLCγ specifi-
`cally binds to the 1175-PY site of
`VEGFR-2 (1173-PY in mice) and fur-
`ther activates PKC, particularly the
`PKCβ pathway.27,28 An 1175-phenylala-
`nine (F) mutant of VEGFR-2 signifi-
`cantly decreases the MAPK pathway
`under stimulation with VEGF and can-
`not efficiently activate the endothelial
`proliferation signal. Furthermore, mice
`with an amino acid knock-in at the 1173
`site from Y to F (VEGFR-2/flk-1 1173F/F
`mutant mice) are embryonic lethal at
`about E9.0 with no formation of blood
`vessels similar to the flk-1–/– mice.29 On
`the other hand, another knock-in mouse
`
`(VEGFR-2/flk-1 1212F/F) was essen-
`tially healthy.
`Taken together, these results strongly
`suggest
`that
`the PLCγ-PKC-MAPK
`pathway initiated from the VEGFR-2
`1175-PY site plays a pivotal role in pro-
`angiogenic signaling from VEGFR-2. A
`spontaneous mutant of zebrafish carry-
`ing the lethal circulatory system abnor-
`mality was shown to have a mutation in
`the fish PLCγ1 gene, indicating that the
`PLCγ-PKC pathway is also important
`for angiogenesis in fish.30 VEGFR-2
`stimulates not only angiogenic signals
`but also the secretion of various proteins
`such as the von Willebrand factor (vWF)
`from endothelial cells. vWF secretion
`was recently reported to be dependent
`on 1175-PY in VEGFR-2.31 In addition,
`Sase et al. indicated that the signaling
`from the 1175-PY site on VEGFR-2 to
`PLCγ is essential for endothelial specifi-
`cation of VEGFR-2–positive vascular
`progenitor cells in embryonic stem (ES)
`cell culture.32 On the other hand, the
`951-PY on VEGFR-2 is important for
`cell migration signals.33
`VEGFR-1 has a much weaker kinase
`activity than VEGFR-2, and the signal-
`ing cascade is not fully understood. The
`1169-Y on VEGFR-1 corresponding to
`1175-Y on VEGFR-2 is a PLCγ activa-
`tion site from VEGFR-1. However,
`we found that 1169-PY is not a major
`autophosphorylation site on VEGFR-1.
`Consistent with this finding, direct pro-
`angiogenic activity from VEGFR-1 is
`usually weak or undetectable.34
`In addition to vascular endothelial
`cells, VEGFR-1 is expressed on macro-
`phage lineage cells and facilitates migra-
`tion of these cells. Recently, we have
`shown that a scaffold protein RACK1 is
`involved in this migration signal and
`that the VEGFR-1-RACK1-PI3K-Akt
`pathway appears to be important for this
`signal.35
`The biological functions of VEGFR-1
`have been the topic of several studies.
`Fong et al. demonstrated that flt-1–null
`mutant mice (flt-1–/– mice) die at
`approximately E8.5 due to an over-
`growth of vascular endothelial cells and
`
`disorganization of blood vessels.36 This
`suggests a negative regulatory role of
`VEGFR-1 (Flt-1) in angiogenesis at
`early
`embryogenesis. To
`examine
`whether the tyrosine kinase of VEGFR-1
`is essential to the negative role of this
`receptor, we generated a mutant mouse
`strain that lacks the tyrosine kinase (TK)
`domain of VEGFR-1 (flt-1 TK–/– mice).
`To our surprise, the mutant mice were
`essentially healthy with an almost nor-
`mal circulatory system, indicating that
`the negative role of VEGFR-1 is inde-
`pendent of its tyrosine kinase activity
`but dependent on the ligand-binding
`domain.37 This flt-1 TK–/– mouse is use-
`ful for clarifying
`the role of
`the
`VEGFR-1 signal and to see whether it is
`important for the progression of diseases
`such as cancer. Using the mutant mice,
`we have found, along with others,
`that flt-1 TK–/– mice show a slower
`tumor growth, a lower level of metasta-
`sis (particularly lung metastasis in the
`carcinogenesis model), and a milder
`inflammation reaction in the rheumatoid
`arthritis model compared with those in
`wild-type mice.38-41 Furthermore, we
`and others have found that the wild-type
`mice carrying the flt-1 TK–/– bone mar-
`row show slower tumor growth similar
`to that in the flt-1 TK–/– mice.38,39 Using
`an anti–VEGFR-1 neutralizing antibody,
`Kaplan et al. indicated that VEGFR-
`1–positive bone marrow precursor cells
`play a significant role in the formation
`of a premetastatic niche, which pro-
`motes tumor metastasis.42 These results
`indicate that VEGFR-1 signaling is
`important for the progression of tumors
`in vivo mostly via bone marrow–derived
`VEGFR-1–positive cells. In addition,
`Wu et al. reported that some human
`tumors, such as breast carcinomas,
`express VEGFR-1 and utilize its signal-
`ing directly for tumor growth.43
`VEGFR-3 has a typical tyrosine
`kinase like other VEGFRs, and upon
`stimulation with VEGF-C, the PKC
`pathway and Ras pathway were reported
`to be activated for lymphangiogenesis.
`However, it remains to be clarified
`which autophosphorylation site(s) on
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`

`VEGF and VEGFR signaling in angiogenesis / Shibuya
`
`Monographs
`
`M
`
`1101
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`the tyrosine residues in VEGFR-3 is
`responsible for these pathways and is
`critical for lymphangiogenesis.
`
`Unique Characteristics of sFlt-1
`The VEGFR-1 (Flt-1) gene expresses 2
`mRNAs: one is a long form of approxi-
`mately 8 kb, and the other is a short form
`of 2.5 to 3.0 kb.44 The short mRNA is
`highly expressed in normal placenta,
`encoding a soluble form of Flt-1 known
`as sFlt-1.44,45 The sFlt-1 contains 6 Ig-
`like domains with a short, 31 amino
`acid–long tail derived from the 5′ region
`of intron 13 and exhibits a strong bind-
`ing ability to VEGF-A, PlGF, and
`VEGF-B.45,46
`trophoblasts
`Within
`the placenta,
`located between the fetal and maternal
`blood vessel systems preferentially
`express sFlt-1 (Fig. 1). Thus, an interest-
`ing possibility is that sFlt-1 functions as
`a biochemical barrier between fetal and
`maternal circulation in the placenta by
`suppressing excess angiogenesis and
`abnormal vascular permeability. From
`this model, the level of sFlt-1 should be
`controlled at an appropriate physiologi-
`cal range because overtrapping of VEGF
`may cause severe problems in the circu-
`latory system of the placenta. Interest-
`ingly enough, abnormal overexpression
`of sFlt-1 in the placenta was observed in
`a major disease in the field of obstetrics.
`In 2003, Maynard et al. and Koga et al.
`reported that patients with preeclampsia
`have abnormally high levels of sFlt-1 in
`serum and plasma.47,48 The trophoblasts
`were the major cell types producing
`large amounts of sFlt-1 in patients with
`this disease. Furthermore, Levine et al.
`demonstrated an intimate relationship
`between the serum levels of sFlt-1 and
`the degree of preeclampsia.49 This
`strongly suggests that abnormal sup-
`pression of VEGF-A by sFlt-1 causes
`hypertension and proteinuria in the
`patients. Supporting this idea, Maynard
`et al. reported that an artificial expres-
`sion of sFlt-1 with a vector system in
`pregnant rats induces symptoms such as
`hypertension and proteinuria, indicating
`
`that sFlt-1 is, at least partly, the cause of
`the preeclamptic syndromes.47
`A podocyte-specific knockout of the
`VEGF-A gene in mice demonstrated
`damage to the glomerular microvascula-
`ture and an induction of proteinuria.
`Thus, a severe block and decrease in the
`level of VEGF-A in the kidney by over-
`expressed sFlt-1 in preeclampsia may
`result in glomerular dysfunction and pro-
`teinuria. Interestingly, similar symptoms
`(hypertension and proteinuria) were
`observed under anti–VEGF-VEGFR
`therapy in cancer patients (see below).
`sFlt-1 was also found to be expressed
`in corneal epithelial cells.50 This strongly
`suggests that sFlt-1 suppresses angio-
`genesis near the lens and maintains the
`transparency of the eye.
`
`Anti–VEGF/VEGFR Therapy
`and Anticancer Therapy
`The VEGF-VEGFR system is unique in
`that it consists of a very limited number
`of molecules that play a central role in
`angiogenesis. The major ligand (VEGF-
`A) is a single gene product, and it uti-
`lizes only 2 TKRs (VEGFR-1 and
`VEGFR-2), although neuropilin-1 is
`used as a coreceptor. Other ligands, such
`as PlGF, VEGF-C, and VEGF-D, and
`the receptor VEGFR-3 appear to be
`partly involved in pathological angio-
`genesis, such as tumor vasculature. On
`the other hand, tumors metastatic to
`lymph nodes express higher levels of
`VEGF-C/D, suggesting that the VEGF-
`C/D and VEGFR-3 system plays an
`important role in lymph vessel–depen-
`dent tumor cell migration into lymph
`nodes. The angiopoietin-Tie system is
`also involved in pathological angiogen-
`esis, but the details of its role in the pro-
`cess of carcinogenesis are not fully
`understood.
`On the basis of these results, anti–
`VEGF-VEGFR drugs such as anti–
`VEGF-A neutralizing antibody and
`tyrosine kinase inhibitors have been
`developed, and bevacizumab
`(anti–
`VEGF-A humanized monoclonal anti-
`body) has been approved for the treatment
`
`of colorectal, breast, lung (non–small cell
`type), and renal cancers as well as for
`glioblastoma patients.3,51 Multikinase
`inhibitors such as sorafenib and sunitinib
`are now approved for renal and hepatic
`cancer patients.
`In addition to these medicines, others
`that target the VEGF-VEGFR system,
`including VEGF-Trap (a fusion protein of
`VEGFR-1 and VEGFR-2 ligand-binding
`domains), anti–VEGFR-1 or anti–VEGFR-
`2 neutralizing antibody, soluble VEGFR-
`3, VEGFR-1 or VEGFR-2 peptide vaccine
`therapy,52 and anti-PlGF antibody,53,54
`have been developed and are undergoing
`preclinical and clinical trials.
`
`The Molecular Basis of
`Anti-Angiogenic Therapy
`In 1993, Kim et al. demonstrated that anti-
`human VEGF-A neutralizing antibody
`efficiently
`suppressed human
`tumor
`growth in immune-deficient mice.55 In this
`case, the antibody only suppressed tumor-
`derived human VEGF-A, but not the host-
`derived mouse VEGF-A, which is secreted
`from mouse bone marrow–derived cells as
`well as tumor-associated fibroblasts. Fur-
`thermore, tumor growth was suppressed
`by the antibody treatment alone, without
`combination with chemotherapy. This
`indicates that the major effect of the anti–
`VEGF-A antibody was to block the forma-
`tion of blood vessels in tumor tissues as
`well as to suppress pre-existing tumor vas-
`culature by inducing apoptotic death of
`endothelial cells by blocking
`tumor-
`derived VEGF-A.
`However, in clinical trials, treatment
`of cancer patients with an anti–VEGF-A
`antibody alone did not produce signifi-
`cant suppression of
`tumor growth,
`except for renal cancer. In contrast to
`murine tumor transplantation models,
`the growth rate of tumors in patients is
`usually slower, and tumor angiogenesis
`may develop more slowly compared
`with the murine tumor models. In clin-
`ics, the vasculature in tumors might be
`more stable than that in the murine sys-
`tem and less sensitive to VEGF-A block-
`ade alone.
`
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`
`

`

`1102
`
`M Monographs
`
`Anti-angiogenic
`therapy
`
`Tumor cell
`
`Cell death due
`to hypoxia and
`low nutrition
`
`Change in metabolism,
`Highsurvivalsignal,
`Increase in invasion
`th due
`
`VEGF etc.
`
`Tumor angiogenesis
`
`Vascular
`normalization
`
`Regression of tumors
`
`Phenotypic change of
`tumors
`
`Figure 3. A possible response of tumor cells to anti-angiogenic therapy: a model. A direct
`suppression of tumor angiogenesis and “vascular normalization” results in the suppression of
`tumor growth. However, after long-term therapy, tumor cells under hypoxia and low nutrition
`double stress acquire a resistant phenotype.
`
`Another hypothesis regarding the
`efficacy of the anti-VEGF antibody and
`anti-VEGFR tyrosine kinase inhibitor
`on tumor growth in patients is termed
`“vascular normalization,” whereby the
`absorption of VEGF-A induces a tran-
`siently normalized vascular structure,
`more stabilized and well covered with
`pericytes with lower vascular permea-
`bility.56 These conditions may result in a
`lower tissue pressure within tumors,
`having a better diffusion of anticancer
`drugs. It is probable that both vascular
`normalization and suppression of new
`tumor angiogenesis can occur in parallel
`within the tumors in patients treated
`with anti–VEGF-VEGFR drugs (Fig. 3).
`
`Side Effects and Refractoriness
`to Anti-Angiogenic Therapy
`A variety of side effects, such as hyper-
`tension, renal dysfunction, proteinuria,
`thrombosis, bleeding, and arrhythmia,
`have been reported in patients under
`anti–VEGF-VEGFR therapy.7,8 Among
`these, the frequency of hypertension and
`proteinuria is higher than that of others,
`suggesting a direct relationship with the
`blockage of VEGF-A in tissues. A
`
`decrease in the level of VEGF-A in the
`kidney could induce damage to vascular
`endothelial cells in glomeruli, and such
`a dysfunction of glomerular microvas-
`culature may cause proteinuria. How-
`ever, the molecular basis of hypertension
`under VEGF-VEGFR blockage remains
`to be clarified.
`Whether tumor cells acquire refracto-
`riness or resistance to anti-angiogenic
`therapy after long-term treatment is an
`important question. In clinical trials, the
`efficacy of anti–VEGF-VEGFR therapy
`on the increase in survival time seems
`sometimes inconsistent. Survival time
`did not increase stably during the course
`of treatment, and in some trials after a
`long period, the efficacy appears to
`decrease, suggesting a resistance or
`refractoriness of tumors to this treat-
`ment. In addition, in some preclinical
`and clinical trials, glioblastoma showed
`an enhanced invasiveness after anti-
`angiogenic therapy57 (Fig. 3).
`Many experimental models could be
`introduced and studied to understand
`this resistance. Casanovas et al. reported
`that gene expression of angiogenic fac-
`tors such as FGF other than VEGF is a
`cause of resistance against anti-VEGF
`
`Genes & Cancer / vol 2 no 12 (2011)
`
`therapy in mice.58 We hypothesized that,
`under a long-term anti-angiogenic therapy,
`tumor cells may receive at least 2 stresses,
`hypoxia and low nutrition conditions. We
`developed a simple in vitro model system
`in which tumor cells were cultured under
`these double stresses (double deprivation
`stress [DDS]). After approximately 10
`cycles of DDS culture, tumor cells showed
`up-regulation of phospho-Akt, a higher
`survival rate, and increased invasiveness.59
`Thus, DDS under anti-angiogenic therapy
`might induce, to some extent, a malignant
`phenotype of tumors. A strategy should be
`developed to overcome this possible
`malignant phenotype after anti-angiogenic
`therapy.
`
`Pro-Angiogenic Therapy
`Ischemic heart failure and cerebral
`attacks with thrombosis or bleeding are
`major diseases in humans. Furthermore,
`recent studies strongly suggest that some
`degenerative diseases, such as neuronal
`degeneration, are due to lower circula-
`tion as well as lower VEGF-VEGFR
`signaling in neuronal cells.60,61
`For the treatment of ischemic heart
`and brain diseases, pro-angiogenic
`therapy could be useful because these
`diseases are essentially due to poor cir-
`culatory conditions. Among the VEGF
`family, VEGF-A plays a crucial role in
`blood vessel formation in embryogene-
`sis and the earlier stages after birth.
`However, in adult stages, VEGF-A stim-
`ulates not only VEGFR-2 but also
`VEGFR-1 (Flt-1), which enhances the
`migration of inflammatory cells such as
`macrophages, resulting in inflammation
`and hypervascular permeability. Several
`articles have previously reported that
`K14 promoter–driven VEGF-A trans-
`genic mice exhibit severe inflammation
`with edema in dermal tissues with angio-
`genesis, this being a model of psoriasis
`vulgaris, which is a chronic inflamma-
`tory skin disease.62 On the other hand,
`we have shown that the VEGFR-2–spe-
`cific ligand VEGF-E induces well-orga-
`nized blood vessels with pericyte
`coverage and maintains normal vascular
`
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`

`

`VEGF and VEGFR signaling in angiogenesis / Shibuya
`
`Monographs
`
`M
`
`1103
`
`VEGF-A
`
`VEGFR-1
`
`VEGFR-2
`
`Vascular
`ll
`l
`d th li
`endothelial cell
`
`VEGFR-1
`
`VEGFR-2
`
`
`
`VEGFR 1VEGFR-1
`
`Monocyte,
`macrophage
`
`Sensory
`neuron
`neuron
`
`Moto-Moto
`
`neuron
`
`Figure 4. A pro-angiogenic therapy using the VEGF-VEGFR system. Recent studies suggest
`that sensory neurons express VEGFR-1 and motoneurons express VEGFR-2. These receptors
`are biologically functional, and therefore, an appropriate ligand, such as VEGF-E, can be used for
`pro-angiogenic therapy as well as for neuron protection therapy.
`
`permeability. No clear inflammatory
`reaction was observed in VEGF-E trans-
`genic mice. K14-P