Materials

Double-stranded siRNA oligonucleotides to rhesus and mouse siRNA (AGS8siRNA#1: sense, 5′-CCCUGCCUGCCAGCUUUCAU-3′; AGS8siRNA#2: sense, 5′-GAAGCCAAGGCACGTGAAACT-3′; AGS8siRNA#3: sense, 5′-GTGAAACTGCTGTCCACTAAA-3′), negative control with scrambled sequences, and Lipofectamine siRNA Max reagent were purchased from Life Technologies (Carlsbad, CA). Antibodies against VEGFR-2 (#2479; 1:1000 dilution for immunoblot, 1:100 dilution for immunoprecipitation), Anti-AKT (#9272), anti-phospho-AKT (Ser473) (#9271), anti-p44/42 MAPK (#9102), anti-phospho-p44/42 MAPK (Thr202/204) (#9101), anti-p38MAPK (#9212), anti-phosphop38MAPK (Thr180/Tyr182) (#9211) were purchased from Cell Signaling Technology (Danvers, MA), and anti phosphotyrosine (PY20; 1:2000 dilution for immunoblot) was obtained from Santa Cruz Biotechnology (Dallas, TX). RF6A cells were obtained from Riken Bioresource Center (Tsukuba, Japan). RF/6A is a immortal endothelial cell line derived from the choroid-retina of a rhesus macaque34, which has been used in biological studies of endothelial cells17,18,19. VEGFR-2 inhibitor was purchased from R&D Systems (Minneapolis, MN).

Animals

C57BL/6N mice were purchased from Japan SLC, Inc. (Hamamatsu, Japan). All animal procedures were approved by The Animal Care and Use Committee of Aichi Medical University (2017-88) and performed in accordance with the Research Guideline for Animal Experimentation of Aichi Medical University. Mice were housed in a room maintained at 23 ± 1 °C with a 12 h light-12 h dark cycle and had free access to food and water.

Choroidal cell culture and siRNA transfection

The choroid-retinal endothelial cell line RF/6A cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Wako Chemicals, Osaka, Japan), supplemented with 10% fetal bovine saline (FBS) (Sigma-Aldrich, St. Louis, MO) and penicillin-streptomycin (Wako Chemicals). For siRNA transfection, RF/6A cells were cultured in six-well plates to subconfluence and treated with 40 nM rhesus AGS8 siRNA or control siRNA with Lipofectamine siRNA Max (Thermo Fisher Scientific., Waltham, MA) for 48 h according to the manufacturer’s instructions.

Tube formation assay

Briefly, 96-well plates were coated with 50 μL of growth-factor-reduced Matrigel (BD Biosciences, San Jose, CA), and polymerized at 37 °C for 1 h. RF/6A cells were starved using DMEM with 1% FBS for 6 h, trypsinized and then suspended in 250 μL of DMEM with 1% FBS. Cells were seeded at 1 × 104 cells/well onto Matrigel and again incubated. Under some conditions, cells were stimulated with 10 ng/mL of VEGF (R&D Systems, Minneapolis, MN). Images were taken at 6 h, and the tube length was determined by the use of ImageJ software35. The total tube length of each image was determined by an observer blinded to the conditions. Data are presented in arbitrary units.

MTT assay

Cell proliferation was analyzed using a Vybrant MTT cell proliferation assay kit (Life Technologies), according to the product manual. Briefly, cells in 6-well plates were transfected with siRNAs and incubated for 24 h. Cells were transferred to 96-well plates at 5 × 103 cells/well. After 24 h, cells were starved with DMEM containing 1% FBS for 6 h and treated with VEGF for 48 h. Cells were then incubated with MTT at 37 °C for 4 h. Medium was removed, and SDS-HCl solution (10% SDS, 0.01 M HCl) was added to each well. Absorbance was measured with a microplate reader after incubation for 18 h at 37 °C.

Cell migration assay

RF/6A cells suspended at 1 × 104 cells/200 μL were seeded on fibronectin coated Transwell inserts with 8 μm pores (BD Biosciences) pre-coated with 3% bovine serum albumin, then stimulated with 10 ng/mL of VEGF in the lower chamber. After 4 h, the Transwell inserts were fixed in 4% PFA and stained with 1% crystal violet. Digital microscopic images of the underside of each Transwell insert were taken at three independent microscopic fields per transwell.

Flow cytometric analysis

Fluorescence-activated cell sorting (FACS) was performed using a FACS Canto II (BD Biosciences, San Jose, CA) as described previously15. RF/6A cells were washed with PBS, harvested with Accutase (Innovative Cell Technologies, San Diego, CA), and fixed with 2% PFA for 30 min. The cells were washed with PBS containing 1% FBS, and labeled with a fluorescein-conjugated anti-human VEGFR-2 antibody (1:100 dilution) or fluorescein-conjugated IgG1 isotype control (1:100 dilution; IC002F; R&D Systems, Minneapolis, MN) for 1 h in the dark on ice. The data were analyzed by FACS DIVA (BD Biosciences) or FlowJo software (FlowJo LLC, Ashland, OR).

Laser-induced experimental CNV Model

Eight-week-old C57BL6/N male mice were used for laser-induced CNV induction according to a previous report36. Briefly, after anesthesia was induced with intraperitoneal injection of 1% pentobarbital sodium (50 mg/kg; Kyoritsu Seiyaku, Tokyo, Japan), the pupils were dilated using 0.5% tropicamide and 0.5% phenylephrine (Santen Pharmaceuticals, Osaka, Japan), followed by topical anesthesia using 0.4% oxybuprocaine hydrochloride (Santen Pharmaceuticals). Bruch’s membrane was injured under a slit-lamp microscope with an argon laser photo coagulator using the following parameters: 514 nm wavelength, 200 mW intensity, 0.1 s duration, and a 100 μm lesion diameter. Equal numbers of laser lesions were generated in the control eye and AGS8 knockdown eye (6–8 spots per eye) in every preparation. On specific days indicated, choroids were isolated and processed for the following experiments after laser induction.

Intravitreal injection of siRNA to knockdown gene expression in choroid

Experiments were essentially performed as previously reported24. Briefly, 100 ng of siRNA was combined with 4.5 μL of Transit-TKO (Takara Bio, Kyoto, Japan) in 50 μL of H 2 O and incubated for 20 min at room temperature. The mixed solution was evaporated under vacuum and the precipitate was dissolved in 5 μL H 2 O. Then, 1 μL of concentrated reagent mixture was injected into the vitreous using a Hamilton syringe with a 32 gauge needle.

Explant culture of choroid and siRNA transfection

Sprouting of choroidal layers was tested as previously described21. Briefly, 8-week-old C57BL/6n male mouse eyes were enucleated immediately after euthanasia. Choroid tissue was isolated and cut into pieces of approximately 1 mm × 1 mm fragments and placed on a coverslip coated with growth factor-reduced Matrigel (20 μL/well; BD Biosciences) in 24-well plates. The plates were then incubated at 37 °C for about 10 min to allow the Matrigel to solidify. Five hundred microliters of EGM-2 (Lonza, Basel, Switzerland) containing VEGF (0.5 ng/mL) was added to each well of the plate and incubated in 5% CO 2 . Images were taken using a phase contrast microscope and merged for further analysis. For siRNA transfection, the explants were transfected with 40 nM mouse AGS8 siRNA or control siRNA with Lipofectamine siRNA Max at days 2 and 3 and cultured until day 4.

Real-time PCR

Isolation of total RNA from cultured RF/6A cells and explant cultures of choroidal tissue and rat choroidal lesions were performed with an RNeasy Mini Kit (Qiagen, Valencia, CA) and cDNA synthesis with ReverTra Ace (Toyobo, Osaka, Japan), respectively. Real-time PCR was carried out with KAPA SYBR Fast qPCR kit (Nippon Genetics, Tokyo, Japan) and STEP one plus (Bio-Rad) according to the manufacturers’ instructions. The primers for real-time PCR were as follows: mouse AGS8 forward, 5′-TCTGGACACACGACTTCTGC-3′; reverse, 5′-GCTGTCTCTCCCATTTGGCT-3′; primer sequences for rhesus AGS8, which agrees with human primer sequence designed previously15, were forward, 5′-TTCCGTAACCCTCTCTCCCG-3′; and reverse, 5′-AACCCACGATCAAGGTCCAC-3′.

Immunohistochemistry

The explants were cultured on glass coverslips (Matsunami Glass, Osaka, Japan) coated by matrigel. The explants with sprouting cells cultured for 4 days were fixed with 4% PFA for 5 min, and choroid explants were removed. Following washing with PBS, the cells sprouted were incubated with 5% normal donkey serum in PBS for 1 h. The cells were incubated with AGS8 antibody13 and fluorescein labeled isolectin B4 (Vector Laboratories, Burlingame, CA) for 18 h, washed with PBS, incubated by HRP-labeled anti-rabbit IgG antibody for 1 h, and followed by TSA signal amplification system (Perkin Elmer, Shelton, CT) for AGS8 detection. The coverslips were mounted on a slide glass (Matsunami Glass) with VECTASHIELD mounting medium (Vector Laboratories, Burlingame, CA, USA). Images were obtained under an LSM710 fluorescence microscope (Zeiss, Oberkochen, Germany).

Immunohistochemistry of RPE-choroidal flat-mounts was performed as described previously36. Mouse eyes were enucleated and fixed in 4% PFA for 30 min on ice. After the cornea, lens, retina and sclera were removed from RPE-choroid in ice-cold PBS, 100% methanol was substituted for PBS for fixation and the tissues were preserved at −20 °C in a freezer. On the day of experiments, methanol was replaced by PBS and 0.5% Triton X (PBST) at room temperature, and PBST with 5% goat serum (Jackson ImmunoResearch, West Grove, PA) blocking buffer for 30 min, before incubation with anti-mouse CD31 antibody (rat, 1:500; Thermo Fisher), or anti-mouse/human AGS8 antibody13 (rabbit, 1:200) overnight at 4 °C. The next day, secondary antibodies, Cy3-conjugated rat anti-rat (1:500; Jackson ImmunoResearch) or Alexa-488 conjugated goat anti-rabbit (1:500; Invitrogen) were incubated for 3 h at room temperature. RPE-choroid was washed with PBST, cut by a razor blade to make flat mounted tissue, and the tissues were covered with a thin layer of VectaMount (Vector Laboratories, Burlingame, CA) and a coverslip before examination with a fluorescence microscope, Keyence BZ-9000 (Osaka, Japan).

Statistical analysis and miscellaneous procedures

All experiments were performed in duplicate, triplicate, or greater and repeated independently at least four times. All statistical analyses were performed with IBM SPSS software. Data were analyzed with one-way or two-way ANOVA followed by post-hoc tests for comparisons of the means. In the case of comparisons in pairs, unpaired t-tests were used. Statistically significant differences between the control and test groups were evaluated if the P value was lower than 0.05 (*) or 0.01 (**) as indicated on the graphs. Error bars in the graphs denote standard errors of the mean (s.e.m). Other miscellaneous procedures have been previously described37.