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Matrix
metalloproteinase-9 and vascular endothelial growth factor expression change in
experimental retinal neovascularization
Yu
Di, Qing-Zhu Nie, Xiao-Long Chen
Department
of Ophthalmology, Shengjing Hospital of China Medical University, Shenyang 110004, Liaoning Province, China
Correspondence to: Yu Di. Department of Ophthalmology, Shengjing Hospital of
China Medical University,
Shenyang 110004, Liaoning Province, China. diyu81@126.com
Received: 2015-10-14 Accepted:
2016-01-15
Abstract
AIM: To investigate the signal transduction mechanism of matrix
metalloproteinase-9 (MMP-9) mediated- vascular endothelial growth factor (VEGF)
expression and retinal neovascularization (RNV) in oxygen-induced retinopathy
(OIR) model.
METHODS: C57BL/6J mice were divided into four groups: control group, OIR group,
OIR control group (phosphate-buffered saline
by intravitreal injection) and treated group [tissue inhibitor of matrix
metalloproteinase-1 (TIMP-1) by intravitreal injection]. OIR model was
established in C57BL/6J mice exposed to 75%±2% oxygen
for 5d. mRNA level and protein expression of MMP-9, TIMP-1 and VEGF were
measured by real-time polymerase chain reaction and Western blotting, and
located by immunohistochemistry.
RESULTS: Levels of MMP-9 and VEGF in retina were significantly increased in
animals with OIR and OIR control group. Levels of TIMP-1 in retina was
significantly reduced in animals with OIR and OIR control group. Furthermore, a
significant correlation was found between MMP-9 and VEGF. Intravitreal injection
of TIMP-1 significantly reduced MMP-9 and VEGF expression of the OIR mouse
model (all P<0.05).
CONCLUSION: These
results demonstrate that MMP-9-mediated up-regulation of VEGF promotes RNV in
retinopathy of prematurity (ROP). TIMP-1 may be a potential target for the
prevention and treatment of ROP.
KEYWORDS: retinal neovascularization; matrix
metalloproteinase-9; vascular endothelial growth factor; oxygen-induced
retinopathy
Citation: Di
Y, Nie QZ, Chen XL. Matrix metalloproteinase-9 and vascular endothelial growth
factor expression change in experimental retinal neovascularization. Int J Ophthalmol 2016;9(6):804-808
INTRODUCTION
Retinal
neovascularization (RNV) causes loss of vision in most of retinal vascular
diseases, including retinopathy of prematurity (ROP), diabetic retinopathy (DR)
and age-related macular degeneration (AMD)[1-3].
Research showed that the production of ocular derived growth factors and cell adhesion
molecules induce RNV, yet the complex interactions are not clear[1,4]. Matrix
metalloproteinases (MMPs) are zinc-dependent enzymes, and MMP-9 is the most
frequently investigated MMPs in the brain and eye because it is relatively easy
to be detected[5-6]. In
particular, MMP-9 promotes endothelial cell migration, survival, and tubule
formation[7]. Tissue
inhibitor of matrix metalloproteinase-1 (TIMP-1) is inhibitor of MMP-9, and the
imbalances of MMP-9 and TIMP-1 cause excessive degradation of extracellular
matrix[8]. In addition to
stimulating angiogenesis[9],
MMP-9/TIMP-1 is implicated in ischemia-induced RNV[10]. Vascular endothelial growth factor (VEGF) is an
important factor to stimulate the proliferation of endothelial cell and
mediate RNV of ROP[11-12],
whereas the correlation of MMP-9 and VEGF in ROP have largely remained elusive.
Therefore, the purpose of this study is to investigate MMP-9-VEGF signaling
pathway in ROP.
MATERIALS AND METHODS
Animals and Groups Oxygen-induced
retinopathy (OIR) mice model was constructed according to the protocol of Smith
et al[13]. Animal experiments were conducted according to
protocols approved by Animal Experimental Committee of China Medical University
(Liaoning Province, China). In brief, C57BL/6J
mice were placed in the hyperoxia chamber set at 75%±2%
oxygen from 7 to 12d then placed in the normal air from 12d to 17d. They were
maintained under conditions of standard lighting (a 12-hour light-dark cycle)
and temperature (68°F).
Mice were
randomly divided into control group (in normal air), OIR group, OIR control
group and treated group (n=60/group).
The treated or OIR control group were treated with 1 μL (500 ng/μL) of TIMP-1
or phosphate-buffered
saline (PBS) by vitreous
cavity injection, and were moved to normal air at 12d. Four groups of mice were
sacrificed at P17 to collect retinas.
Histologic Quantification of
Preretinal Neovascularization Sagittally cutted the whole eyes into serial sections (6 µm) then stained with H&E[14], dehydrated, vitrified, mounted, observed and
imaged using a light microscope. Blindly grouped three reviewers to count the
cells.
Immunohistochemistry Paraffin-embedded
6 μm eye tissue sections were performed by immunohistochemical analysis, treated
with rabbit polyclonal antibodies against mouse MMP-9,
TIMP-1 and
VEGF (1:100 dilution; Boster Bioengineering Co., Wuhan, Hubei Province, China)
at 4℃. PBS instead of the primary antibody. Images were observed
using a light microscope.
Western Blot Protein
in each samples was extracted with lysis buffer. Equal amounts of samples were
loaded on sodium dodecyl sulfate-polyacrylamide gels, electrophoretically
separated and transferred to polyvinylidene fluoride membranes using standard
procedures. Membranes were incubated with primary antibodies (rabbit anti-MMP-9
polyclonal antibody, Abcam, Cambridge, UK; or rabbit anti-TIMP-1, VEGF polyclonal
antibody, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) at 4℃
overnight, then incubated with corresponding secondary
antibodies at 37℃
for 45min. Signals were detected using a chemiluminescence detection system
(Technew Tech. Co. Ltd., Shanghai, China).
Real-time Reverse
Transcription-polymerase Chain Reaction Analysis The samples’ RNA were extracted with TRIzol (Invitrogen,
Carlsbad, CA, USA). Reverse transcription into cDNA with a reverse
transcriptase kit TAKARA 047A according to the protocols. Primers were designed
and purchased from Sangon Biotech Co. Ltd. (Shanghai, China), glyceraldehyde
3-phosphate dehydrogenase (GAPDH) was used as a normalizing controlled. The
sequence of primers is shown in Table 1. Calculated the 2-ΔΔCt to
perform the relative quantification of the gene expression[15].
Table 1 Primers for real-time reverse
transcription-polymerase chain reaction
|
Gene |
Primer sequences (5’-3’) |
Product length (bp) |
Temperature (℃) |
|
|
GAPDH |
Forward |
CCC ATC TAT GAG GGT TAC GC |
150 |
55 |
|
|
Reverse |
TTT AAT GTC ACG CAC GAT TTC |
|
|
|
MMP-9 |
Forward |
GGA GAC CTG AGA ACC AAT CTC |
277 |
55 |
|
|
Reverse |
TCC AAT AGG TGA TGT CGT |
|
|
|
TIMP-1 |
Forward |
CCT TCT GCA ATT CCG ACC TC |
534 |
55 |
|
|
Reverse |
CGG GCA GGA TTC AGG CTA T |
|
|
|
VEGF |
Forward |
CCC GAC AGG GAA GAC AAT |
131 |
55 |
|
|
Reverse |
TCT GGA AGT GAG CCA ACG |
|
|
Statistical Analysis Resulting data was statistically
analyzed using variance (ANOVA) by SPSS15.0 software, with P<0.05 being considered to be significant.
RESULTS
Quantitative Assessment of Retinal
Neovascularization There was an average
of 30.24±0.83 and 31.13±1.21 nuclei per slice in the
OIR and OIR control group compared to 3.27±0.65 in the control group (P<0.05) (Figure 1). The number of preretinal neovascular cells in the
treated group (5.72±1.10) decreased significantly compared with the OIR and OIR
control group (P<0.05). It proved
that there is an inhibitory effect on RNV in the treated group.
Figure 1 Preretinal neovascular cells of P17 mice were
counted aP<0.05 vs the control group, bP<0.05
vs the OIR group, cP<0.05 vs the OIR control group.
Matrix
Metalloproteinase-9-vascular Endothelial Growth Factor Signaling
Pathway Expression by Immunohistochemistry Figure 2 showed that MMP-9 and
VEGF expression was weakly detected only in the ganglion cell layer (GCL) and
inner plexiform layer (IPL) of the control group. Whereas in the control group,
TIMP-1 expression was strongly detected in the GCL, IPL and inner nuclear layer
(INL). In the OIR and OIR control group, MMP-9 and VEGF expression were
strongly detected in the GCL, IPL, INL, outer plexiform layer (OPL) and
neovascularization breaking through the internal limiting membrane (ILM).
Whereas in the OIR and OIR control group, TIMP-1 expression was weakly detected
only in the GCL, IPL, INL and neovascularization breaking through the ILM.
However, MMP-9 and VEGF showed low-level expression in the GCL, IPL and
neovascularization breaking through the ILM of the treated group compared with
the OIR and OIR control group. TIMP-1 showed high-level expression in the GCL,
IPL, INL and neovascularization breaking through the ILM of the treated group
compared with the OIR and OIR control group. The results proved that hypoxia
induced the expression of MMP-9 and VEGF, and TIMP-1 could inhibite the
expression of MMP-9 and VEGF.
Figure 2 MMP-9-VEGF signaling pathway in OIR mice model Protein expression of MMP-9, TIMP-1, and VEGF was determined by immunohistochemistry (magnification: 400×). The arrows indicate MMP-9-, TIMP-1- or VEGF-positive cells.
Inhibitory Effect of Tissue
Inhibitors of Matrix Metalloproteinase-1 Real-time reverse transcription- polymerase chain reaction (RT-PCR) was used to
determine MMP-9, TIMP-1 and VEGF mRNA expression in retina samples. MMP-9
(+135% and +166%) and VEGF (+358% and +299%) expression were increased in the
OIR and OIR control group compared with the control group (all P<0.05) (Figure 3). TIMP-1 (-28.5%
and -38.7%) expression was decreased in the OIR and OIR control group compared
with the control group (P<0.05)
(Figure 3). Compared with the OIR control group, the treated group decreased
MMP-9 and VEGF mRNA expression (-34.6% and -56.1%, respectively, all P<0.05) and increased TIMP-1 mRNA
expression (+50%, P<0.05) (Figure
3).
Western blot
revealed similar results in retina samples (Figure 4). MMP-9 (+217% and +231%)
and VEGF (+100% and +106%) protein expressions were increased in the OIR and
OIR control group compared with the control group (all P<0.05) (Figure 4B). TIMP-1 (-56.4% and -59.1%) protein
expression was decreased in the OIR and OIR control group compared with the control
group (P<0.05) (Figure 4B).
Compared with the OIR control group, the treated group decreased MMP-9 and VEGF
protein expression (-30.7% and -54.4%, respectively, all P<0.05) and increased TIMP-1 protein expression (+154%, P<0.05) (Figure 4B).
Figure 3 TIMP-1 inhibited RNV through the inhibition
of the MMP-9-VEGF signaling in OIR mice model MMP-9, TIMP-1 and VEGF were determined by real-time
RT-PCR (n=9). aP<0.05 vs the control group, cP<0.05 vs the OIR
group, eP<0.05 vs the OIR control group.
Figure 4 TIMP-1 inhibited RNV through
the inhibition of the MMP-9-VEGF signaling in OIR mice model MMP-9, TIMP-1 and VEGF were determined by
western blot (n=9). A: Western blot
assay for protein expression; B: Statistical analysis. aP<0.05 vs the control group, cP<0.05 vs the OIR
group, eP<0.05 vs the OIR control group.
DISCUSSION
RNV is an important pathological event and a main cause
of blindness. Recent studies have demonstrated that retinal laser
photocoagulation, photodynamic therapy and transpupillary thermotherapy (TTT)
were the effective methods for the treatment of RNV. However, these methods may
affect visual function. At present, intravitreal injection of anti-VEGF drug
(bevacizumab or ranibizumab) has become an increasingly popular therapy for ROP[16-18]. Angiogenesis is a
multistep process, and many ways could interfere with its progression[19]. Consequently, the
focus of our research is to clarify the molecular mechanisms of RNV.
MMPs are
important in the development of the central nervous system and retina. Several
research groups have examined high expression of MMPs in RNV. Recent research has
shown that MMPs have dual role in the development of DR; in the early stages of
the disease (pre-neovascularization), MMP-2 and MMP-9 facilitate the apoptosis
of retinal capillary cells, possibly via
damaging the mitochondria, and in the later phase, they help in
neovascularization[9]. Although higher levels of MMPs were
observed in the vitreous of patients with proliferative retinopathy over two
decades ago, their role in the development of this blinding disease has
remained unclear[20]. Das
et al[21] reported that human diabetic epiretinal neovascular
membranes contain high levels of MMP-9 and urokinase. Beránek
et al[22] reported increased MMP-9 expression in the retinas
of proliferative DR. However, there are few reports regarding the interaction
between MMP-9 and VEGF on RNV in the OIR model. Based on these findings, the
present study aimed to determine whether the expression of VEGF was regulated
by MMP-9 on RNV.
We found that increased MMP-9 expression (protein:
+217%; mRNA: +135%) contributed to the increased level VEGF (protein: +100%;
mRNA: +358%) and RNV in OIR. In addition, TIMP-1(inhibitor of MMP-9)
significantly inhibited RNV by reducing the expression of VEGF.
Immunohistochemical analysis indicated that the expression levels of MMP-9 and
VEGF and RNV were prevented by intravitreal injection of TIMP-1. These results
suggested that MMP-9 depletion could reduce VEGF production, which play the
role of anti-angiogenesis. We also found that the anti-angiogenic effect of
TIMP-1 derived from the reduction of VEGF. Thus, these results strongly suggest
that MMP-9 plays an important role in OIR retinas. Furthermore, there was no
inflammatory reaction and cellular toxicity in the treated group, and the
generation of normal retinal blood vessels was not affected, which shows that
intravitreal injection of TIMP-1 may be safe and effective for preventing RNV
in the OIR model. MMP-9-targeted interventions may have an effective
therapeutic effect on ROP.
ACKNOWLEDGEMENTS
Foundations: Supported by National Natural Science
Foundation of China (No.81371045); Science and Technology Project of Shenyang
City, China (No.F13-220-9-37).
Conflicts of Interest: Di Y, None; Nie QZ, None; Chen XL, None.
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