Cent. Eur. J. Biol. • 9(5) • 2014 • 469-475
DOI: 10.2478/s11535-014-0290-5
Central European Journal of Biology
Bevacizumab cured age-related macular
degeneration (AMD) via down-regulate TLR2
pathway
Research Article
Zhi Li Wang1#, Bao di Qiao2#, Guo Xing Li3, Shi Qing Li1, Li Ya Wang1, Ying Li Dong1*
Henan Eye Institute, Henan Eye Hospital,
Zhengzhou, 450003, China
1
The Second People’s Hospital of Zhengzhou,
Zhengzhou, 450006,China
2
The Eye Hospital of Wenzhou Medical University,
Wenzhou, China
3
Received 03 September 2013; Accepted 08 November 2013
Abstract: AMD is the main cause of visual impairment in people over 50 years of age and the most common cause of blindness. In recent years, the
use of bevacizumab to treat neovascular AMD has become a preferred treatment in the United States. However, whether bevacozumab
is available for RPE or AMD patients is unknown. We firstly indicate that Pam3CSK4 (P3C) activates TLR2 pathway during ARPE-19
apoptosis as determined by western blotting. And then, the expression of MyD88, NF-κB, p-IKK in primary RPE cells from AMD patients is
significantly down-regulated after treatment with 50 μg L-1 Bevacizumab. Therefore, our data shows that MyD88 is involved in the TLR2
pathway in ARPE-19 cell apoptosis resulting from Pam3CSK4 (P3C). And more importantly, our findings suggested that Bevacizumab
cured age-related macular degeneration (AMD) via down-regulate Toll—like receptor 2 (TLR2) pathway in RPE from AMD patients.
Keywords: Bevacizumab • AMD • TLR2 • RPE
© Versita Sp. z o.o.
Abbreviations
AMD
RPE
TLR2
P3C
- Age-related macular degeneration
- Retinal pigment epithelium
- The mammalian toll like receptor 2
- Pam3CSK4
1. Introduction
Age-related macular degeneration (AMD) is the most
common cause of blindness and visual impairment in
people over 50 years of age [1,2]. During the pathological
progress of AMD, retinal pigment epithelium (RPE) plays
a very important role in choroidal neovascularisation
(CNV, also called wet AMD) for atrophy of the PRE
resulting from deposits called drusen that form between
the retinal pigment epithelium (RPE) and underlying
choroid [3]. To date, it is commonly proposing that
* E-mail: yinglidong2013@126.com
#
These authors contributed equally to this work.
oxidative damage is involving in RPE dysfunction in
AMD, leading to subepithelial deposits and inflammation
in RPE/choroid interface [4-6]. However, further studies
on the mechanism of RPE dysfunction in AMD are
required.
Bevacizumab (Avastin; Genentech, Inc., South
San Francisco, CA) is a full-length humanized antiVEGF monoclonal IgG1 antibody (molecular weight
149 kDa) that inhibited the action of all isoforms of
vascular endothelial growth factor. The drug was
initially developed as an intravenous agent in the
treatment of metastatic colorectal cancer [5]. In recent
years, clinical trials have established the efficacy
of Bevacizumab for the treatment of AMD [5,6].
Bevacizumab injections with 1.25 mg in a six weekly
variable retreatment regimen are superior to standard
care (pegaptanib sodium, verteporfin, sham).
And then this treatment improved visual acuity on
average at 54 weeks [2]. Furthermore, some groups
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Bevacizumab cured age-related macular degeneration (AMD)
via down-regulate TLR2 pathway
reported that the mammalian toll like receptor 2
(TLR2) pathway is activated early in the process and
drives dedifferentiation of the RPE through reducing
the expression of RPE-characteristic proteins. This
mechanism also occurs in response to a qualitatively
different insult and chemical oxidative damage.
Therefore, inhibiting RPE stress response by blocking
TLR2 activation has beneficial effects on the RPE and
photoreceptors. Thus, understanding the mechanism
by which Bevacizumab cures AMD has become
increasingly critical. However, there is a lack of such
knowledge in RPE or AMD Patients.
In this study, we firstly detected the role of TLR2
pathway for P3C mediated apoptosis of ARPE-19, and
then, we carried out much more studies to make clear
whether the same mechanism in the bevacizumab
cured AMD by in vitro cultured RPE from AMD Patients.
2. Experimental Procedures
2.1 ARPE-19 cells culture
As described by other’s inn previous studies [8], the
ARPE-19 cells were used at passages 24 to 26 and
were maintained in tissue culture flasks in DMEM-F12
(Invitrogen, Carlsbad, CA) containing 2% fetal bovine
serum (FBS; Invitrogen) until they were postconfluence
for several weeks. Then, the cells were harvested and
plated in DMEM-F12, 2% FBS on glass coverslips or
laminin-coated clear filters (Transwell; Fisher Scientific,
Hampton, NH) at a density of 2 x105 cells mL-1. The
filters subdivide the culture dish into two chambers to
enable studies of transmonolayer permeability.
Preparation of Primary RPE Cell Culture from
Postmortem Autopsy Eyes
AMD Donor’s eyes were received through the second
people hospital of Zhengzhou (Zhengzhou, China)
(eyes are collected within a few hours after death and
shipped in sterile physiologic solution). Donors were
60 years and above in age and included both females
and males. The anterior segment from each donor eye
was removed as described in previous reports [9-12].
Briefly, after electron microscopy (EM) examination,
RPE cells were harvested from the RPE-choroid-sclera
using fine forceps. Then the RPE choroid was carefully
separated from the sclera and placed face up in a small
sterile Petri dish dipping with 0.1% dispase contain
Ca2+ and Mg2+-free Hank’s balanced salt solution. After
gently resuspending the cell pellet, the suspended cells
were then transferred to T25 culture flasks and placed
in an incubator at 37°C and 5% CO2 under humidified
atmosphere [6-8].
2.2 DN MyD88 transfection
Dominant negative (DN) cDNA constructs of MyD88
(DN MyD88) was obtained as a gift from Guo Xing
Li nad were prepared as previously described [13].
Luciferase data obtained from the cells transfected
with pcDNA3 empty vector was used to assess the
specificity of DN-MyD88. Briefly, ARPE-19 and primary
RPE cells were plated at a concentration of 50,000
cells/well in 24-well plates and cultured in DMEM-F 12,
2% FBS on glass coverslips or laminin-coated clear
filter overnight. Cells were co-transfected the following
day with FuGene6 Transfection Reagent as per the
manufacturer’s instructions. The Roche Fugene6
transfection system does not affect the cell viability or
induce cytokine production in the transfected cells. The
reporter genes NF-κB -Luciferase (0.5 μg) and either
empty vector or dominant negative mutants of MyD88
(0.1, 0.3 and 0.5 μg) were transfected into the ARPE19 cells. Reporter gene NF-κB -luciferase (0.5 μg) was
transfected to assess the effect of P3C (10 μmol L-1)
on NF-κB expression. pCMV-β- galactosidase cDNA
(0.1 μg) was transfected to normalize the results for
transfection efficiency as described earlier [14-16].
After overnight transfection, the cells were treated with
10 μmol L-1 P3C. The luciferase activity was measured
with a Promega kit (Promega, Madison, WI) and a
luminometer. β-galactosidase activity as determined by
calorimetric method is a well established and accepted
method to assess the transfection efficiency.
2.3 MTT analysis
ARPE-19 and Primary RPE cells were diluted to
2 x105 cells/ ml and seeded at 0.1 ml/well in 96-well flatbottomed plates. They were randomly divided into a
normal control group and two treatment groups treated
with different dosages of P3C (Invivogen, Suite 100 San
Diego, California, USA): 0.5, 1, 5, 10, 20 μmol L-1, or
Avastin (Bevacizumab, Genentech, USA): 5, 10, 20,
50 μg L-1, respectively. The cultures were maintained for
24 h at 37oC with 5% CO2 humidified air. Then, MTT
reagent (5 mg mL-1) was added to each well and incubated
for 4 h. After the incubation, the incubation precipitates
were dissolved with 0.1 ml of SDS. The optical density
(OD) values were measured by spectrophotometry at
570 nm using an ELx800 Microplate Reader (Bio-Tek
Instruments, Inc. Winooski, Vermont, USA).
2.4 Western blotting
After treatment, cells were briefly washed with
phosphate-buffered saline (PBS) and then lysed in
1× sodium dodecylsulfate (SDS) loading buffer on ice.
Lysates were centrifuged at 15,000 x g for 10 min at
4°C. Protein concentrations were determined by the
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Z.L. Wang et al.
Bradford assay with bovine serum albumin as standard
(Bio-Rad, Hercules, CA, USA). Equivalent amounts
of protein (20-50 μg) were separated by 12% SDSpolyacrylamide gels and transferred to polyvinylidene
difluoride membranes (Millipore, Bedford, MA, USA).
Following incubation with PBS containing 0.05% Tween
20 and 5% non-fat dry milk to block non-specific binding,
membranes were incubated with primary antibodies,
then with appropriate secondary antibodies conjugated
to horseradish peroxidase. Immunoreactive bands were
visualized by using chemiluminescence methods. The
blots for detected proteins were quantified using Multi
Gauge Image Analysis software (FujiFilm, Japan) and
were normalized using β-actin as an internal control.
The following antibodies were used: TLR2, Caspase-3,
Cleaved-caspase-3, My D88, NF-κB, IKK, p-IKK and
β-actin (Santa Cruz Biotechnology, Santa Cruz, CA,
USA).
2.5 Statistical analysis
Statistical analyses were performed using the SPSS
software. Differences between groups were evaluated
by one-way analysis of variance (ANOVA) with Tukey’s
test. P values of less than 0.05 were considered to
indicate statistical significance.
3. Results
3.1 ARPE-19 and Primary RPE cells aPoPtosis
in vitro
As shown in Figure 1A, the apoptosis rate of ARPE19 gradually increased when treated with P3C with
an IC50 value of 10 μmol L-1 P3C after 24 h in culture.
However, the apoptosis rate of Primary RPE cells
Figure 1.
gradually decreased when treated with Bevacizumab
dose (Figure 1B).
3.2 P3C active TLR2 Pathway during ARPE-19
aPoPtosis
To examine the role of TLR2 pathway in P3C treated
ARPE-19 in vitro, we used Western blotting analysis
to detect the changes in the TLR2 pathway. As shown
in Figure 2A, the protein expression of TLR2, My D88,
NF-κB, p-IKK in P3C treated group were significantly
up-regulated and higher than the control group
respectively. Furthermore, the expression of Caspase-3
and Cleavead-caspase-3 was significantly up-regulated
and higher than the control group (Figure 2B). Taken
together, these results indicate that P3C active the
TLR2 pathway during ARPE-19 apoptosis.
3.3 DN My D88 inhibit ARPE-19 cells
apoptosis resulting from P3C
TLR2 activation is generally mediated by an
intracellular adaptor, myeloid differentiation primary
response protein 88 (MyD88) [14]. As shown in
Figure 3A inhibition of MyD88 dimerization, using
different doses DN My D88, the high expression
of NF-κB with high luciferase activity induced by
P3C was significantly inhibited (P<0.01, P<0.001,
respectively). Inhibition of MyD88 dimerization
also reduced apoptosis in ARPE-19 cells that
were treated with 10 μmol L -1 P3C (Figure 3B),
In addition, the expression of My D88, NF-κB,
p-IKK is significantly down-regulated when MyD88
dimerization was inhibited (Figure 3C). Furthermore,
after treatment with DN My D88, the expression of
Caspase-3, Cleaved-caspase-3 were markedly
decreased in ARPE-19 cells treated with 10 μmol L -1
ARPE-19 and Primary RPE cells apoptosis in vitro. A. The apoptosis rates of ARPE-19 gradually increases with P3C treatment.
B. Bevacizumab inhibits apoptosis of Primary RPE cells treated with P3C. Columns represent means of triplicates from one representative
experiment; error bars indicate means ± standard error (SE). **P < 0.01, versus the control group.
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Bevacizumab cured age-related macular degeneration (AMD)
via down-regulate TLR2 pathway
Figure 2.
P3C activated TLR2 pathway and caspase-3 during ARPE-19 apoptosis. A. P3C activated TLR2 pathway during ARPE-19 apoptosis.
B. P3C activates caspase-3 during ARPE-19 apoptosis.
Figure 3. DN My D88 inhibits ARPE-19 apoptosis resulting from P3C treatment. A. DN My D88 significantly inhibited the expression of NF-κB shown
as luciferase activity. B. DN My D88 inhibits ARPE-19 cell apoptosis. C. DN My D88 inhibits ARPE-19 apoptosis via TLR2 pathway. D. DN
My D88 inhibits caspase-3 expressions in ARPE-19 cells. Columns represent means of triplicates from one representative experiment;
error bars indicate means ± standard error (SE). **P < 0.01, ***P < 0.001versus the control group.
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Z.L. Wang et al.
P3C (Figure 3D). These findings suggesting that
MyD88 is involved in the TLR2 effect on ARPE-19
cells apoptosis resulting from P3C treatment.
3.4 Bevacizumab inhibit primary RPE Cell
apoptosis from Postmortem Autopsy Eyes
Western blot analysis was used to detect the changes in
TLR2 pathway in primary RPE cells from AMD patients.
Interestingly, the expression of My D88, NF-κB, p-IKK
in primary RPE Cells from AMD patients after treated
with Bevacizumab 50 μg L-1 was significantly downregulated (Figure 4A). However, there is no statistical
different in the expression of TLR2 pathway between
Bevacizumab 50 μg L-1 treated group and DN MyD88
transfection primary RPE Cells from AMD patients and
then Bevacizumab 50 μg L-1 treated group (Figure 4A).
Furthermore, the expression of Caspase-3, Cleavedcaspase-3 were markedly decreased in the same group
(Figure 4B). Therefore, these results suggested that
Bevacizumab cured age-related macular degeneration
(AMD) via down-regulation of Toll—like receptor 2
(TLR2) pathway in retinal pigment epithelium (RPE)
from AMD patients
Figure 4.
4. Discussion
In the current study, we show that MyD88 is involved
in TLR2 pathway and P3C mediated apoptosis in
ARPE-19 cells. And more importantly, our findings
suggest that Bevacizumab cured age-related macular
degeneration (AMD) via down-regulation of Toll—like
receptor 2 (TLR2) pathway in RPE from AMD patients.
During the development of AMD, the RPE in
the macular region undergoes hypopigmentation,
depigmentation, or apparent absence of the RPE in
the macular area [13,14]. Therefore, we used the P3C
mediated apoptosis of ARPE-19 cells (RPE cell line)
as a model to explore the mechanism. In our study, we
found that TLR2 pathway in the ARPE-19 cells treated
with P3C were active and then inhibited by DN MyD88
transfection which is the important protein in the TLR2
pathway. These results suggest that P3C activated TLR2
pathway during ARPE-19 apoptosis. Furthermore, we
found that TLR2 pathway in the primary AMD RPE cells
were active and then inhibited by DN MyD88 transfection
which is the important protein in the TLR2 pathway. Our
findings were consistent with other groups that report
Bevacizumab inhibits primary RPE Cell apoptosis from Postmortem Autopsy Eyes via the TLR 2 pathway. A. Bevacizumab inhibits
primary RPE Cell apoptosis via TLR 2 pathway. B. Bevacizumab inhibits caspase-3 expression in primary RPE Cell.
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Bevacizumab cured age-related macular degeneration (AMD)
via down-regulate TLR2 pathway
TLR2 signaling in RPE cells may play an important role
in innate and adaptive immune responses, apoptosis
within the retina [17-19]. An important finding is that
TLR2 pathway activation will result in RPE apoptosis in
AMD.
Bevacizumab is approved for the treatment of
advanced colorectal cancer (CRC), advanced nonsmall
cell lung cancer (NSCLC), metastatic breast cancer
(MBC), and advanced renal cell cancer and diseases
related to TLR2 pathway [4,20]. However, increasing
number of studies indicated that bevacizumab is
commonly used for the treatment of neovascular
AMD and had equivalent effects on visual acuity
when administered according to the same schedule
compared to ranibizumab [21-23]. On the other hand,
TLR2 activity usually leads to the activation of nuclear
factor (NF)-κB [15]. NF-κB exists in several dimeric
forms, predominantly the p50/p65 heterodimer.
Translocation of NF-kB to the nucleus16 is regulated
by the phosphorylation of I-kappa-B kinase (IKK). In
our studies, we found that the expression of My D88,
NF-κB and p-IKK in primary RPE Cells from AMD
patients after DN MyD88 transfection is significantly
down-regulated. Interestingly, the expression of My
D88, NF-κB and p-IKK in primary RPE Cells from AMD
patients after treatment with Bevacizumab 50 μg L-1
were also significantly down-regulated. Therefore,
our data shown that mammalian toll like receptor 2
(TLR2) pathway is activated early in the process and
drives dedifferentiation of the RPE through reduction
in expression of RPE-characteristic proteins. However,
these results suggest that Bevacizumab cured
age-related macular degeneration (AMD) via downregulate Toll—like receptor 2 (TLR2) pathway in retinal
pigment epithelium (RPE) from AMD patients.
Acknowledgements
The authors thank Dr. Guo xing Li (Eye Hospital,
Wenzhou Medical College. Wenzhou, China) for
providing MyD88 (DN MyD88) as gift.
Conflict of interest statement
Authors ‘ conflict of interest disclosure: The authors
stated that there are no conflicts of interest regarding
the publication of this article. Research support played
no role in the study design; in the collection, analysis,
and interpretation of data; in the writing of the report; or
in the decision to submit the report for publication.
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