REVIEW

Tumor Angiogenesis and Anti-angiogenic Therapy
Yoshiaki Kubota

Center for Integrated Medical Research, School of Medicine, Keio University, Tokyo, Japan(Received for publication on July 23, 2011)(Revised for publication on August 29, 2011)(Accepted for publication on September 15, 2011)

Anti-angiogenic therapy is an anti-cancer strategy that targets the new vessels that grow to provide
oxygen and nutrients to actively proliferating tumor cells. Most of the current anti-cancer reagentsused in the clinical setting indiscriminately target all rapidly dividing cells, resulting in severe adverse
effects such as immunosuppression, intestinal problems and hair loss. In comparison, anti-angiogenic
reagents theoretically have fewer side effects because, except in the uterine endometrium, neoangiogenesis rarely occurs in healthy adults. Currently, the most established approach for limiting tumor angiogenesis is blockade of the vascular endothelial growth factor (VEGF) pathway. In line with the resultsof preclinical studies, significant therapeutic effects of VEGF blockers have been reported in various
types of human cancers, even in patients with progressive/recurrent cancer who could not otherwise betreated. However, some patients are refractory to this treatment or acquire resistance to VEGF inhibitors. Moreover, several studies have shown that VEGF blockade damages healthy vessels and resultsin adverse effects such as hemorrhagic and thrombotic events. In recent research that indicated possible ways to overcome these problems, several VEGF-independent and tumor-selective pro-angiogenic
mechanisms were discovered that could be targeted in combination with or without conventional VEGF
blockade. These findings offer opportunities to greatly improve current anti-angiogenic treatment forcancer. (Keio J Med 61 (2) : 47–56, June 2012)

Keywords: angiogenesis, tumor angiogenesis, VEGF, macrophage, anti-angiogenic therapy

vein or capillary.3 Once these three processes are completed during postnatal development, adult vasculature isstable and rarely proliferates under physiological conditions. However, in pathological situations such as ocular
neovascular diseases and cancer, existing vessels again
start to grow to meet the abnormal requirements for oxygen and nutrients of the pathologically expanding tissues.
Anti-angiogenic therapy targets vascular growth within tumors, with the aim of suppressing tumor growth andmetastasis. Most current anti-cancer chemotherapeutic
agents used in the clinical setting indiscriminately target
all rapidly dividing cells (e.g., at the level of DNA replication and protein synthesis) and therefore can cause severe
adverse effects such as immunosuppression, intestinal
problems and hair loss. In comparison, anti-angiogenic
reagents theoretically have fewer side effects because, except in the uterine endometrium during the menstrual cy-

Introduction
In mammalian development, a vascular network isformed throughout the body (Fig. 1), except in avascular
tissues (e.g., cornea and intervertebral disks), to meet thetissue requirements for oxygen and nutrients.1 Three major processes are necessary to form a complete vascular
network: vasculogenesis, angiogenesis and vascular remodeling.2 Vasculogenesis denotes de novo blood vessel
formation, in which vascular precursor cells (angioblasts)
migrate to sites of vascularization, differentiate into endothelial cells and coalesce to form the initial vascular
plexus. Angiogenesis refers to the budding of new capillary branches from existing blood vessels, whereas vascular remodeling describes a later phase when a newly
formed vessel increases its luminal diameter in response
to increased blood flow and acquires identity as an artery,

Reprint requests: Yoshiaki Kubota, MD, PhD, Center for Integrated Medical Research, School of Medicine, Keio University, 35 Shinanomachi,
Shinjuku, Tokyo 160-8582, Japan, E-mail: ykubo33@a3.keio.jp
Copyright © 2012 by The Keio Journal of Medicine
47

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Kubota Y: Tumor Angiogenesis and Anti-angiogenic Therapy

Fig. 1 A well-organized vascular network is formed throughout the body.
Whole-mount CD31 immunostaining of endothelial cells in normal mouse tissues [brain (A), trachea(B), ear (C) and fat (D)] at postnatal day 30. Scale bar: 200 μm.

cle, neoangiogenesis rarely occurs in healthy adults. This
review discusses the history and current understandingof the molecular and cellular bases of tumor angiogenesis
and anti-angiogenic therapy.
Tumor Angiogenesis and Anti-VEGF TherapyMore than a century ago, early pioneering researchers
observed that dense vascular networks often accompanied human tumors. The existence of tumor-derived angiogenic factors was postulated more than 70 years ago,
and it was proposed that vascular growth in the tumor
relies on such factors.4,5 In 1971, Judah Folkman, whobecame known as the “father of tumor angiogenesis,”first emphasized the importance of tumor vascularity
for tumor growth.6 He described how, if a tumor could
be stopped from growing its own blood supply, it would
wither and die (Fig. 2). Since then, various studies have

led to the discovery of a growing number of anti-angiogenic molecules that limit tumor angiogenesis. Vascularendothelial growth factor (VEGF) was first discovered
by Senger and colleagues as a vascular permeability factor secreted by a guinea pig tumor cell line.7 Various in
vitro and in vivo studies have since uncovered the roleof VEGF as a central player in both physiological andpathological angiogenesis.8 Pathologically expanding tumor tissues rapidly exhaust the available oxygen supply
and become hypoxic. The activation of hypoxia-inducible
factor (HIF) signaling in hypoxia-sensing cells triggers
VEGF expression.9,10 VEGF is secreted not only by tumor cells but also by tumor-associated stromal cells.11 In
turn, secreted VEGF stimulates vascular growth into hypoxic tumor tissues to meet the tumor’s oxygen requirements (Fig. 2).
The use of VEGF blockers to prevent this process isthe most established of the anti-angiogenic modalities.

Keio J Med 2012; 61 (2): 47–56

49

Fig. 2 Association between tumor growth and its vascularity.
Adult vasculature is stable and rarely proliferates under physiological conditions. However, in cancer, existing vessels again start to grow (neoangiogenesis) in response to hypoxia inducible factor (HIF)-driven VEGF expression intumors. Newly formed vessels provide oxygen and nutrients to rapidly expanding tumors.

A number of preclinical studies of angiogenesis inhibition by administration of VEGF blockers have demonstrated significant tumor-suppression effects in various
types of cancers.12,13 In a phase III study of patients withmetastatic colorectal cancer, bevacizumab, a humanizedanti-VEGF monoclonal antibody, showed significant benefits in combination with 5-fluorouracil (5-FU)-based
chemotherapy. In response, the U.S. Food and Drug Administration (FDA) approved bevacizumab for the treatment of metastatic colorectal cancer in combination with
5-FU-based regimens in 2004. Almost simultaneously,
the FDA approved pegaptanib, an aptamer that blocks the
165-amino-acid isoform of VEGF-A, for the treatmentof the wet form of age-related macular degeneration.14
Thereafter, bevacizumab and multi-targeted tyrosine kinase inhibitors (e.g., sorafenib, sunitinib or pazopanib),which block the signaling of pathways such as VEGF,
were approved for clinical use in various types of cancers
including metastatic non-squamous non-small cell lungcancer, metastatic breast cancer, recurrent glioblastoma
multiforme and metastatic renal cell carcinoma. A number of studies have reported their significant therapeutic
efficacy.8,15 In 2007, three years after the approval of bevacizumab by the FDA, the Ministry of Health, Labourand Welfare in Japan approved the use of bevacizumab
for patients with progressive/recurrent colorectal cancer

that cannot be surgically resected.
Problems with Anti-VEGF Therapy
Currently, as described above, the most established approach for limiting tumor angiogenesis is blockade of the
VEGF pathway. In line with preclinical studies, administration of VEGF blockers to patients with various typesof cancers has had significant therapeutic effect. However, some patients are refractory or acquire resistanceto VEGF inhibitors.16 This may be explained, at least inpart, by the administration of often higher doses and different schedules in preclinical mouse studies (10 mg/kg
body weight, twice a week) compared to humans (5 mg/
kg body weight, once every 2 weeks) resulting in more
pronounced effects in mice. Very modest doses are given
to patients, as is usual for other anti-cancer reagents, toavoid possible off-target toxicities. In fact, animal studies have shown that interrupting VEGF blockade induces
rapid vascular regrowth in tumors because the persisting
empty sleeves of the basement membrane provide a scaffold for rapid revascularization.17 This result indicatesthat insufficient doses of VEGF blockers can easily resultin vascular regrowth and limit the tumor-suppression effects.
More importantly, several studies have shown that

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Kubota Y: Tumor Angiogenesis and Anti-angiogenic Therapy

Table 1. Known adverse effects of VEGF blockade
Adverse effectPossible mechanism

Bleeding
Thrombotic events
Hypertension
Proteinuria
Leukopenia, lymphopenia
Hypothyroidism

Endothelial cell apoptosis; loss of integrity of the endothelial vessel lining
Increased platelet activation; exposure of the prothrombotic basement membrane to the circulating blood
Inappropriate balance between arteries and veins; reduced levels of prostaglandin I-2 and nitric oxide
Podocyte dysfunction
Disturbance of hematopoiesis
Regression of capillaries around thyroid follicles

VEGF blockade damages not only tumor vessels but
also healthy vessels and results in severe problems suchas hemorrhagic and thrombotic events (Table 1).18,19
Mice studies indicate that constitutive signaling through
VEGF in both a paracrine and an autocrine manner isrequired for the homeostasis of adult vessels.20–22 VEGF
blockade induces abnormal endothelial apoptosis, resulting in the loss of integrity of the endothelial vessel lining
and increased platelet activation due to exposure of thecirculating blood to the prothrombotic basement membrane. VEGF signaling is required not only for endothelial function but also for the function of podocytes andhematopoietic cells.23,24 Accordingly, VEGF blockade
causes moderate proteinuria, leukopenia and lymphopenia.18,25 At the end of 2010, an advisory panel to the FDA
announced that bevacizumab is neither safe nor effective
against breast cancer, and the FDA decided to revoke
market approval and re-label this drug. Researchers arenow seeking novel anti-angiogenic targets specific for tumor vessels that avoid signaling pathways essential forthe maintenance of healthy vessels.
Potential Mechanisms of Resistance toAnti-VEGF Therapy
Various mechanisms are thought to underlie the resistance to VEGF blockade observed in some patients withcancer. The extent of each mechanism responsible ishighly variable from one cancer to another and differs depending on the type of VEGF blocker used. Understanding the molecular bases of these cancer type-dependent
resistance mechanisms against VEGF blockade offers opportunities to improve anti-angiogenic treatment. Compensation by other pro-angiogenic mechanisms, such as
fibroblast growth factors (FGFs) and angiopoietins, is amajor factor that may contribute to poor responsivenessto VEGF blockade.16,26–29 Recent preclinical studies haveshown that these VEGF-independent pro-angiogenic
mechanisms exert significant effects, particularly in theabsence of VEGF signaling.
VEGF blockade-induced vessel regression causes hypoxia in tumor tissues, and the high rate of tumor cellproliferation in the absence of sufficient oxygen can result in the elimination of a major population of tumor

cells. However, a proportion of hypoxia-tolerant cancer
cells, which are likely to be so-called cancer stem cells,30
survive in poorly oxygenated niches and elicit tumor adaptation to anti-angiogenesis. Some reports suggest thatthe resultant selection of tumor cells renders tumors evenmore invasive and metastatic.31,32 They suggest that theadministration of VEGF blockers alone can aggravatetumor hypoxia and worsen malignancy. However, contradictory results from other preclinical and clinical studies
indicate that the concept of cancer aggravation by VEGF
blockade is still unproven.33,34 Randomized, placebocontrolled phase III studies in 4205 cancer patients did
not support a decreased time to disease progression, increased mortality or altered disease progression pattern
after cessation of bevacizumab therapy.33
Pericytes are essential components of blood vessels
and are necessary for normal development, homeostasis
and the integrity of the blood–brain barrier. Pericyte recruitment is mainly regulated by platelet-derived growth
factor (PDGF)-B/PDGF receptor-β, transforming growth
factor-β (TGF-β) and angiopoietin/Tie signaling.35 In
general, blood vessels found in tumors are immature
with a sparse covering of pericytes.3 However, in somecancers, tumor vessels are covered with a dense pericytecoat, and these vessels are less sensitive to VEGF blockers.36 Interestingly, glioblastoma stem-like cells undergoa vasculogenic process: they differentiate into endothelial
cells and cooperate with the neoangiogenic vessels (vasculogenic mimicry). These tumor cell-derived endothelial cells are less sensitive to VEGF blockade and thus conceivably contribute to the blockade resistance observedin certain types of cancer.37,38 Myeloid cell-driven angiogenesis is also known to contribute to refractoriness orresistance to VEGF blockade. In particular, CD11b+Gr1+
myeloid cells have been shown to render tumors refractory to angiogenic blockade by anti-VEGF antibodies. Thiseffect is mediated by the secreted protein Bv8, which isupregulated by granulocyte colony-stimulating factor.39
CD11b+Gr1− macrophages are also known to have a rolein the VEGF-independent pro-angiogenic mechanism.
Macrophages are currently one of the most promisingtargets for anti-angiogenic cancer therapy (see below).
Chronic exposure of endothelial cells to anti-VEGF
drugs may lead to a stable epigenetic mechanism of resis-

Keio J Med 2012; 61 (2): 47–56

tance. Epigenetic modulation of expression of anti-apoptotic genes such as bcl-2 and survivin could conceivably
lead to resistance to various inhibitors of angiogenesis.40
Indeed, absence of the proapoptotic Bcl-2 homology 3(BH3)-only Bcl-2 family member Bim in endothelial cells
almost completely abolishes the effect of VEGF blockade.41 Enhancer of zeste homolog 2 (EZH2), a memberof the polycomb protein group that contributes to the epigenetic silencing of target genes, is highly upregulated
in tumor endothelial cells.42 EZH2 promotes tumor angiogenesis by methylating and silencing the vasohibin1
gene, an intrinsic negative regulator of angiogenesis.43,44
Macrophages and Tumor Angiogenesis
Macrophages are white blood cells produced throughcolony stimulating factor-1 (CSF-1)-dependent differentiation of monocytes.45,46 They have a role in both non-specific (innate immunity) and specific (adaptive immunity)
defense mechanisms through phagocytosis of cellulardebris and pathogens. Recent evidence demonstrates thatmacrophages are also involved in vascular developmentby promoting angiogenic branching and anastomosis.47,48
Macrophages stimulate vessel sprouting via a soluble factor other than VEGF, rather than through direct contact
with endothelial cells.49 These data suggest that macrophages promote angiogenesis independently of VEGF
signaling. Histological examination of various cancers
reveals a vast accumulation of macrophages, known as
tumor-associated macrophages (TAMs). TAMs, visualized by antibodies against macrophage markers such as
CD11b, F4/80 and CD68, have oval to typical stellatemorphology and are found in both avascular and vascularized areas throughout tumors (Fig. 3A).
TAMs orchestrate various aspects of cancer progression, including the diversion and skewing of adaptive responses, cell growth, matrix deposition and remodeling,
and the construction of metastatic niches where they prepare the tissue for the arrival of tumor cells.50 One current focus in macrophage research is the phenotypic shiftobserved during cancer progression; this shift is called“macrophage polarization” and links molecular pathways
involved in inflammation and cancer (Fig. 3B). Classically activated (M1) macrophages, following exposure
to interferon γ (IFNγ), are tumoricidal, as characterized
by proinflammatory activity, antigen presentation andtumor lysis. During cancer progression, macrophages
acquire features similar to alternatively activated macrophages (M2), which increase angiogenesis, tumor growthand invasion into surrounding tissues and suppress theactivation of dendritic cells, cytotoxic T lymphocytes
and natural killer cells.51 IL-10 signaling interferes with
NF-κB activation and M1 macrophage-induced inflammation. The balance between the activation of M1 macrophage-associated STAT1 and M2 macrophage-associated
STAT3/6 regulates macrophage polarization.52 Recent

51

evidence demonstrates that histidine-rich glycoprotein(HRG) and HIF-1α suppress M2 polarization.53,54 Conversely, Krüppel-like factor 4 (KLF4) and suppressor ofcytokine signaling1 (SOCS1) have been shown to promote M2 polarization.55,56
Promotion of tumor angiogenesis is one of the most important roles of TAMs. Jeffrey Pollard and his colleagues
first established the role of TAMs in the “angiogenic
switch,” which is identified as the formation of a highdensity vessel network and promotes the malignant stateof tumors.57 Blockers of CSF-1 eliminate large populations of macrophages. Our recent study found that M2
macrophages expressed CSF-1 receptor more abundantly
than M1 macrophages did, suggesting that CSF-1 blockade would be more effective on M2 (tumor-promoting)than on M1 (tumor-suppressive) macrophages. Accordingly, in a mouse osteosarcoma model, CSF-1 inhibition
effectively suppressed tumor angiogenesis and disorganized empty sleeves of extracellular matrices.47 Therefore, in contrast to VEGF blockade, interruption of CSF-1
inhibition did not promote rapid vascular regrowth. Continuous CSF-1 inhibition did not affect healthy vascularand lymphatic systems outside the tumor.47 These results
indicate that CSF-1-targeted therapy is a realistic strategy
for limiting tumor angiogenesis. Currently, several drugs
that inhibit CSF-1 signaling are in early clinical trials.50
However, it should be noted that possible side effects of
CSF-1 inhibition are disturbed wound healing and impaired macrophage-related immunity.58
Tumor Endothelium-specific Molecular Signatures
As described above, VEGF signaling is required for the
maintenance of healthy vessels, and its blockade leads toside effects resulting from endothelial damage throughout the body (Table 1). The effects of VEGF blockade in
healthy vessels are apparently more prominent in humans
than in mice. This reflects the considerable differencesin doses and schedules used for mice and humans; thedoses of bevacizumab used in preclinical mouse studies
were much higher than those given to patients. Nevertheless, preclinical mouse studies did not reveal the toxic effects apparent in humans. Therefore, to limit toxicity, it isimportant to find tumor endothelium-specific moleculartargets.
It has been observed that tumor blood vessels differ
morphologically from their normal counterparts; for example, they display inappropriate growth and regression
due to abnormal proliferation and apoptosis in endothelial cells, insufficient pericyte recruitment, enhanced
leakiness, decreased blood flow and abnormalities inthe basement membrane.3,59 These abnormal structures
lead to reduced drug delivery into tumors. Inhibitors ofneuronal NO synthase (nNOS) or prolyl hydroxylase domain protein 2 (PHD2) have been reported to normalize
these structures, thereby enhancing the efficacy of con-

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Kubota Y: Tumor Angiogenesis and Anti-angiogenic Therapy

Fig. 3 Tumor-associated macrophages and their “polarization.”(A) Sectional immunohistochemistry of a B16 melanoma 10 days after cell transplantation into the skin on the back of a syngeneic
mouse. Tumor-associated macrophages (TAMs), visualized by antibodies against F4/80, were found in both avascular and vascularized areas with oval to typical stellate morphology. (B) A scheme for macrophage “polarization.” Classically activated (M1) macrophages have tumoricidal activity. During cancer progression, macrophages shift to alternatively activated macrophages (M2), which
in turn suppress tumor immunity and increase angiogenesis and metastasis. The typical M1 macrophage expression profile includes
CD86, tumor necrosis factor (TNF), IL-12, Nos2 and IL-6, whereas M2 macrophages preferentially express macrophage mannose
receptor (Mrc1), arginase-1, IL-10, VEGF, matrix metalloproteinases (MMPs) and transforming growth factor-β (TGFβ). Several exogenous and intracellular signaling pathways are known to positively or negatively regulate transition from M1 to M2 macrophages.
Scale bar: 50 μm.

ventional anti-cancer therapies.60–62 Gene expression
analysis of endothelial cells derived from human bloodvessels of normal and malignant colorectal tissues revealed forty-six genes that were specifically elevated intumor-associated endothelium.63 A pioneering study by
Hida and colleagues reported genotypic alterations intumor endothelial cells: tumor endothelial cells show
chromosome abnormalities characterized by aneuploidy
and abnormal multiple centrosomes,64 which may lead togenomic instability and activation of the DNA damage
response (DDR). In this context, Chavakis and colleagues
reported that a major DDR molecule, H2AX, is specifically activated in pathological angiogenesis of an ocular
neovascular model and a tumor-xenograft model.65 Lossof H2AX strongly impairs pathological angiogenesis but
does not affect healthy vessels. An antibody against placental growth factor (PlGF), a member of the VEGF family that selectively binds VEGFR-1 and its co-receptors
neuropilin-1 and -2, inhibits growth of VEGF blockaderesistant tumors without affecting healthy vessels.66 Targeting PlGF inhibits not only angiogenesis but also therecruitment of angiogenic macrophages. Macrophage
depletion by CSF-1 blockade can have tumor-selective
anti-angiogenic effects;47 therefore, the main anti-cancer
effects of anti-PlGF therapy could be the result of its im-

pact on macrophages.
Future Directions and Perspectives
The evidence demonstrates that tumor vascularity
mainly depends on VEGF signaling.12,13 However, recent preclinical and clinical studies show that a remarkable degree of compensation by VEGF-independent proangiogenic mechanisms occurs, and this compensation
works, in particular, in the absence of VEGF signaling. It
has also been demonstrated that combining conventionalanti-VEGF therapy with blockade of VEGF-independent
pro-angiogenesis pathways greatly enhances tumor suppression (Fig. 4). For example, blockade of FGFs or angiopoietins enhances and prolongs the anti-angiogenic effects of VEGF blockade.28,67 Some types of perivascular
cells such as macrophages (Kubota et al. 2009; Qian and
Pollard, 2010), pericytes,35 cancer cell-derived endothelial cells37,38 and tissue-resident vascular precursors68 are
known to contribute significantly to tumor angiogenesis
and refractoriness to VEGF blockade. Although further
analysis of the molecular and cellular bases is required,these cells show promise as targets in combination therapy with VEGF blockade.
So far, the efficacy of the anti-angiogenic strategy has

Keio J Med 2012; 61 (2): 47–56

53

Fig. 4 VEGF-independent pro-angiogenic mechanisms: novel targets for anti-angiogenic
therapy.
VEGF blockade enhances the expression in tumors of other pro-angiogenic cytokines suchas fibroblast growth factors (FGFs) and angiopoietins (Angs). Selection of hypoxia-tolerant
tumor cells may elicit tumor adaptation to anti-angiogenesis. Some types of perivascular/
vessel-associated cells such as macrophages, pericytes, cancer stem-like cells and tissueresident vascular precursors contribute to tumor angiogenesis and refractoriness to VEGF
blockade. Inhibition of macrophage function could be achieved by targeting colony-stimulating factor 1 (CSF-1), matrix metalloproteinases (MMPs), Tie-2, Bv8 or placental growth
factor (PlGF). To suppress pericyte recruitment, PDGFRβ or TGFβ signaling could betargeted. The recruitment and function of tissue-resident vascular precursors are mediatedby CXCR4 and Notch signaling.

been validated in solid tumors. Recently, it has gained increasing attention in leukemia therapy. Chronic myeloid
leukemia (CML) is highly vascularized in the bone marrow, and the vessel density has potential as a prognostic
indicator, which suggests that angiogenesis contributes
to leukemogenesis.69 Although anti-VEGF therapy has
limited effectiveness in the treatment of CML,70 a recent
preclinical study indicated that anti-PlGF treatment prolonged survival of patients with imatinib-resistant CML.
This treatment works by inhibiting bone marrow angiogenesis and directly suppressing CML proliferation, inpart independently of oncogenic Bcr-Abl1 signaling.71 In
mouse models of T-cell acute lymphoblastic lymphoma
and pro-B-cell leukemia, the anti-angiogenic effects ofthrombospondins secreted by CD4+ T cells were sufficient to elicit spontaneous oncogene inactivation leading
to tumor regression.72
Overall, the evidence demonstrates that anti-angiogenic therapy (typically blockade of VEGF signaling) has
remarkable therapeutic effects in various types of humancancers. However, the molecular bases of cancer type-de-pendent resistance mechanisms against VEGF blockade,especially VEGF-independent pro-angiogenic mechanisms, now need to be clarified. Targeting these mechanisms would greatly enhance the effects and minimize
the required doses of VEGF blockers. There is no doubtthat effort in this area will yield opportunities to greatly
improve anti-angiogenic treatment.
Acknowledgments
This work was supported by Grants-in-Aid for Specially Promoted Research from the Ministry of Education,
Culture, Sports, Science and Technology of Japan, by aresearch grant from Takeda Science Foundation, and by
the Keio Kanrinmaru Project. I apologize to authors of
relevant papers that could not be cited here due to space
limitations.

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Kubota Y: Tumor Angiogenesis and Anti-angiogenic Therapy

References
1. Carmeliet P: Angiogenesis in life, disease and medicine. Nature
2005; 438: 932–936. [Medline] [CrossRef]
2. Risau W: Mechanisms of angiogenesis. Nature 1997; 386: 671–
674. [Medline] [CrossRef]
3. Jain RK: Molecular regulation of vessel maturation. Nat Med
2003; 9: 685–693. [Medline] [CrossRef]
4. Ide AG, Baker NH, Warren SL : Vascularization of the Brown
Pearce rabbit epithelioma transplant as seen in the transparent ear
chamber. AJR Am J Roentgenol 1939; 42: 891–899.
5. Algire GH, Chalkley HW, Legallais FY, Park HD : Vascular reactions of normal and malignant tissues in vivo. I. Vascular reactions of mice to wounds and to normal and neoplastic transplants.
J Natl Cancer Inst 1945; 6: 73–85.
6. Folkman J: Tumor angiogenesis: therapeutic implications. N Engl
J Med 1971; 285: 1182–1186. [Medline] [CrossRef]
7. Senger DR, Galli SJ, Dvorak AM, Perruzzi CA, Harvey VS,
Dvorak HF : Tumor cells secrete a vascular permeability factor
that promotes accumulation of ascites fluid. Science 1983; 219:
983–985. [Medline] [CrossRef]
8. Ferrara N: VEGF-A: a critical regulator of blood vessel growth.
Eur Cytokine Netw 2009; 20: 158–163. [Medline]
9. Carmeliet P, Dor Y, Herbert JM, Fukumura D, Brusselmans K,
Dewerchin M, Neeman M, Bono F, Abramovitch R, Maxwell P,
Koch CJ, Ratcliffe P, Moons L, Jain RK, Collen D, Keshert E :Role of HIF-1alpha in hypoxia-mediated apoptosis, cell proliferation and tumour angiogenesis. Nature 1998; 394: 485–490. [Medline] [CrossRef]
10. Semenza GL, Agani F, Iyer N, Kotch L, Laughner E, Leung S,
Yu A : Regulation of cardiovascular development and physiology by hypoxia-inducible factor 1. Ann N Y Acad Sci 1999; 874:
262–268. [Medline] [CrossRef]
11. Fukumura D, Xavier R, Sugiura T, Chen Y, Park EC, Lu N, Selig
M, Nielsen G, Taksir T, Jain RK, Seed B : Tumor induction of
VEGF promoter activity in stromal cells. Cell 1998; 94: 715–725.
[Medline] [CrossRef]
12. Kim KJ, Li B, Winer J, Armanini M, Gillett N, Phillips HS, Ferrara N : Inhibition of vascular endothelial growth factor-induced
angiogenesis suppresses tumour growth in vivo. Nature 1993;
362: 841–844. [Medline] [CrossRef]
13. Ferrara N, Kerbel RS: Angiogenesis as a therapeutic target. Nature 2005; 438: 967–974. [Medline] [CrossRef]
14. Gragoudas ES, Adamis AP, Cunningham ET Jr, Feinsod M, Guyer DR : Pegaptanib for neovascular age-related macular degeneration. N Engl J Med 2004; 351: 2805–2816. [Medline] [CrossRef]
15. Carmeliet P, Jain RK: Molecular mechanisms and clinical applications of angiogenesis. Nature 2011; 473: 298–307. [Medline][CrossRef]
16. Bergers G, Hanahan D: Modes of resistance to anti-angiogenic
therapy. Nat Rev Cancer 2008; 8: 592–603. [Medline] [CrossRef]
17. Mancuso MR, Davis R, Norberg SM, O’Brien S, Sennino B, Nakahara T, Yao VJ, Inai T, Brooks P, Freimark B, Shalinsky DR,
Hu-Lowe DD, McDonald DM : Rapid vascular regrowth in tumors after reversal of VEGF inhibition. J Clin Invest 2006; 116:
2610–2621. [Medline] [CrossRef]
18. Verheul HM, Pinedo HM: Possible molecular mechanisms involved in the toxicity of angiogenesis inhibition. Nat Rev Cancer
2007; 7: 475–485. [Medline] [CrossRef]
19. Ratner M: Genentech discloses safety concerns over Avastin. Nat
Biotechnol 2004; 22: 1004–1198. [Medline] [CrossRef]
20. Maharaj AS, Walshe TE, Saint-Geniez M, Venkatesha S, Maldonado AE, Himes NC, Matharu KS, Karumanchi SA, D’Amore
PA : VEGF and TGF-beta are required for the maintenance of thechoroid plexus and ependyma. J Exp Med 2008; 205: 491–501.
[Medline] [CrossRef]

21. Lee S, Chen TT, Barber CL, Jordan MC, Murdock J, Desai S, Ferrara N, Nagy A, Roos KP, Iruela-Arispe ML : Autocrine VEGF
signaling is required for vascular homeostasis. Cell 2007; 130:691–703. [Medline] [CrossRef]
22. Kamba T, McDonald DM: Mechanisms of adverse effects ofanti-VEGF therapy for cancer. Br J Cancer 2007; 96: 1788–1795.
[Medline] [CrossRef]
23. Eremina V, Sood M, Haigh J, Nagy A, Lajoie G, Ferrara N, Gerber HP, Kikkawa Y, Miner JH, Quaggin SE : Glomerular-specificalterations of VEGF-A expression lead to distinct congenital andacquired renal diseases. J Clin Invest 2003; 111: 707–716. [Medline]
24. Gerber HP, Malik AK, Solar GP, Sherman D, Liang XH, Meng G,
Hong K, Marsters JC, Ferrara N : VEGF regulates haematopoietic
stem cell survival by an internal autocrine loop mechanism. Nature 2002; 417: 954–958. [Medline] [CrossRef]
25. Yang JC, Haworth L, Sherry RM, Hwu P, Schwartzentruber DJ,
Topalian SL, Steinberg SM, Chen HX, Rosenberg SA : A randomized trial of bevacizumab, an anti-vascular endothelial growth
factor antibody, for metastatic renal cancer. N Engl J Med 2003;
349: 427–434 The murine mutation osteopetrosis is in the coding
region of the macrophage colony stimulating factor gene. [Medline] [CrossRef]
26. Murakami M, Simons M: Fibroblast growth factor regulation ofneovascularization. Curr Opin Hematol 2008; 15: 215–220. [Medline] [CrossRef]
27. Augustin HG, Koh GY, Thurston G, Alitalo K : Control of vascular morphogenesis and homeostasis through the angiopoietinTie system. Nat Rev Mol Cell Biol 2009; 10: 165–177. [Medline][CrossRef]
28. Koh YJ, Kim HZ, Hwang SI, Lee JE, Oh N, Jung K, Kim M, Kim
KE, Kim H, Lim NK, Jeon CJ, Lee GM, Jeon BH, Nam DH, Sung
HK, Nagy A, Yoo OJ, Koh GY : Double antiangiogenic protein,
DAAP, targeting VEGF-A and angiopoietins in tumor angiogenesis, metastasis, and vascular leakage. Cancer Cell 2010; 18:
171–184. [Medline] [CrossRef]
29. Saharinen P, Eklund L, Pulkki K, Bono P, Alitalo K: VEGF andangiopoietin signaling in tumor angiogenesis and metastasis.
Trends Mol Med 2011; [Epub ahead of print].
30. Mohyeldin A, Garzón-Muvdi T, Quiñones-Hinojosa A : Oxygenin stem cell biology: a critical component of the stem cell niche.
Cell Stem Cell 2010; 7: 150–161. [Medline] [CrossRef]
31. Ebos JM, Lee CR, Cruz-Munoz W, Bjarnason GA, Christensen
JG, Kerbel RS : Accelerated metastasis after short-term treatment
with a potent inhibitor of tumor angiogenesis. Cancer Cell 2009;15: 232–239. [Medline] [CrossRef]
32. Pàez-Ribes M, Allen E, Hudock J, Takeda T, Okuyama H, Viñals
F, Inoue M, Bergers G, Hanahan D, Casanovas O : Antiangiogenic
therapy elicits malignant progression of tumors to increased local
invasion and distant metastasis. Cancer Cell 2009; 15: 220–231.
[Medline] [CrossRef]
33. Miles D, Harbeck N, Escudier B, Hurwitz H, Saltz L, Van Cutsem
E, Cassidy J, Mueller B, Sirzén F : Disease course patterns after
discontinuation of bevacizumab: pooled analysis of randomized
phase III trials. J Clin Oncol 2010; 29: 83–88. [Medline] [CrossRef]
34. Padera TP, Kuo AH, Hoshida T, Liao S, Lobo J, Kozak KR, Fukumura D, Jain RK : Differential response of primary tumor
versus lymphatic metastasis to VEGFR-2 and VEGFR-3 kinase
inhibitors cediranib and vandetanib. Mol Cancer Ther 2008; 7:
2272–2279. [Medline] [CrossRef]
35. Gaengel K, Genove G, Armulik A, Betsholtz C : Endothelialmural cell signaling in vascular development and angiogenesis.
Arterioscler Thromb Vasc Biol 2009; 29: 630–638. [Medline][CrossRef]
36. Hellberg C, Ostman A, Heldin CH : PDGF and vessel maturation.
Recent Results Cancer Res 2010; 180: 103–114. [Medline] [Cross-Keio J Med 2012; 61 (2): 47–56
Ref]
37. Wang R, Chadalavada K, Wilshire J, Kowalik U, Hovinga KE,
Geber A, Fligelman B, Leversha M, Brennan C, Tabar V : 2010
Glioblastoma stem-like cells give rise to tumour endothelium. Nature 2010; 468: 829–833. [Medline] [CrossRef]
38. Ricci-Vitiani L, Pallini R, Biffoni M, Todaro M, Invernici G,
Cenci T, Maira G, Parati EA, Stassi G, Larocca LM, De Maria R: Tumour vascularization via endothelial differentiation of glioblastoma stem-like cells. Nature 2010; 468: 824–828. [Medline][CrossRef]
39. Ferrara N: Role of myeloid cells in vascular endothelial growth
factor-independent tumor angiogenesis. Curr Opin Hematol 2010;
17: 219–224. [Medline]
40. Kerbel RS, Yu J, Tran J, Man S, Viloria-Petit A, Klement G,
Coomber BL, Rak J : Possible mechanisms of acquired resistanceto anti-angiogenic drugs: implications for the use of combination therapy approaches. Cancer Metastasis Rev 2001; 20: 79–86.
[Medline] [CrossRef]
41. Naik E, O’Reilly LA, Asselin-Labat ML, Merino D, Lin A, Cook
M, Coultas L, Bouillet P, Adams JM, Strasser A : Destruction oftumor vasculature and abated tumor growth upon VEGF blockade is driven by proapoptotic protein Bim in endothelial cells. J
Exp Med 2011; 208: 1351–1358. [Medline] [CrossRef]
42. Lu C, Bonome T, Li Y, Kamat AA, Han LY, Schmandt R, Coleman RL, Gershenson DM, Jaffe RB, Birrer MJ, Sood AK : Genealterations identified by expression profiling in tumor-associatedendothelial cells from invasive ovarian carcinoma. Cancer Res
2007; 67: 1757–1768. [Medline] [CrossRef]
43. Lu C, Han HD, Mangala LS, Ali-Fehmi R, Newton CS, Ozbun L,
Armaiz-Pena GN, Hu W, Stone RL, Munkarah A, Ravoori MK,
Shahzad MM, Lee JW, Mora E, Langley RR, Carroll AR, Matsuo
K, Spannuth WA, Schmandt R, Jennings NB, Goodman BW, Jaffe
RB, Nick AM, Kim HS, Guven EO, Chen YH, Li LY, Hsu MC,
Coleman RL, Calin GA, Denkbas EB, Lim JY, Lee JS, Kundra V,
Birrer MJ, Hung MC, Lopez-Berestein G, Sood AK : Regulationof tumor angiogenesis by EZH2. Cancer Cell 2010; 18: 185–197.
[Medline] [CrossRef]
44. Sato Y, Sonoda H: The vasohibin family: a negative regulatory
system of angiogenesis genetically programmed in endothelial
cells. Arterioscler Thromb Vasc Biol 2007; 27: 37–41. [Medline][CrossRef]
45. Yoshida H, Hayashi S, Kunisada T, Ogawa M, Nishikawa S,
Okamura H, Sudo T, Shultz LD, Nishikawa S. Nature 1990; 345:
442–444. [Medline] [CrossRef]
46. Cecchini MG, Dominguez MG, Mocci S, Wetterwald A, Felix R,
Fleisch H, Chisholm O, Hofstetter W, Pollard JW, Stanley ER :Role of colony stimulating factor-1 in the establishment and regulation of tissue macrophages during postnatal development of themouse. Development 1994; 120: 1357–1372. [Medline]
47. Kubota Y, Takubo K, Shimizu T, Ohno H, Kishi K, Shibuya M,
Saya H, Suda T : M-CSF inhibition selectively targets pathological angiogenesis and lymphangiogenesis. J Exp Med 2009; 206:
1089–1102. [Medline] [CrossRef]
48. Fantin A, Vieira JM, Gestri G, Denti L, Schwarz Q, Prykhozhij
S, Peri F, Wilson SW, Ruhrberg C : Tissue macrophages act as
cellular chaperones for vascular anastomosis downstream of
VEGF-mediated endothelial tip cell induction. Blood 2010; 116:
829–840. [Medline] [CrossRef]
49. Rymo SF, Gerhardt H, Wolfhagen Sand F, Lang R, Uv A, Betsholtz C : A two-way communication between microglial cells
and angiogenic sprouts regulates angiogenesis in aortic ring cultures. PLoS ONE 2011; 6: e15846. [Medline] [CrossRef]
50. Qian BZ, Pollard JW: Macrophage diversity enhances tumor progression and metastasis. Cell 2010; 141: 39–51. [Medline] [CrossRef]
51. Mantovani A, Sica A: Macrophages, innate immunity and cancer:
balance, tolerance, and diversity. Curr Opin Immunol 2010; 22:55
231–237. [Medline] [CrossRef]
52. Sica A, Bronte V: Altered macrophage differentiation and immune dysfunction in tumor development. J Clin Invest 2007; 117:
1155–1166. [Medline] [CrossRef]
53. Rolny C, Mazzone M, Tugues S, Laoui D, Johansson I, Coulon
C, Squadrito ML, Segura I, Li X, Knevels E, Costa S, Vinckier
S, Dresselaer T, Åkerud P, De Mol M, Salomäki H, Phillipson
M, Wyns S, Larsson E, Buysschaert I, Botling J, HimmelreichU, Van Ginderachter JA, De Palma M, Dewerchin M, ClaessonWelsh L, Carmeliet P : HRG inhibits tumor growth and metastasis by inducing macrophage polarization and vessel normalization
through downregulation of PlGF. Cancer Cell 2011; 19: 31–44.
[Medline] [CrossRef]
54. Werno C, Menrad H, Weigert A, Dehne N, Goerdt S, Schledzewski K, Kzhyshkowska J, Brüne B : Knockout of HIF-1α intumor-associated macrophages enhances M2 polarization and attenuates their pro-angiogenic responses. Carcinogenesis 2010; 31:
1863–1872. [Medline] [CrossRef]
55. Liao X, Sharma N, Kapadia F, Zhou G, Lu Y, Hong H, Paruchuri
K, Mahabeleshwar GH, Dalmas E, Venteclef N, Flask CA, Kim
J, Doreian BW, Lu KQ, Kaestner KH, Hamik A, Clément K, Jain
MK : Krüppel-like factor 4 regulates macrophage polarization. J
Clin Invest 2011; 121: 2736–2749. [Medline] [CrossRef]
56. Whyte CS, Bishop ET, Rückerl D, Gaspar-Pereira S, Barker RN,
Allen JE, Rees AJ, Wilson HM: Suppressor of cytokine signaling(SOCS)1 is a key determinant of differential macrophage activation and function. J Leukoc Biol 2011; [Epub ahead of print].
57. Lin EY, Li JF, Gnatovskiy L, Deng Y, Zhu L, Grzesik DA, Qian
H, Xue XN, Pollard JW : Macrophages regulate the angiogenic
switch in a mouse model of breast cancer. Cancer Res 2006; 66:11238–11246. [Medline] [CrossRef]
58. Okuno Y, Nakamura-Ishizu A, Kishi K, Suda T, Kubota Y : Bonemarrow-derived cells serve as proangiogenic macrophages but
not endothelial cells in wound healing. Blood 2011; 117: 5264–
5272. [Medline] [CrossRef]
59. Baluk P, Morikawa S, Haskell A, Mancuso M, McDonald DM :Abnormalities of basement membrane on blood vessels and endothelial sprouts in tumors. Am J Pathol 2003; 163: 1801–1815.
[Medline] [CrossRef]
60. Jain RK: Normalization of tumor vasculature: an emerging concept in antiangiogenic therapy. Science 2005; 307: 58–62. [Medline] [CrossRef]
61. Kashiwagi S, Tsukada K, Xu L, Miyazaki J, Kozin SV, Tyrrell JA,
Sessa WC, Gerweck LE, Jain RK, Fukumura D : Perivascular nitric oxide gradients normalize tumor vasculature. Nat Med 2008;
14: 255–257. [Medline] [CrossRef]
62. Mazzone M, Dettori D, Leite de Oliveira R, Loges S, Schmidt T,
Jonckx B, Tian YM, Lanahan AA, Pollard P, Ruiz de AlmodovarC, De Smet F, Vinckier S, Aragonés J, Debackere K, Luttun A,
Wyns S, Jordan B, Pisacane A, Gallez B, Lampugnani MG, Dejana E, Simons M, Ratcliffe P, Maxwell P, Carmeliet P : Heterozygous deficiency of PHD2 restores tumor oxygenation and inhibits
metastasis via endothelial normalization. Cell 2009; 136: 839–5.
[Medline] [CrossRef]
63. St Croix B, Rago C, Velculescu V, Traverso G, Romans KE,
Montgomery E, Lal A, Riggins GJ, Lengauer C, Vogelstein B,
Kinzler KW : Genes expressed in human tumor endothelium. Science 2000; 289: 1197–1202. [Medline] [CrossRef]
64. Hida K, Klagsbrun M: A new perspective on tumor endothelial
cells: unexpected chromosome and centrosome abnormalities.
Cancer Res 2005; 65: 2507–2510. [Medline] [CrossRef]
65. Economopoulou M, Langer HF, Celeste A, Orlova VV, Choi EY,
Ma M, Vassilopoulos A, Callen E, Deng C, Bassing CH, Boehm
M, Nussenzweig A, Chavakis T : Histone H2AX is integral tohypoxia-driven neovascularization. Nat Med 2009; 15: 553–558.
[Medline] [CrossRef]
66. Fischer C, Jonckx B, Mazzone M, Zacchigna S, Loges S, Patta-

56
rini L, Chorianopoulos E, Liesenborghs L, Koch M, De Mol M,
Autiero M, Wyns S, Plaisance S, Moons L, van Rooijen N, Giacca
M, Stassen JM, Dewerchin M, Collen D, Carmeliet P : Anti-PlGF
inhibits growth of VEGF(R)-inhibitor-resistant tumors without
affecting healthy vessels. Cell 2007; 131: 463–475. [Medline][CrossRef]
67. Casanovas O, Hicklin DJ, Bergers G, Hanahan D : Drug resistance by evasion of antiangiogenic targeting of VEGF signaling inlate-stage pancreatic islet tumors. Cancer Cell 2005; 8: 299–309.
[Medline] [CrossRef]
68. Kubota Y, Takubo K, Hirashima M, Nagoshi N, Kishi K, Okuno
Y, Nakamura-Ishizu A, Sano K, Murakami M, Ema M, Omatsu
Y, Takahashi S, Nagasawa T, Shibuya M, Okano H, Suda T : Isolation and function of mouse tissue resident vascular precursors
marked by myelin protein zero. J Exp Med 2011; 208: 949–960.
[Medline] [CrossRef]
69. Aguayo A, Kantarjian H, Manshouri T, Gidel C, Estey E, Thomas
D, Koller C, Estrov Z, O’Brien S, Keating M, Freireich E, Albitar
M : Angiogenesis in acute and chronic leukemias and myelodys-

Kubota Y: Tumor Angiogenesis and Anti-angiogenic Therapy
plastic syndromes. Blood 2000; 96: 2240–2245. [Medline]
70. Li WW, Hutnik M, Gehr G : Antiangiogenesis in haematological malignancies. Br J Haematol 2008; 143: 622–631. [Medline][CrossRef]
71. Schmidt T, Kharabi Masouleh B, Loges S, Cauwenberghs S,
Fraisl P, Maes C, Jonckx B, De Keersmaecker K, Kleppe M, Tjwa
M, Schenk T, Vinckier S, Fragoso R, De Mol M, Beel K, Dias
S, Verfaillie C, Clark RE, Brümmendorf TH, Vandenberghe P,
Rafii S, Holyoake T, Hochhaus A, Cools J, Karin M, Carmeliet
G, Dewerchin M, Carmeliet P : Loss or Inhibition of StromalDerived PlGF Prolongs Survival of Mice with Imatinib-Resistant
Bcr-Abl1(+). Leukemia. Cancer Cell 2011; 19: 740–753. [Medline][CrossRef]
72. Rakhra K, Bachireddy P, Zabuawala T, Zeiser R, Xu L, Kopelman A, Fan AC, Yang Q, Braunstein L, Crosby E, Ryeom S,
Felsher DW : CD4(+) T cells contribute to the remodeling of the
microenvironment required for sustained tumor regression upon
oncogene inactivation. Cancer Cell 2010; 18: 485–498. [Medline][CrossRef]