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IGFBP-rP1-silencing promotes hypoxia-induced angiogenic potential of choroidal endothelial cells via the RAF/MEK/ERK signaling pathway

Insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1) has been reported to have various functions in different cellular contexts. Our previous investigation discovered that IGFBP-rP1 inhibited retinal angiogenesis in vitro and in vivo by inhibiting the pro-angiogenic effect of VEGF and downregulating VEGF expression. Recently, IGFBP-rP1 was confirmed to be downregulated in the aqueous humor of patients with neovascular age-related macular degeneration compared with controls; however, its specific role remains unknown. The present study applied the technique of gene silencing, reverse transcription-quantitative PCR, western blotting, cell viability assays, cell motility assays and tube formation assays. Chemical hypoxic conditions and choroidal endothelial (RF/6A) cells were used to explore the effect of IGFBP-rP1-silencing on the phenotype activation of RF/6A cells under hypoxic conditions and to elucidate the underlying mechanisms. siRNA achieved IGFBP-rP1-silencing in RF/6A cells without cytotoxicity. IGFBP-rP1-silencing significantly restored the viability of RF/6A cells in hypoxia and enhanced hypoxia-induced migration and capillary-like tube formation of RF/6A cells. Furthermore, IGFBP-rP1-silencing significantly upregulated the expression of B-RAF, phosphorylated (p)-MEK, p-ERK and VEGF in RF/6A cells under hypoxic conditions; however, these upregulations were inhibited by exogenous IGFBP-rP1. These data indicated that silencing IGFBP-rP1 expression in RF/6A cells effectively promoted the hypoxia-induced angiogenic potential of choroidal endothelial cells by upregulating RAF/MEK/ERK signaling pathway activation and VEGF expression.


Introduction
Angiogenesis, a process of novel blood vessel formation from the pre-existing vasculature, serves a critical role in numerous human neovascularization diseases, such as diabetic retinopathy, ischemia/reperfusion injury and carcinogenesis (1). Vascular homeostasis is an intrinsically regulated procedure controlled by the balance of angiogenic and anti-angiogenic factors during normal physiological activities (1). Once homeostasis is interrupted, neovascularization is inevitable, becoming a major cause of irreversible vision loss in various clinical eye diseases, including retinopathy of prematurity, diabetic retinopathy and age-related macular degeneration (aMd) (2)(3)(4).
insulin-like growth factor binding protein-related protein 1 (iGFBP-rP1) is a soluble-secreted 36-kda glycoprotein, also known as IGFBP7, mac25, TAF and angiomodulin (10,11). IGFBP-rP1 is distinct from other IGFBPs since it exhibits a low affinity for IGFs and a high affinity for insulin (10,11). Our previous studies have revealed that iGFBP-rP1 suppressed the phenotype activation of retinal endothelial cells induced by VeGF in vitro and inhibited retinal angiogenesis by downregulating VeGF in vivo (12,13). notably, iGFBP-rP1 was demonstrated to be decreased in the aqueous humor of patients with CNV secondary to AMD compared with control patients with cataract; however, its specific role remains unknown (14).
In the present study, IGFBP-rP1 specific small interfering rna (siiGFBP-rP1) was transfected into choroidal endothelial (rF/6a) cells to block the expression of iGFBP-rP1 under hypoxic conditions to investigate the role of iGFBP-rP1-silencing in the hypoxia-induced angiogenic potential of choroidal endothelial cells and the underlying mechanisms.

Materials and methods
Media and reagents. Lipofectamine ® 3000 transfection reagent was purchased from Thermo Fisher Scientific, Inc. PrimeScript reverse transcription (RT) reagent kit and SYBR Premix Ex Taq Real-Time PCR kit were purchased from Takara Bio, Inc. and were used in RT-quantitative (q)PCR assays. Recombinant human IGFBP-rP1 and goat anti-human IGFBP-rP1 polyclonal antibody (cat. no. AF1334) were obtained from R&D Systems, Inc. Rabbit anti-human GAPDH polyclonal (cat. no. 10494-1-AP) and mouse anti-human β-actin monoclonal (cat. no. 66009-1-Ig) antibodies were obtained from ProteinTech Group, Inc. Rabbit anti-human VEGF polyclonal (cat. no. ab150766) antibodies were purchased from Abcam. The other primary antibodies, including rabbit anti-human B-RAF monoclonal (cat. no. 14814), rabbit anti-human phosphorylated-MEK (p-MEK) polyclonal (cat. no. 9121), rabbit anti-human MEK polyclonal (cat. no. 9122), rabbit anti-human phosphorylated-ERK (p-ERK) polyclonal (cat. no. 9101) and rabbit anti-human ERK polyclonal (cat. no. 9102) antibodies were purchased from Cell Signaling Technology, Inc. Horseradish peroxidase (HRP) mouse anti-goat (cat. no. sc2354), goat anti-rabbit (cat. no. sc-2004) and goat anti-mouse (cat. no. sc-2005) secondary antibodies were obtained from Santa Cruz Biotechnology, Inc. The PVDF membranes and Chemiluminescent HRP Substrate reagent were purchased from EMD Millipore. CellTiter 96 ® aQueous one Solution cell Proliferation assay (MTS Assay) was purchased from Promega Corporation. Transwell Permeable Supports with an 8-µm pore polycarbonate filter were purchased from Corning, Inc. Growth factor-reduced Matrigel matrix was purchased from BD Biosciences. All other chemicals were of reagent grade and obtained from Merck KGaA, unless otherwise specified.
Cell line and culture. rF/6a cells, a well-established choroid endothelial cell line for studying CNV pathogenesis (15)(16)(17), were obtained from the Cell Bank of the Chinese Academy of Sciences. The cells were cultured in RPMI-1640 medium (Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin (GE Healthcare Life Sciences). The cells were maintained at 37˚C in a 5% CO 2 -humidified incubator and the medium was changed every 2 or 3 days. Prior to experimental intervention, the medium was replaced with serum-free RPMI-1640 for 12 h.
Following sirna transfection for 24 h, rF/6a cells were subjected to hypoxic conditions and IGFBP-rP1-restored conditions for further study. cobalt chloride (cocl 2 ; Merck KGaA) at a final concentration of 200 µmol/l or 1% O 2 in the presence of 5% CO 2 and 94% N 2 using a ProOx C21 System (BioSpherix, Ltd.) was used to mimic hypoxic conditions. To restore IGFBP-rP1 expression, recombinant human IGFBP-rP1 was added at a final concentration of 200 ng/ml for 24 h in the conditions of the siiGFBP-rP1 duplex 2 group, which has been demonstrated by previous studies (18)(19)(20). All experiments were performed in triplicate.
IGFBP-rP1 gene silencing. sirnas for iGFBP-rP1 were purchased from Guangzhou RiboBio Co., Ltd. The sequences of the positive siIGFBP-rP1 duplex 1 and 2 were 5'-TCC-TCC-TCT-TCG-GAC-ACC-T-3' and 5'-GTC-GCT-ACA-TGC-CCT-GCT-C-3', respectively. RF/6A cells were seeded into six-well plates and transfected with 50 nM siRNA using Lipofectamine ® 3000 transfection reagent. Briefly, 50 nM siRNA in Opti-MEM medium (Thermo Fisher Scientific, Inc.) was mixed with 4 µl Lipofectamine ® 3000 and incubated for 25 min at room temperature prior to adding the mixture to the cells cultured in serum-free medium. The cells were incubated at 37˚C for 5 h. Following this, the medium was replaced with RPMI-1640 complete medium for 48 h before the level of silencing was determined by RT-qPCR and western blotting. Scramble control siRNA, transfection reagent and blank control were used to compare the effects of siiGFBP-rP1.
RNA extraction and RT-qPCR. Total RNA was extracted from cultured rF/6a cells using a rneasy Mini kit (Qiagen, inc.), according to the manufacturer's protocol. Total RNA was eluted in 40 µl nuclease-free water. The concentration and purity of RNA were measured by spectrophotometry (NanoDrop 8000; Thermo Fisher Scientific, Inc.) at 15˚C within 10 sec. In all rna preparations, the ratio of optical density (od) 260 to 280 was 1.9:2.0. Equal amounts of RNA (0.5 µg) were converted into cDNA using PrimeScript RT reagent kit (Takara Bio, Inc.) and qPCR was performed using SYBR Premix Ex Taq real-time PCR kit (Takara Bio, Inc.), according to the manufacturer's protocols. The primers of IGFBP-rP1 and GAPDH were designed and synthesized by Sangon Biotech Co., Ltd. GaPdH served as an internal reference for the control. The primer sequences used were as follows: IGFBP-rP1 forward, 5'-AGC-TGT-GAG-GTC-ATC-GGA-AT-3' and reverse, 5'-CAG-CAC-CCA-GCC-AGT-TAC-TT-3'; and GAPDH forward, 5'-GAG-TCA-ACG-GAT-TTG-GTC-GT-3' and reverse, 5'-GAC-AAG-CTT-CCC-GTT-CTC-AG-3'. The One-Step RT-PCR thermocycling conditions were as follows: 30 min of initial reverse transcription at 50˚C, enzyme heat-activation at 95˚C for 15 min, followed by 35 three-step amplification cycles of denaturation at 94˚C for 40 sec, annealing at 60˚C for 40 sec and extension at 72˚C for 1 min. The qPCR thermocycling conditions were as follows: enzyme heat-activation at 95˚C for 30 sec, followed by 40 two-step amplification cycles of denaturation at 95˚C for 5 sec, annealing and extension at 60˚C for 35 sec. Relative gene expression was calculated using a standard curve (the optical density was determined at 280 nm) and IGFBP-rP1 mRNA levels were normalized to GAPDH.
Cell viability assay. cell viability was evaluated by MTS colorimetric assays, as previously described (12). Briefly, cells were seeded in a 96-well plate (100 µl/well; 5x10 4 cells/ml) and allowed to attach overnight in the serum-free medium. Following this, cells were transfected with sirna duplex 1 and 2 for 6, 12, 24, 48 and 72 h to plot growth curves. The cells transfected with sirna duplex 2 for 24 h were then exposed to cocl 2 mimetic and 1% O 2 -induced hypoxia for 6, 12, 24, 48 and 72 h at 37˚C in a 5% CO 2 -humidified incubator to compare differences between these two types of hypoxic conditions on cell proliferation. Finally, MTS reagent (20 µl) was added to each well for 2 h and the absorbance and od values at 490 nm were recorded using a Wallac Victor 1420 plate reader (PerkinElmer, Inc.).
Cell motility assay. Wound and Transwell assays were performed to assess cell motility. Confluent monolayers of the transfected or untransfected cells (4x10 5 cells/well) in 6-multiwell plates were wounded using a pipette tip and washed with PBS three times. Following this, the plates were incubated in DMEM (1% FBS) containing 200 µmol/l CoCl 2 for 24 h at 37˚C in a 5% CO 2 incubator. Images of the wounded area were immediately recorded at the time of wounding and at 24 h using an inverted microscope (IX50; Olympus Corporation) at a magnification of x400. The relative migration area of cells that had migrated from the edge of the wound was calculated using ImageJ software (version 1.50; National Institutes of Health).
The Transwell assays was performed as previously described (12). Briefly, the transfected or untransfected cells were placed in the upper chamber (6x10 4 cells/chamber) at a final volume of 100 µl of the serum-free RPMI-1640 medium. Following this, the normal or CoCl 2 -induced hypoxic medium was placed in the bottom chamber at a final volume of 600 µl. Following incubation for 24 h, the filters were fixed in 10% paraformaldehyde for 20 min at room temperature and stained with hematoxylin for 15 min at room temperature. Cells on the lower surface of the filters were counted in five randomly selected fields/filter under a light microscope (BX50; Olympus Corporation) at a magnification of x200.
Tube formation assay. Tube formation assays were performed by placing ice-cold growth factor-reduced Matrigel matrix in a prechilled 96-well plate (50 µl/well), followed by incubation at 37˚C for 30 min to allow polymerization. Next, siIGFBP-rP1 transfected cells (8x10 4 cells/well) were seeded on the Matrigel-coated 96-well plate and incubated for 24 h under normal or hypoxic conditions. Enclosed capillary-like tube structures within the Matrigel layer from five randomly selected fields/well were captured under an inverted phase contrast microscope (BX40; Olympus Corporation) at a magnification of x50. The tube length was measured by Image-Pro Plus Analyzer software (version no. 6.0; Media Cybernetics, Inc.).

Statistical analysis.
A one-way ANOVA followed by Dunnett's post-hoc test was used to evaluate differences between the control and intervention groups assessed by different experiments. All tests were performed using SPSS for Windows (version no. 13.0; SPSS Inc.). Data are presented as the mean + standard deviation. P<0.05 was considered to indicate a statistically significant difference.

Results
IGFBP-rP1-silencing in RF/6A cells by siRNA does not exhibit cytotoxicity. Two different siRNA sequences were designed for transfection to attain a higher knockdown efficiency. Firstly, the expression of IGFBP-rP1 was confirmed in rF/6a cells without the specific sirna transfection. Following this, the effectiveness of siiGFBP-rP1 transfection was measured by RT-qPCR and western blotting. IGFBP-rP1 expression exhibited a significant decrease in siIGFBP-rP1 duplex 1-and 2-transfected cells at the mRNA and protein levels (P<0.05 vs. the blank control group; Fig. 1). By contrast, there were no significant differences between the cells cultured in the blank control, transfection reagent and scramble control siRNA groups (P>0.05).
Moreover, the cell growth curve demonstrated that siIGFBP-rP1 duplex 1 and 2 significantly promoted RF/6A cell proliferation at 12, 24 and 48 h compared with the blank control group (P<0.01; Fig. 2). No significant differences in cell viability were observed among the groups at 6 and 72 h (P>0.05). Furthermore, the siIGFBP-rP1 duplex 2 demonstrated a stronger pro-proliferative effect compared with the siIGFBP-rP1 duplex 1 at 24 h (P<0.01). Since the siIGFBP-rP1 duplex 2 exhibited a higher inhibition efficiency and no cytotoxicity, it was selected for the following experiments.
IGFBP-rP1-silencing restores RF/6A cell viability under hypoxia. as revealed in Fig. 3, the od values of rF/6a cells detected by MTS colorimetric assay decreased significantly following 12, 24, 48 and 72 h of culturing in hypoxic environments, as compared with the control group, both at 200 µmol/l cocl 2 and at 1% O 2 compared with the controls (P<0.01) . no significant difference was observed between the two hypoxic conditions (P>0.05). Following transfection with siIGFBP-rP1 for 24 h, the OD values significantly increased following 12, 24 and 48 h of culturing in normoxic conditions compared controls (P<0.01). Although the OD values of transfected cells cultured in hypoxic conditions for 12, 24, 48 and 72 h were lower than those of transfected cells cultured in normoxic conditions, the values were still significantly higher compared with untransfected cells cultured in hypoxic conditions (P<0.01). No significant differences were observed among transfected cells cultured in hypoxic conditions and the control group at any time-point (P>0.05).
IGFBP-rP1-silencing stimulates hypoxia-induced migration and tube formation of RF/6A cells. In order to determine the effect of iGFBP-rP1-silencing on phenotype activation of rF/6a cells under cocl 2 -induced hypoxic conditions, chemotactic motility and tube formation assays were performed on siIGFBP-rP1 transfected and untransfected cells under normal and hypoxic conditions. The scramble control sirna-transfected cells were used as control cells.
The relative migration area of cells that had migrated from the edge of the wound and those that had migrated across the filter toward the lower surface due to hypoxia were ~2.5x and ~2x that of the control, respectively (Fig. 4). siIGFBP-rP1 transfection significantly increased cell mobility compared with non-transfected cells, under both normoxic (P<0.01 vs. the hypoxia group) and hypoxic conditions (P<0.01 vs. the siiGFBP-rPi group).
As presented in Fig. 5, extensive and enclosed networks of capillary-like tubes were observed in the siiGFBP-rP1 and hypoxia + siIGFBP-rP1 groups, while incomplete and sparse tube networks formed by the untransfected cells under normal conditions. Tube formation, as determined by the total tube length, increased ~7-(the siIGFBP-rP1 group vs. the control group) or ~5-fold (the hypoxia + siIGFBP-rP1 group vs. the hypoxic group) following siiGFBP-rP1 transfection, as compared with non-transfection under normal or hypoxic conditions, respectively. Furthermore, tube length was significantly increased in the hypoxia group compared with the control group (P<0.01).
IGFBP-rP1-silen ci ng en h a n ces hyp oxia -i n d u ced RAF/MEK/ERK signaling pathway activation in RF/6A cells. To understand the underlying mechanisms of restored cell viability, enhanced migration and tube formation by siIGFBP-rP1-transfected cells in hypoxic conditions, RF/6A cells cultured in normal conditions and cocl 2 -induced hypoxic conditions with or without siIGFBP-rP1 transfection and exogenous human IGFBP-rP1 were detected to determine the changes of key molecules in the raF/MeK/erK signaling pathway using western blotting. as revealed in Fig. 6, under hypoxic conditions, the expression of B-RAF, p-MEK and p-ERK were significantly increased compared with that under normal conditions (P<0.05). In addition, following IGFBP-rP1-silencing, the phosphorylation level of the raF/MeK/erK signaling pathway was significantly increased, both under normal (the siIGFBP-rP1 group vs. the control group, P<0.05) and hypoxic conditions (the hypoxia + siIGFBP-rP1 group vs. the hypoxia group, P<0.05). Furthermore, with the addition of exogenous human IGFBP-rP1, the upregulation of B-raF, p-MeK and p-erK induced by siiGFBP-rP1 transfection significantly decreased (P<0.01). No significant differences in the expression of MeK and erK were observed between the six groups (data not shown).

IGFBP-rP1-silencing upregulates the hypoxia-induced VEGF expression in RF/6A cells.
Since VeGF expression may be involved in the process of choroidal angiogenesis mediated by IGFBP-rP1 (12), western blotting was performed to detect the distinct protein expression of VeGF under different intervention conditions. as presented in Fig. 6, the expression of VEGF increased ~4-or ~7 fold in the hypoxia group and siIGFBP-rP1 group, respectively, compared with control. When the transfected cells were cultured in hypoxic conditions, the expression of VEGF significantly increased (~8-fold), compared with control. Additionally, the upregulation of VeGF in siiGFBP-rP1-transfected cells under hypoxic conditions was markedly increased compared with transfected cells under normal conditions (P<0.01; data not shown). Similar to the RAF/MEK/ERK pathway, exogenous human iGFBP-rP1 significantly downregulated the expression of VEGF in siIGFBP-rP1-transfected cells, under both normoxic and hypoxic conditions (P<0.01).

Discussion
The number of patients with AMD, a common age-associated eye disease, is increasing worldwide due to exponential population ageing. The projected number of individuals with AMD for 2020 is 196 million, with an expected increase to 288 million in 2040 (21,22). Since 2010, AMD has become the 3rd most common cause of blindness and the 4th leading cause of visual impairment worldwide (23). IGFs, the family of important proteins that regulate various cellular processes, including proliferation, differentiation, apoptosis and neovascularization, may be involved in molecular mechanisms that contribute to the pathogenesis of aMd, involving oxidative stress, mitochondrial dysfunction and impaired resistance to molecular stressors in the choriocapillaris (24)(25)(26)(27)(28)(29)(30). The members of the IGF family, including IGFBP-2, IGFBP-6 and IGFBP-rP1, were demonstrated to be increased in patients with exudative aMd (31). However, further research reported that iGFBP-rP1 was decreased in patients with neovascular aMd (14). This difference in iGFBP-rP1 expression highlighted the possibility of iGFBP-rP1 being an endogenous factor regulating aMd progression.
The IGFBP-rP1 gene is located on chromosome 4q and encodes a 256-amino acid protein (10,11). IGFBP-rP1 is distinct from other IGFBPs, as it can highly bind to insulin yet lowly to IGFs and IGFBP-rP1 has been demonstrated to There was no significant difference between the hypoxic groups (P>0.05). The OD value of the siRNA group were significantly increased at 12, 24 and 48 h compared with controls ( ** P<0.01). Furthermore, the OD values of the transfected cells cultured in hypoxic conditions (cocl 2 + siRNA group and 1% O 2 + siRNA group) for 12, 24, 48 and 72 h were significantly lower compared with the siRNA group; additionally, the values were significantly higher compared with the hypoxic groups ( * P<0.01). No significant differences were identified between the cocl 2 + siRNA, 1% O 2 + siRNA and control groups (P>0.05). OD values are presented as the mean ± standard deviation of 4 wells/group and experiments were performed in triplicate. IGFBP-rP1, insulin-like growth factor binding protein-related protein 1; siIGFBP-rP1, IGFBP-rP1 specific siRNA; cocl 2 , cobalt chloride; OD, optical density.   . Hypoxia significantly promoted cell mobility compared with the controls ( # P<0.01). siIGFBP-rP1 transfection (siIGFBP-rP1 group) further enhanced cell migration, as compared with untransfected cells cultured in hypoxic conditions (hypoxia group; * P<0.01). The transfected cells cultured in hypoxic conditions (hypoxia + siRNA group) exhibited a significantly increased migration ability compared with the transfected cells cultured in normoxic conditions (siIGFBP-rP1 group; ** P<0.01). Values are presented as the mean ± standard deviation of 4 samples/group and experiments were performed in triplicate and are quantified as the ratio relative to the control group. IGFBP-rP1, insulin-like growth factor binding protein-related protein 1; siIGFBP-rP1, IGFBP-rP1 specific siRNA.
have various functions in different cellular contexts (10,11). IGFBP-rP1 was described to have both tumor suppressing and enhancing properties in regard to cell proliferation (32). additionally, iGFBP-rP1 was associated with the senescence and apoptosis of different cell lines, including human breast cancer cell lines and prostate cancer cell lines (33,34). Previous studies revealed a stimulatory effect of IGFBP-rP1 in the adhesion, migration and proliferation of acute myeloid leukemia (35,36). Accumulating evidence has indicated that IGFBP-rP1 downregulation may participate in tumorigenesis, acting as a cancer suppressive factor in various types of cancer, including breast, colorectal, prostate and hepatocellular cancer (37)(38)(39)(40). our previous study revealed that iGFBP-rP1 inhibited the stimulatory effect of VEGF on retinal angiogenesis in vitro (12). it was also revealed that iGFBP-rP1 inhibited ocular neovascularization by downregulating VEGF expression in a mouse model of O 2 -induced retinopathy (13). Furthermore, IGFBP-rP1 was confirmed to be downregulated in the aqueous humor of patients with wet AMD; however, the downregulation of iGFBP-rP1 in the pathogenesis of wet AMD remains unknown (14).
In order to examine the effects of IGFBP-rP1 on the biological behavior of choroidal endothelial cells under a pathological state, gene silencing was performed to interfere with the expression of iGFBP-rP1 in choroidal endothelial cells and hypoxic conditions were used to mimic the internal Figure 6. IGFBP-rP1-silencing upregulates the hypoxia-induced RAF/MEK/ERK signaling pathway activation and VEGF expression. Representative images and quantified data demonstrated that hypoxic stress upregulated B-RAF, p-MEK, p-ERK and VEGF expression in RF/6A cells compared with controls ( # P<0.05). siIGFBP-rP1 transfection significantly promoted hypoxia-induced B-RAF, p-MEK, p-ERK and VEGF expression in RF/6A compared with the hypoxia group ( # P<0.05). IGFBP-rP1 restoration significantly downregulated the expression of B-RAF, p-MEK, p-ERK and VEGF in siIGFBP-rP1-transfected cells, under both normoxic and hypoxic conditions, compared with the siIGFBP-rP1 and hypoxia + siIGFBP-rP1 groups, respectively ( * P<0.01). The membranes were stripped off and probed for the proteins. Values are presented as the mean ± SD of 3 independent experiments with similar results and are presented as the integrated optical density of studied proteins relative to GAPDH. IGFBP-rP1, insulin-like growth factor binding protein-related protein 1; siIGFBP-rP1, IGFBP-rP1 specific siRNA. Lane 1, control; lane 2, siIGFBP-rP1; lane 3, siIGFBP-rP1 + IGFBP-rP1; lane 4, hypoxia; lane 5, hypoxia + siIGFBP-rP1; lane 6, hypoxia + s iIGFBP-rP1 + IGFBP-rP1. environment in patients with AMD. RF/6A cells, a rhesus choroid endothelial cell line widely used in research to simulate ophthalmic micro-endothelial cells and explore the influences of external factors and corresponding mechanisms (15)(16)(17)41,42) were used to observe the influence of IGFBP-rP1-silencing and hypoxia on CNV formation in vitro. rna interference, which allows for the silencing of mammalian genes with great specificity and potency, has been extensively used in several fields to investigate gene functions and design novel therapeutic methods (43)(44)(45)(46)(47)(48). Using this technique, IGFBP-rP1-silencing was successfully accomplished in RF/6A cells. Cell growth curves confirmed that siIGFBP-rP1 duplex 1 and 2 both significantly promoted rF/6a cell proliferation at 12, 24 and 48 h, and siiGFBP-rP1 duplex 2 demonstrated a more significant effect compared with duplex 1 at 24 h. Furthermore, no significant differences were observed between the transfected cells and untransfected cells at 6 and 72 h. These results suggested that there was no significant impairment in cell viability following the silencing of iGFBP-rP1 expression at 6-72 h.
Hypoxia is considered to serve an important role in CNV formation, a critical characteristic of wet AMD and the main cause of visual impairment in patients with subsequent bleeding or fibrosis (6)(7)(8)(49)(50)(51). CoCl 2 -induced chemical hypoxia, a reliable method of mimicking hypoxia (52)(53)(54)(55), was used to imitate the internal environment during choroidal angiogenesis. Since cocl 2 can unexpectedly and markedly decrease pyruvate dehydrogenase phosphorylation and lead to an enhanced glycolytic poise in mammalian cells (56), the different effects between cocl 2 mimetic and 1% O 2 -induced hypoxia on cell proliferation were compared. Both hypoxic conditions significantly decreased cell proliferation and iGFBP-rP1-silencing restored cell viability in these hypoxic conditions. The difference on cell proliferation was not obvious between these two types of hypoxic environment under the current experimental conditions, thus the present study continued to use cocl 2 mimetic hypoxia in subsequent experiments. The results demonstrated that hypoxic conditions significantly enhanced migration and capillary-like tube formation in RF/6A cells. These results indicated that abnormal environments, including hypoxia, serve an important role in the development of CNV formation, which was consistent with previous studies (54,55,57).
The present study further explored the role of iGFBP-rP1-silencing in the angiogenic potential of rF/6a cells under hypoxic conditions. The results demonstrated that the transfected cells under hypoxia were significantly activated to migrate from the edge of the wound, pass across the filter toward the lower surface and form extensive and enclosed networks of capillary-like tubes compared with controls. The migrated area and number of siIGFBP-rP1 transfected cells in hypoxic conditions, as well as total tube length, were significantly higher compared with siIGFBP-rP1 transfected cells in normal conditions. These results indicated that the downregulation of IGFBP-rP1 significantly promoted vascular branching and sprouting under hypoxia, which implied that IGFBP-rP1 may act as an anti-angiogenic regulator of choroidal angiogenesis. during the process of aMd-related choroidal vascular homeostasis deterioration and neovascularization, the downregulation of anti-angiogenic factors has been demonstrated to be a common pathological manifestation (58)(59)(60)(61).
This may explain why the concentration of IGFBP-rP1 in the aqueous humor of patients with wet AMD was significantly lower compared with controls (14). Along with excessively synthesized and released VEGF in the hypoxic eyes of these patients (9,62), the balance was further disrupted by the downregulation of IGFBP-rP1, which may significantly accelerate the occurrence and development of CNV.
The raF/MeK/erK signaling pathway serves a critical role in regulating cell division, proliferation, senescence and apoptosis during physiological and pathological processes, such as adipocyte physiology, insulin signaling, oxidative stress and cancer progression (63)(64)(65)(66). excessive activation of the key molecules of this signaling pathway in the majority of types of cancer results in an unbalanced cell proliferation and apoptosis inhibition or escapement (67,68). Additionally, it has been confirmed that the activation of the RAF/MEK/ERK signaling pathway modulated the role of VEGF in tumor pathogenesis and metastasis (69)(70)(71)(72). The present study demonstrated that the RAF/MEK/ERK signaling pathway was significantly activated when choroidal endothelial cells cultured under hypoxic conditions were transfected with siiGFBP-rP1, while exogenous iGFBP-rP1 downregulated the activation level of this pathway. IGFBP-rP1 may promote the pro-angiogenic effect of VEGF, particularly in pathological conditions, including hypoxia, which was reported by the present results. Furthermore, the results of the present study revealed that the hypoxia-induced VeGF expression was significantly upregulated in rF/6a cells by blocking iGFBP-rP1 expression. The regulation of VEGF expression demonstrated the same trend as the activation of the raF/MeK/erK signaling pathway. additionally, previous studies have indicated that the raF/MeK/erK signaling pathway regulated VeGF expression in diabetic animals (73,74) and hypoxic conditions (75,76). These results suggested that IGFBP-rP1 may regulate VEGF expression in choroidal endothelial cells through the raF/MeK/erK signaling pathway under hypoxia. In combination, the inhibition of iGFBP-rP1 further activated the raF/MeK/erK signaling pathway and upregulated VeGF expression, which may concurrently participate in choroidal endothelial cell phenotype activation, CNV formation and AMD progression under hypoxia. Since patients with partial wet aMd have a poor or no response to anti-VEGF treatment, novel drugs that can resolve this problem are urgently required. IGFBP-rP1 may become a therapeutic target in choroidal angiogenesis due to its potential inhibition of ophthalmic neovascularization.
The present study had limitations. Although there was no significant difference in cell viability between CoCl 2 mimetic and 1% O 2 -induced hypoxia, certain effects induced by cocl 2 may differ from those of actual hypoxia. Therefore, the role of IGFBP-rP1 in RF/6A cells under physical hypoxia requires further investigation. Furthermore, despite the extensive application of rF/6a cells as the closest choroidal endothelial cell line to human species in studies on choroidal angiogenesis in vitro, the signaling circuitry has been altered in this immortalized cell line. Therefore, the exact effect of IGFBP-rP1 on choroidal angiogenesis and the raF/MeK/erK signaling pathway needs to be further explored in human choroidal endothelial cells and in an in vivo model.
in conclusion, the present study revealed that IGFBP-rP1-silencing promoted cell proliferation and stimulated hypoxia-induced migration and tube formation of RF/6A cells. Additionally, the results confirmed that IGFBP-rP1-silencing effectively promoted hypoxia-induced angiogenic potential of choroidal endothelial cells by upregulating VeGF expression, probably by activating the raF/MeK/erK signaling pathway. Further research should be conducted to determine whether iGFBP-rP1 acts as an angiogenesis inhibitor in vivo and its effect on other ocular tissues; the real-world application of iGFBP-rP1 or an associated drug in clinical practice requires further research in future studies.
Acknowledgements not applicable.

Funding
The present study was supported by grants from the National nature Science Foundation for Young Scholars of china (grant no. 81200701), the National Key R&D Program of China (grant nos. 2016YFC0904800 and 2019YFC0840607), the National Science and Technology Major Project of China (grant no. 2017ZX09304010) and the Bethune·Lumitin Research Funding for Young or Middle-aged Ophthalmologists (grant no. BJ-LM2019001J).

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions
SZ and HW conducted the sirna assays, the restoration assays, data acquisition and were major contributors in drafting the original manuscript. MM and ZZha performed the cell viability assays, cell motility assays, tube formation assays, created graphs and were major contributors in drafting the original manuscript. ZZhe and HW performed the signaling pathway assays, analysis and interpretation of data and made major contributions in editing the original manuscript. XX and TS made substantial contributions to conception and design, and made critical revising of the original manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate not applicable.

Patient consent for publication
not applicable.