Chinese Medicine, 2010, 1, 75-83
doi:10.4236/cm.2010.13015 Published Online December 2010 (http://www.SciRP.org/journal/cm)
Inhibitory Actions of Tetrandrine on Tumor Necrosis
Factor -Induced NF-B Activation in Neovascularization
of Cultured Choroidal Explants*
Minoru Kikuchi1, Shusuke Kamimura1, Masaaki Nomura1, Tatsuo Takahashi1,
Nobuyoshi Hagino2, Shinjiro Kobayashi1
1Department of Clinical Pharmacy, Faculty of Pharmaceutical Sciences, Hokuriku University,
3-Ho Kanagawa-Machi, Kanazawa, Japan
2Laboratory of Integrative Medicine, US-Japan Cooperative Biomedical Research Laboratories,
Tulane University Herbert Research Center and Department of Medicine, Tulane University Health
Science Center, School of Medicine, Main Street, Belle Chasse, USA.
Received October 13, 2010; revised November 30, 2010; accepted December 2, 2010
Abstract
Tetrandrine (1 μM), a bis-benzylisoquinoline alkaloid isolated from Stephania tetrandra S Moore, signifi-
cantly decreased tumor necrosis factor alpha (TNFα; 10 ng/ml)-induced increase in the number of micro
vessels that budded from cultured rat choroidal explants. Tetrandrine also decreased the TNFα-induced in-
crease in the number of cells composing the microvessels. Ammonium pyrrolidine dithiocarbamate (APDC;
0.1-0.3 μM), an inhibitor of nuclear factor-B (NF-κB), decreased the TNFα-induced increase in the number
of microvessels in a concentration-dependent manner. TNF increased the phosphorylation and degradation
of inhibitor of NF-κB (IκBα), as well as increasing the DNA-binding activity of NF-B in choroidal explants.
TNF induced an increase of vascular endothelial growth factor (VEGF)-A mRNA, but not VEGF-C mRNA
or VEGF-D mRNA. TNF-induced angiogenic action was inhibited by treatment of VEGF-A antibody in
cultured choroidal capillaries. Tetrandrine inhibited the TNFα-induced increases of phosphorylation and
degradation of IκBα, and reduced the TNFα-induced increase of DNA-binding activity of NF-κB in chor-
oidal explants. In conclusion, tetrandrine inhibits TNF-induced activation of NF-B in the choroidal capil-
laries via inhibition of TNFα-induced phosphorylation of IκBα.
Keywords: Choroidal Neovascularization, Anti-angiogenesis, Tetrandrine, Tumor Necrosis Factor α, NF-κB
Activity, Phosphorylation of IκBα
1. Introduction
The bis-benzylisoquinoline alkaloid tetrandrine is a
component of Stephania tetrandra S. Moore, which is a
herb used in traditional Chinese medicine to treat pa-
tients with silicosis, asthma, hypertension and arthritis
[1-4]. This alkaloid has been shown to be effective in
experimental models of allergic encephalitis, airway mi-
crovascular leakage, subcutaneous air pouch inflamma-
tion, granuloma angiogenesis in chronic inflammation
and proliferation of synovial cells [5-8]. Tetrandrine in-
hibits the production and release of a broad range of in
lammatory mediators and cytokines [9]. It has been re-
ported to block voltage-dependent L-type and T-type
Ca2+ channels in various cells, including retinal cells [10],
and to inhibit nitric oxide (NO) production in endothelial
cells by blocking the Ca2+ release-activated Ca2+ channel
[11]. Tetrandrine also inhibits hypoxia-induced neovas-
cularization of the small pulmonary artery [12], protects
islet beta cells from alloxan-induced injury [13], and
prevents spontaneous development of diabetes mellitus
in BB rats [14]. Moreover, it has been proposed that Ca2+
flux and NO production in endothelial cells are associ-
*A grant (to SK) for the “Academic Frontier” Project for Private Uni-
versities (2005-2009) from the Ministry of Education, Culture, Sports,
Science and Technology of Japan.

76 M. KIKUCHI ET AL.
ated with angiogenesis induced by vascular endothelial
growth factor (VEGF) [15-16]. Administration of tetran-
drine suppresses neovascularization of the choroidal cap-
illaries, which is facilitated by VEGF and platelet-de-
rived growth factor (PDGF)-B in culture [17]. Choroidal
tissue in streptozotocin (STZ)-induced diabetic rats ac-
cumulates N-(carboxymethyl) lysine (CML), one of the
advanced glycation end products (AGEs) (unpublished
data). CML facilitates neovascularization of choroidal
capillaries in culture [18-21]. Tetrandrine inhibits dia-
betic state-induced overproduction of choroidal and reti-
nal neovascularization in culture [17,22,23].
Tumor necrosis factor (TNF) is proinflammatory
cytokine that has been implicated in the pathogenesis of
diabetic retinopathy [24,25]. It is found in the extracellu-
lar matrix, endothelium and vessel walls of fibrovascular
tissue in the eyes of patients with proliferative diabetic
retinopathy [25] and vitreous humor from eyes of pa-
tients with diabetic complications [26]. This cytokine has
been utilized as a potent promoting factor in in vivo and
in vitro angiogenesis models [27-29]. TNF can induce
the expression of many important immune-and angio-
genesis-related genes through TNF receptors. TNF
signaling is required for vessel development in ische-
mia-induced neovascularization [30]. The increased ex-
pression of many inflammatory proteins is mediated at
the level of gene transcription through the activation of
proinflammatory transcription factors, including nuclear
factor kappa B (NF-B) [31].
In the present study, we investigated the effect of
tetrandrine on TNF-induced cell signaling pathways in
neovascularization of choroidal capillaries.
2. Materials and Methods
2.1. Materials
The chemical structure of tetrandrine is shown in Figure
1. Tetrandrine, fibrinogen and thrombin were from
Sigma (St. Louis, MO, USA); -aminocaproic acid, di-
methyl sulfoxide, collagenase and ISOGEN were from
Wako Pure Chemical Industries Ltd. (Osaka, Japan),
dNTPs, RNase inhibitor, M-MLV reverse transcriptase,
fetal bovine serum (FBS), oligo dT12-18 primer and Taq
Figure 1. Chemical structure of tetrandrine.
DNA polymerase were from Invitrogen (Carlsbad, CA,
USA); anti-IB antibody and anti-phospho-IB anti-
body were from Cell Signaling Technology, Inc. (Bev-
erly, MA, USA); TNF and anti-VEGF were from R&D
Systems (Minneapolis, MN, USA); ECL Plus western
blotting detection system was from GE Health Biosci-
ences (Piscataway, NJ, USA); probes for NF-B was
from Promega K. K. (Tokyo, Japan); Dulbecco’s modi-
fied Eagle’s medium (DMEM) was from Nissui (Tokyo);
penicillin/streptomycin was from Lonza (Walkersville,
MD, USA); qPCR Mastermix for SYBR® Green I was
from Eurogentec (San Diego, CA, USA); ammonium
pyrrolidine dithiocarbamate (APDC) was purchased from
Dojin (Osaka).
2.2. Culture of Choroidal Tissues
All of the animal experiments were performed in accor-
dance with the guidelines of the Committee on Animal
Experiments in Hokuriku University. Male Wistar rats
(7-9 weeks of age) were purchased from Nippon SLC
(Shizuoka, Japan). The rats were anaesthetized and killed,
and eyeballs were rapidly isolated under aseptic condi-
tions. Choroidal explants were isolated under an optical
microscope as previously reported [18-20,32]. The size
of isolated explants was approximately 0.16 mm2. The
explants were plated on fibrin gels prepared by mixing 3
mg fibrinogen (0.3 ml), and 1 U thrombin per ml DMEM
containing 30 U/ml penicillin/30 g/ml streptomycin in
24-well plates. Equal volumes of a mixture of the above
concentrations of fibrinogen and thrombin were overlaid
and allowed to solidify. The explants were cultured with
1% FBS-DMEM (0.5 ml) containing 30 U/ml penicillin/
30 g/ml streptomycin and 300 g/ml -aminocaproic
acid in a CO2 incubator at 37°C for 8 days. In some ex-
periments, tetrandrine (0.1 and 1 M), APDC (0.1 and
0.3 M), anti-VEGF antibody (0.3 g/ml) or TNF (10
ng/ml) were added to tissue culture.
2.3. Assessment of Neovascularization
The number of micro vessel-like structures newly bud-
ded from choroidal explants cultured as described in the
previous section was counted under a CKS microscope
equipped with a camera (Olympus, Tokyo). The mi-
crovessel-like structures have been demonstrated previ-
ously to have the features of immature microvessels,
consisting of vascular endothelial progenitor cells, such
as angioblasts [18]. The number of microvessel-like
structures per explant was used as an index of in vitro
neovascularization.
Choroidal tissue from eyeballs of male Wistar rats
(7-9 weeks of age) was sampled with a biopsy punch (1

M. KIKUCHI ET AL.
77
mm; Kai Industries Co. Ltd., Gifu, Japan). The choroidal
explants (0.785 mm2) were cultured in fibrin gel with 1%
FBS-DMEM containing antibiotics and -aminocaproic
acid in a CO2 incubator at 37°C for 8 days as described
in the previous section. The primary choroidal explants
were removed, and microvessel-like structures were di-
gested with 0.75% collagenase (1 ml) in phosphate-
buffered saline (PBS) in a CO2 incubator at 37°C for 30
min. The digested cells were transferred to 1.5 ml cen-
trifuge tubes, and centrifuged at 16,000 xg for 5 min.
Total cell number was counted using a Burker-Turk
blood cell counting plate (Erma Inc., Tokyo) under a
CKZ microscope (Olympus).
2.4. DNA Binding Activity of NF-κB
Choroidal tissues isolated from eyeballs of male Wistar
rats (7-9 weeks of age) were cut into pieces of approxi-
mately 0.16 mm2, and incubated in 1% FBS-DMEM con-
taining TNF (10 ng/ml) with or without tetrandrine (1
M) for 120 min in a CO2 incubator at 37°C. The chor-
oidal tissues were lysed with 50 l of buffer A (10 mM
HEPES; pH 7.9, 10 mM KCl, 0.1% Triton X-100, 50 M
DTT, 1.5 mM MgCl2) for 15 min on ice. After centrifuga-
tion at 250 xg for 5 min at 4°C, 20 l of buffer A was
added to the pellets, and the mixture was incubated for 5
min on ice and centrifuged again at 8,000xg for 20 min at
4°C. The pellets were incubated in buffer B (20 mM
HEPES; pH 7.9, 420 mM NaCl, 1% CA-630, 25% glyc-
erol, 500 M DTT, 1.5 mM MgCl2, 0.2 mM EDTA) for
60 min on ice. After centrifugation at 16,000 xg for 5 min
at 4°C, the supernatants were harvested as nuclear protein
extracts and adjusted to 1 mg/ml proteins. The DNA
binding reaction was performed with 3 g of nuclear pro-
tein and 15,000 cpm of a 32P-labeled oligonucleotide con-
taining NF-B-binding element (5’-AGT TGA GGG
GAC TTT CCC AGC C-3’; Promega) as described previ-
ously [33,34]. The samples were separated on 5% poly-
acrylamide gels, which were analyzed with a Typhoon
9410 image analyzer (Amersham Biosciences).
2.5. Western Blotting
Choroidal tissues isolated from eyeballs of male Wistar
rats (7-9 weeks of age) were cut into pieces of ap-
proximately 0.16 mm2, and incubated in 1%
FBS-DMEM containing TNF (10 ng/ml) with or
without tetrandrine (1 M) for 5 min in a CO2 incuba-
tor at 37°C. The choroidal tissues were extracted with
200 l of triple-deter-gent buffer (50 mM Tris-HCl; pH
8.0, 150 mM NaCl, 0.02% sodium azide, 0.1% sodium
dodecyl sulfate (SDS), 100 g/ml phenylmethylsul-
fonyl fluoride (PMSF), 1 g/ml aprotinin, 1% Nonidet
P-40, 0.5% sodium deoxycholate for 30 min on ice,
and homogenized using an UltraS Homogenizer VP-5S
(Taitec, Saitama, Japan). After centrifugation at 16,000
xg for 5 min at 4°C, the supernatants were harvested as
samples for Western blotting; results were normalized
with respect to untreated controls. Western blotting
analysis was performed using antibodies against IB
and its phosphorylated proteins as described previously
[33,34]. Antibody-bound proteins were detected using
an ECL-Plus Western Blotting Kit (GE Health Biosci-
ences) and analyzed with a Typhoon 9410 image ana-
lyzer.
2.6. Real-Time PCR
Total RNAs from the cultured choroidal explants were
extracted using ISOGEN according to the manufac-
turer’s instructions. Then, reverse transcription reaction
was performed for 1 h at 37°C in a final volume of 20 l,
containing 3 g of total RNA (previously denatured for
10 min at 65°C), 0.5 mM dNTPs, 25 ng/ml oligo dT12-18
primer and 200 units of M-MLV reverse transcriptase.
Real-time PCR was optimized with each set of oligonu-
cleotide primers, which were designed for rat VEGF-A
(Gene Bank accession No. NM_031836; forward, 5’-
GCA CTG GAC CCT GGC TTT ACT-3’; reverse, 5’-
CGC TGG TAG ACG TCC ATG AA-3’; amplicon size
119 bp), rat VEGF-C (Gene Bank accession No.
NM_053653; forward, 5’-CAC AAT CAG TTT TGC
CAA TCA CA-3’; reverse, 5’-CAC ACG TAG TTT
GCT GGA CAA GTC-3’; amplicon size 127 bp), rat
VEGF-D (Gene Bank accession No. AY032728; forward,
5’-CAC CGA GCA GTG AAG GAT GTT-3’; reverse,
5’-CAG TCC TCA GAG TGT GCG ACT T-3’; ampli-
con size 124 bp) and rat -actin (Gene Bank accession
No. NM_031144; forward, 5’-AGG GAA ATC GTG
CGT GAC AT-3’; reverse, 5’-GAA CCG CTC ATT
GCC GAT AG-3’; amplicon size 149 bp). Primers were
designed with the help of the software Primer Express
Ver. 3.0 (Applied Biosystems Japan, Tokyo). The reac-
tions were performed using qPCR Mastermix for
SYBR® Green I in a 25 l reaction volume containing
10 l of cDNA, 10 pmol of each primer and 12.5 l of
the 2x Mastermix in an ABI PRISM 7500 (Applied
Biosystems Japan). The program profile used for ampli-
fication was: 50°C for 2 min (stage 1), 95°C for 10 min
(stage 2), 40 cycles of denaturation at 95°C for 15 sec
and annealing at 65°C for 1 min (stage 3), 95°C for 15
sec (stage 4), 60°C for 1 min (stage 5), and 95°C for 15
sec (stage 6). The real-time amplified PCR products were
checked on polyacrylamide gel to demonstrate that, un-
der the specified real-time conditions, the expected am-
p
licon sizes are produced.

M. KIKUCHI ET AL.
78
Figure 2. Effect of tetrandrine on the increase in the number of microvessels induced by TNFα in rat choroidal explants. a,
The choroidal explants were cultured in the presence or absence of TNFα (10 ng/ml) and/or tetrandrine (1 μM) for 8 days. b,
The explants were incubated with tetrandrine (0, 0.1 and 1 μM) in the presence (right) or absence (left) of TNFα (10 ng/ml)
on the 6th day in culture. Values are expressed as means ± S.E.M. of at least 3 data. #P < 0.05 vs. the corresponding control
without TNFα nor tetrandrine. *P < 0.05 vs. TNFα alone without tetrandrine.
3.2. Inhibitory Effect of APDC on
TNFα-Induced Increase of Choroidal
Neovascularization
2.7. Statistical Analysis
Values were expressed as means ± S.E.M. of at least 3
data. Statistical analysis was carried out using Stu-
dent’s t-test and Welch’s t-test at P = 0.05 or P = 0.01.
A value of P < 0.05 was considered statistically sig-
nificant.
To study the participation of NF-B activity in signal
transduction of TNF, the effect of APDC, an inhibitor
of NF-B, on TNF-induced choroidal neovasculariza-
tion in culture was investigated. APDC (0.1-0.3 M)
significantly reduced the TNF (10 ng/ml)-induced in-
crease in the number of microvessels that budded from
choroidal capillaries from the 4th day in culture, and the
inhibitory action was in a concentration-dependent man-
ner. However, APDC did not affect the number of mi-
crovessels in the absence of TNF (Figure 4). This
3. Results
3.1. Inhibitory Effects of Tetrandrine on
TNFα-Induced Increase of Choroidal
Neovascularization and Cell Proliferation
in Choroidal Microvessels in Culture
We have previously reported that TNF increased ne-
ovascularization of cultured choroidal explants in nor-
mal Wistar rat (Kobayashi et al., 2005). Tetrandrine (1
M) in Figure 1 suppressed 10 ng/ml TNF-induced
increase in the number of microvessels that budded
from choroidal capillaries from the 4th day in culture,
and this inhibitory action was in a concentration-de-
pendent manner (Figure 2). TNF (10 ng/ml) increased
the number of cells in microvessels of choroidal ex-
plants at the 8th day in culture (Figure 3). Tetrandrine
(1 M) completely reduced the TNF-induced increase
in the number of cells in microvessels budded on the 8th
day in culture to the levels of control with tetrandrine
(Figure 3). However, tetrandrine (1 M) did not sig-
nificantly affect the number of microvessels (Figure 2)
and cell number of microvessels (Figure 3) in the ab-
sence of TNF, respectively.
Figure 3. Effect of tetrandrine on the increase of the cell
number in microvessels induced by TNFα in rat choroidal
explants. Microvessels that budded from the explants in the
presence or absence of TNFα (10 ng/ml) and/or tetrandrine
(1 μM) for 8 days in culture were digested with collagenase
for 30 min. The cell number in the microvessels per explant
was counted. Values are expressed as means ± S.E.M. of at
least 3 data. #P < 0.05 vs. control without agents, *P < 0.05
s. TNFα alone without tetrandrine. v

Chinese Medicine, 2010, 1, 75-83
doi:10.4236/cm.2010.13015 Published Online December 2010 (http://www.SciRP.org/journal/cm)
Figure 4. Effect of APDC on the increase in the number of microvessels induced by TNFα in rat choroidal explants. a, The
choroidal explants were cultured in the presence or absence of TNFα (10 ng/ml) and/or APDC (0.3 μM) for 8 days. b, The
explants were incubated with APDC (0, 0.1 and 0.3 μM) in the presence (right) or absence (left) of TNFα (10 ng/ml) on the 6th
day in culture. Values are expressed as means ± S.E.M. of at least 3 data. # P < 0.05, ##P < 0.01 vs. the corresponding control
without TNFαor APDC. *P < 0.05, **P < 0.01 vs. TNFα alone without APDC.
effect of APDC showed a similar pattern to that of
tetrandrine.
3.3. Inhibitory Effect of Tetrandrine on the
TNFα-Induced Increase of DNA Binding
Activity of NF-κB
The effect of tetrandrine on the TNF-induced increase of
DNA binding activity of NF-B was investigated using
the nuclear fraction of choroidal explant incubated for 120
min. Tetrandrine (1 M) reduced the TNF (10 ng/ml)
-induced increase of DNA binding activity, though not to
the level seen in the absence of TNF (Figure 5). The
NF-B specific bands were confirmed by the competition
experiments with unlabeled NF-B binding oligonucleo-
tides (related competitor) and unlabeled AP-1 binding
oligonucleotides (unrelated competitor) (data not shown).
Figure 5. Effect of tetrandrine on the TNFα-induced in-
crease of DNA binding activity of NF-κB. The choroidal
explants were preincubated with or without tetrandrine (1
μM) for 30 min and then TNFα (10 ng/ml) was added for
120 min. DNA binding activity of NF-κB was determined by
means of gel shift assay.
3.4. Effect of Tetrandrine on the TNFα-Induced
Increases of Phosphorylation and
Degradation of IkBα
To study the mechanism of tetrandrine’s action on NF- B
activation by TNF, the effect of tetrandrine on phos-
phorylation of IB and on the content of IB in choroidal
capillary cells treated with TNF was investigated. TNF
(10 ng/ml) increased the content of phosphorylated IB
and decreased the content of total IB in the choroidal
explants. Tetrandrine (1 M) inhibited both actions of
TNF on phosphorylated IB and total IB (Figure 6).
3.5. Effect of Anti-VEGF Antibody on
TNFα-Induced Choroidal
Neovascularization
To examine whether production of VEGF is involved in
Figure 6. Effect of tetrandrine on the TNFα-induced increase
of phosphorylation and degradation of IkBα in choroidal
explants. The choroidal explants were preincubated with or
without tetrandrine (1 μM) for 30 min and then TNFα (10
ng/ml) was added for 30 min. The contents of phosphorylated
IκBα and total IκBα were determined by Western blotting.

80 M. KIKUCHI ET AL.
the TNF-induced increase of choroidal neovasculariza-
tion, the effect of anti-VEGF antibody on the neovascu-
larization was investigated in culture. Simultaneous
treatment of anti-VEGF antibody (0.3 g/ml) signifi-
cantly reduced the TNF (10 ng/ml) - induced increase
of choroidal neovascularization on the 6th and 8th days in
culture (Figure 7).
3.6. Expression of VEGF mRNA
during TNFα- Induced Choroidal
Neovascularization
The levels of mRNAs of VEGF-A, VEGF-C and VEGF-
D in choroidal explants were directly measured on the
6th day in culture by means of real-time PCR and nor-
malized by -actin mRNA levels. Expression of nor-
malized VEGF-A mRNA in choroidal explants was al-
most 5-time greater than those of VEGF-C and VEGF-D.
TNF (10 ng/ml) significantly increased expression of
VEGF-A mRNA 3-time greater. However, TNF unaf-
fected VEGF-C mRNA expression and decreased
VEGF-D mRNA expression in choroidal explants, re-
spectively (Table 1).
4. Discussion
The present study was designed to investigate the action
of tetrandrine on TNF-induced cell signaling associated
with neovascularization of choroidal capillaries in cul-
ture. Tetrandrine inhibited 1) the TNF-induced increase
of choroidal neovascularization (Figures 2 and 3), 2) the
TNF-induced increase of DNA binding activity of
Figure 7. Effect of anti-VEGF antibody on the TNFα-in-
duced increase in the number of microvessels in choroidal
explants in culture. The choroidal explants were cultured in
the presence or absence of TNFα (10 ng/ml) and/or anti-
VEGF antibody (0.3 μg/ml) for 8 days. Values are expressed
as means ± S.E.M. of at least 3 data. ##P < 0.01 vs. the cor-
responding control without TNFα or tetrandrine. *P < 0.05,
**P < 0.01 vs. TNFα alone without anti-VEGF antibody at
the corresponding day.
Table 1. mRNA Levels of VEGF-A, VEGF-C and VEGF-D
in choroidal explants cultured in the presence of TNFα.
VEGF-A VEGF-C VEGF -D
TNF 0.1664 ± 0.0237*0.0018 ± 0.0010 0.0008 ± 0.0005*
Control0.0531 ± 0.01130.0023 ± 0.0005 0.0025 ± 0.0014
The choroidal explants were cultured in fibrin gel with 1% FBS-DMEM
with or without TNFα (10 ng/ml) for 6 days. VEGF-A, VEGF-C and
VEGF-D mRNA levels ( △ C) in choroidal explants were determined by
means of the protocol for real-time reverse transcription- polymerase chain
reaction and normalized by β-actin mRNA levels. Values were expressed as
means ± S.E.M. of at least 3 data. *P < 0.05 vs. the corresponding control
without TNFα.
NF-B (Figure 5), 3) the TNF-induced increase of
phosphorylation of IB(Figure 6) and 4) the TNF-
induced increase of degradation of IB in choroidal
explants (Figure 6). These results indicate that tetran-
drine inhibits TNF-induced neovascularization through
NF-B inactivation in choroid. The site of action of
tetrandrine is upstream of TNF-induced phosphoryla-
tionin the process of NF-B activation. Therefore, tetran-
drinedecreases translocation of NF-B heterodimers into
the nucleus of choroidal capillary cells. This inhibitory
action of tetrandrine is similar to that of APDC in chor-
oidal neovascularization. APDC prevented release of IB
from NF-B in intact cells, but did not inhibit the DNA
binding activity in the nucleus in vitro or the inducibility
of AP-1 by PMA. APDC has at least two chemical prop-
erties that may be related to the inhibition of NF-B ac-
tivity: one is a chelating activity for heavy metals, and
the second is an antioxidant activity [35].
We have previously reported that nifedipine, an L-type
Ca2+ channel blocker, inhibits TNF-induced neovascu-
larization of choroidal capillaries [32]. Nifedipine also
inhibits basic fibroblast growth factor-induced prolifera-
tion of endothelial cells by causing cell-cycle arrest at
G0/G1 through blocking the Ca2+ channel [36]. There are
also evidences that nifedipine results in increased release
of NO [37-38]. It has been proposed that Ca2+ flux and
NO production in endothelial cells are associated with
angiogenesis induced by VEGF [15-16]. Tetrandrine also
blocks voltage-dependent L-type and T-type Ca2+ chan-
nels in various cells, including retinal cells [10], and in-
hibits NO production in endothelial cells by blocking
Ca2+ release-activated Ca2+ channels [11]. The action of
tetrandrine on TNF-induced cell signaling in neovas-
cularization may therefore be associated with the inhibi-
tion of NF-B activity through reducing Ca2+ flux and
NO production in choroidal capillaries. Angiogenic ac-
tion of TNF has been reported to be associated with

M. KIKUCHI ET AL.
81
increased expression of many immune-and angiogene-
sis-related genes, mediated by TNF receptors. TNF
signaling induces NF-B activation and regulates VEGF
expression and consequently VEGF- mediated neovas-
cularization [30,39,40]. On the contrary NF-B also in-
duces formation of vascular endothelial growth inhibitor
(VEGI), which induces apoptosis to proliferating cells
while enforcing a growth arrest in quiescent cells [41]. In
the present study, TNF accelerated cell proliferation in
newly formed microvessels, as well as number of mi-
crovessels budded from cultured choroidal capillaries
(Figures 2 and 3), indicating that TNF may induce ex-
pression of VEGF rather than expression of VEGI in our
angiogenesis model of choroidal explants. We have addi-
tional data that TNF (10 ng/ml) inhibits cleavage of
poly (ADP-ribose) polymerase by caspase-3 (data not
shown), supporting that TNF is angiogenic activator
but not inducer of apoptosis in the choroidal explants.
The angiogenic action of TNF on choroidal neovascu-
larization was inhibited by APDC, an inhibitor of NF-B
[35] (Figure 4), supporting that NF-B is involved in the
mechanism of TNF’s action on choroidal neovascu-
larization. The present investigations also indicated that
TNF facilitated phosphorylation of IBand degrada-
tion (decrease of content) of IB (Figure 6). The deg-
radation of IB results from release of NF-B het-
erodimers to translocate to the nucleus, where they bind
to nuclear DNA (Figure 5), leading to activation of spe-
cific subsets of angiogenic genes in the choroidal ex-
plants. We have now plans to identify cellular sources of
NF-B and I-B in our choroidal explant lysates.
The present study directly demonstrates that TNF
increases expression of mRNA of VEGF-A, but not
VEGF-C or VEGF-D in choroid (Table 1). Angiogenic
action of TNF was reduced by anti-VEGF-A antibody
in choroidal explant (Figure 7). These results indicate
that TNF stimulates the production and release of
VEGF-A via activation of NF-B in choroidal explant
and increases neovascularization. VEGF-C and VEGF-D
interact with VEGF receptor (VEGFR)-2 and VEGFR-3
and have a role for the growth of lymphatic vessels [42].
TNF did not increase mRNA of VEGF-C and VEGF-D,
suggesting that TNF does not increase lymph angio-
genesis. Tetrandrine (1 M) inhibited TNF-induced cell
signaling of neovascularization in cultured choroidal
explant. However, this concentration of tetrandrine did
not affect basal neovascularization or cell proliferation in
the absence of TNF (Figures 2 and 3). Tetrandrine (1
M) inhibits VEGF-induced neovascularization in chor-
oidal explant [17], indicating that tetrandrine reduces
both of TNF-induced release of VEGF-A and the action
of VEGF-A in neovascularization of choroidal explant.
5. Conclusion
Tetrandrine inhibits TNF-induced activation of NF-B
in the choroidal capillaries via inhibition of TNF-in-
duced phosphorylation of IBin choroidal neovascu-
larization.
6. Acknowledgements
This work was supported in part by a grant (to SK) for
the “Academic Frontier” Project for Private Universities
(2005-2009) from the Ministry of Education, Culture,
Sports, Science and Technology of Japan, Japan.
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