Systemic anti-vascular endothelial growth factor therapies induce a painful sensory neuropathy

BRAINA JOURNAL OF NEUROLOGY

Systemic anti-vascular endothelial growth factortherapies induce a painful sensory neuropathyAn Verheyen,1,2,3 Eve Peeraer,3,4 Rony Nuydens,3 Joke Dhondt,1,2 Koen Poesen,1,2

Isabel Pintelon,5 Anneleen Daniels,3 Jean-Pierre Timmermans,5 Theo Meert,3

Peter Carmeliet2,6 and Diether Lambrechts1,2

1 Laboratory for Translational Genetics, Department of Oncology, University of Leuven, B-3000 Leuven, Belgium

2 Vesalius Research Centre, VIB, B-3000 Leuven, Belgium

3 Department of Neuroscience, Janssen Research and Development, A Division of Janssen Pharmaceutica N.V., B-2340 Beerse, Belgium

4 Biomedical Research Institute, University of Hasselt, B-3590 Diepenbeek, Belgium

5 Laboratory of Cell Biology and Histology, Department of Veterinary Sciences, University of Antwerp, B-2020 Antwerp, Belgium

6 Laboratory of Angiogenesis and Neurovascular Link, University of Leuven, B-3000 Leuven, Belgium

Correspondence to: Diether Lambrechts, PhD,

Vesalius Research Centre,

University of Leuven,

Campus Gasthuisberg,

Herestraat 49,

B-3000 Leuven,

Belgium

E-mail: [email protected]

Systemic vascular endothelial growth factor inhibition, in combination with chemotherapy, improves the outcome of patients

with metastatic cancer. Peripheral sensory neuropathies occurring in patients receiving both drugs are attributed to the chemo-

therapy. Here, we provide unprecedented evidence that vascular endothelial growth factor receptor inhibitors trigger a painful

neuropathy and aggravate paclitaxel-induced neuropathies in mice. By using transgenic mice with altered neuronal vascular

endothelial growth factor receptor expression, systemic inhibition of vascular endothelial growth factor receptors was shown to

interfere with the endogenous neuroprotective activities of vascular endothelial growth factor on sensory neurons. In vitro,

vascular endothelial growth factor prevented primary dorsal root ganglion cultures from paclitaxel-induced neuronal stress and

cell death by counteracting mitochondrial membrane potential decreases and normalizing hyperacetylation of a-tubulin. In

contrast, vascular endothelial growth factor receptor inhibitors exerted opposite effects. Intriguingly, vascular endothelial

growth factor or vascular endothelial growth factor receptor inhibitors exerted their effects through a mechanism whereby

Hdac6, through Hsp90, controls vascular endothelial growth factor receptor-2-mediated expression of the anti-apoptotic Bcl2.

Our observations that systemic anti-vascular endothelial growth factor therapies interfere with the neuroprotective activities of

vascular endothelial growth factor may have important implications for the application of anti-vascular endothelial growth factor

therapies in cancer patients.

Keywords: anti-angiogenesis; histone deacetylase 6; neuropathy; vascular endothelial growth factor

Abbreviations: VEGF = vascular endothelial growth factor

doi:10.1093/brain/aws145 Brain 2012: 135; 2629–2641 | 2629

Received December 8, 2011. Revised April 2, 2012. Accepted April 18, 2012. Advance Access publication June 25, 2012

� The Author (2012). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved.

For Permissions, please email: [email protected]

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IntroductionTumours critically depend on the formation of new blood vessels

for their outgrowth and metastasis. Numerous studies have high-

lighted vascular endothelial growth factor (VEGF) as a key inducer

of this process and have shown that VEGF interference impedes

vessel growth and starves tumours (Carmeliet, 2005; Jain et al.,

2007). This resulted, in 2004, in the approval of a neutralizing

VEGF antibody (bevacizumab) as an effective treatment for

advanced colorectal, breast and lung cancer (Hurwitz et al.,

2004; Miller et al., 2005; Sandler et al., 2006). Soon thereafter,

VEGF receptor inhibitors, such as sorafenib and sunitinib proved

equally successful in other cancers (Demetri et al., 2006; Llovet

et al., 2008).

Following extensive clinical use of bevacizumab, a distinct pro-

file of side-effects, mostly mild to moderate in severity (hyperten-

sion and proteinuria) or occurring rather uncommonly (wound

healing complications and gastro-intestinal perforations) was es-

tablished (Gordon and Cunningham, 2005). Emerging evidence

suggests, however, that bevacizumab may also be associated

with increased incidence of sensory neuropathies. Indeed, when

administered in combination with FOLFOX (fluorouracil, leucov-

orin and oxaliplatin) the incidence of severe neuropathies induced

by bevacizumab increases from 9.3% to 16.3% (Giantonio et al.,

2007). Peripheral sensory neuropathies also frequently occur as a

common side-effect of chemotherapy and severely impact the

patient’s quality of life, leading to dose reduction or premature

termination of the cytostatic regimen (Mantyh, 2006). Since bev-

acizumab is delivered together with chemotherapy and patients

receiving both drugs exhibit prolonged progression-free survival,

neuropathies occurring in patients receiving chemotherapy and

bevacizumab are often attributed to prolonged exposure to

chemotherapy. It is unknown, however, whether bevacizumab

also directly induces or aggravates sensory neuropathies.

Likewise, sunitinib and sorafenib may also affect the PNS. Both

therapies are frequently associated with hand–foot syndrome in

42% and 8.9% of the patients, respectively (Lipworth et al.,

2009). Although hand–foot syndrome is considered a cutaneous

condition, characterized by palmoplantar erythema, oedema and

loss of skin integrity, patients developing hand–foot syndrome

usually first note a tingling sensation, which subsequently pro-

gresses to a burning pain, suggesting that the PNS is also affected

by these inhibitors (Lipworth et al., 2009).

Several studies revealed that VEGF also exerts strong neuropro-

tective effects. The first neuroprotective effects of VEGF were

described by Sondell et al. (2000) who reported that VEGF pro-

motes axonal outgrowth of primary dorsal root ganglia. More

convincing in vivo evidence for VEGF-mediated neuroprotection

was provided by studies in knock-in mice, in which reduced ex-

pression of VEGF caused adult-onset progressive degeneration of

motor neurons (Oosthuyse et al., 2001; Storkebaum et al., 2005).

Meanwhile, studies injecting VEGF inhibitors directly into the

nervous system have also implicated VEGF in other neurological

disorders such as depression and Parkinson’s disease (Warner-

Schmidt and Duman, 2007). Although these findings triggered

some initial concerns about the use of anti-VEGF therapies in

cancer patients, none of these studies could convincingly demon-

strate that systemic delivery of VEGF (receptor) inhibitors also dir-

ectly contributes to these disorders.

Since clinical studies suggest that anti-VEGF therapies may in-

crease the incidence of neuropathies in cancer patients, and since

evidence that anti-VEGF therapies interfere with VEGF-mediated

neuroprotection in cancer patients is still lacking, here we carefully

assess the role of VEGF (inhibition) in the sensory nervous system.

Materials and methods

Paclitaxel-induced neuropathy andbehavioural testingMice were injected intraperitoneally on four alternate days with pacli-

taxel (Bristol Myers Squibb; 1 mg/kg in saline). All mice were always

tested in behavioural assays before paclitaxel injections. The following

behavioural assays were used: the Von Frey test to evaluate mechan-

ical (tactile) allodynia and the Hargreaves’ Paw Flick test to evaluate

thermal hyperalgesia. A more detailed description of these tests is

given in the online Supplementary material. The local ethical commit-

tee approved all experiments.

Mice overexpressing a wild-type or truncated murine VEGF

receptor-2 or Flk1 transgene in post-natal neurons were generated

by pronuclear injection of a mouse Thy1.2 expression cassette.

ThyFlk1WT mice were further intercrossed with FVB mice to obtain

heterozygous litters, while ThyFlk1DN mice were bred homozygous

to achieve maximal expression levels of the Flk1DN transgene.

Isolation and treatment of dorsal rootganglion neuronsDorsal root ganglia (all levels, unless explicitly specified) were dissected

from the spinal column and collected in PBS containing 1 g/l glucose.

Ganglia were then enzymatically dissociated by incubating them in

medium containing 0.5% collagenase followed by 0.25% trypsin.

Subsequently, ganglia were mechanically dissociated into single cells.

The cell suspension was placed in a Petri dish coated with foetal calf

serum for 90 min at 37�C. Dorsal root ganglion neurons were plated in

poly-L-lysine coated 96-well plates in Neurobasal� medium supple-

mented with B27 (Gibco). Glial cell line-derived neurotrophic factor

(GDNF; PeproTech EC), VEGFA (Supplementary material), SU5416

(Sigma-Aldrich) or DC101 were added to the culture medium 4 h

prior to paclitaxel addition.

ImmunocytochemistryDorsal root ganglion neurons were fixed using 0.5% TritonTM X-100

(Sigma) and 0.5% glutaraldehyde dissolved in PHEM buffer (buffer

containing PIPES, double Hanks, EGTA and MgCl2). Cells were per-

meabilized with 0.5% TritonTM X-100 and incubated in 1 mg/ml

NaBH4 in PHEM. Primary antibodies used are polyclonal anti-ATF3

(Santa Cruz Biotechnology 1:800) and monoclonal anti-neurofilament

SMI32 (Sternberger Monoclonals Incorporated, 1:1000). For tubulin

stainings, anti-acetylated and anti-total tubulin antibodies (Sigma,

1:300) were combined with anti-rabbit b3-tubulin (Covance;

1:1000). Subsequently, cells were incubated with Alexa Fluor� 488

and Alexa Fluor� 555 secondary antibodies (Invitrogen). The percent-

age of ATF3-positive neurons was determined using fluorescence

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microscopy and by analysing 4100 cells per well. The percentage of

acetylated or total tubulin was determined by general segmentation

(KS300 image analysis software) on pictures taken with a fluorescence

microscope (Zeiss). Single cell analysis was used to calculate the mean

fluorescence of the cytoplasm of b3-tubulin stained neurons. To exam-

ine cell death, neurons were paraformaldehyde-fixed and labelled with

anti-b3-tubulin followed by TUNEL staining (terminal deoxynucleotidyl

transferase dUTP nick end labelling; Roche). The percentage of

TUNEL-positive neurons was determined in each specific culture con-

dition by using fluorescence microscopy. More than 100 neurons were

counted per well, each well was analysed three times and at least two

wells per condition were determined.

Western blotWhole L4-5-6 dorsal root ganglia were isolated, snap-frozen and pro-

teins were extracted with tissue protein extraction reagent. Dorsal root

ganglion cultures were lysed in mammalian protein extraction reagent

buffer or directly in SDS sample buffer. Proteins were loaded on Novex

Bis–Tris gels (4–12%) (Invitrogen) and blotted on nitrocellulose.

Membranes were blocked with PBS-Tween (PBS-T) containing 5%

bovine serum albumin and incubated with primary antibodies over-

night at 4�C. Primary antibodies used are mouse anti-acetylated or

total tubulin (Sigma), rabbit anti-HDAC6 (Millipore), rabbit anti-

HSP90 (StressMarq) and mouse anti-BCL2 (Santa Cruz). Primary anti-

bodies were detected using horseradish peroxidase-labelled secondary

antibodies via West Dura� enhanced chemiluminescence (Pierce,

Thermoscientific). Signals were captured and quantified by a

Lumi-imaging system (Roche Diagnostics).

Mitochondrial membrane potentialmeasurementMouse dorsal root ganglia were cultured overnight in 96-well plates.

The next day, 4 h after preincubation, neurons were loaded with 2 mM

JC-1 (Molecular Probes) in PBS ( + Ca2 + , Mg2 + and 1 g/l glucose) for

30 min at 37�C. After washing, mitochondrial membrane potential was

measured with a Zeiss LSM 510 confocal microscope. A total of

15 images was taken, paclitaxel (10mM, diluted in PBS) or PBS was

added after the second image and FCCP (10 mM), as positive control,

after Image 8. Red to green ratios were calculated and normalized to

the first value of each cell. Pre-values and values after treatment were

averaged.

StatisticsData are shown as mean � SEM. To calculate differences between

groups, unpaired Student’s t-tests or univariate ANOVA considering

equal variances was used. For the Von Frey measurements, overall

differences between groups were calculated using the repeated meas-

urement ANOVA test. Significance was defined as P5 0.05.

Results

VEGF receptor inhibitors induce apainful neuropathyTo assess the possibility that VEGF receptor inhibitors trigger

adverse effects in the sensory nervous system, we first assessed

whether their systemic administration affects sensory nerve func-

tion in mice. Two inhibitors were used: a small-molecule VEGF

receptor tyrosine-kinase (TK) inhibitor (SU5416) and a monoclonal

antibody directed against the murine Flk1 receptor (�Flk1 or

DC101) (Tessler et al., 1994). The latter was chosen because the

neutralizing monoclonal antibody for murine VEGF is not commer-

cially available. Furthermore, since Flk1 is the main ‘angiogenic’

receptor of VEGF, �Flk1 should closely mimic bevacizumab.

When delivering both inhibitors intraperitoneally at doses that suc-

cessfully inhibit tumour angiogenesis (Klement et al., 2000; Bergers

et al., 2003), mice dose-dependently developed tactile allodynia

(Fig. 1A and B). Monitoring the hind limb paw by the Von Frey

aesthesiometer revealed that allodynia was mild upon administra-

tion of a daily dose of 12.5 mg/kg SU5416, but more severe after

delivery of 25 mg/kg SU5416 (Fig. 1A). A similar effect was

observed when mice were treated with 20 and 40 mg/kg �Flk1

three times per week (Fig. 1B). Notably, tactile allodynia developed

rapidly, within days of the first injection, but the effect was only

transient, as mice gradually recovered after the last injection. Mice

treated with �Flk1 also developed thermal hyperalgesia, as assessed

by the Paw Flick test (Supplementary Fig. 1).

VEGF receptor inhibitors aggravatepainful paclitaxel-induced neuropathiesIn clinical practice, bevacizumab is combined with a reference

chemotherapy such as paclitaxel. The intriguing question therefore

arises whether VEGF receptor inhibitors—in particular �Flk1 as it

most closely mimics bevacizumab—also aggravate paclitaxel-

induced neuropathies. In a first set of experiments, �Flk1 was

delivered together with paclitaxel. Mice receiving paclitaxel or

�Flk1 alone exhibited a similar withdrawal response in the Von

Frey test, whereas mice receiving both paclitaxel and �Flk1

developed more tactile allodynia than mice receiving paclitaxel

alone (Fig. 1C). Alternatively, when �Flk1 was given after halting

paclitaxel injections, i.e. to mice that had already developed a

painful neuropathy due to paclitaxel, mice treated with �Flk1

failed to recover. In contrast, mice receiving paclitaxel and rat

IgG quickly recovered after halting paclitaxel injections (Fig. 1D).

VEGF exerts direct neuroprotectiveeffects on isolated dorsal rootganglion neuronsSince VEGF can affect both vessels and nerves, it is possible that

VEGF receptor inhibitors—in addition to their established effects

on the vasculature—also directly affect dorsal root ganglion neu-

rons. To assess this intriguing hypothesis, we first characterized the

neuroprotective effects of VEGF on primary dorsal root ganglion

neurons. Since pilot studies revealed that paclitaxel did not induce

cell death of dorsal root ganglion neurons, but rather increased

expression of the neuronal stress marker ATF3 (activating tran-

scription factor 3, Supplementary Fig. 2A and B), primary dorsal

root ganglion cultures were challenged with a low concentration

of paclitaxel (10 nM), which increased ATF3 without inducing cell

death (Supplementary Fig. 2). VEGF administration to primary

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dorsal root ganglion neurons exposed to paclitaxel dose-

dependently reduced ATF3 immunoreactivity and was as effective

as GDNF in exerting these effects (Fig. 2A).

Next, we assessed whether VEGF also counteracts paclitaxel-

induced decreases in the mitochondrial membrane potential

(��m) as one of the earliest markers of apoptosis (Bernardi

et al., 1999). To monitor ��m, we exposed the cell-permeable

mitochondrial probe JC-1 to dorsal root ganglion cultures and

applied two-colour confocal microscopy. At a high ��m, JC-1 is

detectable as a red fluorescent signal, but as ��m drops, the fluor-

escence emitted by JC-1 changes to green. The ��m can thus be

monitored as the ratio of red over green mean fluorescent inten-

sity. Administration of paclitaxel (10mM, Supplementary Fig. 2C)

caused a significant reduction in ��m and this reduction could

effectively be inhibited by pretreating cultures with VEGF (Fig. 2B).

Finally, we also assessed whether VEGF affects paclitaxel-

induced cell death of primary dorsal root ganglion neurons.

Rather than exposing dorsal root ganglia to low levels of

paclitaxel, which were used to assess the effects on ATF3 immu-

noreactivity, higher levels of paclitaxel were applied to assess the

effect on cell death (Supplementary Fig. 2D). As expected, VEGF

also protected dorsal root ganglion neurons against 10 mM

paclitaxel-induced cell death, as assessed by TUNEL staining

(Fig. 2C). Overall, this confirms that VEGF exerts direct neuropro-

tective effects on primary dorsal root ganglion neurons, by redu-

cing neuronal stress and protecting against cell death.

VEGF counteracts paclitaxel-inducedincreases in tubulin acetylationPaclitaxel directly binds to microtubules, thereby preventing de-

polymerization of �-tubulin and inducing a stable, hyper-

acetylated tubular state (Schiff et al., 1979). We therefore also

Figure 1 Systemic delivery of VEGF receptor inhibitors induces and aggravates a painful neuropathy. (A) SU5416 dose-dependently

induces mechanical allodynia, as measured with a Von Frey test [P = 0.49 and P50.001 for low and high doses of SU5416 versus DMSO

(dimethylsulphoxide); n = 10–10]. *P50.05 high dose SU5416 versus DMSO at individual days. (B) Low and high doses of DC101

induce a similar mechanical allodynia as SU5416 (P = 0.073 and P = 0.023 for low and high doses of DC101 versus rat IgG; n 410 per

group). *P50.05 for high dose of DC101 versus rat IgG at individual days. (C) Co-delivery of paclitaxel and DC101, on alternate days,

induces more mechanical allodynia than paclitaxel or rat IgG alone (P50.001; n = 16–16). *P50.05 for DC101 + paclitaxel versus rat

IgG + paclitaxel. (D) Rat IgG and DC101 are delivered to mice, in which systemic paclitaxel was used to induce a painful neuropathy. Mice

receiving rat IgG recover very quickly and exhibit a normal withdrawal response 1 day after the first injection (P = 0.15 versus vehicle

treatment; n = 8–10). In contrast, DC101-treated mice exhibit a reduced withdrawal response for the total duration of the treatment

(P = 0.004 and P = 0.042 after 1 and 2 weeks of DC101 versus rat IgG; n = 8–8). *P50.05 DC101 versus rat IgG-treated mice on

individual days.



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Figure 2 VEGF and VEGF receptor inhibitors directly affect primary dorsal root ganglion neurons through Flk1. (A) Measurement of

neuronal stress in primary dorsal root ganglion cultures by quantifying ATF3 reactivity after paclitaxel administration. VEGF dose-

dependently reduces paclitaxel-induced ATF3 reactivity (r2 = 0.999 after a sigmoidal fit, n = 6 wells from three rats per condition) similarly

as glial cell-derived neurotrophic factor (GDNF). ***P5 0.001 versus vehicle; #P50.05, ##P5 0.01 and ###P50.001 versus paclitaxel.


(continued)


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assessed whether VEGF is capable of counteracting these effects,

as a measure of protection against paclitaxel-induced neurotox-

icity. To this extent, we immunocytochemically quantified fluores-

cence intensities of acetylated and total �-tubulin in the cytoplasm

of dorsal root ganglion neurons using multichannel fluorescence

microscopy. As expected, paclitaxel increased acetylated �-tubulin

levels dose-dependently (Supplementary Fig. 2E). This increase

was counteracted by the addition of VEGF (Fig. 2D). Representa-

tive images are shown in Fig. 2E. Furthermore, to observe the

general state of tubulin acetylation in dorsal root ganglion cultures

after paclitaxel and VEGF, a complimentary western blot analysis

for acetylated and total tubulin was performed to confirm these

effects (Fig. 2F and G). Since paclitaxel is known to upregulate

various isoforms of tubulin, representing the cell’s attempt to re-

plenish the supplies of tubulin monomers that are depleted due to

paclitaxel (Stargell et al., 1992), total �-tubulin levels were also

quantified. Paclitaxel increased total �-tubulin levels, whereas

VEGF failed to affect total �-tubulin levels (Supplementary

Fig. 3A–C). Similar results were obtained using an independent

method to quantify acetylated and total �-tubulin levels (Supple-

mentary Fig. 3D), thus confirming that VEGF selectively reduces

acetylated but not total �-tubulin levels when combined with

paclitaxel.

Flk1 mediates the neuroprotectiveactivities of VEGFSince VEGF induces potent neuroprotective activities on primary

dorsal root ganglion neurons and VEGF receptor inhibitors, pos-

sibly by interfering with the direct neuroprotective activities of

VEGF, induce a painful neuropathy, the role of the VEGF recep-

tors, Flk1 and Flt1, in mediating VEGF’s neuroprotective activities

was studied in more detail. To this end, various transgenic mouse

strains with altered expression of Flk1 or Flt1 were used, including

mice overexpressing, under control of the neuron-specific Thy1.2

promoter, a wild-type (Thy:Flk1WT mice) or dominant-negative

Flk1 (Thy:Flk1DN mice). Quantification of Flk1 messenger RNA

transcripts confirmed that Flk1 was overexpressed in whole

dorsal root ganglia (number of Flk1 copies per 103 �-actin

copies: 7.32 � 0.30, 855.40 � 81.99 and 3.91 � 0.28 for

ThyFlk1WT, ThyFlk1DN and wild-type mice, respectively,

P50.005 versus wild-type mice) and primary dorsal root ganglion

cultures (number of Flk1 copies per 103 b-actin copies:

11.88 � 1.04, 134.83 � 25.45 and 4.34 � 0.65 for ThyFlk1WT,

ThyFlk1DN and wild-type mice, respectively, P50.01 versus

wild-type mice). Additionally, knock-in mice expressing a TK

dead Flt1 receptor (Flt1-TK�/�) were also used.

When isolating primary dorsal root ganglion cultures from

wild-type, Thy:Flk1WT and Thy:Flk1DN mice under baseline condi-

tions, ATF3 levels did not differ in wild-type and Thy:Flk1WT dorsal

root ganglia. On the other hand, ATF3 levels were slightly

increased in Thy:Flk1DN dorsal root ganglia (Fig. 2H), presumably

due to a lack of baseline Flk1 activation. Upon challenge with a

low concentration of paclitaxel, Thy:Flk1WT dorsal root ganglion

cultures exhibited less pronounced increases in ATF3 levels

than wild-type or Thy:Flk1DN cultures. When cultures were subse-

quently treated with VEGF prior to exposure to paclitaxel, ATF3

levels decreased in treated wild-type cultures, but Thy:Flk1DN cul-

tures failed to respond (Fig. 2H). Notably, Thy:Flk1WT, but not

Thy:Flk1DN dorsal root ganglion cultures, also exhibited less

hyperacetylation of �-tubulin, but not total tubulin, when chal-

lenged with paclitaxel (Supplementary Fig. 4). On the other

hand, dorsal root ganglion cultures with a defective Flt1 receptor

(Flt1-TK�/�) exhibited decreased ATF3 and hyperacetylation of

Figure 2 Continued(B) Paclitaxel reduces the mitochondrial membrane potential (��m) relative to PBS (n420 neurons from three mice). Pretreatment (4 h)

of dorsal root ganglion cultures with VEGF protects against a decrease in ��m. **P50.01 versus PBS; ##P5 0.01 and ###P50.001

versus paclitaxel. (C) Mouse dorsal root ganglion cultures treated with paclitaxel for 24 h contain more dead neurons than untreated dorsal

root ganglion cultures. Pretreatment with VEGF protects neurons against paclitaxel-induced cell death (n = 6 wells from three mice).

***P50.001 versus vehicle; #P5 0.05 and ##P5 0.01 versus paclitaxel. (D and E) Tubulin acetylation as determined by quantitative

microscopy is increased by paclitaxel. This increase is partly counteracted by the addition of VEGF (n4 40 neurons from two mice).

***P50.001 versus vehicle; ##P5 0.01 versus paclitaxel. Representative images are shown in (E). Acetylated tubulin (red), b3 tubulin

(green) and DAPI (blue) expression in a representative dorsal root ganglion neuron from a primary dorsal root ganglion culture.

Treatments are indicated on each panel. Scale bar = 25 mm. (F and G) Western blots (F) for acetylated tubulin on dorsal root ganglion

neurons treated with vehicle, VEGF, with and without paclitaxel confirming that VEGF reduces the amount of acetylated tubulin

(P = 0.024, n = 4 mice). **P50.01 versus PBS and #P5 0.05 versus paclitaxel. (H) At baseline, ATF3 is increased in Thy:Flk1DN neurons.

Paclitaxel induces only minimal neuronal stress in Thy:Flk1WT neurons compared with wild-type (WT) and Thy:Flk1DN neurons. VEGF

reduces ATF3 levels in wild-type neurons, whereas Thy:Flk1DN neurons do not respond to VEGF treatment (P = 0.84) (n4 6 wells from at

least three mice per genotype). *P50.05 and ***P50.001 versus respective vehicles; #P5 0.05 and ##P5 0.01 versus paclitaxel. (I)

VEGF receptor inhibitors dose-dependently increase ATF3 reactivity in primary wild-type dorsal root ganglion cultures (r2 = 0.836 and

0.968 for DC101 and SU5416, respectively; n = 6 wells per condition). **P50.01 and ***P50.001 versus vehicle. (J and K) Exposure to

DC101 or paclitaxel causes hyperacetylation of tubulin in dorsal root ganglion cultures (n445 neurons from three mice). Notably, DC101

combined with paclitaxel causes an additional increase in acetylated tubulin. *P50.05 and ***P50.001 versus vehicle and ###P50.001

versus paclitaxel. Representative images are shown (K). Acetylated tubulin (red), b3 tubulin (green) and DAPI (blue) expression in a

representative dorsal root ganglion neuron from a primary dorsal root ganglion culture. Treatments are indicated on each panel. Scale

bar = 25 mm. (L) Western blot for acetylated tubulin on dorsal root ganglion neurons treated with rat IgG, DC101, with and without

paclitaxel confirming that DC101 increases the amount of acetylated tubulin (P = 0.03, n = 3 mice) alone and in combination with

paclitaxel (P = 0.018). *P50.05 and ***P50.001 versus rat IgG and #P50.05 versus paclitaxel. (M) Acetylated/total tubulin ratio is

increased in L4–L6 dorsal root ganglia from DC101-treated mice compared with dorsal root ganglia from rat IgG-treated mice (n = 4).

*P5 0.05 and **P5 0.01 versus rat IgG.



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�-tubulin after VEGF exposure (data not shown), indicating that

Flt1 was not crucially involved in mediating VEGF’s neuroprotec-

tive activities.

VEGF receptor inhibitors negativelyaffect primary dorsal rootganglion neuronsSince VEGF potently protected primary dorsal root ganglion neu-

rons through Flk1, we assessed whether VEGF receptor inhibitors

also directly affect primary dorsal root ganglion neurons and exert

similar effects as paclitaxel. When exposing primary dorsal root

ganglion neurons to �Flk1 and SU5416, both inhibitors

dose-dependently increased ATF3 levels (Fig. 2I) without affecting

cell death. Additionally, �Flk1 caused a significant increase in

acetylated �-tubulin (Fig. 2J) without affecting total �-tubulin

levels (Supplementary Fig. 5). When �Flk1 was combined with

paclitaxel, acetylated �-tubulin levels also increased compared to

�Flk1 or paclitaxel alone (Fig. 2J). Representative images confirm-

ing these effects are shown in Fig. 2K. A complimentary western

blot on dorsal root ganglion cultures treated with �Flk1 with or

without paclitaxel confirmed this effect (Fig. 2L and

Supplementary Fig. 5). Similar effects on acetylated tubulin levels

were observed for SU5416 (data not shown). Overall, these data

illustrate that VEGF receptor inhibitors exert direct effects on pri-

mary dorsal root ganglion neurons and amplify the neurotoxic

effects of paclitaxel. Notably, upon systemic delivery, �Flk1 also

increased expression of Atf3 in dorsal root ganglia as determined

by real-time PCR (RT-PCR) expression analysis of whole dorsal

root ganglia. Likewise, we found that �Flk1 increased acetylated

versus total �-tubulin levels (Fig. 2M), thereby reinforcing the hy-

pothesis that VEGF receptor inhibitors may directly affect the sen-

sory nervous system.

VEGF receptor inhibitors interfere withthe neuroprotective effects of Flk1To further assess the in vivo relevance of Flk1-mediated neuro-

protection, we characterized sensory nerve function in Thy:Flk1WT,

Thy:Flk1DN and wild-type mice under normal conditions and after

exposure to paclitaxel. Thy:Flk1WT, Thy:Flk1DN and wild-type mice

appeared healthy and fertile and exhibited normal densities of

PGP9.5 + axons or perfused vessels in their paws

(Supplementary Fig. 6). At baseline, Thy:Flk1WT and wild-type

mice also responded similarly to mechanical pressure applied by

the Von Frey meter. Intriguingly, Thy:Flk1DN mice showed clear

signs of tactile allodynia (Fig. 3A). When assessing thermal hyper-

algesia, Thy:Flk1DN were also hypersensitive to heat compared to

Thy:Flk1WT and wild-type mice, which behaved normally in the

Paw Flick assay (Supplementary Fig. 1).

When Thy:Flk1WT, Thy:Flk1DN and wild-type mice were subse-

quently challenged with systemic paclitaxel, Thy:Flk1WT mice de-

veloped a less painful neuropathy compared with wild-type mice,

as assessed by the Von Frey test (Fig. 3A). In Thy:Flk1DN mice,

tactile allodynia did not increase substantially, such that on the last

day of paclitaxel injections, Thy:Flk1DN and wild-type mice showed

a similar withdrawal response. However, after halting paclitaxel

injections, tactile allodynia disappeared in wild-type mice, whereas

Thy:Flk1DN mice failed to recover (Fig. 3A). When quantifying

acetylated and total �-tubulin levels in whole dorsal root ganglia

at the time of the last paclitaxel injection, as a measure of induced

neurotoxicity, wild-type and Thy:Flk1DN dorsal root ganglia ex-

hibited increased acetylated levels relative to total �-tubulin

levels. On the other hand, these levels remained unaltered in

Thy:Flk1WT mice (Fig. 3B and C). Overall, these data indicate

that neuron-specific overexpression of Flk1 protects against

paclitaxel-induced neuropathy, whereas dominant-negative inhib-

ition of neuronal Flk1, similar to the systemic delivery of �Flk1,

induces a painful sensory neuropathy.

In a next set of experiments, we measured tactile allodynia in

wild-type, Thy:Flk1WT and Thy:Flk1DN mice treated with systemic

�Flk1. Thy:Flk1WT mice did not develop allodynia after �Flk1

(Fig. 3D), presumably due to residual activation of overexpressed

Flk1 receptors that could not be blocked by �Flk1. On the other

hand, Thy:Flk1DN mice failed to develop additional signs of allo-

dynia and were as sensitive to a tactile stimulus as wild-type mice

treated with �Flk1 (Fig. 3E). Similar effects were observed when

thermal hyperalgesia was assessed (Supplementary Fig. 1). In con-

clusion, since Thy:Flk1WT mice treated with �Flk1 did not develop

a painful neuropathy, these data indicate that �Flk1 interferes with

the neuroprotective activities of Flk1 to sensitize sensory nerves.

The neuroprotective activities of Flk1are Hdac6-dependentSince neuronal Flk1 was essential for normal sensory nerve func-

tion and potently protected against a paclitaxel-induced neur-

opathy, we assessed downstream signalling activation of Flk1

after VEGF stimulation. VEGF stimulation and Flk1 overexpression

both decreased acetylated �-tubulin levels after paclitaxel expos-

ure, thereby suggesting that histone deacetylase 6 (HDAC6;

Hubbert et al., 2002), which is responsible for deacetylation of

�-tubulin, is involved in mediating these effects. Two HDAC in-

hibitors were used to assess this hypothesis: trichostatin A, which

inhibits class I and II mammalian HDACs, and tubacin, which spe-

cifically inhibits HDAC6. As expected, trichostatin A (30 nM) and

tubacin (100 nM) increased acetylated �-tubulin levels in primary

dorsal root ganglia (Fig. 4A and B) without affecting total

�-tubulin levels. VEGF decreased acetylated �-tubulin levels in

paclitaxel-treated cultures but failed to reduce acetylated �-tubulin

when combined with trichostatin A or tubacin (Fig. 4A and B). We

then assessed whether the neuroprotective activities of VEGF were

affected by HDAC6 inhibition. As expected, VEGF was protective

against a paclitaxel-induced increase in ATF3. However, in com-

bination with trichostatin A or tubacin, VEGF failed to reduce

ATF3 expression (Fig. 4C and D). Likewise, although trichostatin

A and tubacin individually did not affect ��m, VEGF combined

with either trichostatin A or tubacin failed to prevent a

paclitaxel-induced decrease in ��m (Fig. 4E and F). Finally,

VEGF also failed to provide protection against paclitaxel-induced

cell death when combined with trichostatin A or tubacin (Fig. 4G

and H), thus indicating that Hdac6 plays an essential role within



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the pathway of VEGF-mediated neuroprotection against

paclitaxel-induced toxicity.

Hsp90 and Bcl2 as mediators ofFlk1-driven neuroprotectionTo explore how Hdac6 regulates Flk1-mediated neuroprotection,

we assessed whether VEGF by directly affecting Hdac6 activity

reduces acetylation of �-tubulin and protects against paclitaxel-

induced toxicity. We failed, however, to detect any change in

Hdac6 messenger RNA or protein expression levels in primary

dorsal root ganglion cultures after VEGF exposure (data not

shown). Moreover, VEGF did not affect acetylation of �-tubulin

in primary dorsal root ganglion neurons not exposed to paclitaxel

(Supplementary Fig. 3D), suggesting that there is no direct inter-

action between VEGF and Hdac6. Overall, this finding supports

previous observations that hyperacetylation of �-tubulin represents

a marker rather than a cause of microtubule hyperstability (Zhang

et al., 2003).

Intriguingly, HDAC6 is also involved in the regulation of various

other non-histone proteins. For instance, HDAC6-mediated deace-

tylation activates Hsp90, which is an important regulator of cell

signalling (Aoyagi and Archer, 2005). Additionally, in leukaemia

cells, VEGF promotes survival through Hsp90-mediated induction

of Bcl2 expression and inhibition of apoptosis (Dias et al., 2002).

We therefore assessed whether Hdac6, through the regulation of

Hsp90 and Bcl2, could mediate the neuroprotective effects of

Figure 3 VEGF receptor inhibitors interfere with the neuroprotective effects of Flk1. (A) Systemic paclitaxel-treated Thy:Flk1WT mice are

protected against mechanical allodynia [n = 9–11; P = 0.045 for Thy:Flk1WT mice versus wild-type (WT) mice]. Thy:Flk1DN mice have

mechanical allodynia at the start of the experiment and do not recover over time (P = 0.005 versus wild-type mice; n = 11–18). *P50.05

and #P50.05 for Thy:Flk1WT and Thy:Flk1DN mice versus wild-type mice, respectively. (B and C) Systemic paclitaxel increases the

acetylated/total tubulin ratio in wild-type and Thy:Flk1DN dorsal root ganglia, but not in Thy:Flk1WT dorsal root ganglia (P = 0.83) (n = 6

mice per group). A representative western blot is shown. *P5 0.05 versus untreated mice. (D) Thy:Flk1WT mice receiving DC101 are

resistant to hypersensitivity (P = 0.39 versus rat IgG Thy:Flk1WT mice). *P50.05 DC101 Thy:Flk1WT mice versus DC101 wild-type mice at

individual days. (E) At baseline, Thy:Flk1DN mice display a mechanical allodynia, which initially is only mild (P = 0.024 at baseline versus

wild-type mice) but then aggravates over time (P50.001 at Day 22). DC101 does not cause any additional hypersensitivity (P = 0.93

versus rat IgG Thy:Flk1DN mice). Mechanical allodynia of DC101-treated wild-type mice is comparable to that in Thy:Flk1DN mice

(P = 0.81 for rat IgG Thy:Flk1DN mice versus DC101 wild-type on Day 8; n = 8–8). *P50.05 for rat IgG Thy:Flk1DN mice versus rat IgG

wild-type mice on individual days.



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Figure 4 The neuroprotective effects of Flk1/VEGF are Hdac6-dependent. (A and B) Trichostatin A (TSA) and tubacin cause an increase

in acetylated tubulin. Trichostatin A additionally increases acetylated tubulin in combination with paclitaxel. VEGF fails to reduce

paclitaxel-induced hyperacetylation in combination with trichostatin A (P = 0.3) or tubacin (P = 0.9) (n460 neurons from four mice).

*P5 0.05 and ***P50.001 versus vehicle; #P50.05 and ##P50.01 versus paclitaxel. (C and D) Treatment of dorsal root ganglion

neurons with trichostatin A or paclitaxel causes neuronal stress. VEGF does not protect against paclitaxel-induced neuronal stress in

combination with trichostatin A (P = 0.67) or tubacin (P = 0.35) (n = 6 wells from three rats per condition). **P50.01 and ***P50.001

versus vehicle; #P50.05 versus paclitaxel. (E and F) Pretreatment of dorsal root ganglion cultures with VEGF protects against

paclitaxel-induced depolarization, but not when VEGF is combined with trichostatin A or tubacin (P = 0.30 and P = 0.37, respectively;

n414 neurons from at least three mice). *P50.05 and **P50.01 versus PBS; #P5 0.05 versus paclitaxel. (G and H) VEGF reduces

neuronal cell death caused by paclitaxel, but not in combination with trichostatin A (P = 0.14) or tubacin (P = 0.14) (n = 6–8 wells from

three mice). ***P50.001 versus vehicle; #P50.05 and ##P50.01 versus paclitaxel. NS = not significant.



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VEGF against paclitaxel-induced toxicity. Whole dorsal root gang-

lia isolated from Thy:Flk1WT mice showed increased expression of

BCL2 and HSP90 proteins (Fig. 5A). Likewise, VEGF stimulation

of primary dorsal root ganglion cultures increased expression of

BCL2 and HSP90 (Fig. 5B). However, when VEGF stimulation was

combined with the HDAC6-specific inhibitor tubacin, expression of

BCL2 failed to be upregulated (Fig. 5B and C), whereas expression

of HSP90 was unaffected (Fig. 5B and D), suggesting that failure

of deacetylating Hsp90 by Hdac6 inhibits VEGF-induced expres-

sion of Bcl2. Since quantification of acetylated HSP90 after immu-

noprecipitation of total HSP90 was technically not feasible in

primary dorsal root ganglion cultures, we could not, however,

formally prove that deacetylation of HSP90 by Hdac6 indeed

mediated these effects. Application of the Hsp90 inhibitor

17-AAG (0.5 mM) in combination with VEGF and paclitaxel in-

hibited the neuroprotective effects of VEGF (Fig. 5E and F),

thereby reinforcing the role of HSP90. When applying the Bcl2

inhibitor ABT-737 (1 mM), at concentrations that failed to induce

cell death (Supplementary Fig. 7), together with VEGF and pacli-

taxel, the neuroprotective activities of VEGF on cell death

(Fig. 5G) and tubulin deacetylation (Fig. 5H) were also inhibited.

Overall, these data suggest that VEGF-mediated neuroprotection

against paclitaxel-induced toxicity depends at least partially on a

mechanism (Fig. 5I), whereby Hdac6 mediates VEGF-induced

upregulation of Bcl2.

Finally, we also observed that BCL2 expression was drastically

decreased in primary dorsal root ganglion cultures exposed to

�Flk1 (41.1 � 7.8% reduction in BCL2 relative to control cultures

by western blotting, P = 0.0016). To show that reduced BCL2

expression after �Flk1 was mediated by HDAC6, we also transi-

ently expressed a HDAC6-GFP construct in endothelioma E2 cells

(the latter cells were used since transfection in primary dorsal root

ganglion cultures was technically not feasible). Overexpression of

HDAC6 at least partially inhibited the increase in acetylated tubu-

lin levels induced by �Flk1 as well as the reduction in BCL2 ex-

pression relative to untransfected cells (Supplementary Fig. 8).

Similar effects were observed for the other VEGF receptor inhibitor

SU5416 (Supplementary Fig. 8), thereby reinforcing the role of

HDAC6 in Flk1-mediated neuroprotection and the neurotoxic ef-

fects of the VEGF receptor inhibitors.

DiscussionThe most important finding of the study is that systemic applica-

tion of VEGF (receptor) inhibitors triggers undesired effects in the

sensory nervous system. Indeed, when systemically delivering

�Flk1 or SU5416, mice dose-dependently developed mechanical

allodynia and thermal hyperalgesia, and also suffered from more

severe paclitaxel-induced neuropathies. Remarkably, wild-type

mice receiving �Flk1 developed an allodynia that was very similar

to that of Thy:Flk1DN mice under baseline conditions, whereas

Thy:Flk1WT mice were completely resistant to �Flk1. These obser-

vations clearly indicate that VEGF receptor inhibitors interfere with

the endogenous neuroprotective effects of VEGF.

Are there any reasons to be wary of similar effects when

applying anti-VEGF (receptor) therapies to humans? Several

observations suggest that caution should be warranted. When de-

livered simultaneously with systemic paclitaxel, �Flk1 aggravated

paclitaxel-induced neuropathy in mice. Combined treatment regi-

mens of bevacizumab and taxanes are effectively being used in

the clinic. The fact that increased incidence of sensory neuropa-

thies in combination therapies has already been reported, suggests

that anti-VEGF therapies also contribute to neuropathies in

humans (Miller et al., 2007). Increasing evidence indicates

that anti-VEGF therapies are being delivered for longer periods.

Indeed, due to their efficacy in delaying disease progression,

cancer patients receive chemotherapy and bevacizumab for

much longer periods and neuropathies induced by both substances

may therefore accumulate or aggravate over time. Clinical trials

assessing the efficacy of adjuvant anti-angiogenic therapies, which

implicates prolonged treatment schedules with bevacizumab, have

recently also been initiated (Allegra et al., 2011). Although VEGF

receptor inhibitors, sorafenib and sunitinib, have proven

single-agent activity in patients with solid tumours, combination

therapies with chemotherapy are currently also being considered

(Hauschild et al., 2009). Based on our findings, the long-term

effects of anti-VEGF therapies in the sensory nervous system of

patients receiving such prolonged combination treatments should

carefully be monitored. Importantly, VEGF is not only neuropro-

tective for sensory neurons—it also potently affects other types of

neurons. Therefore, it is possible that the long-term delivery of

anti-VEGF (receptor) therapies will also interfere with these activ-

ities, thereby inducing or accelerating the onset of other neuro-

logical disorders. In this respect, it is striking that a considerable

fraction of glioblastoma patients treated with bevacizumab

develop severe optic neuropathies (Sherman et al., 2009).

Since systemic VEGF inhibition—alone or in combination with

paclitaxel—interferes with the neuroprotective activities of VEGF,

we carefully studied the mechanisms of VEGF-mediated neuropro-

tection in sensory neurons. We found that Hdac6 regulates

Flk1-driven upregulation of the anti-apoptotic BCL2 protein, re-

sulting in protection against paclitaxel-induced toxicity. HDAC6

can regulate deacetylation of various non-histone proteins, includ-

ing Hsp90 (Kovacs et al., 2005), cortactin (Zhang et al., 2007) and

b-catenin (Li et al., 2008). Although we could not provide any

formal proof, HDAC6 could participate in the proposed pathway

by deacetylating Hsp90. VEGF has been shown to stimulate the

survival of various cell types through a Bcl2 dependent mechanism

(Nor et al., 1999; Pidgeon et al., 2001; Hwang et al., 2009). Our

study is the first, however, to report that VEGF regulates Bcl2

expression through an Hdac6- and Hsp90-dependent mechanism.

Interestingly, Bcl2 has also been shown to protect against

paclitaxel-induced toxicity by directly interacting with microtubules

and preventing polymerization (Nuydens et al., 2000). In addition

to this, paclitaxel can also functionally mimic the endogenous

Bcl2 ligand (Nur77) and directly bind to Bcl2 (Ferlini et al.,

2009). A potential secondary mechanism of VEGF-induced neuro-

protection against paclitaxel could thus rely on enhanced neutral-

ization of paclitaxel through upregulation of Bcl2. Since we found

that VEGF receptor inhibitors also affect BCL2 expression, pacli-

taxel and VEGF receptor inhibitors could both exert their neuro-

toxic activities by affecting BCL2 expression. In the case of

paclitaxel, which exerts a much broader neurotoxicity profile



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Figure 5 Hsp90 and Bcl2 mediate VEGF/Flk1-induced neuroprotection. (A) Western blot for BCL2 and HSP90 on whole dorsal root

ganglia from Thy:Flk1WT mice and wild-type (WT) littermates. Thy:Flk1WT mice have 47.37 � 16.02% more BCL2 and 22.42 � 4.56%

more HSP90 in their dorsal root ganglia compared with wild-type mice (n = 3–4 mice per genotype). *P50.05 versus wild-type mice. (B–

D) Western blot on dorsal root ganglion cultures for BCL2 and HSP90 (B) reveals that VEGF causes a 38.22 � 11.68% significant increase

in BCL2 but not when combined with tubacin (P = 0.22 versus untreated cultures; n = 3). VEGF also increases the expression of HSP90

with 33.49 � 12.78%, which is not affected by tubacin (P = 0.91 versus VEGF-treated cultures; n = 6). Representative blots for BCL2 (C)

and HSP90 (D) experiments are shown. *P50.05. (E) VEGF prevents paclitaxel-induced cell death, but not when VEGF is combined with


(continued)


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than VEGF receptor inhibitors, it should be noted, however, that

BCL2 represents only one of the many molecules or pathways that

may be affected.

Interestingly, the role of HDAC6-driven deacetylase activity in

the context of neurodegenerative diseases is incompletely under-

stood. HDAC6 deficiency may lead to autophagosome maturation

failure and protein aggregate build-up, thereby promoting a neu-

rodegenerative disease process (Kawaguchi et al., 2003). Depletion

of HDAC6 enhances loss of dopaminergic neurons, retinal degen-

eration and locomotor dysfunction caused by ectopic expression of

alpha-synuclein (Du et al., 2010). HDAC6 activity also promotes

outgrowth and regeneration of neurons (Tapia et al., 2010). On

the other hand, HDAC6 activity may also trigger a number of

detrimental effects in neurons, the precise reasons for these oppos-

ite effects still require further elucidation (Dompierre et al., 2007;

Parmigiani et al., 2008). Our current findings reveal for the first

time that HDAC6 activity is required in the context of neuropro-

tection. In particular, our data provide unprecedented evidence

that HDAC6 regulates VEGF-mediated neuroprotection against

paclitaxel. The extent to which this proposed mechanism is also

relevant for other neurotrophic growth factors or other causes of

neurotoxicity remains outstanding and a most intriguing question.

FundingInstitute for the Promotion of Innovation by Science and Technology

(IWT) in Flanders (to J.D. and K.P.); Geneeskundige stichting

Koningin Elisabeth (to P.C.); ‘Long term structural Methusalem fund-

ing’ by the Flemish Government (to P.C.); Fonds Wetenschappelijk

Onderzoek in Flanders (G.0210.07 to P.C.) and governmental

Institute for Science and Technology (IWT) for the promotion of re-

search between universities (‘Research & Development’ grant to

D.L., partial) and industry (‘Research & Development’ grant to

T.M. and R.N., partial), Stichting Tegen Kanker (to D.L).

Supplementary materialSupplementary material is available at Brain online.

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Systemic anti-vascular endothelial growth factor therapies induce a painful sensory neuropathy