Imidazole-based alkaloid derivative LCB54-0009 suppresses ocular angiogenesis and lymphangiogenesis in models of experimental retinopathy and corneal neovascularization

Article abstract of DOI:10.1111/bph.13177

Background and Purpose Abnormally induced angiogenesis and lymphangiogenesis are associated with human diseases, including neovascular eye disease. Substances that inhibit these processes may have potential as an attractive therapeutic strategy for these

Full text of DOI:10.1111/bph.13177

DOI:10.1111/bph.13177
www.brjpharmacol.org
British Journal of
Pharmacology
BJP
Correspondence
Tae-Yoon Kim, Department of
Dermatology, College of
RESEARCH PAPER
Medicine, The Catholic
Imidazole-based alkaloid
derivative LCB54-0009
suppresses ocular
University of Korea, 222
Banpo-daero, Seocho-gu, Seoul
137-701, Korea. E-mail:
tykimder@catholic.ac.kr
----------------------------------------------------------------
*Present address: Department of
Pharmacology and Biomedical
Science Project (BK21PLUS), Seoul
National University College of
Medicine, Seoul, Korea.
angiogenesis and
----------------------------------------------------------------
†Present address: Department of
Ophthalmology, Asan Medical
Center, College of Medicine,
University of Ulsan, Ulsan, Korea.
----------------------------------------------------------------
lymphangiogenesis in
models of experimental
retinopathy and corneal
neovascularization
Received
20 October 2014
Revised
8 April 2015
Accepted
18 April 2015
Byung-Hak Kim1,*, Junyeop Lee2,†, Jun-Sub Choi3, Dae Young Park2,
Ho Young Song4, Tae Kyo Park4, Chung-Hyun Cho5, Sang-Kyu Ye5,
Choun-Ki Joo3, Gou Young Koh2 and Tae-Yoon Kim1
1Department of Dermatology, College of Medicine, The Catholic University of Korea, Seoul,
Korea, 2Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science
and Technology (KAIST), Daejeon, Korea, 3Catholic Institute for Visual Science and Department
of Ophthalmology and Visual Science, College of Medicine, The Catholic University of Korea,
Seoul, Korea, 4LegoChem Biosciences, Inc., Daejeon, Korea, and 5Department of Pharmacology
and Biomedical Sciences, Seoul National University College of Medicine, Seoul, Korea
BACKGROUND AND PURPOSE
Abnormally induced angiogenesis and lymphangiogenesis are associated with human diseases, including neovascular eye
disease. Substances that inhibit these processes may have potential as an attractive therapeutic strategy for these diseases.
EXPERIMENTAL APPROACH
In vitro and in vivo angiogenesis and/or lymphangiogenesis were assessed in VEGF- or hypoxia-stimulated endothelial and
retinal cells and in animal models of oxygen-induced retinopathy (OIR), streptozotocin-induced diabetic retinopathy (SIDR),
suture-induced inflammatory corneal neovascularization (SICNV) and silver nitrate-induced corneal neovascularization. HUVECs
and retinal cells were cultured under hypoxic conditions or incubated with VEGF to identify the molecular mechanisms
involved.
KEY RESULTS
The imidazole-based alkaloid derivative LCB54-0009 inhibited capillary-like tube formation in VEGF-induced HUVECs without
inducing cytotoxic effects. Intravitreal injection of LCB54-0009 into retinas suppressed the formation of the pathological
neovascular tufts and increased vascular permeability in both OIR of mice and SIDR of rats. Furthermore, subconjunctival
© 2015 The British Pharmacological Society
British Journal of Pharmacology (2015) 172 3875–3889 3875

B-H Kim et al.
BJP
injection of LCB54-0009 into the cornea suppressed corneal inflammation and inflammation-associated angiogenesis and
lymphangiogenesis in SICNV of mice and silver nitrate cauterization of rats. These pharmacological activities were associated
with effects on HIF-1α protein stability and HIF-1α/NF-κB redox sensitivity through its antioxidant activities. LCB54-0009 also
inhibited the hypoxia-induced expression of angiopoietin-2, and VEGF-induced VEGFR-2 activation and downstream
signalling, resulting in the down-regulation of the expression of pro-angiogenic factors and pro-inflammatory mediators and
an up-regulation of the expression of anti-angiogenic factors.
CONCLUSIONS AND IMPLICATIONS
LCB54-0009 is a potential candidate molecule for blocking pathological angiogenesis and lymphangiogenesis mediated by
HIF-1α- angiopoietin-2 expression and VEGFR-2 activation.
Abbreviations
Ang, angiopoietin; DPPH, 2,2-diphenyl-1-picrylhydrazy; HIF, hypoxia-inducible factor; LCB54-0009,
3-(2-((3,4-dihydroxy-phenyl)-[4-(2-dimethylamino-ethyl)-imidazol-1-yl]-methyl)-benzo[1,3]dioxol-5-yl)-propionic acid;
LEC, lymphaticendothelial cell; OIR, oxygen-induced retinopathy; PHD, prolyl hydroxylase; pVHL, von Hippel Lindau
protein; ROS, reactive oxygen species; SICNV, suture-induced inflammatory corneal neovascularization; SIDR,
streptozotocin-induced diabetic retinopathy; TIMP, tissue inhibitor of metalloproteinase
Tables of Links
TARGETS
LIGANDS
Catalytic receptorsa Enzymesb
Angiopoietin-2
Angiostatin
Biotin
IL-1β
IL-4
L-cysteine
LPS
CD11b
TIE-2
COX-2
iNOS
PHD1 (HIF-2)
PHD2 (HIF-1)
IL-6
TIMP1
TIMP2
TNF-α
VEGF-A
TLR2
MMP-1
MMP-2
MMP-3
MMP-9
PHD3
Histamine
Histidine
IFN-γ
IL-8
TLR4
Plasminogen (angiostatin)
IL-12
IL-13
VEGFR-2
Src
TSP-1
IL-1α
These Tables list key protein targets and ligands in this article which are hyperlinked to corresponding entries in http://
www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Pawson et al., 2014) and are
permanently archived in the Concise Guide to PHARMACOLOGY 2013/14 (a,bAlexander et al., 2013a,b).
some tissues with a unique structure and function are partly
or completely deficient in these vessels under normal
Introduction
Angiogenesis and lymphangiogenesis are complex processes
by which endothelial cells (ECs) and lymphatic endothelial
cells (LECs) proliferate and migrate from pre-existing blood
and lymphatic vessels to form new blood and lymphatic
vessels (Adams and Alitalo, 2007; Cao et al., 2013). These
newly formed vessels are essential for normal physiological
and vital processes, such as growth, development, reproduc-
tion, tissue repair, granulation tissue formation, homeostasis,
metabolism and immunity. However, pathophysiological
angiogenesis and lymphangiogenesis are associated with
numerous human diseases, including cancer, vascular disease,
inflammatory disease and neovascular eye diseases (Carmeliet
and Jain, 2000; Regenfuss et al., 2012). Therefore, drugs that
can affect pathological angiogenesis and lymphangiogenesis
have potential therapeutic benefits in the prevention of
tumour progression and the treatment of vascular, inflamma-
tory and neovascular eye diseases.
conditions. The cornea is one of these rare tissues and an
appropriate hypoxic, avascular state is maintained by anti-
haemangiogenic factors such as angiostatin, endostatin and
thrombospondin 1 (TSP-1; Regenfuss et al., 2012). In con-
trast, the retina is one of the most metabolically active tissues
in the body, and it needs large amounts of oxygen and nutri-
ents to produce metabolic energy. Therefore, ill-timed oxygen
levels in the eyes are associated with ocular diseases, such as
pathological corneal and retinal haemangiogenesis, lym-
phangiogenesis, diabetic retinopathy, retinopathy of prema-
turity (ROP), age-related macular degeneration, high-altitude
retinopathy, glaucoma and retinitis pigmentosa, which ulti-
mately lead to vision loss (Arjamaa and Nikinmaa, 2006;
Grimm and Willmann, 2012).
Molecular oxygen (O2) is utilized for the synthesis of ATP.
ATP synthesis is essential for metabolic processes, including
tissue development, homeostasis and normal physiological
function. The cellular O2 concentration is well-controlled by
the vascular system in mammals. However, acute decreases in
Although blood and lymphatic vessels are essential for
supplying oxygen and nutrients between tissues and cells,
3876 British Journal of Pharmacology (2015) 172 3875–3889

Anti-angiogenic and anti-lymphangiogenic activities of LCB54-0009
BJP
these levels cause cellular dysfunction and pathogenesis due
Preparation of primary retinal cells
to insufficient ATP production, excess reactive oxygen species
(ROS) production and hypoxia (Semenza, 2007). Exposure to
hypoxia transcriptionally induces a broad range of genes
through the induction of oxygen-sensitive hypoxia-inducible
factors (HIFs; Kietzmann and Görlach, 2005; Gordan and
Simon, 2007; Majmundar et al., 2010). HIF-1α is a master
transcription factor in the maintenance of oxygen homeosta-
sis and cellular responses to hypoxia. Short- and long-term
exposures to hypoxia induce various genes that are associated
with human diseases, including ischaemic cardiovascular
disease, hypertension, organ transplant rejection, colitis,
ocular neovascularization, hereditary erythrocytosis, inflam-
matory disease, angiogenesis and cancer (Konisti et al., 2012;
Semenza, 2014). In addition, chronic exposure to hypoxia
influences drug delivery by altering the expression of drug
transporters and the functional activity of carrier transporters
(Takagi et al., 1998; Nelson et al., 2003; Wasa et al., 2004;
Fradette et al., 2007; Kadam et al., 2013). Therefore, targeting
HIFs may be a potential therapeutic strategy for the treatment
of many human diseases, including angiogenesis- and
lymphangiogenesis-related vascular and neovascular eye
diseases.
Primary retinal pigmented epithelial cells were isolated from
the eyes of C57BL/6J mouse as described previously
(Grozdanov et al., 2010). Briefly, the retina was isolated from
the eye of 6-week-old male C57BL/6J mouse, digested in the
digestion solution containing 17 U·mL−1 papain (Sigma-
Aldrich, St. Louis, MO, USA) and 0.3 g·mL−1 L-cysteine
(Sigma-Aldrich) in DMEM for 30 min at 37°C, and triturated
by sucking it up and down with a Pasteur pipette, followed
by removal of the lens, cornea and ciliary body under an
inverted microscope. After centrifugation at 600× g for
5 min, the cell pellet was suspend in fresh DMEM and cul-
tured at 37°C and 5% CO2 to maintain the primary retinal
cells.
Antioxidant assays
For the antioxidant assays, total antioxidant capacity (TAC),
2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging
activity, superoxide radical scavenging activity, nitric oxide
(NO) production and peroxynitrite scavenging activity were
performed, and the detailed information on experimental
procedures is in the Supporting Information.
In addition to histidine, histamine, biotin and alkaloids,
the heterocyclic ring of imidazole is an important biological
building block. In this study, we identified an imidazole-
based alkaloid derivative, 3-(2-((3,4-dihydroxy-phenyl)-[4-
(2-dimethylamino-ethyl)-imidazol-1-yl]-methyl)-benzo[1,3]
dioxol-5-yl)-propionic acid (LCB54-0009), which exhibited
anti-angiogenic and anti-lymphangiogenic activities. This
compound effectively inhibited capillary-like tube formation
without inducing cytotoxic effects in ECs. In addition, in vivo
treatment with LCB54-0009 decreased pathological angio-
genesis, inflammation and inflammation-associated lym-
phangiogenesis in animal models of retinopathy and corneal
neovascularization. These results were associated with its
antioxidant activity, which resulted in the regulation of
HIF-1α protein stability and HIF-1α/NF-κB redox sensitivity
as well as the inhibition of angiopoietin (Ang) expression and
VEGFR-2 signalling cascades. These findings suggest that
LCB54-0009 may be a potential molecule for blocking patho-
logical angiogenesis and lymphangiogenesis.
Tube formation assays
HUVECs were seeded onto the Matrigel (BD Biosciences, San
Jose, CA, USA) and incubated with vehicle (0.1% DMSO) or
LCB54-0009 (20 μM) in the presence or absence of VEGF
(20 ng·mL−1) for 14 h in serum-free conditions. Capillary-like
tube formation was assessed using an inverted microscope
(Olympus, Japan).
Western blot analysis
Protein samples were separated by SDS-PAGE, and transferred
onto PVDF membranes. The membranes were blocked in a
blocking buffer containing 5% skimmed milk and incubated
with specific primary antibodies for the target molecules
overnight at 4°C. Antibodies specific for HIF-1α (ab1), and
VEGF (ab46154) were purchased from Abcam (Cambridge,
UK). Anti-phospho-VEGFR-2 (#2478), VEGFR-2 (#2479),
phospho-p125FAK (#3284), p125FAK (#13009), phospho-Src
(#2101), Src (#1587) and GAPDH (#2118) were purchased
from Cell Signaling Technology (Danvers, MA, USA), and
anti-VHL antibody (SC-17780) was obtained from Santa Cruz
Biotechnology (Santa Cruz, CA, USA). The membranes were
then incubated with horseradish peroxidase-conjugated sec-
ondary antibodies (A24518 and A24537, Life Technologies,
Grand Island, NY, USA), and signals were detected using an
enhanced chemiluminescence detection kit (iNtRON Bio-
technology, Seongnam, Korea).
Methods
Cell culture
Primary human umbilical vein endothelial cells (HUVECs)
were maintained in EBM-2 medium containing EGM-2 Sin-
gleQuots (Clonetics, Walkersville, MD, USA). The spontane-
ously arising human retinal pigment epithelial cell line
ARPE-19 (CRL-2302) was obtained from American Type
Culture Collection (ATCC, Manassas, VA, USA) and main-
tained in DMEM/F-12 medium supplemented with 10% heat-
inactivated FBS, 2.5 mM L-glutamine, 15 mM HEPES buffer
and antibiotics (100 U·mL−1 penicillin and 100 μg·mL−1 strep-
tomycin; Invitrogen, Carlsbad, CA, USA). Cells were incu-
bated at 37°C with 5% CO2 atmosphere in a humidified
incubator. Hypoxia was induced in a hypoxic modular incu-
bator chamber with a 94:5:1 mixture of N2 : CO2 : O2, as pre-
viously described (Kim et al., 2011).
Quantitative real-time and semi-quantitative
reverse transcription PCR
Total RNA was isolated using an RNeasy Mini Kit, and cDNA
was synthesized using QuantiTect Reverse Transcription Kit
(Qiagen, Valencia, CA, USA). Quantitative real-time PCR
(qRT-PCR) was performed using the KAPA SYBR fast qPCR Kit
(KAPA Biosystems, Woburn, MA, USA), and the data were
normalized to the GAPDH expression. The PCR conditions
were one cycle at 95°C for 5 min, followed by 35 cycles of
British Journal of Pharmacology (2015) 172 3875–3889 3877

B-H Kim et al.
BJP
denaturation at 96°C for 20 s, annealing at 56°C for 20 s, and
extension at 72°C for 20 s, and ending with one cycle at 72°C
for 5 min. Primers used in this experiment were purchased
from Qiagen. Semi-quantitative reverse transcription PCR
(RT-PCR) was performed using an RNA PCR kit (Bioneer,
Korea), according to the manufacturer’s protocols. Total RNA
was reverse-transcribed at 42°C for 1 h, and PCR was per-
formed with 25 cycles at 94°C for 30 s, 60°C for 30 s and 72°C
for 60 s, and it ended with one cycle at 72°C for 5 min. The
products were resolved on agarose gels with DNA SafeStain
(Lamda Biotech, St. Louis, MO, USA) by electrophoresis.
Primers used in this experiment are summarized in Support-
ing Information Table S1.
per 10 μL) was subconjunctivally injected on the day of
suture placement. After 2 weeks, the mice were killed.
Silver nitrate-induced corneal
neovascularization model
Sprague-Dawley male rats weighing 250 g (n = 5) were anaes-
thetized by an i.p. injection of ketamine hydrochloride
(20 mg·kg−1) and xylazine hydrochloride (2.5 mg·kg−1). The
centres of the right corneas were then cauterized with a silver
nitrate applicator stick (75% silver nitrate and 25% potassium
nitrate), which was held in contact with the cornea (1 mm
diameter) for 10 s. Three days after cauterization, vehicle (1%
DMSO per 20 μL), LCB54-0009 (50 μg per 20 μL) or Avastin
(200 μg per 20 μL) was injected into the subconjunctiva. The
inhibitory effect was observed under an ophthalmic micro-
scope (Carl Zeiss, Jena, Germany) with a digital camera. The
images captured were analysed using ImageJ software.
Immunoprecipitation
HUVECs were incubated with deferoxamine (DFX, 200 μM)
for 6 h in the presence or absence of LCB54-0009. Whole-cell
lysates were pre-cleared with protein A/G-sepharose (Upstate,
New York, NY, USA) for 2 h at 4°C and then incubated with
2 μg antibodies in TEG buffer [20 mM Tris–HCl (pH 7.4),
1 mM EDTA, 10% glycerol and 1 mM dithiothreitol] for over-
night at 4°C. The immune complexes were precipitated by
protein A/G-sepharose beads and washed with TEG buffer
containing 0.1% Triton X-100 and then subjected Western
blot analysis with appropriate antibodies.
Histological analysis
Immunohistochemistry in whole-mounted retinas or corneas
was performed as previously described (Bock et al., 2007). The
retina was incubated with hamster anti-CD31 monoclonal
antibody (Millipore, Billerica, MA, USA) or with one or more
of the following antibodies: rat anti-TER119 monoclonal
antibody (clone TER-119, BD Biosciences), rabbit anti-
lymphatic vessel endothelial receptor (LYVE)-1 polyclonal
antibody (AngioBio, Del Mar, CA, USA) or rat anti-CD11b
monoclonal antibody (clone M1/70, BD Biosciences). After
several washes, the samples were incubated for 4 h at room
temperature with cyanine 3 (Cy3)-conjugated anti-hamster
immunoglobulin G (IgG), Cy5-conjugated anti-rat IgG, or
FITC-conjugated anti-rabbit IgG (Jackson ImmunoResearch,
West Grove, PA, USA). Flat-mount stained retinas or corneas
were visualized, and digital images were obtained using a
Zeiss LSM 510 confocal microscope equipped with argon and
helium-neon lasers (Carl Zeiss).
Oxygen-induced retinopathy (OIR) model
All animal care and experimental procedures were conducted
in accordance with the National Institute of Health Guide for
the Care and Use of Laboratory Animals and were approved
by the Animal Care Committee of Korea Advanced Institute
of Science and Technology and the Catholic University of
Korea. Mice and rats were handled in accordance with the
ARVO Statement for the Use of Animals in Ophthalmic and
Vision Research. All studies involving animals are reported in
accordance with the ARRIVE guidelines for reporting experi-
ments involving animals (Kilkenny et al., 2010; McGrath
et al., 2010). All animals were bred and housed in our
pathogen-free animal facility, and they were fed with a stand-
ard normal diet (PMI Lab diet) ad libitum with free access to
water. The OIR mouse model was generated, as previously
described (Lee et al., 2013). Briefly, newborn C57BL/6J mice
(n = 5) at P7 and their nursing mothers were exposed to 75%
oxygen in a hyperoxic chamber (ProOx Model 110, Bio-
spherix, Lacona, NY, USA) for 5 days, after which they were
returned to room air for 5 days.
Morphometric analysis
Morphometric analysis on the retina and cornea was per-
formed using the ImageJ software or LSM Image Browser (Carl
Zeiss). Neovascular tufts (NVTs) and avascular areas in OIR
retinas were measured using the Lasso tool of the Adobe
Photoshop software, as previously described (Connor et al.,
2009). The numbers of extravasated Ter119+ erythrocytes
were counted in four random 0.2 mm2 areas of each retina.
Retinal vascular permeability was determined using FITC-
labelled dextran. The area extravasated dextran was calcu-
lated as the FITC-dextran-positive area divided by the total
retinal area in each quadrant of retinal mount, and it is
presented as a percentage. Vessel diameters on the cornea
were averaged from all CD31 positive blood vessels that were
located at the halfway point between the outer point of
suture placement and the limbus. The blood or lymphatic
vascular density in whole-mounted corneas was calculated as
the area of CD31 positive blood vessels or LYVE-1 positive
lymphatic vessels divided by the total corneal area measured
per 0.2 mm2 field, and it is presented as a percentage. The
macrophage infiltration area was calculated by measuring the
CD11b positive area per 0.02 mm2 field of each site of suture
placement.
Suture-induced inflammatory corneal
neovascularization (SICNV) model
The SICNV model was generated according to a previous
report (Bock et al., 2007) with some modifications. Briefly,
8-week-old male C57BL/6J mice (n = 5) were anaesthetized
with an i.m. injection of ketamine (40 mg·kg−1) and xylazine
(12 mg·kg−1). One 10-0 nylon suture (Ethicon, Somerville, NJ,
USA) was placed intrastromally. The outer point of suture
placement was placed near the limbus, and the inner suture
point was positioned near the corneal centre that was equi-
distant from the limbus. Vehicle (0.1% DMSO per 10 μL),
LCB54-0009 (100 μg per 10 μL) or VEGF trap (100 μg
3878 British Journal of Pharmacology (2015) 172 3875–3889

Anti-angiogenic and anti-lymphangiogenic activities of LCB54-0009
BJP
Statistics
LCB54-0009 exhibits anti-angiogenic activity
Values are presented as mean
SD of three independent
in vitro
experiments (n = 3). Statistical differences were determined by
one-way ANOVA followed by Dunnett’s multiple comparison
tests. Differences were considered to be statistically signifi-
cant at P < 0.05.
We first performed a tube formation assay to identify the in
vitro anti-angiogenic activity of LCB54-0009 because this phe-
nomenon is a well-known, essential step for angiogenesis.
Capillary-like tube formation was increased in VEGF-
stimulated HUVECs, compared with the vehicle-treated
group. LCB54-0009 effectively inhibited this angiogenic
activity (Figure 1B). In parallel with this effect, LCB54-0009
reduced the mRNA levels of pro-angiogenic factors, including
VEGF-A, MMPs (MMP-1, MMP-2, MMP-3 and MMP-9) and
cluster of differentiation 31 (CD31). In contrast, LCB54-0009
increased the mRNA levels of the tissue inhibitors of metal-
loproteinases, TIMP-1 and TIMP-2, which are anti-angiogenic
factors (Figure 1C). Interestingly, treatment with 100 μM
LCB54-0009 over a 72-h period did not affect the viability of
HUVECs, ARPE-19 and primary retinal cells isolated from the
eyes of C57BL/6J mouse (Figure 1D and Supporting Informa-
tion Fig. S7A).
Results
Synthesis of imidazole-based alkaloids
Only a small population of alkaloids contains the imidazole
nucleus. However, these imidazole-based alkaloids have been
shown to exhibit multiple biological activities, such as anti-
tuberculosis, antibacterial, anti-fungal and anti-inflammation
(Rivers and Mancera, 2008; Duan et al., 2013; Kong et al.,
2013; Castelli et al., 2014). To develop novel compounds
possessing pharmacological properties that exhibit anti-
angiogenic and anti-lymphangiogenic activities, we
synthesized imidazole-based alkaloid derivatives from 3,4-
dihydroxybenzaldehyde (Supporting Information Fig. S1,
≥97% purity). The synthetic procedures, 1H- and 13C-NMR
and LC-MS spectra of intermediates and LCB54-0009 are
shown in Supporting Information Fig. S2–S4 and have been
previously described (Song et al., 2012). The functional
groups of L and M are –H, –OH or –OR, and R is C1–4 alkyl. The
X and Y are carbon or nitrogen, and m is 0∼4. The R1 is –H,
–OH, –OR3, –NR3R4, –NR3(C(O)R4), –NR3(SO2R4), –NR3(CO2R4),
–NR3(C(O)NR3R4), –CN, –CO2R3 or –C(O)NR3R4, and R3 and R4
are –H or C1–6 alkyl. The R2 is –H, –F, –Cl, –Br, –OH, –OR5,
–OSO3R5, –CF3, –OCF3, –C(O)Me, –CN, –CO2R5, –C(O)NR5R6,
–SO3R5, –SO2NR5R6, –(CH=CH)CO2R5, –(CH=CH)C(O)NR5R6,
–(CH=CH)SO3R5, –(CH=CH)CH2OH, –R5, –R5OR6, –R5OSO3R6,
–R5CO2R6, –R5C(O)NR6R7, –R5SO3R6 or –R5SO2NR6R7, and R5, R6
and R6 are –H, C1–6 alkyl, or C2–6 alkylene. All of the com-
pounds contain two chiral carbons and LCB54-0002 and
LCB54-0003, LCB54-0008 and LCB54-0009, and LCB54-0016
and LCB54-0017 are identified as isomers. We performed in
vitro and in vivo experiments to identify potential candidate
molecules. The data show that LCB54-0007, LCB54-0009 and
LCB54-0012 exhibited effective anti-angiogenic activity in
models of streptozotocin-induced diabetic retinopathy (SIDR)
and silver nitrate-cauterized corneal neovascularization (Sup-
porting Information Fig. S5). In addition, these three mol-
ecules exhibited more potential antioxidant activity than
other molecules, while all of the compounds exhibited mar-
ginal to weak peroxynitrite scavenging activity (Supporting
Information Fig. S6). We also determined their effects on
hypoxia-induced increased levels of HIFs and LCB54-0009
exhibited the strongest inhibition of the increase in HIF-1α
without affecting HIF-2α levels (Supporting Information
Fig. S7b). We therefore selected LCB54-0009 (Figure 1A) as a
potential target molecule for further studies and to elucidate
the mechanisms involved in its anti-angiogenic activity.
Structure–activity relationship analysis suggested that the
number of hydroxyl groups in the catechol plays an impor-
tant role in its antioxidant activity and the methyl group
in the amine as well as the carboxylic group in the
benzo[1,3]dioxole may be important factors for determining
their biological activities.
LCB54-0009 suppresses pathological
angiogenesis and vascular leakage in vivo
To investigate the in vivo anti-angiogenic activity of LCB54-
0009 in the retina, we generated the OIR mouse model,
which mimics human retinopathy of prematurity (ROP) and
certain aspects of proliferative diabetic retinopathy (Frank,
2004; Sapieha et al., 2010). The retinas of OIR mice developed
extensive vascular regression in the central region and patho-
logical NVTs in the middle and peripheral regions. Intravit-
real injection of LCB54-0009 (10 μg) or VEGF trap (10 μg)
effectively suppressed NVT formation, compared with that of
fragment crystallizable (Fc)-injected group (Figure 2A–C).
To measure vascular leakage, we injected FITC-dextran i.v.
into OIR mice, 1 h before they were killed. Some extravasated
FITC-dextran was detected within the retina near the NVTs in
control mice. The regions where FITC-dextran was most
abundant matched the locations of extravasated Ter119+
erythrocytes, indicating ongoing leakage at the sites of haem-
orrhage. The degree of FITC-dextran leakage was markedly
decreased by the intravitreal injection of LCB54-0009 or
VEGF trap (Figure 2D–F), which suggests that LCB54-0009
suppresses pathological angiogenesis and vascular leakage in
the OIR mouse model. These results indicate that LCB54-
0009 significantly decreases retinal vascular permeability in
pathogenic angiogenesis.
LCB54-0009 suppresses corneal
neovascularization and
inflammation-associated lymphangiogenesis
in vivo
The aforementioned results encouraged us to determine
whether LCB54-0009 may also affect corneal inflammation
and neovascularization. We generated a SICNV mouse model,
which develops extensive blood and lymphatic vessel prolif-
erations that originate from the limbus to the suture site
(Bock et al., 2007). Haemangiogenesis and lymphangiogen-
esis were induced by suture-induced corneal inflammation.
Subconjunctival injection of LCB54-0009 (100 μg) or VEGF-
trap (100 μg) effectively suppressed haemangiogenesis, lym-
British Journal of Pharmacology (2015) 172 3875–3889 3879

B-H Kim et al.
BJP
Figure 1
LCB54-0009 inhibits VEGF-induced angiogenesis in vitro. (A) Chemical structure of LCB54-0009 (C24H27N3O6, MW = 453.49, ≥97% purity). (B)
Serum-starved HUVECs were seeded onto the Matrigel and incubated with vehicle (0.1% DMSO) or LCB54-0009 (20 μM) in the presence or
absence of VEGF (20 ng·mL−1) for 14 h. Capillary-like tube formation was viewed by using an inverted microscope. (C) HUVECs were incubated
with vehicle (0.1% DMSO) or LCB54-0009 (20 μM) in the presence or absence of VEGF (20 ng·mL−1) for 48 h. The mRNA levels were determined
by quantitative real-time PCR. (D) HUVECs were incubated with various concentrations of LCB54-0009 for 24 to 72 h and cell viability was
measured using the WST-1 reagent. Results are expressed as the mean SD of three independent experiments (n = 3). #P < 0.05 versus
vehicle-treated group; *P < 0.05 and **P < 0.005 versus VEGF-stimulated group.
phangiogenesis and the infiltration of CD11b positive
inflammatory cells, compared with the Fc-treated group
(Figure 3A–E). This indicates that LCB54-0009 suppresses
corneal inflammation and inflammation-associated angio-
genesis and lymphangiogenesis.
The effects of LCB54-0009 were also examined in a rat
corneal angiogenesis model, in which the centres of the right
corneas were cauterized with a silver nitrate applicator stick.
After 2 weeks of cauterization, new vessels covered the full
cornea. However, new vessels were observed in the quarter-
cornea region in the LCB54-0009 (50 μg)- and Avastin
(200 μg)-treated corneas. The mean percentages of the neo-
vascularized area were greatly reduced in the LCB54-0009
treated groups compared to the control (Figure 3F and G).
These findings indicate that LCB54-0009 has an inhibitory
effect on corneal neovascularization and inflammation.
lymphangiogenesis, metastasis and inflammation (Cao,
2005; Tammela and Alitalo, 2010). Our preliminary results
revealed that among the derivatives, LCB54-0009 exhibited
the strongest inhibition of HIF-1α expression without affect-
ing HIF-2α levels (Supporting Information Fig. S7B). To elu-
cidate the molecular mechanisms of the anti-angiogenic
and anti-lymphangiogenic activities of LCB54-0009, we first
determined whether LCB54-0009 could inhibit the expres-
sion of oxygen-sensitive master transcription factors HIFs.
The levels of HIF-1α were markedly elevated in HUVECs,
ARPE-19 and primary retinal cells under hypoxic conditions.
LCB54-0009 inhibited these elevated levels of HIF-1α in
a
concentration-dependent manner, ultimately resulting
in the down-regulation of VEGF levels. However, HIF-1α
levels were restored in the presence of the protease inhibitor,
carbobenzoxy-Leu-Leu-leucinal (MG-132) (Figure 4A–C and
Supporting Information Fig. S7C). HIFs can activate the tran-
scription of numerous pro-angiogenic factors and pro-
inflammatory mediators (Kelly et al., 2003; Simon et al., 2008;
Semenza, 2013). We therefore performed quantitative
real-time PCR to identify the effects of LCB54-0009 on the
LCB54-0009 inhibits HIF-1α levels
Low oxygen levels induce a wide range of genes through the
up-regulation of HIFs that are associated with angiogenesis,
3880 British Journal of Pharmacology (2015) 172 3875–3889

Anti-angiogenic and anti-lymphangiogenic activities of LCB54-0009
BJP
Figure 2
LCB54-0009 suppresses pathological angiogenesis and vascular leakage. (A–C) Intravitreal injection of vehicle (0.1% DMSO), LCB54-0009 (10 μg)
or VEGF-trap (10 μg) in OIR mice (n = 5 per group). Vascular regression in the central region (A), pathological NVTs (B) and avascular area (C)
were measured in the middle and peripheral regions of the retinas. Scale bars = 200 μm. (D–F) FITC-dextran was injected i.v. 1 h before the OIR
mice were killed (D). Extravasated FITC-dextran (E) and extravasated Ter119+ cells (F) were measured within the retina near the NVT. Scale bars
= 100 μm. *P < 0.05 and **P < 0.005 versus control mice. CTL, control; LCB54, LCB54-0009; V-trap, VEGF trap.
expression of angiogenic factors and inflammatory media-
tors. LCB54-0009 significantly inhibited the mRNA levels of
pro-angiogenic factors, including VEGF-A and MMPs
(Figure 4D and Supporting Information Fig. S8) and pro-
inflammatory molecules, including IL-1α, IL-4, IL-6, IL-8,
IL-12, IL-13, COX-2 and inducible NOS (iNOS), compared
with those of hypoxia-stimulated HUVECs, ARPE-19 and
primary retinal cells. The expression levels of these factors
and mediators were restored in the presence of MG-132
(Figure 4E).
LCB54-0009 on HIF-1α protein levels is associated with tran-
scription levels and/or HIF-1α/NF-κB redox sensitivity. Inter-
estingly, although the mRNA levels of HIF-1α were not
affected by LCB54-0009, this compound inhibited the mRNA
levels of PHD3, but not PHD1 and PHD2 (Figure 5A and
Supporting Information Fig. S9A), suggesting that it may
regulate HIF-1α stability by pVHL. pVHL is a tumour suppres-
sor protein that targets HIF-1α for oxygen-dependent prote-
olysis (Maxwell et al., 1999). LCB54-0009 increased the
interaction between HIF-1α and pVHL, compared with DFX
(hypoxia-mimetic agent)-treated cells (Figure 5B). In addi-
tion, the ubiquitinated levels of HIF-1α protein were
increased in LCB54-0009-treated cells (Figure 5C). We also
observed that LCB54-0009 inhibited the mRNA levels of Toll-
like receptors (TLR2 and TLR4) and NF-κB subunits p50 and
p65, while these levels were restored in the presence of
MG-132 (Figure 5D), indicating that LCB54-0009 affects
HIF-1α protein stability and HIF-1α/NF-κB redox sensitivity.
LCB54-0009 regulates HIF-1α stability and
HIF-1α/NF-κB redox sensitivity
HIF-1α stability is regulated by oxygen levels, which affect the
activity of prolyl hydroxylases (PHDs) and von Hippel Lindau
protein (pVHL; Lee et al., 2004; Ke and Costa, 2006). In addi-
tion, many stimuli, including growth factors, LPS, cytokines,
thrombin and insulin can induce HIF-1α expression under
normoxic conditions through ROS production and redox-
sensitive transcription factor NF-κB (Zelzer et al., 1998;
Richard et al., 2000; Görlach et al., 2001; Haddad and Land,
2001; Blouin et al., 2004; Bonello et al., 2007; Görlach and
Bonello, 2008). We examined whether the inhibitory effect of
LCB54-0009 exhibits antioxidant activity
Hypoxia increases ROS levels, which perturb the balance
between cellular antioxidants and pro-oxidants. Antioxidants
exhibit anti-angiogenic activity by affecting HIF-1α stability
British Journal of Pharmacology (2015) 172 3875–3889 3881

B-H Kim et al.
BJP
Figure 3
LCB54-0009 inhibits corneal neovascularization and inflammation-associated lymphangiogenesis. (A–E) Proliferation of blood vessels (BV) and
lymphatic vessels (LV) were generated in the SICNV mouse model (n = 5 per group). Vehicle (0.1% DMSO), LCB54-0009 (100 μg) or VEGF trap
(100 μg) was injected subconjunctivally on the day of corneal suture placement. Corneal angiogenesis, lymphangiogenesis and inflammation were
determined by staining with CD31 (red), LYVE-1 (green) and CD11b (blue) antibodies respectively (A). BV diameter (B), BV density (C), LV density
(D) and the infiltration area of inflammatory cells (E) were measured. Scale bars = 500 μm. (F and G) Corneal neovascularization was induced by
the subconjunctival injection of silver nitrate to cauterize the cornea of rats (n = 5 per group). Vehicle (1% DMSO), LCB54-0009 (50 μg) or Avastin
(200 μg) was injected subconjunctivally three days after silver nitrate cauterization and measured using an ophthalmic microscope with a digital
camera. Original magnification was ×100. Avastin was used as a positive control. *P < 0.05 and **P < 0.005 versus control mice. CTL, control;
LCB54, LCB54-0009; V-trap, VEGF trap.
and HIF-1α/NF-κB redox sensitivity (Haddad et al., 2000; Gao
et al., 2007; Park et al., 2010; Sceneay et al., 2013). To inves-
tigate the possible antioxidant effects of LCB54-0009, we first
measured the total antioxidant capacity (TAC). LCB54-0009
exhibited effective TAC activity with an IC50 value of 15.8 μM
(Figure 6A). In addition, the imidazole-based alkaloid deriva-
tives all exhibited DPPH radical scavenging activity, except
for LCB54-0016 and LCB54-0017. Among them, LCB54-0009
exhibited the strongest DPPH radical scavenging activity with
an IC50 value of 8.3 μM (Supporting Information Fig. S6 and
Figure 6B). To determine the ROS scavenging activities, we
performed a non-enzymatic assay of superoxide radicals and
assessed their radical scavenging activities. LCB54-0009 scav-
enged superoxide radicals with an IC50 value of 15.5 μM
(Figure 6C). To further investigate whether LCB54-0009 could
affect the production of reactive nitrogen species (RNS), we
performed the Griess reaction to measure NO production.
The levels of nitrites, the stable NO metabolites, were
increased by more than fivefold by stimulation of HUVECs
for 36 h with LPS, TNF-α, IL-1β and IFN-γ; LCB54-0009 exhib-
ited an inhibitory effect against NO production with an IC50
value of 10.5 μM; this effect was mediated via inhibition of
the levels of iNOS, an inducible enzyme producing large
amounts of NO (Figure 6D and Supporting Information
Fig. S9B). However, our synthetic derivatives, including
LCB54-0009 (up to 50 μM) did exhibit weak peroxynitrite
scavenging activity (Supporting Information Figs S6A and
S9C). These results clearly indicate that the antioxidant
activities of LCB54-0009 result in its inhibitory effects on
HIF-1α stability and HIF-1α/NF-κB redox sensitivity.
LCB54-0009 inhibits Ang-2 levels and
VEGFR-2-signalling cascades
The Ang/Tie pathway also regulates angiogenesis, and Ang-2
is increased by exposure to hypoxia (Yuan et al., 2000; Felcht
et al., 2012; Fagiani and Christofori, 2013). Although, Ang-2
is known to inhibit angiogenesis by binding with TIE-2,
which interacts with integrins to induce angiogenesis (Felcht
3882 British Journal of Pharmacology (2015) 172 3875–3889

Anti-angiogenic and anti-lymphangiogenic activities of LCB54-0009
BJP
Figure 4
LCB54-0009 inhibits hypoxia-induced increased levels of HIF-1α, angiogenic factors and inflammatory mediators. HUVECs were incubated with
LCB54-0009 for 6 h in hypoxia. (A–C) HIF-1α levels were determined by Western blotting (A and B) or immunofluorescence (C). GAPDH served
as a loading control. (D and E) The mRNA levels of angiogenic factors (D) and inflammatory mediators (E) were determined by quantitative
real-time PCR. Results are expressed as the mean SD of three independent experiments (n = 3). #P < 0.05 versus vehicle-treated group; *P < 0.05
†
and **P < 0.005 versus hypoxia-induced group; P < 0.05 and ††P < 0.005 versus LCB54-0009-treated group.
British Journal of Pharmacology (2015) 172 3875–3889 3883

B-H Kim et al.
BJP
Figure 5
LCB54-0009 regulates HIF-1α stability and HIF-1α/NF-κB redox sensitivity. (A and D) HUVECs were incubated for 6 h in hypoxia and the mRNA
levels were determined by quantitative real-time PCR. Results are expressed as the mean SD of three independent experiments (n = 3). #P < 0.05
versus vehicle-treated group; *P < 0.05 and **P < 0.005 versus hypoxia-induced group; †P < 0.05 and ††P < 0.005 versus LCB54-0009-treated
group. (B and C) HUVECs were incubated with DFX (200 μM) in the presence or absence of LCB54-0009 (20 μM) in hypoxia for 6 h. Anti-VHL
or anti-HIF-1α immunoprecipitates were analysed by Western blot analysis.
et al., 2012). Targeting Ang/Tie with VEGF signalling path-
ways has recently emerged to have valuable therapeutic
potential for anti-angiogenic therapy. Ang-2 expression was
increased in either hypoxia- or VEGF-treated HUVECs,
whereas Ang-1 expression was increased by VEGF stimula-
tion. Treatment with LCB54-0009 effectively decreased the
levels of Ang-2, which resulted in the inhibition of VEGF-A
levels (Figure 7A–C). The inhibition of hypoxia-induced
Ang-2 levels by LCB54-0009 suggests that this molecule may
affect the binding of integrins with TIE-2 rather than that of
Ang-2 and TIE-2. Interestingly, LCB54-0009 inhibited the
tyrosine phosphorylation of VEGFR-2 without altering its
mRNA and protein levels in either hypoxia- or VEGF-
stimulated HUVECs, thus resulting in the inhibition of its
downstream signalling targets, such as p125FAK and Src in
VEGF-stimulated HUVECs (Figure 7C and D). However, this
compound did not inhibit VEGFR-2 kinase activity (data not
shown). The results indicate that the inhibitory effect of
LCB54-0009 on Ang-2 expression and VEGFR-2 activation
may also contribute to its anti-angiogenic activity.
oped for different pharmacological effects, including anti-
fungal, antibacterial, analgesic, anti-inflammatory, anti-
cancer, cardiovascular, anti-neoplastic, anti-tuberculosis and
enzyme-inhibitory activities (Rivers and Mancera, 2008;
Duan et al., 2013; Kong et al., 2013; Castelli et al., 2014). In
this study, we identified an imidazole-based alkaloid
derivative LCB54-0009 as a potential inhibitor molecule for
pathological angiogenesis and inflammation-associated
lymphangiogenesis.
HIFs are oxygen sensors that are induced under low
oxygen levels. They induce the transcription of numerous
signalling factors, including pro-angiogenic and pro-
inflammatory molecules (Kelly et al., 2003; Simon et al.,
2008; Semenza, 2013); these molecules, by interacting with
their cognate receptors on blood vessel endothelial cells,
LECs, bone marrow-derived cells and inflammatory cells, lead
to inflammation, inflammation-associated angiogenesis and
lymphangiogenesis, and tumour metastasis (Cao, 2005;
Tammela and Alitalo, 2010). In addition, lymphangiogenesis
exacerbates inflammatory diseases and graft rejections (Cueni
and Detmar, 2006; Norrmén et al., 2011; Semenza, 2013).
Therefore, HIFs are important therapeutic targets for the pre-
vention and treatment of pathogenic angiogenesis and lym-
phangiogenesis. We synthesized a series of imidazole-based
alkaloid derivatives to identify small molecule inhibitors
that target HIF-mediated signalling cascades to exert anti-
Discussion and conclusions
Large numbers of imidazole derivatives have been synthe-
sized in the field of medicinal chemistry and are being devel-
3884 British Journal of Pharmacology (2015) 172 3875–3889

Anti-angiogenic and anti-lymphangiogenic activities of LCB54-0009
BJP
Figure 6
LCB54-0009 exhibits antioxidant activities. (A) The mixtures of hypoxia-stimulated HUVEC lysates, LCB54-0009 and Cu2+-containing working
solution were incubated at room temperature for 1.5 h. Total antioxidant capacity (TAC) was measured as absorbance at 570 nm. (B) LCB54-0009
was mixed with DPPH solution and the mixtures were incubated at 25°C for 30 min. DPPH radical scavenging activity was measured as absorbance
at 517 nm. (C) Superoxide radical was produced by the NADH/PMS system and the scavenging activity was measured as absorbance at 567 nm,
followed by incubation at 25°C for 5 min. (D) HUVECs were incubated with LCB54-0009 in combination with stimuli (LPS 1 μg·mL−1, TNF-α
10 ng·mL−1, IL-1β 5 U·mL−1 and IFN-γ 100 U·mL−1) for 36 h. Amounts of NO were measured using a Griess reaction. Results are expressed as the
mean SD of three independent experiments (n = 3). *P < 0.05 and **P < 0.005 versus control group (A–C); #P < 0.05 versus vehicle-treated
group; *P < 0.05 and **P < 0.005 versus stimuli-induced group (D).
angiogenic and anti-lymphangiogenic activities. Among the
derivatives, LCB54-0009 significantly inhibited the levels of
HIF-1α protein in hypoxia-induced HUVECs. This resulted in
the down-regulation of VEGF levels and capillary-like tube
formation in either hypoxia- or VEGF-stimulated HUVECs,
mediated through the suppression of the mRNA levels of
various pro-angiogenic and pro-inflammatory molecules and
the up-regulation of the mRNA levels of anti-angiogenic mol-
ecules. We further observed that LCB54-0009 suppressed
pathological angiogenesis and inflammation-associated lym-
phangiogenesis in animal models of hypoxia-induced retin-
opathy, diabetic retinopathy and corneal neovascularization.
The stability of HIF-1α protein is regulated by oxygen
balance. HIF-1α protein undergoes rapid degradation under
normoxic conditions by being processed by the PHD/pVHL-
mediated ubiquitin-proteasome pathway, whereas it is pro-
tected from degradation under hypoxic conditions (Lee et al.,
2004; Ke and Costa, 2006). Numerous growth factors and
cytokines as well as NO have been reported to stabilize HIF-1α
under normoxic conditions (Zelzer et al., 1998; Richard et al.,
2000; Görlach et al., 2001; Haddad and Land, 2001; Blouin
et al., 2004; Lee et al., 2004; Ke and Costa, 2006; Bonello et al.,
2007; Görlach and Bonello, 2008). In addition, ROS released
from a functional electron transport chain stabilize HIF-1α
protein (Haddad and Land, 2001; Hagen, 2012), indicating
the importance of controlling intracellular oxygen, ROS and
RNS levels for HIF-1α stabilization. The factors that regulate
HIF-1α stabilization are associated with activation of the tran-
scription factor NF-κB, which binds to a distinct element in
the proximal promoter of the HIF-1α gene (Bonello et al.,
2007; Görlach and Bonello, 2008). These findings imply that
one possible mechanism underlying HIF-1α expression and
stabilization is regulated by antioxidants and at the transcrip-
tion level by the redox-sensitive transcription factor NF-κB.
Interestingly, our results revealed that LCB54-0009 is able to
mediate antioxidant activities, which may contribute to
the regulation of HIF-1α stability and HIF-1α/NF-κB redox
sensitivity.
The formation of blood and lymphatic vessels under well-
controlled angiogenic and lymphangiogenic processes is
important in normal physiological conditions and performs a
vital functions. However, when abnormal or dysfunctional,
these processes are associated with numerous human diseases
(Carmeliet and Jain, 2000; Regenfuss et al., 2012). LCB54-
British Journal of Pharmacology (2015) 172 3875–3889 3885

B-H Kim et al.
BJP
Figure 7
LCB54-0009 inhibits Ang-2 levels and VEGFR-2-signalling cascades. (A–C) HUVECs were incubated for 6 h in hypoxia or 48 h with VEGF
(20 ng·mL−1) in the presence or absence of LCB54-0009 (20 μM). The mRNA levels were determined by quantitative real-time PCR (A and B) or
reverse transcription PCR (C). Results are expressed as the mean SD of three independent experiments (n = 3). #P < 0.05 versus vehicle-treated
group; *P < 0.05 versus hypoxia- or VEGF-induced group; †P < 0.05 versus LCB54-0009-treated group. (D) HUVECs were incubated with
LCB54-0009 for 1 h and then stimulated with VEGF (20 ng·mL−1) for 15 min. Western blotting was performed from the cell lysates. GAPDH served
as a loading control.
0009 also suppressed angiogenesis and inflammation-
associated lymphangiogenesis, as demonstrated by its
inhibition of capillary-like tube formation in VEGF-
stimulated HUVECs and retinopathy and corneal neovascu-
larization in animal models of inflammation. VEGF is a
well-known, pro-angiogenic factor that binds to its receptor
to increase vascular permeability and promote vasculogenesis
and angiogenesis by stimulating EC sprouting and prolifera-
tion. There are five members of VEGFRs: VEGFR-1, VEGFR-2,
VEGFR-3, neuropilin-1 and neuropilin-2. Of these, VEGFR-2
is the most important receptor for VEGF-A-induced angiogen-
esis in vascular ECs (Ferrara et al., 2003; Roskoski, 2007). The
phosphorylation of VEGFR-2 at specific tyrosine residues by
VEGF-A activates its down-signalling cascades (Olsson et al.,
2006; Holmes et al., 2007). LCB54-0009 effectively inhibited
the phosphorylation of VEGFR-2, but not its kinase activity,
in VEGF-stimulated ECs, which resulted in the inhibition of
its down-signalling cascades. Furthermore, we found that
LCB54-0009 inhibited Ang-2 expression in either hypoxia- or
VEGF-stimulated ECs, suggesting that the Ang-2-mediated
anti-angiogenic activity of this molecule may be associated
with inhibition of the interaction with integrins in ECs.
Ocular diseases that are associated with inflammation-
associated angiogenesis and lymphangiogenesis can lead to
serious problems in the eyes. VEGF and HIF-1α play impor-
tant roles in the development of pathological corneal and
retinal diseases, and they can also alter the expression of drug
transporters and the functional activity of carrier transporters
(Takagi et al., 1998; Nelson et al., 2003; Wasa et al., 2004;
Fradette et al., 2007; Kadam et al., 2013). These events
decrease drug delivery and make treatment difficult, thus
indicating the importance of regulating inflammation-
associated angiogenesis and lymphangiogenesis in eye dis-
eases. Importantly, we observed that in vivo treatment with
LCB54-0009 effectively suppressed pathological angiogenesis
and vascular leakage in retina, but also inflammation-
associated neovascularization and lymphangiogenesis in
cornea.
In summary, LCB54-0009 is a potent inhibitor of angio-
genesis and lymphangiogenesis and the possible mechanisms
of its effects are summarized in Figure 8. This compound is an
imidazole-based alkaloid that inhibited the levels of HIF-1α
and Ang-2 and down-regulated VEGFR-2-signalling cascades.
These events resulted in a reduction in the levels of angio-
genic factors and inflammatory mediators, leading to inhibi-
tion of the capillary-like tube formation in ECs. In addition,
LCB54-0009 affected HIF-1α protein stability and HIF-1α/
NF-κB redox sensitivity via its antioxidant activities. Further-
more, LCB54-0009 exhibited anti-inflammatory, anti-
angiogenic and anti-lymphangiogenic effects in animal
models of retinopathy and corneal neovascularization in vivo.
Altogether, these results suggest
a potential therapeutic
3886 British Journal of Pharmacology (2015) 172 3875–3889

Anti-angiogenic and anti-lymphangiogenic activities of LCB54-0009
BJP
Alexander SPH, Benson HE, Faccenda E, Pawson AJ, Sharman JL,
Spedding, M et al. (2013a). The Concise Guide to
PHARMACOLOGY 2013/14: catalytic receptors. Br J Pharmacol 170:
1676–1705.
Alexander SPH, Benson HE, Faccenda E, Pawson AJ, Sharman JL,
Spedding M et al. (2013b). The Concise Guide to PHARMACOLOGY
2013/14: enzymes. Br J Pharmacol 170: 1797–1867.
Arjamaa O, Nikinmaa M (2006). Oxygen-dependent diseases in the
retina: role of hypoxia-inducible factors. Exp Eye Res 83: 473–483.
Blouin CC, Pagé EL, Soucy GM, Richard DE (2004). Hypoxic gene
activation by lipopolysaccharide in macrophages: implication of
hypoxia-inducible factor 1alpha. Blood 103: 1124–1130.
Bock F, Onderka J, Dietrich T, Bachmann B, Kruse FE, Paschke M
et al. (2007). Bevacizumab as a potent inhibitor of inflammatory
corneal angiogenesis and lymphangiogenesis. Invest Ophthalmol
Vis Sci 48: 2545–2552.
Bonello S, Zähringer C, BelAiba RS, Djordjevic T, Hess J, Michiels C
et al. (2007). Reactive oxygen species activate the HIF-1alpha
promoter via a functional NFkappaB site. Arterioscler Thromb Vasc
Biol 27: 755–761.
Figure 8
Proposed modes of action of LCB54-0009. LCB54-0009 inhibits ROS-
mediated signalling cascades, resulting in the inhibition of HIF-1α,
TLR/NF-κB and VEGF/VEGFR-2 signalling pathways. LCB54-0009 also
inhibits VEGF-induced Ang-1/Tie-2 signalling cascades. LCB54,
LCB54-0009.
Cao Y (2005). Opinion: emerging mechanisms of tumour
lymphangiogenesis and lymphatic metastasis. Nat Rev Cancer 5:
735–743.
Cao Z, Shang B, Zhang G, Miele L, Sarkar FH, Wang Z et al. (2013).
Tumor cell-mediated neovascularization and lymphangiogenesis
contrive tumor progression and cancer metastasis. Biochim Biophys
Acta 1836: 273–286.
effect of LCB54-0009 in the treatment of neovascular eye
diseases that are mediated by pathological angiogenesis and
lymphangiogenesis.
Carmeliet P, Jain RK (2000). Angiogenesis in cancer and other
diseases. Nature 407: 249–257.
Acknowledgements
Castelli MV, Butassi E, Monteiro MC, Svetaz LA, Vicente F,
Zacchino SA (2014). Novel antifungal agents: a patent review (2011
– present). Expert Opin Ther Pat 24: 323–338.
This study was supported by a grant from the Future-Based
Technology Development program through the National
Research Foundation (NRF) of Korea funded by the Ministry
of Education, Science, and Technology (2012–0006131).
Connor KM, Krah NM, Dennison RJ, Aderman CM, Chen J, Guerin
KI et al. (2009). Quantification of oxygen-induced retinopathy in
the mouse: a model of vessel loss, vessel regrowth and pathological
angiogenesis. Nat Protoc 4: 1565–1573.
Cueni LN, Detmar M (2006). Insights into the molecular control of
the lymphatic vascular system and its role in disease. J Invest
Dermatol 126: 2167–2177.
Author contributions
B. H. Kim and T. Y. Kim participated the hypothesis and
design; B. H. Kim, J. Lee, J. S. Choi and D. Y. Park performed
the experiments; H. Y. Song and T. K. Park synthesized com-
pounds; C. H. Cho, S. K. Ye, C. K. Joo, G. Y. Koh and T. Y. Kim
provided experimental materials and reagents; B. H. Kim, J.
Lee, J. S. Choi and T. Y. Kim analysed the data, discussion of
the results and wrote the manuscript.
Duan YT, Wang ZC, Sang YL, Tao XX, Zhu HL (2013). Exploration
of structure-based on imidazole core as antibacterial agents. Curr
Top Med Chem 13: 3118–3130.
Fagiani E, Christofori G (2013). Angiopoietins in angiogenesis.
Cancer Lett 328: 18–26.
Felcht M, Luck R, Schering A, Seidel P, Srivastava K, Hu J et al.
(2012). Angiopoietin-2 differentially regulates angiogenesis through
TIE2 and integrin signaling. J Clin Invest 122: 1991–2005.
Ferrara N, Gerber HP, LeCouter J (2003). The biology of VEGF and
its receptors. Nat Med 9: 669–676.
Conflict of interest
The authors declare no competing financial interest in rela-
tion to the work described.
Fradette C, Batonga J, Teng S, Piquette-Miller M, du Souich P
(2007). Animal models of acute moderate hypoxia are associated
with a down-regulation of CYP1A1, 1A2, 2B4, 2C5, and 2C16 and
up-regulation of CYP3A6 and P-glycoprotein in liver. Drug Metab
Dispos 35: 765–771.
Frank RN (2004). Diabetic retinopathy. N Engl J Med 350: 48–58.
References
Gao P, Zhang H, Dinavahi R, Li F, Xiang Y, Raman V et al. (2007).
HIF-dependent antitumorigenic effect of antioxidants in vivo.
Cancer Cell 12: 230–238.
Adams RH, Alitalo K (2007). Molecular regulation of angiogenesis
and lymphangiogenesis. Nat Rev Mol Cell Biol 8: 464–478.
British Journal of Pharmacology (2015) 172 3875–3889 3887

B-H Kim et al.
BJP
Gordan JD, Simon MC (2007). Hypoxia-inducible factors: central
regulators of the tumor phenotype. Curr Opin Genet Dev 17:
71–77.
Lee JW, Bae SH, Jeong JW, Kim SH, Kim KW (2004).
Hypoxia-inducible factor (HIF-1)alpha: its protein stability and
biological functions. Exp Mol Med 36: 1–12.
Görlach A, Bonello S (2008). The cross-talk between NF-kappaB and
HIF-1: further evidence for a significant liaison. Biochem J 412:
e17–e19.
Majmundar AJ, Wong WJ, Simon MC (2010). Hypoxia-inducible
factors and the response to hypoxic stress. Mol Cell 40: 294–309.
Maxwell PH, Wiesener MS, Chang GW, Clifford SC, Vaux EC,
Cockman ME et al. (1999). The tumour suppressor protein VHL
targets hypoxia-inducible factors for oxygen-dependent proteolysis.
Nature 399: 271–275.
Görlach A, Diebold I, Schini-Kerth VB, Berchner-Pfannschmidt U,
Roth U, Brandes RP et al. (2001). Thrombin activates the
hypoxia-inducible factor-1 signaling pathway in vascular smooth
muscle cells: role of the p22(phox)-containing NADPH oxidase. Circ
Res 89: 47–54.
McGrath J, Drummond G, McLachlan E, Kilkenny C, Wainwright C
(2010). Guidelines for reporting experiments involving animals: the
ARRIVE guidelines. Br J Pharmacol 160: 1573–1576.
Grimm C, Willmann G (2012). Hypoxia in the eye: a two-sided
coin. High Alt Med Biol 13: 169–175.
Nelson DM, Smith SD, Furesz TC, Sadovsky Y, Ganapathy V, Parvin
CA et al. (2003). Hypoxia reduces expression and function of
system A amino acid transporters in cultured term human
trophoblasts. Am J Physiol Cell Physiol 284: C310–C315.
Grozdanov V, Müller A, Sengottuvel V, Leibinger M, Fischer D
(2010). A method for preparing primary retinal cell cultures for
evaluating the neuroprotective and neuritogenic effect of factors on
axotomized mature CNS neurons. Curr Protoc Neurosci Chapter 3
(Unit 3.22): 3.22.1–3.22.10.
Norrmén C, Tammela T, Petrova TV, Alitalo K (2011). Biological
basis of therapeutic lymphangiogenesis. Circulation 123:
1335–1351.
Haddad JJ, Land SC (2001). A non-hypoxic, ROS-sensitive pathway
mediates TNF-alpha-dependent regulation of HIF-1alpha. FEBS Lett
505: 269–274.
Olsson AK, Dimberg A, Kreuger J, Claesson-Welsh L (2006). VEGF
receptor signalling – in control of vascular function. Nat Rev Mol
Cell Biol 7: 359–371.
Haddad JJ, Olver RE, Land SC (2000). Antioxidant/pro-oxidant
equilibrium regulates HIF-1alpha and NF-kappa B redox sensitivity.
Evidence for inhibition by glutathione oxidation in alveolar
epithelial cells. J Biol Chem 275: 21130–21139.
Park SY, Jang WJ, Yi EY, Jang JY, Jung Y, Jeong JW et al. (2010).
Melatonin suppresses tumor angiogenesis by inhibiting HIF-1alpha
stabilization under hypoxia. J Pineal Res 48: 178–184.
Hagen T (2012). Oxygen versus reactive oxygen in the regulation of
HIF-1α: the balance tips. Biochem Res Int 2012: 436981.
Pawson AJ, Sharman JL, Benson HE, Faccenda E, Alexander SP,
Buneman OP et al.; NC-IUPHAR. (2014) The IUPHAR/BPS Guide to
PHARMACOLOGY: an expert-driven knowledgebase of drug targets
and their ligands. Nucl Acids Res 42 (Database Issue):
D1098–D1106.
Holmes K, Roberts OL, Thomas AM, Cross MJ (2007). Vascular
endothelial growth factor receptor-2: structure, function,
intracellular signalling and therapeutic inhibition. Cell Signal 19:
2003–2012.
Kadam RS, Ramamoorthy P, LaFlamme DJ, McKinsey TA, Kompella
UB (2013). Hypoxia alters ocular drug transporter expression and
activity in rat and calf models: implications for drug delivery. Mol
Pharm 10: 2350–2361.
Regenfuss B, Bock F, Cursiefen C (2012). Corneal angiogenesis and
lymphangiogenesis. Curr Opin Allergy Clin Immunol 12: 548–554.
Richard DE, Berra E, Pouyssegur J (2000). Nonhypoxic pathway
mediates the induction of hypoxia-inducible factor 1alpha in
vascular smooth muscle cells. J Biol Chem 275: 26765–26771.
Ke Q, Costa M (2006). Hypoxia-inducible factor-1 (HIF-1). Mol
Pharmacol 70: 1469–1480.
Rivers EC, Mancera RL (2008). New anti-tuberculosis drugs with
novel mechanisms of action. Curr Med Chem 15: 1956–1967.
Kelly BD, Hackett SF, Hirota K, Oshima Y, Cai Z, Berg-Dixon S et al.
(2003). Cell type-specific regulation of angiogenic growth factor
gene expression and induction of angiogenesis in nonischemic
tissue by a constitutively active form of hypoxia-inducible factor 1.
Circ Res 93: 1074–1081.
Roskoski R Jr (2007). Vascular endothelial growth factor (VEGF)
signaling in tumor progression. Crit Rev Oncol Hematol 62:
179–213.
Kietzmann T, Görlach A (2005). Reactive oxygen species in the
control of hypoxia-inducible factor-mediated gene expression.
Semin Cell Dev Biol 16: 474–486.
Sapieha P, Joyal JS, Rivera JC, Kermorvant-Duchemin E, Sennlaub F,
Hardy P et al. (2010). Retinopathy of prematurity: understanding
ischemic retinal vasculopathies at an extreme of life. J Clin Invest
120: 3022–3032.
Kilkenny C, Browne W, Cuthill IC, Emerson M, Altman DG (2010).
Animal research: Reporting in vivo experiments: the ARRIVE
guidelines. Br J Pharmacol 160: 1577–1579.
Sceneay J, Liu MC, Chen A, Wong CS, Bowtell DD, Möller A (2013).
The antioxidant N-acetylcysteine prevents HIF-1 stabilization under
hypoxia in vitro but does not affect tumorigenesis in multiple breast
cancer models in vivo. PLoS ONE 8: e66388.
Kim Y, Kim BH, Lee H, Jeon B, Lee YS, Kwon MJ et al. (2011).
Regulation of skin inflammation and angiogenesis by EC-SOD via
HIF-1α and NF-κB pathways. Free Radic Biol Med 51: 1985–1995.
Semenza GL (2007). Life with oxygen. Science 318: 62–64.
Kong TT, Zhang CM, Liu ZP (2013). Recent developments of p38α
MAP kinase inhibitors as antiinflammatory agents based on the
imidazole scaffolds. Curr Med Chem 20: 1997–2016.
Semenza GL (2013). Cancer-stromal cell interactions mediated by
hypoxia-inducible factors promote angiogenesis,
lymphangiogenesis, and metastasis. Oncogene 32: 4057–4063.
Konisti S, Kiriakidis S, Paleolog EM (2012). Hypoxia – a key
regulator of angiogenesis and inflammation in rheumatoid arthritis.
Nat Rev Rheumatol 8: 153–162.
Semenza GL (2014). Oxygen sensing, hypoxia-inducible factors, and
disease pathophysiology. Annu Rev Pathol 9: 47–71.
Lee J, Kim KE, Choi DK, Jang JY, Jung JJ, Kiyonari H et al. (2013).
Angiopoietin-1 guides directional angiogenesis through integrin
αvβ5 signaling for recovery of ischemic retinopathy. Sci Transl Med
5: 203ra127.
Simon MP, Tournaire R, Pouyssegur J (2008). The angiopoietin-2
gene of endothelial cells is up-regulated in hypoxia by a HIF
binding site located in its first intron and by the central factors
GATA-2 and Ets-1. J Cell Physiol 217: 809–818.
3888 British Journal of Pharmacology (2015) 172 3875–3889

Anti-angiogenic and anti-lymphangiogenic activities of LCB54-0009
BJP
Song HY, Kim SJ, Park SH, Lee HS, Park TK, Woo SH et al. (2012).
ured using an ophthalmic microscope with a digital camera.
Avastin was used as a positive control. *P < 0.05 and **P <
0.005 versus control rats. Among the compounds, LCB54-
0009 exhibited the strongest suppression of retinal vascular
permeability and corneal neovascularization in vivo, suggest-
ing a potential candidate molecule of LCB54-0009 on patho-
logical angiogenesis.
Imidazole-based alkaloid derivatives which have angiogenesis
inhibition and antioxidant effects and production method thereof.
PCT/KR/003103.
Takagi H, King GL, Aiello LP (1998). Hypoxia upregulates glucose
transport activity through an adenosine-mediated increase of
GLUT1 expression in retinal capillary endothelial cells. Diabetes 47:
1480–1488.
Figure S6 Antioxidant activity of imidazole-based alkaloid
derivatives. (A) DPPH radical (black bars) and peroxynitrite
(grey bars) scavenging activities of each compound were
measured by absorbance using a microplate reader. Data are
represented as the inhibition % at 30 μM (DPPH) or 50 μM
(peroxynitrite), and the values in the bars indicate IC50
values. (B and C) DPPH radical (B) and peroxynitrite (C)
scavenging activities of LCB54-0007, LCB54-0009 and
LCB54-0012 were represented. Data are represented as the
mean SD of three independent experiments (n = 3).
Figure S7 Inhibition of the HIFs expression by imidazole-
based alkaloid derivatives. (A) ARPE-19 (top) and primary
retinal cells (down) were incubated with various concentra-
tions of LCB54-0009 for 24 to 72 h. Cell viability was meas-
ured using the WST-1 reagent and the data are represented as
the mean SD of three independent experiments (n = 3). B.
HUVECs were incubated with each compound (20 μM) for
6 h in hypoxic conditions and Western blot analysis was
performed. Among the compounds, LCB54-0009 exhibited
the strongest inhibition of HIF-1α expression. (C) ARPE-19
and primary retinal cells were incubated with various con-
centrations of LCB54-0009 for 6 h in hypoxic conditions. The
levels of the HIF-1α and HIF-2α were determined by Western
blot analysis. GAPDH served as a loading control.
Tammela T, Alitalo K (2010). Lymphangiogenesis: molecular
mechanisms and future promise. Cell 140: 460–476.
Wasa M, Wang HS, Shimizu Y, Okada A (2004). Amino acid
transport is down-regulated in ischemic human intestinal epithelial
cells. Biochim Biophys Acta 1670: 49–55.
Yuan HT, Yang SP, Woolf AS (2000). Hypoxia up-regulates
angiopoietin-2, a Tie-2 ligand, in mouse mesangial cells. Kidney Int
58: 1912–1919.
Zelzer E, Levy Y, Kahana C, Shilo BZ, Rubinstein M, Cohen B
(1998). Insulin induces transcription of target genes through the
hypoxia-inducible factor HIF-1alpha/ARNT. EMBO J 17: 5085–5094.
Supporting information
Additional Supporting Information may be found in the
online version of this article at the publisher’s web-site:
http://dx.doi.org/10.1111/bph.13177
Figure S1 Chemical structures of the imidazole-based alka-
loid derivatives. Twelve derivatives were synthesized and des-
ignated as LCB54 series.
Figure S8 LCB54-0009 exhibits anti-angiogenic activity in
vitro. ARPE-19 (A) and primary retinal cells (B) were incubated
with either vehicle (0.1% DMSO) or LCB54-0009 (20 μM) for
6 h in hypoxic conditions or 72 h in the presence or absence
of VEGF (20 ng·mL−1). The mRNA levels were determined by
quantitative real-time PCR and the data are represented as the
mean SD of three independent experiments (n = 3). #P <
0.05 versus vehicle-treated group; *P < 0.05 and **P < 0.005
versus VEGF- or hypoxia-induced group; ††P < 0.005 versus
LCB54-0009-treated group.
Figure S2 Synthetic scheme of LCB54-0009. LCB54-0009
was synthesized from 3,4-dihydrobenzylaldehyde by sequen-
tial synthetic procedures. The core-structure of imidazole-
based alkaloid derivatives is shown in the box. The detailed
synthetic methods and properties of each compound were
referred in the Supporting Information and have been previ-
ously reported (Song et al., 2012).
Figure S3 1H-NMR spectra of the intermediate compound
alcohol I (A), ketone II (B), bromide III (C) and compound IV
(D).
Figure S9 LCB54-0009 inhibits iNOS expression. A. HUVECs
were incubated with either vehicle (0.1% DMSO) or LCB54-
0009 (20 μM) for 6 h in hypoxic conditions. The mRNA levels
of PHD1 and PHD2 were determined by quantitative real-
time PCR and the data are represented as the mean SD of
three independent experiments (n = 3). #P < 0.05 versus
vehicle-treated group. B. HUVECs were incubated with
LCB54-0009 in combination with stimuli (LPS 1 μg·mL−1,
TNF-α 10 ng·mL−1, IL-1β 5 U·mL−1 and IFN-γ 100 U·mL−1) for
36 h. Western blot analysis was performed to determine iNOS
expression. GAPDH served as a loading control. C. In vitro
peroxynitrite scavenging activity was measured as absorbance
at 542 nm, followed by addition with Pyrogallol Red.
Table S1 The primer sequences and product sizes used for
end-point RT-PCR.
Figure S4 LC-MS spectra of the intermediate compound V
1
(A) and H-NMR (B), 13C-NMR (C) and LC-MS (D) spectra of
the LCB54-0009.
Figure S5 In vivo anti-angiogenic activity of imidazole-based
alkaloid derivatives. (A) Eight-week-old SD male rats were
injected intravitreally with streptozotocin (60 mg·kg−1) to
induce experimental diabetic retinopathy and retinal blood
vessel leakage was measured by extravasated FITC-dextran
staining using fluorescence microscopy, followed by injection
with either vehicle (1% DMSO) or each compound (100 μM)
into the vitreous body. (B) Corneal neovascularization was
induced by the subconjunctival injection of silver nitrate to
cauterize the corneas of SD rats. Vehicle (1% DMSO), each
compound (50 μg) or Avastin (200 μg) was injected subcon-
junctivally 3 days after silver nitrate cauterization and meas-
British Journal of Pharmacology (2015) 172 3875–3889 3889