Synergistic Inhibition of Endothelial Cell Proliferation,
Tube Formation, and Sprouting by Cyclosporin A and
Benjamin A. Nacev1,2, Jun O. Liu1,3*
1Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America, 2Medical Scientist
Training Program, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America, 3Department of Oncology, Johns Hopkins University
School of Medicine, Baltimore, Maryland, United States of America
Pathological angiogenesis contributes to a number of diseases including cancer and macular degeneration. Although
angiogenesis inhibitors are available in the clinic, their efficacy against most cancers is modest due in part to the existence
of alternative and compensatory signaling pathways. Given that angiogenesis is dependent on multiple growth factors and
a broad signaling network in vivo, we sought to explore the potential of multidrug cocktails for angiogenesis inhibition. We
have screened 741 clinical drug combinations for the synergistic inhibition of endothelial cell proliferation. We focused
specifically on existing clinical drugs since the re-purposing of clinical drugs allows for a more rapid and cost effective
transition to clinical studies when compared to new drug entities. Our screen identified cyclosporin A (CsA), an
immunosuppressant, and itraconazole, an antifungal drug, as a synergistic pair of inhibitors of endothelial cell proliferation.
In combination, the IC50dose of each drug is reduced by 3 to 9 fold. We also tested the ability of the combination to inhibit
endothelial cell tube formation and sprouting, which are dependent on two essential processes in angiogenesis, endothelial
cell migration and differentiation. We found that CsA and itraconazole synergistically inhibit tube network size and sprout
formation. Lastly, we tested the combination on human foreskin fibroblast viability as well as Jurkat T cell and HeLa cell
proliferation, and found that endothelial cells are selectively targeted. Thus, it is possible to combine existing clinical drugs
to synergistically inhibit in vitro models of angiogenesis. This strategy may be useful in pursuing the next generation of
Citation: Nacev BA, Liu JO (2011) Synergistic Inhibition of Endothelial Cell Proliferation, Tube Formation, and Sprouting by Cyclosporin A and Itraconazole. PLoS
ONE 6(9): e24793. doi:10.1371/journal.pone.0024793
Editor: Marc Tjwa, University of Frankfurt - University Hospital Frankfurt, Germany
Received September 10, 2010; Accepted August 19, 2011; Published September 28, 2011
Copyright: ? 2011 Nacev, Liu. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported in part by NCI [Grant RO1 CA122814], the National Center for Research Resources (NCRR), a component of the National
Institutes of Health (NIH) and NIH Roadmap for Medical Research [Grant UL1 RR 025005], the Flight Attendant Medical Research Institute Fund, the Keck
Foundation, the Walsh Prostate Cancer Fund, and the Commonwealth Foundation (J.O.L.), and by the NIH Medical Scientist Training Program [Grant T32GM07309]
(B.A.N). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Angiogenesis, the process of new blood vessel growth and
development, underlies a number of human diseases including
cancer, macular degeneration, psoriasis, rheumatoid arthritis,
diabetic retinopathy, and pulmonary hypertension . Inhibitors
of angiogenesis such as the anti-VEGF antibody bevacizumab are
used clinically to treat cancer. However, the experience with
existing therapies has been mixed. While they have shown efficacy,
their effects in terms of halting disease progression and improving
survival have been modest and often involve side effects including
hypertension and increased risk of stroke . Hence, although the
promise of angiogenesis inhibitors has been demonstrated, there is
a clear need for more effective anti-angiogenic therapies.
On average, the development of a clinically viable drug requires
an investment of roughly $800 million and takes about 12 years .
One strategy to accelerate drug development is to re-purpose
existing drugs . Because re-purposed drugs have already been
approved for clinical use, their pharmacodynamic and pharmaco-
kinetic properties are well established. In addition, existing drugs
have acceptable levels of toxicity and in many cases they have
known mechanisms, which makes their pharmacology amenable to
detailed molecular study. Thus, by focusing on existing drugs, many
hurdles in drug development are already cleared. The end result is a
discovered to have new applications. We have previously adopted
this approach when we assembled and screened the Johns Hopkins
Drug Library (JHDL) for inhibitors of angiogenesis and other
activities [5–9]. Presently, the JHDL contains ,3,300 drugs
approved by the US Food and Drug Administration or foreign
equivalent. The initial screen for angiogenesis inhibitors identified
221 compounds with .50% inhibition of human umbilical vein
endothelial cell (HUVEC) proliferation at a 10 mM dose.
A number of these hits had IC90doses above the peak plasma
level obtained under clinical dosing regimens or had dose-limiting
toxicities. One way to expand the clinical applicability of these
hits, we reasoned, was to find synergy between them, thereby
reducing the doses needed for those synergistic pairs to inhibit
angiogenesis in vivo. We have thus conducted a screen for synergy
among 741 binary combinations of 39 clinical drugs that were hits
PLoS ONE | www.plosone.org1September 2011 | Volume 6 | Issue 9 | e24793
from the initial screen. In addition to lowering the necessary dose
of otherwise toxic agents, combination therapy is often used to
limit the potential for drug resistance and to achieve synergistic
inhibition of multiple independent pathways that converge on a
single essential molecular process. For these reasons, the
simultaneous use of multiple drugs has been an effective strategy
to overcome diseases intractable to single agent therapies. For
instance, combination therapy is now the standard of care in
treating HIV infection and many neoplasms .
Synergy, or superadditivity, is often observed upon the inhibition
of multiple pathways that converge to promote a single biological
process such as proliferation. Thus, it is hypothetically possible that
synergistic inhibition of angiogenesis may be possible given that
signaling in angiogenesis is complex and involves multiple pathways
including those downstream of growth factors such as vascular
endothelial growth factor (VEGF) and basic fibroblast growth factor
(bFGF) [11,12]. In fact, the limitations of anti-VEGF therapy have
been attributed to the existence of redundant alternative pathways
in the in vivo pro-angiogenic signaling network . Thus, a more
effective strategy to inhibit angiogenesis may be to simultaneously
target multiple pathways. Just as anticancer regimens have evolved
to simultaneously utilize drugs with multiple mechanisms to achieve
synergy, so might anti-angiogenic regimens have to evolve to
provide additional efficacy. Thus, we sought to determine whether
there exist clinical drugs that synergistically inhibit endothelial cell
proliferation and tube formation.
Materials and Methods
Reagents and materials
Pooled HUVEC and EGM-2 bullet kit media were purchased
from Lonza. Jurkat T cells (a human acute T cell leukemia line)
and HeLa cells (a human cervical adenocarcinoma line) were from
the American Type Tissue Collection. Low and high glucose
DMEM, RPMI 1640, fetal bovine serum, and penicillin/
streptomycin were from Gibco. Recombinant human VEGF165
and bFGF146were purchased from R&D systems and reconsti-
tuted in 0.1% BSA in PBS as 100 mg/mL and 10 mg/mL stocks,
respectively. Methyl cellulose (4 cP) was purchased from Sigma
and used to prepare methocel as previously described .
Itraconazole (Ita) (Sigma), cyclosporin A (CsA) (LC labs), and
sunitinib (LC labs) were stored frozen in DMSO and added to cells
from 2006stocks. Calcein AM and Alamar Blue were purchased
from Invitrogen and [3H]-thymidine was from PerkinElmer. Glass
filtermats were obtained from Wallac. Phenol red free Matrigel
and rat tail collagen type I were from BD biosciences.
All cells were grown at 37uC with 5% CO2in a humidified
environment. HUVEC were grown in EGM-2 bullet kit media
and used between passages 2 and 8. Jurkat T cells were grown in
RMPI 1640 (+10% FBS, 1% penicillin/streptomycin), HeLa were
grown in low glucose DMEM (+10% FBS, 1% penicillin/
streptomycin), and HFF in high glucose DMEM (+10% FBS,
2000 HUVEC or HeLa/well or 16104Jurkat T cells/well were
seeded in a 96-well plate (Costar) in 199 mL media. After an
overnight recovery, drugs were added. For CsA+Ita combinations
the molar ratio was always 10:1. Following a 24-h incubation, cells
were pulsed with 0.9 mCi of [3H]-thymidine for 6 h, washed once
with PBS, trypsinized, and transferred to filtermats (Wallac) using
a Mach III M Harvester 96 (Tomtec). For Jurkat T cells, the
PBS wash and trypsinization steps were omitted. After drying,
[3H]-thymidine retention on the filtermats was determined by
scintillation counting using a 1450 Microbeta apparatus (Wallac).
Counts were normalized to that of control cells treated with
vehicle only. GraphPad Prism (v4.03) software was used to
determine IC50values using a four parameter logistic regression.
In the case of growth factor-dependent proliferation assays, the
cells were first seeded as above but in basal EBM-2 basal media
(Lonza) with 2% FBS added (hereafter referred to as ‘basal’
media). After an overnight recovery, the media was replaced with
either basal media, standard EGM-2 media, basal media with
100 ng/mL VEGF165or basal media with VEGF165vehicle alone.
Drugs were then added and the assay was continued as described
above. In processing the data for VEGF-dependent proliferation,
the [3H] counts for basal proliferation at each dose was subtracted
from that of basal+VEGF proliferation prior to normalizing the
data to vehicle control.
Cell Viability Assay
HUVEC and HeLa were plated at 2000 cells/well and HFF at
2500 cells/well in a 96-well plate in 199 mL of media. Cells were
incubated with drug for 30 h and then washed with 200 mL of PBS
and incubated for 30 min at 37uC with 100 mL of 1 mM Calcein
AM diluted from a 1 M stock in DMSO. The excess dye solution
was then aspirated and 100 mL PBS was added prior to reading
with a fluorescence plate reader (Fluostar Optima). The
fluorescence of drug treated samples was normalized to DMSO
controls. Three independent experiments were performed.
An Alamar Blue assay for Jurkat viability was used since it does
not require the media to be removed prior to analysis, which is
advantageous for cells grown in suspension. 16104Jurkat cells were
seeded per well of a 96-well plate in 200 mL of media. Following a
24-h incubation with drug, 20 mL of Alamar blue was added and
cells were incubated for an additional 6 h. The florescence of each
well was measuredat590 nmaftera 544 nmexcitation.Mediaonly
wells were included as blanks. Three independent experiments with
multiple technical replicates were performed.
Tube formation assay
For the synergy analysis, 230 mL of ice cold, phenol red-free
matrigel was added to a 24-well plate using a chilled pipet tip.
Following a 30–40 min incubation at 37uC, 76104HUVEC were
added in 500 mL of media. Cells were treated with drug for 18 h.
The cells were then gently washed with 500 mL PBS and followed
by incubation with 300 mL of 2 mM calcein AM (diluted in PBS
from a 1 M DMSO stock) for 30 min. The calcein AM was
replaced with 500 mL PBS and the tube network was photographed
using fluorescent microscopy. For the comparison of sunitinib and
CsA+Ita, the above experiment was scaled down to a 96-well plate
format. 50 mL of ice cold matrigel was used and 1.56104HUVEC
in 200 mL of media were seeded. Following an 18-h drug treatment,
the cells were washed with 150 mL of PBS and incubated for
30 minutesat 37uCwith100 mLofCalceinAMwhichwas replaced
with100 mL of PBS prior to imaging. In both cases, the imageswere
inverted and equal areas of the central or best focused region of the
image were cropped out using Adobe Photoshop (v 9.0.2). The tube
networks were quantified using Angioquant .
This assay was based on work by Korff and Augustin . We
modified the procedure by first coating the bottom of the tissue
culture surface with a collagen, growth factor, and drug solution to
VEGF165(30 ng/mL) and bFGF (30 ng/mL) or vehicle (0.1% BSA
in PBS) was added to a methocel:media (42:58) solution, followed by
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drug or vehicle addition and mixing. This was then mixed 1:1
(0.5 mL total volume) with a working collagen solution [3.5 mg/mL
collagen (10 equivalents) and ice cold 106 PBS (1 equivalent)
premixed with 1 N NaOH (0.23 equivalents), kept on ice].
Immediately after mixing, the matrix was added to a pre-warmed
24-well plate (37uC) and returned to 37uC. Meanwhile, HUVEC
which were induced to form spheroids of 1000 cells each by
suspension in 25 mL media:methocel (4:1) hanging drops in uncoated
sterile petri dishes for 18 h were washed from the plates in PBS and
pelleted at 1506g, no brake. The spheroids were suspended in serum
free EGM-2 (,55 spheroids/mL) and transferred to Eppendorf tubes
in 1-mL aliquots and pelleted. The supernatant was carefully
aspirated and the tubes were gently scraped to release the pellet.
The media:methocel solution with growth factors was layered over
the pellet (0.25 mL) followed by the addition of 0.25 mL working
collagen stock and then drugs or vehicle. After mixing, the solution
was immediately transferred to the plates containing the base coat of
matrix. Assuming a 100% yield, approximately 55 spheroids were
seeded per well. After a 30-minute incubation, 150 mL of EGM-2
with 2% FBS was added to the surface of each well. After a 24-h
with a 206 phase I contrast objective. Eight spheroids in 4
independent experiments were measured for each condition.
Cumulative sprout length (per spheroid) was quantitated using
Volocity software (v 5.4.1; PerkinElmer). Quantitation was optimized
with a package of Volocity modules for intensity filtering (to highlight
the sprouts), exclusion of objects ,100 pixels2, hole filling, noise
reduction (very coarse filter), skeleton measurement and exclusion of
sprouts fragments smaller than 60.5 mm.
Synergy calculations and Statistical analysis
The combination indices and dose reduction index (DRI) values
were calculated using CompuSyn. In the case of the prolifera-
tion assays, the synergy parameters were skewed towards zero.
Therefore, the data was log transformed to normalize the
distribution prior to performing Student’s t-test. Otherwise,
Student’s t-test was applied to untransformed data. In comparing
the potency of CsA + Ita with the same dose of sunitinib in tube
formation assays conducted in parallel, paired t-tests were used. In
all hypothesis testing for synergy, H0=additive or antagonistic
interactions. For DRI, H0=unchanged or increased dosing
requirements. For other applications, the t-test was 2-sided.
Design of the Synergy Screen
For the synergy screen, we selected a pool of thirty-nine drugs
from among the initial list of 221 hits generated by a prior screen
of the JHDL for HUVEC proliferation inhibition (Table 1) [5,6].
This pool was chosen to exclude topical or generally cytotoxic
drugs, and to represent a large number of different drug classes in
order to increase diversity. This was done to maximize the
coverage of targeted molecular pathways so as to increase the
probability of identifying unpredicted connections between drug
targets. In all, twenty different drug classes were chosen to include
classes as diverse as antiviral, vasodilator, and antihistamine drugs
The pool was combined in 741 unique two-drug combinations
and screened for the ability to synergistically inhibit the
proliferation of HUVEC. Since in vivo angiogenesis requires
endothelial cell proliferation and cell proliferation is amenable to
high-throughput analysis, proliferation is often used as a proxy for
angiogenesis. The screening was conducted in multiple phases
(Figure S1). In the first phase, the potency of each drug
concentration was determined in duplicate for combinations of
Table 1. Collection of clinical drugs screened in binary combinations for synergistic inhibition of HUVEC proliferation.
Albendazole Clomiphene FluvastatinMiconaozoleSimvastatin
Amphotericin BCyclosporin A OH-progesteroneMycophenolic acid Terconazole
AtorvastatinDigoxin Hypericin NimodipineTerfenadine
b-estradiol Ethinyl esterdiolItraconazoleProgesteroneTribromosalan
Bromocriptine FelodipineLovastatinRaloxifene Zidovudine
Table 2. Distribution of Drug Classes.
Bone Metabolism Modulator1
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the drugs at 1-, 0.5-, and 0.25-fold of the rough IC50doses of each
drug alone. Proliferation was measured by the incorporation of
[3H]-thymidine during a 6-h pulse following a 24-h drug
treatment. The presence of synergy was determined using the
Chou-Talalay analysis, which required the side-by-side generation
of the individual drug dose response curves along with the
combination proliferation data . The Chou-Talalay method
uses the median effect equation to determine if the combination of
two drugs produces an effect which exceeds that predicted by the
simple addition of the individual drug effects [17,18]. The key
parameter returned by this analysis is the combination index (CI).
A CI of 1 indicates pure additivity; a CI greater than 1 indicates
antagonism; and a CI between 0 and 1 indicates synergy. The
initial screening phase identified 47 pairs with a CI in the
In Phase 2 of the screen, we attempted to validate the initial 47
hits by performing complete dose-response curves consisting of
seven doses for each combination. This allowed for a more
compete characterization of the synergy since a broader dose
range produces data that is resistant to random fluctuations in the
high throughput format. The Chou-Talalay analysis was again
applied and drug combinations that produced statistically
significant synergy (i.e. p,0.05) across a range of effect levels
(i.e. IC30–IC60) were considered validated hits. Forty-six of the
initial 47 hits did not meet this validity test and were discarded as
Figure 1. Cyclosporin A and itraconazole are synergistic inhibitors of HUVEC proliferation. Chemical structures of cyclosporin A (A) and
itraconazole (B). The CI plot for CsA+Ita indicated CI in the synergistic range across a wide range of effect levels (C). The IC50dose of both Ita (D) and
CsA (E) is significantly reduced in combination. This is also reflected in the dose reduction index (F). Bars, standard error of the mean (SEM). n=8;
* p,0.1; ** p,0.05; # p,0.005; ## p,0.0001.
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Cyclosporin A and Itraconazole Synergistically Inhibit
Endothelial Cell Proliferation
The second phase of screening identified two commonly used
clinical drugs, cyclosporin A (CsA), an immunosuppressant, and
itraconazole (Ita), an antifungal drug, as synergistic inhibitors of
HUVEC proliferation (Fig. 1, A and B). The CI value is
dependent on the effect level. Therefore we calculated the CI
value across a range of effects and performed a test for statistical
significance for each data point (Fig. 1C; Table 3). The CsA + Ita
combination was synergistic over a range from IC30to IC90with
the strongest statistical significance correlating with points in the
Effective drug combinations result in a marked decrease in the
concentration of each drug necessary to produce the desired effect
when compared the doses of each drug to produce the same effect
as a single agent. This dose reduction is amplified in the case of a
synergistic interaction. Based on the HUVEC proliferation IC50
doses for CsA, Ita, and the combination regimen, the combination
of the two drugs resulted in a statistically significant 4-fold and 3-
fold reduction in the CsA and Ita dose, respectively (Fig. 1, D and
E). This resulted in combination IC50doses of 540 nM (CsA) and
54 nM (Ita). We validated these findings using a modification of
the CI equation to yield the Dose Reduction Index (DRI), which
also indicates the fold reduction of a drug dose in combination
. In this analysis, the DRI for CsA was 7.1-fold (p,0.05) and
for Ita was 8.2-fold (p,0.1) (Fig. 1F).
Having shown that the CsA+Ita combination was synergistic for
inhibition of HUVEC proliferation, we compared the combina-
tion to sunitinib, an FDA-approved antiangiogenic drug (Fig. 2)
. Both the combination and sunitinib inhibited HUVEC
proliferation with an IC50in the single-digit micromolar range
(Fig. 2A), which was unexpected because sunitinib is known to
inhibit VEGFR2, the major receptor for VEGF-mediated
angiogenic signaling, with a Kiof 9 nM . However, a recently
study concluded that a number of growth factors, some of which
are present in standard HUVEC media, including FGF2 and
EGF, can rescue the effects of sunitinib on HUVEC proliferation
. Thus, in standard media the discrepancy between the
HUVEC proliferation IC50and the Kifor the VEGFR2 receptor
was likely due to the influence of growth factors other than VEGF.
Presumably, the less potent activity of sunitinib against these
growth factor receptors and other off-target effects dictated the
To address this possibility, we made a further comparison
between the potency of CsA+Ita and sunitinib for inhibiting
HUVEC first grown in basal media (EBM-2+2% FBS) and then
stimulated with 100 ng/mL VEGF165(Fig. 2, B, C and E). As a
control, cells grown in basal media alone or grown first in basal
media and then standard media were also tested (Fig. 2, B and D).
In the absence of exogenous growth factors, the IC509s for CsA +
Ita and sunitinib were remarkably similar and in the single-digit
micromolar range as in the experiments with standard media. This
is consistent with the findings of several other groups in similar
experiments [22,23]. However, when HUVEC were treated in
media supplemented with VEGF, the potency of CsA + Ita was
lower than that of sunitinib (Fig. 2E). Interestingly, the dose-
response curve for sunitinib had two apparent EC50s, one
corresponding to the IC50seen in either standard or basal media
and a second much lower EC50. This mid-curve plateau has also
been observed by other groups . We reasoned that the
observed curve was the superimposition of sunitinib’s effects on
both VEGF-dependent and -independent HUVEC proliferation.
Thus, we subtracted the proliferation in basal media at each dose
of both sunitinib and CsA + Ita from that in VEGF-supplemented
basal media to obtain the dose response curve for VEGF-
dependent growth (Fig. 2F). The sunitinib IC50 for VEGF-
dependent proliferation was 4.6 nM (95% CI, 2.6 nM, 8.0 nM),
which is consistent with previous reports . In comparison,
the IC50for CsA + Ita was roughly 700-fold higher, and was
essentially unchanged from that observed in standard media.
Thus, although less potent, the activity of CsA + Ita was
independent of VEGF signaling.
Cyclosporin A and Itraconazole Synergistically Inhibit
Endothelial Tube Formation and Sprouting
Cell proliferation is a simplified measure of angiogenesis,
representing only one aspect of angiogenesis in vivo. Alternative
measures of drug efficacy against angiogenesis are the tube
formation and sprouting assays, which entail cell migration,
intercellular interactions, and differentiation. Thus, to comple-
ment the original screen, we utilized both these assays in further
assessing the effect of the CsA + Ita combination. In the tube
formation assay, HUVEC are grown on the surface of extracel-
lular matrix on which they form a network of capillary-like
structures. Under normal conditions, these networks are complex
and highly branched, resembling capillary beds in vivo.
To test whether or not CsA and Ita synergized at the level of
tube formation, HUVEC were seeded in a 24-well plate on a layer
of pre-solidified matrigel in the presence or absence of CsA and Ita
alone or in combination. After an 18-h treatment, the cells were
stained to highlight the tube network and photographed (Fig. 3A).
Using semi-automated software, several parameters of the tube
network were quantified . These included total tube size,
which measures both the total length and thickness of the network,
total tube length, which measures the linear length of the tubes,
and lastly the number of junctions in the tube network (Fig. 3B–
3D). The combination led to a decrease in tube network length,
size, and junctions greater than that resulting from the single drugs
The Chou-Talalay method for synergy analysis requires a
complete dose-response curve for the single drugs. However, while
it is relatively simple to generate a dose-response curve in the
context of a proliferation assay, it is difficult to do so in a tube
formation experiment since the distribution of any given drug
between the matrix and the aqueous media is difficult to predict
over the wide concentration range necessary. Thus, to analyze
synergy in the tube formation assay, we utilized Webb’s Fractional
Product model, a modification of the Bliss Independence model
for synergy, which does not require the full dose-response curve
[24–26]. We found that for total network size the combination of
CsA and Ita exhibited significant synergism (p,0.05). For total
Table 3. Combination Indices and p-values versus effect level
for CsA and Ita combination treatment.
IC Level CI Valuep-value
90 0.79 0.104
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tube length, the synergism was more moderate in both degree and
significance (p,0.1). In the case of junction formation, the
combination trended towards synergy but did not reach
significance. Together, these results suggest that the architecture
of the network was not affected, but instead there was synergistic
inhibition of the stability and viability of the tubes. For visual
reference, the non-interaction value (i.e. the effect predicted by a
simple additive interaction per Webb’s Fractional Product model)
Figure 2. The potency of cyclosporin A and itraconazole against HUVEC proliferation is independent of VEGF signaling. (A) The
proliferation of HUVEC grown in standard media in the presence of the indicated doses of CsA + Ita or sunitinib was determined. (B) HUVEC were
seeded in either standard media (standard), or basal media (EBM-2+2% FBS), which was changed the next day to either fresh basal media with
100 ng/mL VEGF (basal + VEGF) or VEGF vehicle (basal), or standard media (basal + standard). The cells were then treated with drug vehicle for 24 h
and the incorporation of [3H]-thymidine after a 6 h pulse was determined. The dose response curves CsA + Ita and sunitinib were determined in basal
media (C), basal + standard media (D), and basal media + VEGF (E). (F) The dose-response curve for VEGF-dependent proliferation (basal media + VEGF
proliferation minus basal media proliferation) was also determined. Bars=SEM, n=3.
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is indicated by a horizontal line on Figure 3A–D. As a control,
sunitinib and CsA + Ita were compared at several doses including
the synergistic dose established in Figure 3 (Figure S2). Sunitinib
significantly inhibited tube formation at 8.8 mM and 880 nM,
whereas CsA + Ita only showed significant inhibition at the highest
dose (8.8 mM). While sunitinib demonstrated greater potency in all
parameters measures, these differences were mostly non-significant
In the sprout formation assay, spheres of endothelial cells were
embedded in a three-dimensional collagen matrix and were
induced to form sprouts by the application of growth factors
including VEGF165and bFGF. While neither CsA nor Ita alone
were able to reduce cumulative sprout length, the CsA + Ita
combination inhibited sprout formation by 43% (p,0.05) (Fig. 3E
and 3F) and led the severe fragmentation of the sprouts. This was
similar to the fragmentation observed in the tube formation assay.
Thus, per Webb’s Fractional Product model, CsA and Ita were
also synergistic in the sprouting assay for angiogenesis.
The combination of Cyclosporin A and Itraconazole does
not cause general toxicity
In some cases combining two drugs can lead to a synergistic
toxicity as well as synergism of the desired effect. To evaluate
whether or not general cellular toxicity was occurring in the case of
the CsA-Ita combination, we compared the effects of the
combination on the viability of HUVEC and another primary cell
type, human foreskin fibroblasts (HFF) (Fig. 4A). We found that
while the combination impaired the viability of HUVEC at higher
doses, there was a negligible effect on the viability of HFF (Fig. 4A).
In addition, we examined the effect of the combination on the
proliferation of HeLa and Jurkat T cells in comparison to HUVEC
(Fig. 4B). There was a window of roughly an order of magnitude
between the potency of the combination against HUVEC and the
other two cell types. The IC50doses for HUVEC proliferation
caused no effect on either HeLa or Jurkat T cells. In a subsequent
experiment, the effect of CsA+Ita on HeLa and Jurkat viability was
determined at the three highest doses used in the comparison of
HUVECandHFFviability. HeLaviability was reduced byless than
25% at the highest dose and by less than 12% at the lower two
doses.Jurakat viabilitywasnot affectbymore than12%atanydose.
In this work, we took a novel approach to angiogenesis inhibition
by screening a collection of clinically approved drugs for synergistic
inhibition of endothelial cell proliferation. We undertook this
approach both to circumvent the high cost involved in the
Figure 3. Cyclosporin A and itraconazole synergistically inhibit endothelial cell tube formation and sprout formation. (A) HUVEC
were grown on a matrigel coated plates in the presence of vehicle only (DMSO), CsA (8 mM), Ita (800 nM), or a combination of the same CsA and Ita
doses. The tube networks were stained with Calcein AM. Total tube length (B), number of junctions (C), and total network size (D) were calculated
using AngioQuant (n=3). (E) HUVEC spheres were embedded in a collagen matrix in the presence of basal media (EGM-2+2% FBS) (2GF), or
complete media supplemented with VEGF165and bFGF (+GF). The spheroids were treated with drug as in (A) (n=4; scale bar=50 mm). Insets are
magnified views of the tube architecture. Cumulative sprout length was quantified (F). The non-interaction value is denoted by the horizontal line in
B–D and F. Error bars, SEM; * p,0.1; ** p,0.05.
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development new drugs and to take advantage of the increase in
potency resulting from synergism. Screening of 741 binary drug
combinations resulted in the identification of a synergistic
interaction between CsA and Ita in HUVEC proliferation, which
was also observed in endothelial cell tube formation and sprouting
assays. Importantly, this combination was selective for endothelial
cells. To our knowledge, this is the first screen for synergy among
clinical drugs for inhibition endothelial cell proliferation.
CsA is a natural product drug discovered in the 1970s and has
been used widely as an immunosuppressant . The ability of
CsA to inhibit endothelial cell proliferation and angiogenesis has
also been known for some time, but CsA has not been used as a
clinical angiogenesis inhibitor due to its immunosuppressive
properties and nephrotoxic side effects at high doses [28,29].
Interestingly, while the mechanism for immunosuppression by
CsA is due to inhibition of the protein phosphatase calcineurin in
T cells, the antiangiogenic properties of the drug are independent
of calcineurin [30,31].
Itraconazole is a member of the triazole class of antifungal
drugs. We previously identified itraconazole as an angiogenesis
inhibitor in an initial screen of the JHDL . Itraconazole inhibits
lanosterol 14-a-demethylase (14DM), a key enzyme in the fungal
ergosterol and human cholesterol biosynthetic pathways. Howev-
er, it is unclear whether or not Ita can inhibit 14DM in humans
and there is evidence that 14DM is not the relevant target for
antiangiogenesis [32,33]. We have shown that supplementation of
HUVEC with cholesterol can only partially rescue the effects of
itraconazole treatment and that there is a lack of correlation
between fungal and endothelial cell proliferation for a series of
stereoisomers of itraconazole [5,34,35]. Interestingly, itraconazole
was shown by Heitman and colleagues to synergistically inhibit
fungal growth with CsA, but this activity was shared by other azole
antifungals suggesting that 14DM plays an important role in
antifungal synergy . In addition, we previously found that Ita
inhibits mTOR in HUVEC and causes a defect in intracellular
cholesterol trafficking .
Since its inception nearly four decades ago, the field of
angiogenesis had been growing in complexity. While VEGF was
the initial proangiogenic growth factor to be isolated, it is now
known that multiple factors including FGF, PIGF, and PDGF all
play roles in stimulating angiogenesis in vivo. Given that the
induction of angiogenesis requires not only proliferation but also
differentiation and migration, the complexity of signaling down-
stream of these growth factors is high. Although initially studied as
independent units, there is now evidence that pro-angiogenic
signaling pathways are integrative, branched networks of parallel
pathways. This is evidenced by the finding that decreasing the
expression levels of FGF and VEGF in vivo synergistically inhibited
tumorangiogenesis.Theoretically,sucha networkisa potential
target for synergistic inhibition by a combination of small molecules
which targetdifferentbranches. Theidentification of CsA andIta as
synergistic inhibitors of endothelial cell proliferation, tube forma-
tion, and sprouting provides proof of principle for synergistic
inhibitionasa potentialantiangiogenicstrategy.The modestclinical
Figure 4. The combination of cyclosporin A and itraconazole does not cause general toxicity. (A) HUVEC and HFF were treated with a
combination of CsA and Ita for 30 hours. Cell viability was measured by Calcein AM staining (n=3). (B) Proliferation of HeLa and Jurkat T cells treated
with a combination of CsA and Ita was compared to that of HUVEC after a 30 hour incubation (n=3). Total combined drug dose shown (the ratio of
CsA to Ita was 10:1 as in other experiments). Bars, SEM; ** p,0.05; dashed lines indicate 95% confidence bands. (C) The viability of HeLa was
determined as in (A) and Jurkat viability was determined after a 24 h drug dose followed by a 6 h incubation with Alamar blue. Bars=SEM; n=3.
Synergistic Inhibition of Endothelial Cells
PLoS ONE | www.plosone.org8 September 2011 | Volume 6 | Issue 9 | e24793
effects of angiogenesis inhibitors like bevacizumab, which targets
only a single node in the complex proangiogenic network, motivates
pursuing a synergistic approach to antiangiogenesis . That the
potency of CsA+Ita against HUVEC proliferation was independent
of VEGF signaling suggests an intriguing difference from bevaci-
zumab, sunitnib, and other existing therapies.
In addition to the clinical implications, the identification of a
synergistic drug combination may provide insight into the
underlying molecular mechanisms of angiogenesis. Pharmacologic
synergy is the chemical biology equivalent of synthetic lethality in
classical genetics. Synthetic lethality implies that the two
interacting alleles being tested work in either parallel arms of the
same pathway or in two compensatory pathways . Likewise, if
the chemical perturbation of two distinct proteins by small
molecules results in synergistic inhibition of angiogenesis, it may
imply that the two protein targets are involved in similar or
compensatory pathways responsible for angiogenesis. This knowl-
edge can then be used to draw previously unexpected connections
between pathways. A benefit of searching for synergy between
clinical drugs is that a high proportion has known protein targets
compared to non-drug chemical libraries. This increases the
practicality of a mechanistic study of the observed synergy, which
may potentially uncover novel interactions among angiogenesis
In summary, we have provided proof of principle that
identifying synergistic inhibition of endothelial cell proliferation
between clinical drug combinations is possible in a high-
throughput format. In addition the synergy between CsA and
Ita has generated a launching off point to explore novel
interactions between the relevant drug targets. Further screens of
collections of drugs both with and without individual activity
against HUVEC proliferation will likely lead to additional hits
with potential clinical and basic science implications.
combinations of 39 drugs were screened in duplicate using three doses
centered around the IC50of each drug. From this initial round, 47
combinations (6.3% preliminary hit rate) were identified as synergistic
per a Chou-Talalay analysis. In the second phase, the initial hits were
rescreened across a greater concentration range to generate full dose-
response curves for the drugs both alone and in combination. The
A and itraconazole (0.13% validated hit rate).
Screening design. In the first phase of screening, 741
A+ +itraconazole combination in tube formation assays.
(A) HUVEC were seeded on matrigel in the presence of the
indicated compounds. Following an 18 h incubation, the tube
networks were visualized with Calcein AM and photographed.
Micrographs from one of four independent experiments are
shown. (B) Total tube length, network size, and number of
junctions were determined using Angioquant. Bars=SEM; n=4;
** p,0.05; # p,0.005.
Comparison of sunitinib and the cyclosporine
We wish to thank Dr. Wade Gibson (JHU) for providing HFFs and the
members of the Liu lab for stimulating discussions and technical advice as
well as Dr. Noy Bassik for helpful comments on the manuscript. We also
wish to thank Drs. TC Chou (Memorial Sloan-Kettering) and Paul Talalay
(JHU) for advice and assistance.
Conceived and designed the experiments: BAN JOL. Performed the
experiments: BAN. Analyzed the data: BAN JOL. Wrote the paper: BAN
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