Vessel abnormalization: another hallmark of cancer?Molecular mechanisms and therapeutic implications

Available online at www.sciencedirect.com

Vessel abnormalization: another hallmark of cancer?Molecular mechanisms and therapeutic implicationsKatrien De Bock1,2, Sandra Cauwenberghs1,2 and Peter Carmeliet1,2

As a result of excessive production of angiogenic molecules,

tumor vessels become abnormal in structure and function. By

impairing oxygen delivery, abnormal vessels fuel a vicious

cycle of non-productive angiogenesis, which creates a hostile

microenvironment from where tumor cells escape through

leaky vessels and which renders tumors less responsive to

chemoradiation. While anti-angiogenic strategies focused on

inhibiting new vessel growth and destroying pre-existing

vessels, clinical studies showed modest anti-tumor effects. For

many solid tumors, anti-VEGF treatment offers greater clinical

benefit when combined with chemotherapy. This is partly due

to a normalization of the tumor vasculature, which improves

cytotoxic drug delivery and efficacy and offers unprecedented

opportunities for anti-cancer treatment. Here, we overview key

novel molecular players that induce vessel normalization.

Addresses1 Vesalius Research Center, K.U.Leuven, Leuven, Belgium2 Vesalius Research Center, VIB, Leuven, Belgium

Corresponding author: Carmeliet, Peter

([email protected])

Current Opinion in Genetics & Development 2011, 21:73–79

This review comes from a themed issue on

Genetic and cellular mechanisms of oncogenesis

Edited by Chris Marshall and Karen Vousden

Available online 22nd November 2010

0959-437X/$ – see front matter

# 2010 Elsevier Ltd. All rights reserved.

DOI 10.1016/j.gde.2010.10.008

IntroductionAngiogenesis promotes tumor growth and malignancy. In

contrast to the healthy vasculature, tumor vessels are,

however, highly abnormal structurally and functionally

[1–3]. This is the result of an uncontrolled, relentless

production of angiogenic stimulators, in excess of inhibi-

tors, which tips the balance in favor of hyperactive vessel

growth (Figure 1). These abnormal tumor vessels are

characterized by a mal-shaped, irregular, disorganized

and tortuous architecture with a highly dysfunctional

and leaky endothelial cell (EC) layer [1,3]. The abnormal

tumor vasculature exhibits remarkable spatiotemporal

heterogeneity. In certain regions, ECs with irregular

shape are stacked upon each other and obstruct blood

flow by extending multiple protrusions, while in other

sites, ECs move away or die and leave behind gaps. Also,

they are often loosely connected, have wider junctions

www.sciencedirect.com

and are covered by fewer and abnormal mural pericytes

(PCs) [1,2,4��]. These changes not only impair drug

delivery and perfusion, but also convert the tumor into

a hostile hypoxic and acidic milieu, from where cancer

cells escape through leaky vessels [5,6]. Such an unnatural

milieu also promotes a vicious cycle of non-productive

angiogenesis and stimulates pro-malignant reprogram-

ming of tumor cell metabolism (Figure 1). In addition,

it hampers the anti-tumor immune defense and highjacks

inflammatory cells for angiogenesis, enhances tumor tis-

sue swelling (potentially life-threatening in brain tumors)

and makes chemoradiotherapy less efficient [1]. Paradoxi-

cally thus, even though tumors crave for oxygen, they

stimulate a non-productive process of angiogenesis extre-

mely, so that abnormal tumor vessels deliver less — rather

than more — oxygen to the hypoxic cancer cells, which in

turn continues to fuel the cycle. Thus, tumor vessel

abnormalization promotes tumor invasiveness, dissemi-

nation and overall malignancy.

Current anti-angiogenic therapies are based on the con-

cept of starving and depriving the tumor from its nutrient

supply by destroying existing vessels and preventing new

vessel growth (anti-angiogenic ‘vessel pruning’) [7,8].

However, despite successes, clinical trials with VEGF-

targeted monotherapy have shown a more modest pro-

longation of progression-free or overall survival of cancer

patients than anticipated [8,9]. Aside from the benefit of

anti-VEGF monotherapy in glioblastoma and renal cell

carcinoma, other solid cancers (breast, lung and color-

ectal) showed a greater therapeutic effect when anti-

VEGF was combined with conventional chemotherapy.

Recent findings have resolved the paradox of how an anti-

angiogenic vessel pruning strategy (which would be

expected to impede vascular supply of cytotoxic drugs)

can, in fact, improve chemotherapy by partially normal-

izing the tumor vasculature and thereby enhancing drug

delivery [1]. This has not only fostered the novel concept

that judicious use of vessel pruning agents may induce

vessel normalization by restoring the angiogenic balance,

but also raised the question whether more selective anti-

angiogenic ‘vessel normalization’ strategies could be

developed. In this review, we will briefly illustrate some

examples of both strategies.

Targeting the VEGF-family for vesselstabilizationVEGF stimulates EC migration, proliferation, survival,

permeability and lumen formation [10] and is indispensa-

ble for physiological angiogenesis [11]. Given its import-

ance in cancer and numerous other angiogenic disorders,


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http://dx.doi.org/10.1016/j.gde.2010.10.008

74 Genetic and cellular mechanisms of oncogenesis

Figure 1

Oncogenes - inflammation

Angiogenic stimulators

Angiogenic inhibitors

Vessel abnormalization

InvasionMetastasis

Tumor tissueswelling

Hypoxia

Acidosis

chemoradiationtherapy

+++

immuneresponse

serumdeprivation

Current Opinion in Genetics & Development

Mechanism for tumor vessel abnormalization. During tumor

development, oncogenic mutations and inflammation foster the

relentless production of angiogenic growth factors, in excess of

angiogenic inhibitors. The angiogenic dysbalance favors hyperactive

vessel growth and vessel abnormalization, which impairs tumor

perfusion, and renders it more hypoxic and acidic. This creates a

continuous self-perpetuating cycle of non-productive angiogenesis that

hampers the anti-tumor immune defense, enhances tumor tissue

swelling, and makes chemoradiation therapy less efficient. Moreover,

the leaky, abnormal vasculature promotes tumor invasiveness and

dissemination.

VEGF has become a prime target for anti-angiogenic

therapy [9,12]. Apart from its effects on pruning pre-exist-

ing vessels and inhibiting growth of new vessels, blockage

of VEGF or its signaling pathways also induces vessel

normalization [1] (Figure 2). For instance, switching off

VEGF expression in experimental tumors prunes imma-

ture vessels, while increasing PC coverage and vessel

maturation [13]. Aside from inducing similar effects,

pharmacological neutralization of VEGF also decreases

tumor vessel permeability and interstitial fluid pressure,

improves tumor perfusion and restores the hydrostatic

pressure gradient across the vascular wall, which leads to

a deeper penetration of drugs and chemotherapy [14,15].

The resulting ‘normalization window’ increases tumor

oxygenation and improves radiation therapy in most but

not all tumor studies [16,17]. This normalization window is

transient, since prolonged VEGF-inhibitor therapy ulti-

mately prunes the large majority of tumor vessels, including

the normalized ones.

Molecularly, VEGF inhibition causes vessel normaliza-

tion, in part by the upregulation of angiopoietin-1 (see

below) [17], matrix metalloproteinases (which remodel

the vascular basement membrane and surrounding

matrix) [17], and activating PDGFRb signaling (see

below) [18]. Interestingly, myeloid production of VEGF

is important, as deletion of VEGF from these inflamma-

tory cells promotes vessel normalization and maturation


[19]. Though ablation of myeloid-cell VEGF accelerates

initial tumor progression, tumors are more sensitive to

chemotherapy [19]. Of all preclinical anti-angiogenic

vessel normalization factors identified, clinical evidence

for efficacy in cancer patients has been only provided for

anti-VEGF agents so far. For instance, VEGF blockage

prolongs the survival of glioblastoma patients and mice by

alleviating intracerebral edema through restoration of the

blood–brain barrier as part of the vessel normalization

process, even despite continuous tumor growth [20,21��].Anti-VEGF treatment also enhances tumor vessel PC

coverage while reducing interstitial fluid pressure and

induces other signs of vessel normalization in patients

with rectal cancer [22].

Placental growth factor (PlGF), another member of the

VEGF family, is redundant for vascular growth in de-

velopment and health, but contributes to the angiogenic

switch in numerous diseases by acting as a multi-tasking

pro-angiogenic cytokine [23]. Although not all agents

show efficacy [24], PlGF blockage by other antibodies

reduces tumor growth and metastasis partly via vessel

pruning [25,26�]. Since anti-PlGF is a weaker vessel-

pruning agent than anti-VEGF and improves vessel

maturation, it does not cause severe tumor hypoxia, yet

improves chemotherapy, indicating that it induces vessel

normalization [25,26�]. More recent data confirm that

PlGF blockage partially normalizes tumor vessels in

hepatocellular carcinoma (HCC) models by counteracting

tumor-induced capillarization of sinusoidal vessels and

coverage with vaso-constrictive PC-like hepatic stellate

cells, and by reducing vessel tortuosity and the formation

of hypo-perfused string vessels; overall, these changes

decrease tumor hypoxia and malignancy [25,26�].Additional proof for structural and functional tumor vessel

normalization (including improved tumor perfusion and

oxygenation) was observed in a more recent study upon

lowering PlGF in tumors [27]. Since PlGF is upregulated

in tumor-bearing mice and patients and in ocular models

of neovascularization receiving VEGF-targeted therapy

[23], and because PlGF-blockage enhances VEGF-tar-

geted therapy without causing adverse effects [25,26�], a

combination of both strategies warrants further study.

Role of PDGFs and angiopoietins in vesselnormalizationPDGFB, secreted by sprouting ECs, is a well character-

ized recruitment signal for PCs, expressing PDGF re-

ceptor-b (PDGFRb) [28]. Ablation of PDGFB or its

receptor, or removal of its retention signal (required for

PDGFB to bind to PCs) leads to abnormal vessel leakage,

dilatation and tortuosity in the embryonic and tumor

vasculature [28], while overexpression increases PC cov-

erage and maturation [29,30] (Figure 3). Blocking this

PC-recruitment signal causes, however, variable effects

on tumor progression and metastasis. Since cancer-associ-

ated fibroblasts contract in response to PDGFB, this


Mechanisms of vessel abnormalization in cancer De Bock, Cauwenberghs and Carmeliet 75

Figure 2

Anti-VEGF treatment normalizes the tumor vasculature. VEGF inhibition prunes the immature vasculature and stabilizes the remaining vessels by

enhancing their pericyte coverage, a process that is mediated via induction of Ang-1 and PDGFRb signaling. Also, VEGF neutralization decreases

tumor vessel permeability and improves tumor perfusion and oxygenation, which leads to a deeper penetration of drugs and enhanced

chemoradiotherapeutic efficacy.

growth factor increases the interstitial fluid pressure,

which impairs drug delivery, explaining in part why

PDGFB blockage improves chemotherapy [31]. Also,

some but not all studies show that blockage of PDGFB

renders tumor vessels more sensitive to VEGF inhibitors

[32,33]. However, these beneficial effects may be offset

by the risk of increased metastasis of tumor cells, which

intravasate more readily through PC-deficient vessels

[34]. By inducing the formation of a complex between

VEGFR2 and PDGFRb, which inhibits downstream

signaling of the latter, VEGF promotes vessel abnorma-

lization [18].

Various angiopoietin family members, which bind to their

Tie2 receptor, also regulate vessel normalization [35]

(Figure 3). Ang-1 promotes vessel normalization and

counteracts the VEGF-induced vascular permeability

by establishing more inter-endothelial junctions

[36,37], and favors tumor vessel maturation through PC

recruitment [35,38]. Part of the normalization activity of

anti-VEGF relies on upregulating Ang-1 expression [15].


Ang-2, by contrast, induces opposite effects and renders

tumor vessels abnormal. Hence, loss or inhibition of Ang-

2 promotes tumor vessel PC coverage and vessel quies-

cence [35,39�,40], though its effects on tumor vasculature

are often contextual. In line with findings that Ang-2

plasma levels correlate with metastatic cancer and are

upregulated in cancer patients receiving VEGF blockers

[41], combined targeting of VEGF and Ang-2 exhibits

superior anti-cancer efficacy [42].

Role of PHD2 oxygen sensor in tumor vesselnormalizationOne of the essential functions of blood vessels is to

supply oxygen. In order to adequately sense and respond

to changing oxygen tensions, cellular oxygen sensing is

performed by the HIF-prolyl hydroxylases (PHD),

which control the stability of the hypoxia-inducible

factors (HIFs), that orchestrate the hypoxic response

[43,44]. Activation of HIF-1a in tumor cells by

oncogenes, hypoxia and inflammatory signals upregu-

lates VEGF production and thereby promotes vessel



Figure 3

Pericyte Ang-1 PDGFRβ

HIF1

Ang-2 Tie2 PDGFβ

Endothelial cell

Angiopoietins in cancer

*Ang-1 decreases vessel leakage *Ang-1 promotes tightening endothelial junctions *Ang-1 enhances pericyte recruitment

*Ang-2 inhibits Ang-1 *Ang-2 reduces pericyte recruitment and vessel maturation *Ang-2 levels correlate with poor patient prognosis *Loss of Ang-2 reduces early stage tumor growth

PDGF signaling in cancer

*Ablation of PDGF(R) abnormalizes the tumor vasculature *Overexpression of PDGFB enhances tumor vessel maturation *PDGFB promotes vessel pericyte coverage *VEGF reduces PDGFR signaling

Current Opinion in Genetics & Development

Role of PDGFs and angiopoietins in cancer. By binding to its Tie2 receptor, Ang-1 promotes pericyte coverage and the establishment of tight

intercellular junctions. Ang-2, whose expression is controlled by HIF, inhibits Ang-1 and thus renders the tumor vessels abnormal. PDGFB is secreted

by endothelial cells and signals through PDGFRb to stimulate pericyte recruitment. The role of both Ang as well as PDGF(R) signaling in vessel

(ab)normalization is overviewed in the inserted boxes.

abnormalization. Endothelial PHD2, via regulation of

HIF-2a, does not affect physiological angiogenesis and

vessel branching, but improves tumor oxygenation by

sensing and readapting oxygen supply in conditions of

oxygen deprivation [4��]. Haplodeficiency of PHD2 does

not affect tumor vessel density and area, turtuosity or

lumen size, but induces normalization of the EC lining.

Tumor vessels of heterozygous PHD2 deficient mice are

lined by a single monolayer of regular, orderly formed,

polarized, cobblestone ECs with few fenestrations.

These changes do not affect primary tumor growth

but improve tumor perfusion and oxygenation, thereby

preventing a metastatic switch and overall prolonging

survival [4��].

Mechanistically, PHD2 haplodeficient ECs exhibit a

‘phalanx’ phenotype: they are more quiescent, and exhi-

bit lower proliferation and migration responses to VEGF

[4��]. Their molecular signature includes the upregula-

tion of soluble VEGFR1, a molecular VEGF trap, which

reduces VEGF signaling, induces vessel quiescence and

shapes vessel sprouts [45,46]. Moreover, the normalized

EC layer in heterozygous PHD2 deficient mice forms a


tighter barrier, not only because ECs express more junc-

tional proteins (such as ZO-1 and VE-cadherin) but also

because they are surrounded by more PCs and a stable

basement membrane [4��]. VE-cadherin, a HIF-2a de-

pendent gene, induces a ‘normalized and quiescent’

endothelial layer by redirecting VEGF signaling towards

EC survival [47,48]. Consistent with the identified role of

HIF-2a downstream of PHD2, reduced HIF-2a levels

cause improper remodeling of nascent vessels into larger

conduits in the embryo [49], and the formation of an

aberrant vascular network in tumors due to impaired

remodeling [50]. In addition, EC specific deletion of

HIF-2a increases vascular leakage while reducing tumor

neovascularization [51�]. Interestingly, the Ang-1 induced

normalization of immature vessels is associated with

reduced PHD2 expression [52]. Intriguingly, the effect

of PHD2 is dose-dependent, as homozygous loss of

PHD2 increases vessel branching [53].

Does Notch contribute to the ‘phalanx cell’phenotype?Another candidate that recently emerged as a mediator of

vessel quiescence is Notch, an EC receptor for Dll4.



Since Dll4/Notch signaling controls vessel branching by

suppressing the formation of endothelial tip cells (so-

called, because they lead the branch at the forefront)

[54], inhibition of this pathway induces the formation of

more but hypoperfused vessels [55,56]. Conversely, over-

expression of Dll4 by tumor cells reduces vessel density,

while enhancing lumen size and perfusion, overall

improving tumor oxygenation [57]. However, chronic

blockage of its ligand Dll4 in healthy animals resulted

in a dose-dependent formation of vascular neoplasms

characterized by irregularly shaped blood vessels [58��].These vascular abnormalities are likely due to reduced

EC quiescence and suggest a role for Dll4/Notch in the

maintenance of the phalanx cell phenotype. Indeed,

inactivation of RBP-J, a transcription factor implicated

in Notch signaling, in ECs leads to widespread spon-

taneous angiogenesis [59], while stimulation of ECs with

Dll4 induces a quiescent phenotype that is reminiscent of

the phalanx cell signature, including a reduction of

VEGF-responses because of the downregulation of

VEGFR2 [54]. Aside from effects on ECs, Dll4 signaling

from ECs to Notch-expressing mural cells upregulates

PDGFRb expression and thereby stimulates vessel matu-

ration. In accordance, Dll4 expression in patient tumor

biopsies correlates with vessel maturation [60].

ConclusionsThe abnormal structure and function of the tumor vas-

culature help to explain many fundamental hallmarks of

cancer and challenges of anti-cancer treatment. While

anti-angiogenic vessel pruning strategies bear the risk of

rendering the tumor micro-environment further abnormal

and thereby fueling tumor invasiveness and metastasis

[61,62], anti-angiogenic vessel normalization strategies

offer the opportunity of converting a malignant invasive,

metastatic cancer into a more benign, encapsulated,

metabolically less aggressive, and poorly invasive/meta-

static tumor, which also responds better to conventional

anti-cancer chemoradiation therapy. This novel vessel

normalization concept has recently proven its applica-

bility in the clinic. Understanding the molecular basis of

vessel abnormalization not only in malignant but also in

inflammatory or ischemic diseases is therefore of great

importance. This review has highlighted some key prin-

ciples and players of this process, but other factors such as

Rgs5 [63], nitric oxide [64], and EGF receptor [64] also

participate. Discovering additional molecules with vessel

normalization activity therefore promises to offer unpre-

cedented novel opportunities to improve anti-cancer

therapy.

AcknowledgementsKDB is supported by the Fund for Scientific Research in Flanders (FWO).PC is supported by long-term structural funding (Methusalem funding bythe Flemish Government), Interuniversity attraction pole (Grant P60/30,funded by the Belgian Government, BELSPO), FWO G.0692.09 (FlemishGovernment) and a research grant by the Belgian ‘‘Foundation againstCancer’’, GAO 2006/11–K.U.Leuven.


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