the vascular-disrupting agent combretastatin impairs splitting and sprouting forms of physiological...

The Vascular-Disrupting Agent Combretastatin ImpairsSplitting and Sprouting Forms of PhysiologicalAngiogenesis

ARIF HUSSAIN,* MANUEL STEIMLE,� HANS HOPPELER,� OLIVER BAUM,� AND STUART EGGINTON**Department of Physiology, University of Birmingham Medical School, Birmingham, UK; �Institut fur Anatomie, Universitat Bern, Bern,

Switzerland

Address for correspondence: Dr. Stuart Egginton, Angiogenesis Research Group, Department of Physiology, University of Birmingham, Birmingham

B15 2TT, UK. E-mail: [email protected]

Received 11 May 2011; accepted 4 January 2012.

ABSTRACT

Objective: Vascular-disrupting agents like combretastatin (CA-4-P),

used to attenuate tumor blood flow in vivo, exert anti-mitotic and

anti-migratory effects on endothelial cells in vitro. We tested

whether anti-vascular or anti-angiogenic effects of CA-4-P are

evident with physiological angiogenesis in skeletal muscle (EDL)

due to sustained hyperemia (intraluminal splitting) and chronic

muscle overload (abluminal sprouting).

Methods: CA-4-P was given i.v. (25 mg ⁄ kg on alternate days for

14 days) to mice subjected to angiogenic stimuli (prazosin or

synergist extirpation). The responses of femoral artery blood flow

as well as capillarity, capillary ultrastructure, and levels of Rho

GTPase were measured.

Results: Blood flow was unaffected in the sprouting angiotype,

but decreased in the splitting angiotype, by CA-4-P. In contrast,

CA-4-P attenuated the capillarity increase in both models,

associated with reduced lamellipodia and filopodia formation.

Muscle overload, but not hyperemia, was accompanied by an

increase in Rho GTPase with CA-4-P.

Conclusions: CA-4-P impaired the angiogenic response in both

experimental models. This inhibitory effect was associated with a

lower increase in femoral blood flow in splitting, whereas

sprouting angiogenesis was accompanied by higher Rho activity

consistent with the interruption of actin polymerization. Thus,

CA-4-P may exert context-dependent anti-vascular and anti-

angiogenic effects in vivo under physiological conditions.

Key words: blood flow, capillaries, electron microscopy, stereology

Abbreviations used: ANOVA, analysis of variance; C:F, capillary

to fiber ratio; CA-4-P, combretastatin; EC, endothelial cell; EDL,

extensor digitorum longus; FBF, femoral blood flow; GTPase,

guanosine triphosphate hydrolase; l-NNA, NG-nitro-l-arginine;

NO, nitric oxide; NOS, nitric oxide synthase; PLSD, protected

least square difference; TA, tibialis anterior; VDAs, vascular-

disrupting agents; VEGF, vascular endothelial growth factor;

VSMC, vascular smooth muscle cell.

Please cite this paper as: Hussain A, Steimle M, Hoppeler H, Baum O, Egginton S. The vascular-disrupting agent combretastatin impairs splitting and sprout-

ing forms of physiological angiogenesis. Microcirculation 19: 296–305, 2012.

INTRODUCTION

In order to support a high metabolic rate by adequate per-

fusion, solid tumors generate pro-angiogenic growth fac-

tors. Angiogenesis is therefore an essential component

fueling cancer development. Consequently, disrupting the

tumor vasculature (e.g., with anti-vascular endothelial

growth factor [VEGF] antibodies) is a growing component

of clinical therapy [11,20]. To date, however, the transla-

tion of anti-angiogenesis therapies developed in animal

models has proved disappointing in the clinic. This has

wider implications, both for the treatment of other diseases

such as diabetic retinopathy, and revealing inadequate

knowledge about the normal physiological control mecha-

nisms.

An alternative anti-cancer strategy using VDAs was

identified when colchicine and tubulin-binding agents were

shown to damage tumor vasculature [6]. Two distinct

groups of VDA are in clinical development: (i) those pri-

marily affecting tubulin, and (ii) synthetic flavonoids [23].

The combretastatins bind to microtubules and interfere

with the cyclic formation of tubulin dimers. They are

chemically related to colchicine, and were first isolated

from the African willow tree Combretum caffrum [27].

DOI:10.1111/j.1549-8719.2012.00160.x

Original Article

296 ª 2012 John Wiley & Sons Ltd

The Official Journal of the Microcirculatory Society, Inc. and the British Microcirculation Society

Combretastatin A-1 (CA-1) and A-4 (CA-4) are the most

active forms, the latter being more potent in vitro. The

derivative disodium combretastatin A-4 3¢-O-phosphate

(CA-4-P) overcame limited aqueous solubility, and this

prodrug is cleaved to the active CA-4 by endogenous non-

specific phosphatases [28]. In contrast to colchicine and

other tubulin-binding agents, the anti-vascular effects of

CA-4-P are apparent at well below its maximum tolerated

doses in animal models, offering a wide therapeutic win-

dow [5]. CA-4-P is currently in phase 2 ⁄ 3 clinical trials, in

combination with conventional chemotherapy and radio-

therapy.

CA-4-P destabilization of the mitotic spindle impairs

cell division [15], hence CA-4-P shows a dose- and time-

dependent cytotoxic and anti-proliferative effect on both

tumor cells and endothelial cells (ECs) in culture [2,5].

After exposure, EC undergo contractions, an indication of

rapid alterations of the actin cytoskeleton [17], and lose

their characteristic cobblestone morphology [14]. Disrup-

tion of the junctional adhesion molecule vascular endothe-

lial-cadherin (VE-cadherin) in vitro and in vivo in mice,

and an increase in EC monolayer permeability to

macromolecules have also been observed after CA-4-P

administration [21]. In addition, a rapid and direct vaso-

constrictive effect on tumor-supplying arterioles has also

been reported [33]. This leads to a rapid and significant

reduction in tumor blood flow [23], whereas the extent of

tumor damage is increased due to the self-trapping of

CA-4-P that occurs as the blood flow is decreased [34].

The rapid collapse of tumor blood flow in vivo in

response to CA-4-P treatment is accompanied by massive

necrosis of all but a narrow rim of tumor tissue by

24 hours [5].

It remains unclear why vessels around the tumor

periphery are spared, as this leads to a rapid regrowth of

the tumors after VDA treatment ends [3]. Two possibili-

ties exist: the periphery is drained by nontumor capillar-

ies, or survives and expands due to another VDA-

resistant angiogenesis mode. Indeed, if VDA treatment is

truly anti-angiogenic, then these ‘‘normal’’ vessels should

also be affected, suggesting a differential sensitivity

between physiological and pathological angiogenesis.

Whereas in pathological angiogenesis the vascular growth

process is usually chemically driven, adaptive (physiologi-

cal) angiogenesis is largely controlled by the local

mechanical environment. This gives rise to at least two

forms of capillary growth, shear-stress dependent split-

ting and stretch-dependent sprouting, involving both

common and specific signaling pathways [7,10]. We

therefore examined the effect of combretastatin on these

two well-characterized, distinct forms of physiological

angiogenesis to test their differential sensitivity to vascu-

lar disruption.

METHODS

AnimalsMale C57 ⁄ BL10 mice weighing 25 ± 3 g (Charles River,

Margate, UK) were used for all procedures, and were

housed at 21�C with a 12:12 light ⁄ dark cycle, with access

to food and water ad libitum. All work were carried out in

accordance with the UK Animals (Scientific Procedures)

Act 1986.

Chronic HyperemiaMice were given prazosin (Tocris, Bristol, UK; 50 mg ⁄ L)

dissolved in drinking water for a period of 14 days. Each

mouse received approximately 175 lg ⁄ day, based on the

average water consumption, which was monitored through-

out the experiment [36].

Muscle OverloadUnilateral extirpation of the m. tibialis anterior was per-

formed as previously described (34), resulting in hyperplasia

and hypertrophy of the synergist m. extensor digitorum lon-

gus (EDL). Briefly, mice were anesthetized with 10 mL ⁄ kg

hypnorm ⁄ hypnovel (NVS, Stoke-on-Trent, UK) anesthetic,

supplemented with inhalation anesthetic (0–2% halothane;

ICI, Macclesfield, UK) as necessary. Topical antibiotics

(Duplocillin LA; NVS, Stoke-on-Trent, UK) and systemic

analgesia (2.5 mL ⁄ kg bupenorphine, s.c.; NVS, Stoke-

on-Trent, UK) were administered peri-operatively. Mice

were left to recover for a period of 14 days with normal

food and water. As control, experimental data of the over-

load group (blood flow, capillarity, ultrastructural, gene

expression) were related to those of the contralateral TA.

Drug TreatmentAbout 25 mg ⁄ kg disodium combretastatin A-4 3¢-O-phos-

phate (CA-4-P) has been documented as the lowest effec-

tive dose in mouse models [30], with concentrations of up

to 100 mg ⁄ kg used in other studies, and 50 mg ⁄ kg was

adopted as a compromise for use in this study for 14 days.

Furthermore, periodic doses of CA-4-P have been docu-

mented to be more effective than a single large bolus dose,

so an i.v. injection via the tail vein was given every other

day for duration of the experiment.

Blood FlowBlood flow to the hindlimb was recorded in mice anesthe-

tized with ketamine (0.1 mg ⁄ kg; Pharmacia, Milton Keynes,

UK) and xylazine (0.01 mg ⁄ kg; Millpledge Pharmaceuticals,

Clarborough, UK). A perivascular flow probe (0.7 V with

T106 meter; Transonic Systems, Ithaca, NY, USA) was placed

on the upper portion of the femoral artery. Core temperature

was controlled with a heating plate and monitored with a

thermistor (Fluke S2 KLS; ISS, Knutsford, UK).

Combretastatin and Physiological Angiogenesis

ª 2012 John Wiley & Sons Ltd 297

HistologyThe mice were euthanized; the EDL carefully dissected and

frozen in liquid nitrogen-cooled isopentane. Ten-microme-

ter transverse cryostat sections were stained with FITC-con-

jugated Griffonia simplicifolia lectin-1 (Vector Labs,

Peterborough, UK; 20 lg ⁄ mL in PBS for 30 minutes at RT

out of direct light) to label capillaries. Digital images were

taken (·200, Zeiss Axioplan with MRc camera Zeiss, Wel-

wyn Garden City, UK), and capillaries and fibers in an

unbiased counting frame were identified to calculate a cap-

illary to fiber ratio (C:F), used as an indication of angio-

genesis when compared with controls.

Rho GTPase AssayThe levels of Rho GTPase in skeletal muscle were deter-

mined by a Rho Activation Assay Kit (Upstate Cell Signal-

ling Solutions, Millipore, Watford, UK) according to the

manufacturer’s instructions. The EDL was powdered under

liquid nitrogen and extracted in lysis buffer. After centrifu-

gation (five minutes, 16 g), the supernatants were normal-

ized for protein concentration using a colorimetric DC

protein assay (Bio-Rad, Hemel Hempstead, UK). Equal

amounts of protein from the supernatants (200 lg) were

incubated with anti-active RhoA mouse monoclonal anti-

body. The bound active RhoA was then pulled down by

protein A ⁄ G agarose. The precipitated active RhoA was

then detected by immunoblot analysis using anti-RhoA

rabbit polyclonal antibody. In addition, other aliquots of

the supernatants (50 lg protein) were subjected also to

immunoblotting in order to demonstrate total Rho expres-

sion as loading control. Enhanced chemiluminescence

(Amersham, Little Chalfont, UK) and horseradish peroxi-

dase-conjugated secondary antibody (1:500; Invitrogen

Ltd., Paisley, UK) were used for the development of the

immunoblots. Blots were scanned and analyzed by densi-

tometry in ScionImage (Scion Corp., Frederick, MD, USA).

The integrated density of each band was calculated from at

least three independent experiments.

Electron MicroscopyFrom each of the eight experimental groups (n = 4–5 mice

each), the EDL was fixed by in situ superfusion with 2.5%

glutaraldehyde (Agar Scientific, Stansread, UK) in 0.1 M

phosphate buffer (pH 7.4) to maintain muscle dimensions

and minimize tissue shrinkage. Excised tissue was

immersed in fresh fixative for 30 minutes and trimmed to

expose the medial portion of muscle and returned to fresh

fixative overnight. The tissue was postfixed in buffered

osmium tetroxide, dehydrated in an ethanol series, and

cleared in propylene oxide, then vacuum-embedded in

Mollenhauer resin (Agar Scientific, Stanstead, UK). Embed-

ded muscle blocks were selected at random from each

animal, trimmed, and sectioned at 90 nm and collected

upon formvar-coated copper slotted grids. Sections were

stained with 30% uranyl acetate in methanol followed by

Reynolds lead citrate, using standard protocols for viewing

under a transmission electron microscope (Jeol 100 CX II;

Jeol GmbH, Echling, Germany).

Stereological AnalysesFrom each experimental group, electron micrographs of

25 capillaries per mouse were taken in a systematic ran-

dom manner at an initial magnification of ·15,000 and

subjected to stereological analyses [36]. A number of

parameters sensitive to the level of angiogenic activity [9]

were evaluated by nominal scoring, including the number

of cell boundaries identified by the presence of a zone of

tight junctions (and hence ECs) per capillary, the inci-

dence of pericapillary pericytes, and fibroblasts adjacent

to capillaries. We counted those cells within the intersti-

tium as fibroblasts that were solitary, showing elongated

cytoplasm with organelle content as usually described,

and were pericapillary located without contact to the vas-

cular basement membrane. In addition, the frequency of

intraluminal projections (lamellipodia), abluminal sprouts

(filopodia), and cytoplasmic vacuoles within EC were

quantified (for further definitions and illustrations, see

[8,10,37,38]). Subsequently, a counting grid (d = 285

points) was laid over each micrograph to determine the

volume density of EC cytoplasm and nucleus, capillary

lumen, and perivascular pericytes; respective surface den-

sities were calculated by intersections with horizontal grid

lines (l = 0.378 lm), as well as estimating the extent of

pericyte coverage [9].

Statistical AnalysisAll data are presented as mean ± SEM. Statistical signifi-

cance between groups was performed using factorial

ANOVA with post hoc comparisons among groups using

PLSD, and a 5% significance level.

RESULTS

Blood flowCA-4-P administration to control mice did not result in

any significant changes in femoral blood flow (FBF), nor

was any difference evident in the contralateral leg of the

extirpation group (Figure 1). Prazosin administration

resulted in a 43% increase in FBF from untreated mice

(0.59 ± 0.04 vs. 0.36 ± 0.02 mL ⁄ min), but when CA-4-P

was administered, the FBF was 47% lower than with prazo-

sin alone (0.46 ± 0.02 mL ⁄ min). No significant effects were

seen on FBF with muscle overload (0.39 ± 0.02 vs.

0.44 ± 0.02 mL ⁄ min without and with CA-4-P, respec-

tively; Figure 1).

A. Hussain et al.


CapillarityAs shown in Figure 2, angiogenesis in EDL was induced by

both prazosin and overload, 35% (1.62 ± 0.04 vs.

1.20 ± 0.01 in untreated mice, p £ 0.05) and 11%

(1.55 ± 0.01 vs. 1.40 ± 0.02 compared with the contralat-

eral leg, p £ 0.05) increase in capillary to fiber ratio (C:F),

respectively. C:F was significantly raised with CA-4-

P + prazosin treatment (1.41 ± 0.07, p < 0.01), although

this was only 38% of the levels seen with prazosin treat-

ment alone (1.62 ± 0.04). CA-4-P administration in the

extirpation model resulted in a C:F only 29% of that with

extirpation alone (1.35 ± 0.07), which was not significant

from CA-4-P contralateral controls (1.42 ± 0.12).

CA-4-P treatment alone produced no significant effect

on C:F of control mice (1.25 ± 0.03) compared with

untreated animals, nor did it produce any significant differ-

ence in the contralateral leg of the extirpation group

(1.42 ± 0.05), demonstrating that CA-4-P has little or no

effect on an intact vasculature (Figure 2).

Vascular Fine StructureCapillaries from untreated animals had the expected phe-

notype, and CA-4-P had little effect on the appearance of

capillaries from control animals. Drug treatment was asso-

ciated with increased endothelial translucency and thicken-

ing with both prazosin and extirpation, but greater EC

disruption was evident in the latter group (Figure 3). All

the major variables implicated in the angiogenesis response

were similar to previous values for muscle from control,

prazosin, and extirpation groups of mice [36]. Capillary

cross-sectional area was consistent across all groups at

�15 lm2, with no effect of CA-4-P, but the drug reduced

the number of ECs per capillary profile in all groups

(p < 0.05; Figure 4). Although there was no effect of CA-4-

P on lumen volume density (Figure 4), nor other indices of

lumen surface area (Figure S1), there was a significant

reduction in the extent of intraluminal projections (previ-

ously defined as lamellipodia [38]) following angiogenic

stimulation, and with prazosin with a reduction in pericyte

coverage and abluminal projections (or filopodia [37])

(Figure 4). There appeared to be little effect of CA-4-P on

other variables (Figure S1), and little evidence could be

seen for EC blebbing in micrographs from any CA-4-P

treatment group (Figure 3).

Given the presumed action of CA-4-P on the macrocir-

culation, it was important to obtain an impression of the

terminal arterioles to compliment the main focus of the

study, the capillaries. Unfortunately, arteriolar density was

too low to attempt statistically valid quantification, with

<20 distinct profiles per group, although smaller profiles

with thickened walls ⁄ narrower lumen were evident follow-

ing drug treatment (Figure 5). We believe this is the first

observation of such a response in nonpathological, skeletal

muscle samples that suggests similar responses to those

observed in pathological, tumor samples.

Rho GTPaseA 55% increase in Rho GTPase levels was seen in EDL

taken from CA-4-P controls compared with untreated mice

(p < 0.05). A 16% decrease (n.s.) in Rho GTPase activity

occurred with prazosin treatment, and a 39% decrease

(n.s.) following synergist extirpation compared with control

mice, but neither of these results achieved statistical signifi-

cance due to high data variance (Figure 6). Although there

was a 48% increase in the CA-4-P-treated extirpation

group compared with extirpation alone (p < 0.05), this was

ControlCombrestatin

1.6

1.8

*

-

-*

* ***

**

1.2

1.4

C:F

ratio

-

-

0.0

-

-

-

-

-

Figure 2. Capillary to fiber ratios (C:F) in EDL are shown for CA-4-P-

treated mice (filled columns) with control mice and prazosin and

extirpation groups (open columns).

Control

Combrestatin0.6

0.8

*****

0.2

0.4

Blo

od fl

ow (m

L/m

in)

0.0

Figure 1. Femoral artery blood flow to the hindlimb is shown for

combretastatin (CA-4-P)-treated mice (filled columns) compared with

control, prazosin, and extirpation groups (open columns). *p < 0.05,

**p < 0.01 between groups indicated.



still 46% lower than CA-4-P controls (p < 0.05). No signif-

icant changes were seen with combined prazosin and CA-

4-P treatment compared with prazosin treatment alone,

but a decrease of 91% (p < 0.05) was seen compared with

CA-4-P controls (Figure 6).

DISCUSSION

Although anti-angiogenic approaches aim to prevent

growth of the neovasculature, anti-vascular approaches are

designed to cause a rapid and selective shutdown of the

existing tumor vascular bed, leading to secondary tumor

cell death due to the resultant ischemia [21]. A leading

VDA is the tubulin depolymerizing agent combretastatin-

A4 3¢-O-phosphate (CA-4-P). This drug binds to microtu-

bules close to the colchicine binding site and interferes with

the cyclic formation of tubulin dimers. EC microtubules

and the actin cytoskeleton are essential for migration,

structural support, and intracellular vesicular transport.

Due to their structural and regulatory interactions, the dis-

ruption of microtubule assembly may modulate the actin

cytoskeleton function, including the control of physiologi-

cal angiogenesis through coordinated lamellipodial or filop-

odial formation seen in capillary splitting and sprouting,

respectively [10]. As a consequence, VDAs exhibit both

anti-angiogenic and anti-vascular effects in tumors [5], but

it is not known whether they may exhibit differential effects

on physiological angiogenesis.

Blood FlowA dose- and time-dependent decrease in blood flow to ani-

mal tumors was seen in response to CA-4-P administration

[3], reducing the perfused vascular volume of a murine

P22 tumor to <10% by six hours after the treatment, with

no recovery over 24 hours [34]. Hypertension in rats one

hour after CA-4-P treatment was mainly due to an increase

in flow resistance, with the perfusion pressure remaining

unchanged or increasing in normal tissues [34], whereas

the blood flow reduction in tumors was larger and gener-

ally longer lasting than for any other tissue studied [18]. In

contrast, CA-4-P administration without angiogenic stimu-

lus did not change FBF in the current study, suggesting

A B

C D

E F

Figure 3. Representative electron micrographs of capillaries from control (A), prazosin (C), and extirpation (E) groups treated with CA-4-P (B, D, F,

respectively). Scale bar = 1 lm. Annotations: N, EC nucleus; Lu, capillary lumen; P, perivascular pericyte; L, intraluminal projections (lamellipodia).

A. Hussain et al.


that the skeletal muscle vasculature may be particularly

resistant to microtubule disruption unless subjected to

physiological or pathological perturbation.

Anti-vascular Effects of CA-4-P is Dominant inSplitting AngiogenesisChronic peripheral dilatation by prazosin and muscle over-

load by extirpation are associated with sustained hyperemia

and little or no change in blood flow, respectively [10].

Previous studies have shown that splitting angiogenesis

occurs due to an increased luminal shear stress in capillaries,

whereas sprouting angiogenesis occurs in situations where

the influence on the microcirculation is abluminal, with no

evidence for a flow-mediated stimulus [10]. CA-4-P in com-

bination with prazosin caused an inhibition of flow by

almost half, but had no significant effect on FBF following

overload. Due to low rates of EC proliferation in response

to chronic hyperemia [37,38], inhibition of the capillary

splitting angiotype with CA-4-P treatment may be domi-

nantly due to a blunting of the usual prazosin-induced

increase in blood flow to skeletal muscle. In this case, the

partial inhibition of angiogenesis seems to be largely due to

the anti-vascular effects of CA-4-P, reducing the shear

stress-dependent angiogenesis.

The nitric oxide (NO) component of vasodilatation is

proportional to any increase in blood flow [13], and NO

production by tumors offers some protection from the

vascular damaging effect of CA-4-P [34], possibly through

an inhibitory effect on neutrophil activity [26]. Systemic

NOS inhibition led to enhanced vascular damage in

tumors while having little effect on normal tissue perfu-

sion [34], and co-administration of a NOS inhibitor (L-

NNA) led to enhanced MPO activity in murine tumors

following CA-4-P treatment [26]. Other pathways may

include the interaction of NO with tubulin, and a possi-

ble role for NO in inhibiting CA-4-P-induced contraction

of the actin cytoskeleton [26]. This would be consistent

with a flow-induced vasodilatation that occurs with praz-

osin administration leading to upstream release of NO

[1,4], thus attenuating the effects of CA-4-P in the split-

30 3

20 -

-

*

* **

2 -

-

**

*

0 -

10 -

Cap

illa

ry a

rea

(µm

2)

0 -

1 -

End

oth

elia

l cel

ls p

er c

apil

lary

0.6 -

0.8 -

***

**

0.20 -

0.25 -

**

0.2 -

0.4 -

Vv

(lu

men

) (%

)

0.05 -

0.10 -

0.15 -

Per

icyt

cov

erag

e (%

)

0.0 - 0.00 -

4 -

**

**

***

0.3 -

2 -

3 -

**

**

**

*

0.1 -

0.2 -

**

0

Control

Prazosin

Extirpatio

n

Extirpatio

n

contrala

tera

l -

1 -

Lam

elli

pod

ia p

er c

apil

lary

**

0.0 -

Filo

pod

ia p

er c

apil

lary

****

**

**

*

*

**

*

Control

Prazosin

Extirpatio

n

Extirpatio

n

contrala

tera

l

A B

C

E F

D

Figure 4. Stereological analysis of capillary fine structure. (A) Capillary cross-sectional area, lm2; (B) number of ECs per capillary profile; (C) lumen

volume density; (D) relative pericyte coverage; (E) occurrence of intraluminal lamellipodia; (F) occurrence of abluminal filopodia. *p < 0.05,

**p < 0.01 between groups indicated (either untreated controls or contralateral muscles, as appropriate).



ting, compared with the sprouting form of capillary

growth.

Anti-angiogenic Effects of CA-4-P is Dominant inSprouting AngiogenesisWe observed a significant reduction in C:F not only of

the splitting form but also of the sprouting form of phys-

iological angiogenesis in response to CA-4-P. With over-

load, any slight increase in flow (compared with that of

controls) is likely a product of angiogenesis rather than a

cause, such that an attenuated increase in capillarity seems

to be largely due to the anti-angiogenic effects of CA-4-P,

rather than any anti-vascular effects. Whether this differ-

ential effect is due to physical differences, such as involve-

ment of microtubules and number of proliferating EC in

the different angiotypes or response to angiogenic stimu-

lus, remains unclear. The extent of EC proliferation varies

markedly between these angiotypes, being relatively high

with sprouting and unusually low in splitting forms of

angiogenesis [8], suggesting that the former would be

more sensitive to the anti-mitotic action of CA-4-P.

Destabilization of the tubulin cytoskeleton and mitotic

spindle disrupts cell division, and a number of studies

have demonstrated a dose- and time-dependent anti-pro-

liferative and cytotoxic effect of CA-4-P on EC in culture

[21,32]. Indeed, it appears to preferentially damage newly

divided EC, which may explain its supposed selectivity for

tumor vasculature. However, the mechanism responsible

for the selective vascular damage remains unknown [32],

although the examination of vascular fine structure may

offer a clue.

Vascular Fine StructureEC migration requires complex and dynamic interactions

between the cell and its extracellular environment, involv-

ing spatial and temporal coordination of physical and

chemical signals. Microtubules and F-actin are both

required for directed cell motility, acting by different mech-

anisms [29]. CA-4-P inhibits migration and tube formation

of proliferating EC, predominantly through disruption of

the VE-cadherin ⁄ b-catenin ⁄ Akt signaling pathway, thereby

leading to rapid vascular collapse [32,35]. Mitotic-spindle

disruption in EC is observed in vitro following drug expo-

sure at very low concentrations (7.5 nM) [21,32,35]. A dis-

ruption of this structural interaction should affect the

sprouting form of angiogenesis more than the splitting

form, as a much greater degree of directional migration is

required to form intercapillary anastomoses. In contrast,

capillary splitting may occur independently of microtu-

bule ⁄ microfilament pathway inhibition, and therefore be

insensitive to CA-4-P effects, as directional migration may

not be required because the lamellipodia have only to cross

the lumen, probably in response to shear-mediated actin

polymerization, cf. [19]. There was a proportionately

A B

C D

Figure 5. Light microscope images of arterioles from semithin sections from muscles of control (A), prazosin (B), extirpation (C), and prazosin + CA-

4-P (D) mice. Note the disrupted endothelium on combretastatin treatment (arrow).

A. Hussain et al.


greater luminal than abluminal response in both groups,

presumably reflecting the different cytoskeletal elements

involved.

Newly formed EC may be more sensitive than mature

cells as the latter have a more highly developed actin cyto-

skeleton, which maintains the cell shape despite depolymer-

ization of the tubulin cytoskeleton [32], perhaps explaining

the minimal effect of CA-4-P on quiescent EC in vitro.

Signs of EC disruption may be reduced as the rate of capil-

lary growth maximizes at approximately seven days,

whereas the maximal capillarity is found at the 14-day time

point chosen for this study [36]. In addition, CA-4-P is a

reversible inhibitor of tubulin polymerization with a rela-

tively short plasma half-life [34], which would limit its

anti-proliferative effects and enable other effects of destabi-

lizing of tubulin to become evident. Microtubules also help

resist the shortening of vascular smooth muscle cells

(VSMC), and thus their depolymerization will make vessels

more sensitive to vasoconstriction, although the presence

of smooth muscle cells inhibits CA-4-P-mediated disrup-

tion [35]. The effect of CA-4-P on VSMC and EC provide

a route for its differential effect on the response to hyper-

emia (anti-vascular) and overload (anti-angiogenic), respec-

tively. Other evidence for the role of actin filament

disruption may be obtained from changes in regulatory

pathways.

Rho GTPaseThe effects of CA-4-P do not seem to act solely by disrup-

tion of the structural interaction of microtubules and

F-actin, as actin reorganization induced by CA-4-P in vitro

are inhibited by co-administration of Rho ⁄ Rho kinase

inhibitors, indicating an upregulation of this pathway on

CA-4-P administration [21,32]. Rho cycles between its GDP

bound form to its active GTP form and is a key contributor

along with Rac and Cdc42 in actin dynamics. As RhoA acti-

vates myosin through a Rho-kinase dependent phosphoryla-

tion of myosin light chain, this raises the possibility that

Rho GTPases regulates both microtubule- as well as actin-

based proteins. The activity of Rho GTPase therefore pro-

vides insight into the regulatory interactions governing cell

migration during the course of physiological angiogenesis,

in this case an increase leading to impaired sprout forma-

tion. A further effect of CA-4-P in vitro is the accumulation

of F-actin via polymerization generated by Rho ⁄ Rho-kinase

activity into surface blebs of EC, where stress fibers misas-

semble into a spherical network surrounding the cytoplasm

[21]. However, surface blebbing rarely occurs in vivo, sug-

gesting that either this is an artifact of cell culture, or the in

situ environment opposes this tendency.

Rho is responsible for stress fiber formation, Rac for lam-

ellipodial formation, and Cdc42 for orientation of microtu-

bules and actin cytoskeleton [12]. Rac and Cdc42 have been

shown to be upregulated by shear stress, and this provides a

possible differential regulation of the cytoskeletal systems

between the two physiological phenotypes, via integrin-med-

iated development of filopodia and the progressive forma-

tion of focal adhesions [31]. VEGF is reported to cause

increased Rho and Rac activity [24,25], whereas CA-4-P

raises RhoGTPase expression, presumably by feedback con-

trol, which is decreased a little by overload and abolished by

hyperemia. Interestingly, C:F ratio did not increase for the

control group treated with CA-4-P, so increased RhoGTPase

levels do not show a simple correlation with angiogenesis.

Although mechanical strain can increase the activity of Rho

GTPases in muscle cells [22], suggesting that the effects seen

may not be uniquely endothelial, angiogenesis likely pro-

vides an antagonistic response to elevated RhoGTPase levels

as a result of microtubule disruption. Thus, CA-4-P may

exert an inhibitory influence on sprouting angiogenesis in

part through attenuating EC capacity for migration. A simi-

lar disruption was shown for MMP inhibition, but in this

case there were no additional anti-mitotic effects [16].

CONCLUSIONS

CA-4-P leads to a partial angiogenic inhibition in the capil-

lary splitting phenotype that is proportional to its inhibi-

tory effects on hindlimb blood flow, with little effect on

Rho activity levels, suggesting that in this case the drug

150

200Control

Combrestatin*

-

- *

**

50

100

-

-

0

Control

Prazosin

Extirpation

(% o

f tot

al rh

o)R

ho G

TPas

e co

ncen

trat

ion -

-

-

-

-

ControlControlComb CombCombExtirp

ExtirpPraz

Praz

Rho GTPase

Total Rho

Figure 6. Top panel: Rho GTPase bands indicating levels of Rho

GTPase in control, CA-4-P control, extirpation, prazosin, CA-4-

P + extirpation, and CA-4-P + prazosin samples. Total Rho levels for

each of the various interventions are also shown. Bottom panel: Rho

GTPase levels in comparison with total Rho levels are given for CA-4-P-

treated mice (filled columns), and control and angiogenic models (open

columns). Mean ± SEM (n = 5). *p < 0.05 between groups indicated.



primarily exerts an anti-vascular effect. In the abluminal

sprouting phenotype that requires EC proliferation, CA-4-P

appears to have a largely anti-angiogenic action, associated

with increased Rho activity. The effect of combretastatin in

vivo therefore varies according to the form of angiogenesis

involved, which may prove useful in targeting the vessels in

tumors from normal tissue around the periphery. Indeed,

preclinical and clinical data further indicate that CA-4-P

can effectively be combined with chemotherapy or radio-

therapy [32].

PERSPECTIVE

Inadequate treatment of pathologies is often predicated by

inadequate knowledge of basic physiological process that

have become dysfunctional. Here, we show that a com-

pound developed for use in tumor therapy has value in dis-

criminating between different forms of physiological

angiogenesis, opening up new approaches to identifying

important control points.

ACKNOWLEDGMENTS

This work was funded by a grant from the British Heart

Foundation to SE. We would like to thank Franziska Grab-

er and Adolfo Odriozola (University of Bern) for their

skillful technical support with the TEM, Dr. Zubair Ahmed

(University of Birmingham) for performing the Rho

GTPase assays, and Prof. G. Tozer (University of Sheffield)

for the gift of combretastatin.

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SUPPORTINGINFORMATION

Additional Supporting Information

may be found in the online version of

this article at http://www.microcircu-

lationjournal.com

Figure S1. Stereological analysis of

capillary fine structure.

Please note: Wiley-Blackwell are not

responsible for the content or func-

tionality of any supporting materials

supplied by the authors. Any queries

(other than missing material) should

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