ANALYTICAL AND QUANTITATIVE CYTOPATHOLOGY AND HISTOPATHOLOGY

1. 16
Analytical and Quantitative Cytopathology and Histopathology®
0884-6812/19/4101-0016/$18.00/0 © Science Printers and Publishers, Inc.
Analytical and Quantitative Cytopathology and Histopathology®
OBJECTIVE: To investigate the effects of autophagy
on vascular endothelial growth factor (VEGF) expres­
sion in human retinal pigment epithelial cells (RPE-19)
under the condition of hypoxia in vitro.
STUDY DESIGN: RPE-19 cells were divided into 3
groups: control group, hypoxia group (the final con­
centration of CoCl2 in the medium was 125 μmol/L),
and autophagy inhibitor group (cells were pretreated
with 3-methyladenine for 12 hours and then cultured
with 125 μmol/L CoCl2). After 24 hours, enzyme-linked
immunosorbent assay (ELISA) and western blotting
were used to detect the expression levels of VEGF. The
expression of autophagy correlation protein Beclin 1,
LC3 (LC3-II/LC3-I ratio), and p62 were examined by
western blotting. Then, we increased autophagy level
using rapamycin to detect its effect on VEGF in the
condition of normal oxygen culture (cells were pre-
treated by 500 nM rapamycin for 12 hours); the expres­
sion level of VEGF was detected by the above method.
RESULTS: Compared with the control group, expres-
sion of VEGF, Beclin-1, and LC3II/I protein were in-
creased while p62 was decreased under hypoxia. VEGF
protein expression and secretion were decreased in the
autophagy inhibitor group. Furthermore, we found that
rapamycin also promoted the expression and secretion of
VEGF protein.
CONCLUSION: Autophagy promoted the expression of
VEGF in RPE-19 cells. Targeting autophagy is expected
to upregulate VEGF levels in RPE cells. (Anal Quant
Cytopathol Histpathol 2019;41:16–22)
Keywords: autophagy; Beclin-1; choroidal neovas­
cularization; hypoxia; hypoxia in vitro; intraocular
neovascularization; neovascularization; ophthalmic
diseases; retinal pigment epithelial; VEGF; vascular
endothelial growth factor; vision loss.
The formation of intraocular neovascularization
Autophagy Promotes the Expression of Vascular
Endothelial Growth Factor in Human Retinal
Pigment Epithelium Cells
Xinhong Chai, B.S., Xia Li, M.D., Mengyang Kang, B.S., Xiaoye Wang, B.S., and
Junhui Du, M.D.
From the Departments of Ophthalmology and of Internal Medicine, Xi’an Ninth Hospital Affiliated to Medical College of Xi’an Jiaotong
University, Xi’an, Shaanxi Province; the Department of Ophthalmology, Guangzhou Special Service Recuperation Center of PLA Rocket
Force, Guangzhou, Guangdong Province; and the Department of Clinical Medicine, Xi’an Medical University, Xi’an, Shaanxi Province,
China.
Ms. Chai is Ophthalmologist, Department of Ophthalmology, Xi’an Ninth Hospital Affiliated to Medical College of Xi’an Jiaotong
University.
Dr. Li is Ophthalmologist, Department of Ophthalmology, Guangzhou Special Service Recuperation Center of PLA Rocket Force.
Ms. Kang is Medical Undergraduate, Department of Clinical Medicine, Xi’an Medical University.
Ms. Wang is Physician, Department of Internal Medicine, Xi’an Ninth Hospital Affiliated to Medical College of Xi’an Jiaotong University.
Dr. Du is Ophthalmologist, Department of Ophthalmology, Xi’an Ninth Hospital Affiliated to Medical College of Xi’an Jiaotong
University.
This work was supported by grants from the Natural Science Foundation of Xi’an Science Technology Bureau (No. SF1508 [3]) and the
Natural Science Foundation of Shaanxi Province (No. 2016JM8018).
Address correspondence to: Junhui Du, M.D., Department of Ophthalmology, Xi’an Ninth Hospital Affiliated to Medical College of
Xi’an Jiaotong University, Xi’an 710054, Shaanxi Province, China (djh79918@163.com).
Financial Disclosure: The authors have no connection to any companies or products mentioned in this article.

2. Volume 41, Number 1/February 2019 17
Autophagy Promotes VEGF Expression in RPE Cells
is an important cause of vision loss in ophthalmic
diseases such as proliferative diabetic retinopathy,
age-related macular degeneration (AMD), central
retinal vein occlusion, retinopathy of prematur­
ity, etc. Choroidal neovascularization (CNV) is the
main pathological change of AMD and is one of
the major causes of severe visual loss in people
aged over 50 years old. Vascular endothelial
growth factor (VEGF) is the key cytokine to pro­
mote the formation of neovascularization.1 It pro­
motes endothelial cell division and proliferation
and increases the permeability of blood vessels.
In clinical studies it has been found that inhibi-
tion of VEGF can inhibit CNV effectively. How­
ever, the main problem in anti-VEGF therapy is
the recurrence of CNV. Some studies have found
that anti-VEGF drugs can cause autophagy activa­
tion,2 while other studies revealed that the acti­
vation of autophagy promotes the formation of
new blood vessels. Thus, whether the activation
of autophagy caused by anti-VEGF therapy leads
to the recurrence of CNV or not is still unclear,
and the mechanism needs to be further studied. In-
creasing evidence has shown that hypoxia con­
tributes to the development of CNV, and retinal
pigment epithelial (RPE) cells play a causative
role in VEGF production during the formation of
CNV.3 Thus, we conducted this study to try to
investigate the influence of autophagy activation
on VEGF expression in RPE cells.
Materials and Methods
RPE-19 cell line was purchased from Type Cul-
ture Collection of Chinese Academy of Sciences.
The anti-LC3B and anti-p62 were purchased
from Santa Cruz Biotechnology, USA (Cat Nos
sc-376404 and sc-48402, respectively); anti-Beclin-1
and β-actin were purchased from Bioworld Tech­
nology, USA (Cat Nos AP0768 and BS1002). 3-MA,
a potent autophagy inhibitor, was purchased from
Sigma, USA (Cat No M9281). Rapamycin was
purchased from Sigma, USA (Cat No V900930).
The VEGF antibody was purchased from BD Bio­
sciences (Cat No 555036). Human VEGF ELISA Kit
was purchased from ExCell Biology Inc., Shang-
hai, China (Cat No EH015-96). CoCl2 was pur-
chased from Sigma, USA (Cat No C8661). The
other cell culture reagents were purchased from
Invitrogen.
Cell Culture
RPE-19 cell line was obtained from Type Culture
Collection of Chinese Academy of Sciences. RPE-
19 cells were incubated in Dulbecco’s modified
Eagle’s medium (DMEM) supplemented with 10%
fetal bovine serum, 100 U/mL penicillin, and
100 U/mL streptomycin at 37°C in a humidified
95% room air with 5% CO2. Culture medium was
replaced after 24 hRPE-19 cells were randomly
divided into different groups: control group,
hypoxia group (treated with 125 μmol/L CoCl2
in medium), hypoxia and autophagy inhibition
group (pretreated with 10 mM 3-MA for 12 hours
and then treated with 125 μmol/L CoCl2), and
autophagy activation group (cells were pretreated
by 500 nM rapamycin for 12 hours).
Western Blotting
RPE-19 cells were lysed and processed for deter­
mination of LC3-II/LC3-I ratio, Beclin-1, and p62.
After treatment, cell lysate (30 μg proteins) was
loaded onto and separated by 12% sodium dode-
cyl sulfate polyacrylamide (SDS-PAGE) gel elec-
trophoresis. The separated proteins were trans­
ferred to nitrocellulose membranes, which were
then blocked with Tris-buffered saline–T buffer
con­
taining 5% nonfat milk, incubated with prima­
ry antibodies overnight directed against β-actin
(1:500), LC3 (1:500), Beclin-1 (1:500), and p62 (1:500)
and then incubated with a horseradish peroxidase–
conjugated secondary antibody for 1 hour at room
temperature. The labeled bands were visualized
and quantified using a chemiluminescence imag-
ing system (CliNX, Shanghai, China). CliNX anal­
ysis software was used to scan and count the gray
value. The ratio of the target protein gray value/
β-actin was represented as the relative expression
level of target protein.
Enzyme-Linked Immunosorbent Assay
VEGF was determined with human enzyme-linked
immunosorbent assay (ELISA) kits used according
to the manufacturers’ instructions: install stan-
dard well, test sample well, and blank well as in-
structed. Then 100 mL of horseradish peroxidase–
conjugate reagent was added to each well covered
with an adhesive strip and they were incubated for
60 minutes at 37°C. Then chromogen solution A
and B were added to each well and they were
incubated for 15 minutes at 37°C. Stop solution
was then added to each well to induce a colored
reaction product. The product was read by a mi-
croplate reader. The intensity of the produced col-
ored product was directly proportional to the con­

3. 18 Analytical and Quantitative Cytopathology and Histopathology®
Chai et al
centration of VEGF present in the samples. Each
assay was performed in duplicate (coefficient of
variation was <5%).
Statistical Analysis
Statistical analyses were performed using the SPSS
13.0 software package. All quantitative data were
representative of at least 3 independent experi­
ments. The data were presented as the mean with
standard deviation. Between-group differences
were tested by analyses of variance (ANOVA). The
least significant difference procedure was used
for pairwise comparisons. A two-tailed p value of
p<0.05 was considered significant.
Results
RPE-19 Cell Morphology Under the Inverted Phase
Contrast Microscope
After 12 hour cell culture, the morphology of RPE-
19 cells in different groups were observed under
inverted phase contrast microscope (Figure 1). The
cell morphology of RPE-19 cells was typical as
shown in the control group. The number of cells
in the hypoxic group was significantly reduced,
and the cell morphology was slightly changed. In
the hypoxic+MA group, the cell number was in-
creased and the morphology was more typical as
compared with the hypoxic group.
Hypoxia Activates Autophagy in RPE-19 Cells
To investigate autophagic activity in RPE-19 cells,
we measured the expression of autophagy bio­
markers LC3, Beclin-1, and p62 protein by western
blotting. As shown in Figure 2, CoCl2 significant­
ly increased expression of Beclin-1 and LC3II/I,
while 3-MA, an autophagy inhibitor, reduced the
expression in RPE-19 cells. p62 protein was sig-
nificantly decreased under hypoxia and was up-
regulated by 3-MA.
Effects of Autophagy on the Expression of VEGF in
RPE Cells Under Hypoxia
In order to investigate the effect of autophagy
on the expression of VEGF in RPE cells under
hypoxia condition, we detected the expression of
VEGF protein by western blotting (Figure 3) and
ELISA (Figure 4). Our results demonstrated that
the level of VEGF expression and secretion were
both increased when RPE-19 cells were treated
with CoCl2 and were decreased with 3-MA (au-
tophagy inhibitor).
Effect of Autophagy Activation on the VEGF
Expression and Secretion in RPE Cells
Our results showed that hypoxia induced the
activation of autophagy and promoted VEGF ex-
pression. Then, it is necessary to clarify the rela­
tionship between autophagy level and VEGF ex-
pression in RPE cells. Rapamycin as an autophagy
activator was used to treat RPE cells under nor-
moxia condition. To investigate autophagic activ­
ity in RPE-19 cells, expression levels of LC3 and
Beclin-1 were measured by western blotting. As
shown in Figure 5, the expression of Beclin-1,
LC3II/I, and VEGF were all promoted when RPE
cells were treated by rapamycin, and the secretion
level of VEGF was improved as well (Figure 6).
Discussion
Our results suggested that hypoxia upregulated
the level of autophagy in RPE cells and promoted
VEGF expression. The expression of VEGF can be
Figure 1 RPE-19 cell morphology under the inverted phase contrast microscope.

4. Volume 41, Number 1/February 2019 19
Autophagy Promotes VEGF Expression in RPE Cells
inhibited by autophagy inhibitors. Further study
on the expression of VEGF affected by the acti­
vation of autophagy under normal oxygen has
also been carried out. These results indicated that
the activation of autophagy improved the level of
VEGF expression. Our results revealed that the
activation of autophagy promoted the secretion
and expression of VEGF in RPE cells, which may
be involved in the process of CNV recurrence.
In recent years increasing attention has been
paid to the effect of autophagy on RPE cells. The
dysfunction of autophagy causes damage to RPE
cells, which promotes the formation of lipofuscin
and takes part in the accumulation of drusen.4,5
These studies indicated that autophagy had a
certain role in protecting RPE cells’ function and
delaying atrophic AMD. However, the function of
autophagy in the neovascular type of AMD is still
unclear. It has been found that the activation of
autophagy contributes to the migration and tube
formation of vascular endothelial cells,6 which
suggested that autophagy may play a role in the
neovascular type of AMD. In this study, autoph­
agy was found to be critical in stimulating VEGF
Figure 2 Effects of hypoxia on autophagy-related proteins Beclin-1, LC3I/II, and p62 in RPE-19 cells. **p<0.01 vs. control. ##p<0.01 vs.
CoCl2 group.

5. 20 Analytical and Quantitative Cytopathology and Histopathology®
Chai et al
expression and therefore possibly participating in
promoting angiogenesis in AMD.
Hypoxia is one of the important pathways of
autophagy activation. Hypoxia-inducible factor 1
(HIF-l) is an important protein regulating cellular
oxygen concentration, and it plays an important
role in the process of autophagy activation.7 In
recent years the role of autophagy in the forma­
tion of neovascularization has attracted much at-
tention.8,9 It has been confirmed that the inhibition
of autophagy inhibits bovine aortic endothelial cell
migration and tube formation and can also inhibit
VEGF-induced neovascularization.8 Our previous
study also found that hypoxia activated autoph-
agy in retinal vascular endothelial cells and pro­
moted cell migration and tube formation. Inhibi­
tion of autophagy prevented neovascularization
in vitro.6 These findings suggested that autophagy
plays an important role in angiogenesis under
hypoxia.
The level of autophagy can be assessed by
detecting autophagy-related genes and proteins.
LC3, Beclin-1, and p62 were commonly used to
detect autophagy levels. LC3II/I and Beclin-1 were
positively correlated with autophagy level,10-12
while the expression of p62 is negatively corre­
lated to autophagy.13 We used these 3 proteins
to monitor autophagy levels in our experiments.
Previous studies have found that the activation
of autophagy in smokers’ RPE cells promotes the
expression of VEGF, which may play an impor­
tant role in protecting RPE cells and reducing
their apoptosis.14 In RPE-19 cells, malondialde­
hyde (MDA) treatment induced VEGF expression
alternation, and the MDA-induced VEGF increase
was inhibited by autophagy-lysosomal inhibi-
tors.15 However, in podocyte related studies the
activation of autophagy was found to contribute
Figure 3 Effects of autophagy on the expression of VEGF protein in RPE cells under hypoxia condition. **p<0.01 vs. control. ##p<0.01
vs. CoCl2 group.
Figure 4 Effects of autophagy on the level of VEGF secretion
in RPE cells under hypoxia condition. **p<0.01 vs. control.
#p<0.05 vs. CoCl2 group.

6. Volume 41, Number 1/February 2019 21
Autophagy Promotes VEGF Expression in RPE Cells
to VEGF reduction, while autophagy inhibition
increased VEGF level. Podocytes are the major
sites of VEGF production in kidneys. High glu-
cose was found to induce VEGF expression and
reduce the viability of podocytes. After treatment
with rapamycin, an autophagy activator, the ex-
pression of VEGF was significantly reduced. In
contrast, when treated with 3-MA, the expression
of VEGF has been increased.16 Furthermore, a
study has found that VEGF upregulated the ex-
pressions of Beclin1 and LC3B both in vivo and
in vitro.17 These results suggest that VEGF in-
creases autophagy function. Another study has
found that the VEGF-C/NRP-2 axis is involved in
the activation of autophagy, which helps cancer
cell survival following treatment.18
Figure 5 Effects of autophagy activation on VEGF expression in RPE cells. *p<0.05 vs. control. **p<0.01 vs. control.

7. 22 Analytical and Quantitative Cytopathology and Histopathology®
Chai et al
In conclusion, our study found that the activa­
tion of autophagy may lead to the increase of
VEGF expression in RPE cells under the condi-
tion of hypoxia, which may contribute to protect-
ing RPE cells. However, the increase of VEGF
expression may have negative effects such as
increasing the vascular permeability and neovas­
cularization. We confirmed that autophagy may
play a dual role in AMD. In addition, further
studies are still needed to clarify the role of au-
tophagy in the neovascular diseases.
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Figure 6 Effects of autophagy activation on the level of VEGF
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