Intravitreal Short-Hairpin RNA Attenuated Adeno-Associated Virus–Induced Knockdown of Amphiregulin and Axial Elongation in Experimental Myopia

Abstract

Background: Epidermal growth factor (EGF) and its family members have been reported to be involved in myopic axial elongation. We examined whether short hairpin RNA attenuated adeno-associated virus (shRNA-AAV)–induced knockdown of amphiregulin, an EGF family member, has an influence on axial elongation.

Methods: Three-week-old pigmented guinea pigs underwent lens-induced myopization (LIM) without additional intervention (LIM group; n = 10 animals) or additionally received into their right eyes at baseline an intravitreal injection of scramble shRNA-AAV (5 × 10¹⁰ vector genome [vg]) (LIM + Scr-shRNA group; n = 10) or of amphiregulin (AR)–shRNA-AAV (5 × 10¹⁰ vg/5 µL) (LIM + AR-shRNA-AAV group; n = 10), or they received an injection of AR-shRNA-AAV at baseline and three weekly amphiregulin injections (20 ng/5 µL) (LIM + AR-shRNA-AAV + AR group; n = 10). The left eyes received equivalent intravitreal injections of phosphate-buffered saline. Four weeks after baseline, the animals were sacrificed.

Results: At study end, interocular axial length difference was higher (P < 0.001), choroid and retina were thicker (P < 0.05), and relative expression of amphiregulin and p-PI3K, p-p70S6K, and p-ERK1/2 was lower (P < 0.05) in the LIM + AR-shRNA-AAV group than in any other group. The other groups did not differ significantly when compared with each other. In the LIM + AR-shRNA-AAV group, the interocular axial length difference increased with longer study duration. TUNEL assay did not reveal significant differences among all groups in retinal apoptotic cell density. In vitro retinal pigment epithelium cell proliferation and migration were the lowest (P < 0.05) in the LIM + AR-shRNA-AAV group, followed by the LIM + AR-shRNA-AAV + AR group.

Conclusions: shRNA-AAV–induced knockdown of amphiregulin expression, in association with suppression of epidermal growth factor receptor signaling, attenuated axial elongation in guinea pigs with LIM. The finding supports the notion of EGF playing a role in axial elongation.

Controlled elongation of the ocular optical axis is the main part of the process of emmetropization, changing hyperopia in the newborn to emmetropia in the adolescent and, if overshooting, to myopia in the adolescent and adult. Several substances have been reported to be associated with axial elongation. These include molecules such as dopamine, atropine, transforming growth factor β, fibroblast growth factor, hepatocyte growth factor, insulin-like growth factor, amphiregulin, and other epidermal growth factor (EGF) family members.^1–9 Recent studies conducted in young guinea pigs with or without lens-induced myopization (LIM) showed that the repeated intravitreal application of antibodies against EGF family members, such as amphiregulin, neuregulin-1, and EGF itself, resulted in a decrease in axial elongation, while repeated intravitreal injections of EGF family members—namely, amphiregulin, neuregulin-1, and EGF—were associated with an increase in axial elongation.^10–12 As a corollary, the intravitreal application of EGF receptor antibodies was associated with a reduction in axial elongation in young guinea pigs.¹³ Based on the observations made in the experimental studies and in histomorphometric and clinical investigations, the hypothesis was formulated that EGF and its family members, potentially produced by cells in the retina, may stimulate the retinal pigment epithelium (RPE) in the midperiphery of the fundus to locally enlarge Bruch's membrane.¹⁴ The RPE contains receptors for EGF.^15–17 

Based on the results of the previous studies and to further explore the hypothesis of an association of the EGF family with the process of axial elongation, we examined here whether knockdown of amphiregulin as a member of the EGF family is associated with a change in axial elongation in guinea pigs. To achieve the knockdown, we used an intravitreally injected short hairpin RNA attenuated adeno-associated virus (shRNA-AAV). We chose amphiregulin out of the EGF family since it had been the first EGF family member for which an influence on axial elongation had been reported and the association of which with axial elongation was one of the strongest as compared with the other EGF family members.^10–13 

Methods

Animals and Experimental Designs

Animal treatment and care were approved and supervised by the Ethics Committee of Beijing Tongren Hospital. All research protocols and procedures followed and adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The study included male pigmented guinea pigs (Cavia porcellus) with an age of 3 weeks. They were kept at a room temperature of 25°C with ad libitum access to food and water in a 12-hour light (450–500 lux) and 12-hour dark (∼0 lux) cycle. The animals were randomly divided into five groups: 

• - The normal control group without LIM and without any intraocular injection (n = 10 animals)
• - The LIM group with bilateral LIM and without any intraocular injection (n = 10 animals)
• - The LIM + Scr-shRNA group with bilateral LIM and an intravitreal injection of scramble shRNA-AAV (5 × 10¹⁰ vector genome [vg]/5 µL) into the right eyes at baseline (n = 10 animals)
• - The LIM + AR-shRNA-AAV group with bilateral LIM and an intravitreal injection of amphiregulin (AR)–shRNA-AAV (5 × 10¹⁰ vg/5 µL) into the right eyes at baseline (n = 10 animals)
• - The LIM + AR-shRNA-AAV + AR group with bilateral LIM, an intravitreal injection of AR-shRNA-AAV (5 × 10¹⁰ vg/5 µL) into the right eyes at baseline, and additional intravitreal applications of 20 ng/5 µL amphiregulin weekly injected into the right eyes three times after week 1 (n = 10 animals)

Lens-Induced Myopia

Goggles with a refractive power of −10.0 diopters (polymethyl methacrylate; diameter: 12.7 mm) were applied to the orbital rim of the guinea pigs as described in detail previously.^13,18 The goggles were taped onto the orbital rims of both eyes, with care being taken to ensure that the guinea pigs could freely open their eyes and blink while wearing the goggles. The goggles were examined daily for cleanliness and correct position; otherwise, they were detached, cleaned, and reattached. The goggles were removed during the biometric examinations and retaped after the examinations. 

AAV Vector Production

Recombinant AAV8 vectors were produced as described previously.¹⁹ In brief, the AAV vector, rep/cap packaging plasmid, and adenoviral helper plasmid were added to human embryonic kidney 293T cells (HCL4517; Thermo Fisher Scientific, Waltham, MA, USA). Twenty-four hours after transfection, the supernatant was collected for the AAV8 preparations. The supernatant was centrifuged at 7000 × g for 10 minutes and resuspended in virus buffer (150 mM NaCl and 20 mM Tris, pH 8.0). RT-PCR and protein gels were run to determine the virus titers. 

Before the start of the main study, we first designed three sequences of amphiregulin-shRNA (referred to as AREG-shRNA1, AREG-shRNA2, and AREG-shRNA3) and selected the sequence with the most marked inhibition of the expression of amphiregulin and amphiregulin mRNA to be used in the main study (Supplementary Figs. S1, S2). 

Reagents, Drug Preparation, and Intravitreal Injection

A total of 5 µL AR-shRNA-AAV containing AAV-viral particles (1 × 10¹³ vg/mL for a total dose of 5 × 10¹⁰ vg) was intravitreally injected at baseline into the right eyes. For external amphiregulin, 20 ng murine recombinant amphiregulin was injected weekly after week 1. The intravitreal injections were performed under topical anesthesia using 0.5% proxymetacaine hydrochloride eye drops. The scleral entry for the injection needle was created by a 30-gauge needle puncture 1.5 mm posterior to the limbus. Using a Hamilton microsyringe (Hamilton Microliter syringe; Sigma-Aldrich, St. Louis, MO, USA), we delivered 5 µL of the solution into the right eyes through the scleral entry. The contralateral left eyes received an injection of 5 µL PBS. 

Axial Length and IOP Measurement

The axial length and IOP were measured at baseline (day 1), day 8, day 15, day 22, and day 29. The axial length measurement was performed using ocular sonographic biometry (A/B mode scan; oscillator frequency: 11 MHz; Quantel Co., Les Ulis, France) under topical anesthesia. The predicted sound velocities were 1557.5 m/s for aqueous humor, 1723.3 m/s for lens, and 1540 m/s for vitreous humor. For each measurement, 10 readings were taken. If the standard deviation of the 10 readings was less than 0.1 mm, the mean values of the readings were used for further statistical analysis. IOP was determined by tonometry (Tono-Pen; Reichert, Inc., Depew, NY, USA) at 4 pm, and the mean value of three measurements was taken for further statistical analysis. 

Optical Coherence Tomography

At the end of the study, three animals in each group were randomly selected to undergo optical coherence tomography (OCT) examination. Before the examination, all animals were anesthetized with an intraperitoneal injection of 1% pentobarbital sodium (cat. 4579; R&D Systems, Bio-Techne Co., Minneapolis, MN, USA) at a dose of 4 mg pentobarbital/100 g body weight. The pupils of both eyes were dilated by mydriatic eye drops (2.5% tropicamide eye drops; Bausch and Lomb, Rochester, NY, USA). A custom bracket was used to fix the animals, and a custom lid speculum was applied to keep the eyelids open. Artificial tear eye drops (Hycosan; Ursapharm Arzneimittel GmbH, Saarbrücken, Germany) were topically applied to keep the cornea moist. Each animal underwent bilateral swept source OCT (VG200D SVision Imaging, Henan, China) with a central wavelength of 1050 nm. If the quality of the scans was not sufficient, OCT imaging was repeated. In the horizontal sections running through the center of the optic nerve head, the thickness of the posterior retina and choroid was measured at a location 1000 µm temporal to the optic disc. 

Tissue Collection

Guinea pigs were anesthetized by an intraperitoneal injection of urethane (1000 mg/kg). After sacrificing the animals, the eyes were enucleated, and the cornea, lens, and vitreous were removed. The retinal tissue was harvested under a microscope. All samples were immediately stored in liquid nitrogen and translocated into a −80°C refrigerator. The frozen tissues were analyzed within 1 week. For histopathologic examination, the enucleated globes were fixed for 24 hours in 10 mL FAS Eyeball Fixative Solution (Wuhan Servicebio Technology Co. Ltd., Wuhan, China) and embedded in paraffin. 

Hematoxylin and Eosin and TUNEL Staining

The details for hematoxylin and eosin (H&E) and TUNEL staining have been described previously.² After 24 hours of FAS fixation, histologic slides (thickness: 6 µm) of three animals were prepared in a routine process. The slides were stained with H&E and examined under a light microscope (Olympus Co., Tokyo, Japan). The thickness of the retina at various regions was measured using Slideviewer software (3DHISTECH Ltd., Budapest, Hungary). We performed TUNEL staining (Cell Death Detection kit; Kaiji Biotechnology Co. Ltd., Jiangsu, China) to detect and count apoptotic cells in the retina. The histologic sections were incubated with 1% Triton X-100 for 15 minutes and rinsed in PBS three times at room temperature. To inactivate the enzymes, two drops of 3% hydrogen peroxide-methanol solution were applied to each section for 10 minutes, and the sections were rinsed in PBS three times. The sections were incubated with 10% terminal deoxyribonucleotidyl transferase enzyme solution at 37°C for 1 hour and washed in PBS solution three times. The slides were then incubated in horseradish peroxidase solution at 37°C for another 30 minutes and washed in PBS three times. After that, diaminobenzidine solution was applied for staining, and distilled water was used to clean the sections. Three sections from each group were photographed, and TUNEL-positive cells in the retina were counted at the posterior pole. The mean of the counts obtained from the three images was recorded and taken for statistical analysis. 

RPE Cell Harvest and Culture

Primary RPE cells from guinea pigs were harvested according to a previously described protocol.³ Male pigmented guinea pigs aged 4 weeks were sacrificed, and the eyes were enucleated. The cornea, lens, vitreous, and retina were removed under a stereomicroscope. Trypsin was then added to each eye cup. After incubation for 10 minutes at 37°C, the trypsin solution was removed and substituted with a trypsin-EDTA solution for 45 minutes at 37°C. The RPE cells were gently pipetted off Bruch's membrane and were collected and washed with an ICell Primary Epithelial Cell Culture System (PriMed-iCell-001; Emelca Bioscience, Shanghai, China). All cells were grown at 37°C in 5% CO₂. The culture medium was replaced twice a week. 

RPE Cell Counting Kit-8 Assay

Primary RPE cells were trypsinized, centrifuged, and resuspended in ICell Primary Epithelial Cell Culture medium in 96-well plates. Each well contained 5 × 10⁴ cells/100 µL medium. After 24 hours, the medium was replaced with fresh RPE cell medium, and scramble-shRNA, AR-shRNA, and AR-shRNA with external AR (1000 ng/L) were added. After another 24 hours of incubation, 10 µL CCK-8 labeling reagent was added according to the manufacturer's instructions (Dojindo-CK04; Dojindo Molecular Technologies Inc., Rockville, MD, USA). A scanning spectrophotometer (MULTISKAN MK3; Thermo Fisher Scientific, Waltham, MA, USA) was used to measure the absorbance of the formazan product at a wavelength of 450 nm. The results were expressed as the mean optical densities, which indicated the proliferation and viability of the cells. The experiments were repeated five times. The ratio of the optical density of the experimental sample and that of the control was calculated. 

RPE Cell Migration Assay

The scratch assay determined the migratory ability of the RPE cells. We seeded 1.5 × 10⁵ primary RPE cells in a 6-well plate. Three wounds were scratched in the confluent cell layer with a toothpick, and the cells were washed with RPE culture medium to remove detached cells. Microscopic brightfield images of three spots were taken. The medium was replaced with fresh RPE cell medium, and scramble-shRNA, AR-shRNA, and AR-shRNA with external AR (1000 ng/L) were added. After 5 hours of transfection, the medium was replaced with fresh RPE cell medium for 48 hours, and microscopic brightfield images of three spots were taken. 

RT-PCR

Primary RPE cells, taken from treatment-naive guinea pigs, were harvested at the end of the designated periods, and total RNA was isolated using TRIzol reagent (TIANGEN Biotech Co., Beijing, China) in accordance with the manufacturer's instructions. The RNA purity was examined by measuring the optical density value (UV-2450; SHIMADZU, Kyoto, Japan). Two to three micrograms of RNA was reverse transcribed using the Reverse Transcription System kit (Thermo Fisher Scientific). Quantitative real-time PCR was performed in a 20-µL reaction with SYBR Green (Kapa Biosystems, Wilmington, MA, USA) and 1 µL cDNA, 10 µL SYBR Green Mix, 8.2 µL ddH₂O, and 0.4 µL of each specific primer at 500 nM. The cycling parameters were as follows: 95°C for 15 seconds, 60°C for 20 seconds, and 72°C for 40 seconds. 

Western Blotting

Retinal tissues were lysed in cold lysis buffer (Amresco 0754, Solon City, OH, USA) supplemented with protease inhibitors (11697498001; Roche, Basel, Switzerland) and phosphatase inhibitors (04906837001; Roche). The tissue extracts were separated on 8% SDS-PAGE gels and transferred to nitrocellulose membranes according to a standard protocol. The membranes were blocked with 5% skim milk in Tris-buffered saline with Tween 20 for 2 hours and sequentially incubated with primary antibodies overnight and with secondary antibodies for 2 hours the following day. Signals were assessed with an ECL kit (Millipore, Boston, MA, USA), and images were taken with Total Lab Quant V11.5 (Newcastle upon Tyne, UK). The target bands were quantified and analyzed using ImageJ (National Institutes of Health, Bethesda, MD, USA) with β-tubulin as an internal control. The antibody details are listed in Supplementary Table S1. 

Statistical Analysis

The statistical analysis was conducted using the SPSS 27.0 software program (IBM Corp., Armonk, NY, USA) and the program GraphPad Prism 9.3.1 (GraphPad Software, San Diego, CA, USA). Unless stated otherwise, continuous variables are presented as the means ± standard deviations. For the comparison of the intereye difference in axial length and other parameters between the study groups in dependence of the time point, we applied a two-way ANOVA. Two-tailed P values <0.05 were considered statistically significant. 

Results

Amphiregulin Expression

The relative retinal amphiregulin expression was significantly (P < 0.05) lower in the LIM + AR-shRNA-AAV group than in the LIM group, the LIM + Scr-shRNA group, and the LIM + AR-shRNA-AAV + AR group (Fig. 1a). Correspondingly, the relative expression of amphiregulin mRNA was the lowest in the AREG-shRNA2 group, followed by the AREG-shRNA3 and AREG-shRNA1 groups (Supplementary Figs. S1, S2). 

Figure 1.

 

Knocked-down AR expression attenuated axial elongation in guinea pigs with LIM. *P < 0.05 compared to LIM. **P < 0.01 compared to LIM. (a) Intravitreal injection of AR-AAV significantly downregulated AR expression in the retina. (b) The interocular difference in axial length (right eye minus left eye) in guinea pigs with different interventions. LIM group: bilateral LIM without any intraocular injection (n = 10); LIM + Scr-shRNA group: bilateral LIM and intravitreal injection of scramble short hairpin RNA attenuated adeno-associated virus (shRNA-AAV) (5 × 10¹⁰ vg) into the right eyes at baseline and PBS into left eyes; LIM + AR-shRNA-AAV group: bilateral LIM and intravitreal injection of AR-shRNA-AAV injected into the right eyes at baseline and PBS into the left eyes; LIM + AR-shRNA-AAV + AR group: bilateral LIM and intravitreal injection of AR-shRNA-AAV into the right eyes at baseline, amphiregulin weekly into the right eyes, and PBS into left eyes.

Axial Elongation

The interocular difference in axial length did not vary significantly (all P > 0.05) between the control group (−0.00 ± 0.04 mm), the LIM group (0.00 ± 0.02 mm), the LIM + Scr-shRNA group (0.01 ± 0.03 mm), and the LIM + AR-shRNA-AAV + AR group (−0.00 ± 0.03 mm) (Fig. 1b, Table, Supplementary Tables S2, S3). In the LIM + AR-shRNA-AAV group, axial length was significantly (P < 0.001) shorter in the right eyes than in the left eyes (8.63 ± 0.03 vs. 8.77 ± 0.02 mm), so that the interocular difference in axial length was significantly (P < 0.05) higher in that group (−0.13 ± 0.04 mm) than in any other group (Table). The difference between the groups increased significantly with longer study duration (all P values <0.05). The IOP did not differ significantly between the groups during the study (Supplementary Table S4). 

Table.

 

Biometric Measurements in Each Group (Mean ± SD)

 

Choroidal and Retinal Thickness

The thickness of the choroid and retina as measured by OCT did not differ significantly between any group except for the LIM + AR-shRNA-AAV group, in which the choroid and retina were significantly (both P values <0.05) thicker than in any other group (Fig. 2). In a similar manner, the thickness of the posterior retina as measured histomorphometrically by light microscopy did not differ significantly between any group except for the LIM + AR-shRNA-AAV group, in which the retina was significantly (all P values <0.05) thicker than in any other group (Fig. 3a). 

Figure 2.

 

In vivo measurement of the retina and choroid in guinea pigs. (a) OCT image of a guinea pig. (b) Thickness of the posterior retina measured at 1000 µm temporal to the optic disc. (c) Thickness of the posterior choroid measured at 1000 µm temporal to the optic disc. LIM group: bilateral LIM without any intraocular injection (n = 10); LIM + Scr-shRNA group: bilateral LIM and intravitreal injection of scramble short hairpin RNA attenuated adeno-associated virus (shRNA-AAV) (5 × 10¹⁰ vg) into the right eyes at baseline and PBS into the left eyes; LIM + AR-shRNA-AAV group: bilateral LIM and intravitreal injection of AR-shRNA-AAV injected into the right eyes at baseline and PBS into the left eyes; LIM + AR-shRNA-AAV + AR group: bilateral LIM and intravitreal injection of AR-shRNA-AAV into the right eyes at baseline, amphiregulin weekly into the right eyes, and PBS into the left eyes.

Figure 3.

 

Effects of AR knockdown on retinal cells. **P < 0.01 compared to the LIM group. (a) The thickness of the retina in the LIM group and transfected groups. (b) TUNEL test assessing the toxicity of AAV-ShRNA in retinal cells (200×). LIM group: bilateral LIM without any intraocular injection (n = 10); LIM + Scr-shRNA group: bilateral LIM and intravitreal injection of scramble short hairpin RNA attenuated adeno-associated virus (shRNA-AAV) (5 × 10¹⁰ vg) into the right eyes at baseline and PBS into the left eyes; LIM + AR-shRNA-AAV group: bilateral LIM and intravitreal injection of AR-shRNA-AAV injected into the right eyes at baseline and PBS into the left eyes; LIM + AR-shRNA-AAV + AR group: bilateral LIM and intravitreal injection of AR-shRNA-AAV into the right eyes at baseline, amphiregulin weekly into the right eyes, and PBS into left eyes.

Apoptosis Assessment

The TUNEL assay did not reveal any significant difference between all groups in the density of apoptotic cells (Fig. 3). 

Relative Expression of EGF Pathway Downstream Signaling Molecules

The relative expression of p-PI3K, p-p70S6K, and p-ERK1/2 was significantly lower in the LIM + AR-shRNA-AAV group than in any other group, which did not differ significantly in that parameter, indicating suppression of epidermal growth factor receptor (EGFR) signaling in the LIM + AR-shRNA-AAV group (Fig. 4). 

Figure 4.

 

Knockdown of AR suppressed EGFR signaling in LIM guinea pigs. *P < 0.05 compared to LIM. LIM group: bilateral LIM without any intraocular injection (n = 10); LIM + Scr-shRNA group: bilateral LIM and intravitreal injection of scramble short hairpin RNA attenuated adeno-associated virus (shRNA-AAV) (5 × 10¹⁰ vg) into the right eyes at baseline and PBS into the left eyes; LIM + AR-shRNA-AAV group: bilateral LIM and intravitreal injection of AR-shRNA-AAV injected into the right eyes at baseline and PBS into the left eyes; LIM + AR-shRNA-AAV + AR group: bilateral LIM and intravitreal injection of AR-shRNA-AAV into the right eyes at baseline, amphiregulin weekly into the right eyes, and PBS into the left eyes.

RPE Cell Proliferation and Migration

The in vitro RPE cell proliferation and migration were the lowest (all P values <0.05) in the LIM + AR-shRNA-AAV group, followed by the LIM + AR-shRNA-AAV + AR group, in which it was significantly lower (both P values <0.05) than in the remaining two other groups (Fig. 5). 

Figure 5.

 

Knockdown of AR suppressed RPE cell proliferation and migration in vitro. (a) CCK-8 assay revealed cell viability. (b, c) Scratch wound healing assay revealed cell migration. **P < 0.01 compared to NC. NC, normal control. The RPE cells were taken from treatment-naive young guinea pigs.

Discussion

In this experimental study on young guinea pigs, eyes with shRNA-AAV–induced knockdown of amphiregulin expression, in association with suppression of EGFR signaling, showed less marked ocular elongation than contralateral eyes with PBS injections. Subsequently, the interocular difference in axial length was higher and increased with longer study duration in the LIM + AR-shRNA-AAV group than in any other group. If amphiregulin was added to the eyes with a knockdown of amphiregulin expression, axial elongation did not differ significantly compared to the eyes without a knockdown. The in vitro RPE cell proliferation and migration were the lowest in the eyes with a knockdown of the amphiregulin expression as compared to any other group. 

The findings agree with observations made in previous studies in which young guinea pigs with or without LIM showed an increased axial elongation after the intravitreal application of EGF family members. As a corollary, guinea pig eyes with intraocular injections of antibodies against EGF family members and the EGF receptor showed a reduction in axial elongation.^10–13 Similar results were obtained with young monkeys, which, without LIM, showed a decreased axial elongation after intravitreal injections of amphiregulin antibodies.²⁰ Correspondingly, eyes of young guinea pigs with LIM and no further intervention showed upon immunohistochemistry an increased intraretinal expression of amphiregulin.^10–12 These observations and clinical and histologic findings led to the hypothesis that EGF and its family members, potentially produced by cells in the retina, may stimulate the RPE in the midperiphery of the fundus to locally enlarge Bruch's membrane in the midperiphery of the fundus.¹⁴ The hypothesis is supported by other findings. The elastic modulus of Bruch’s membrane is comparable to or higher than that of the sclera. The thinning of the posterior choroid might be explained by a backward movement of the posterior Bruch’s membrane. A primary backward expansion of the sclera alone cannot explain the posterior choroidal thinning. Axial elongation is associated with a thinning of the retina and a reduction in the RPE cell density in the fundus midperiphery while both parameters measured at the posterior pole are not associated with axial length. The thickness of the sclera and choroid at the posterior pole decreases with longer axial length while their total volume is independent of axial length. The thickness of Bruch's membrane is independent of axial length, so that the volume of Bruch's membrane increases with longer axial length. Parapapillary gamma zone in the temporal region in moderately myopic eyes can be explained by a shift of Bruch's membrane opening (BMO) into the direction of the macula. Such a BMO shift can be induced by an enlargement of Bruch's membrane in the fundus midperiphery.^21–27 The potential role of the RPE in the process of axial elongation, as expressed in the hypothesis, is supported by the finding that the in vitro RPE cell proliferation and migration were the lowest in the eyes with a knockdown of the amphiregulin expression. The hypothesis is contrary to the assumption that the sclera is the primary structure involved in achieving elongation of the eye.^28,29 

Parallel to the reduced axial elongation, the eyes with a knockdown of the amphiregulin expression showed, as compared to the eyes of the other groups, a thicker sclera and choroid as measured intravitally by OCT and postmortem by histomorphometry (Figs. 2 and 3). This finding fits with the results of previous clinical and experimental studies in which axial elongation was strongly associated with a decrease in the thickness of the posterior choroid. The thinning of the retina in guinea pigs physiologically not having a macula is paralleled by a thinning of the retina in the midfundus midperiphery in human eyes, in which the macular region does not show a retinal thinning in association with axial elongation.^14,22 

Interestingly, the density of apoptotic cells did not differ between any of the study groups, which may suggest that the intravitreally applied substances, including AR-shRNA-AAV, did not have a markedly toxic effect on the retina within the study period. Future studies may address whether this finding eventually opens the possibility of a knockdown of the expression of amphiregulin or other EGF family members in myopic patients to prevent further axial elongation. The observation that the in vitro retinal pigment epithelium cell proliferation and migration were reduced in the LIM + AR-shRNA-AAV group agrees with findings made in previous studies in which RPE cell migration and proliferation could be increased by adding EGF to the cell culture medium and reduced by adding EGF receptor blockers to the cell culture medium. 

The observations made in our study may be of clinical interest. Studies have found that axial elongation usually stops in the third decade of life in about two-thirds of moderately myopic individuals.³⁰ In one-third of these individuals, however, axial elongation can continue, usually at a low rate.³⁰ In particular, highly myopic adult patients with myopic maculopathy can experience further axial elongation, which is a main risk factor for the progression of myopic maculopathy.^31–36 Since other risk factor for progression of myopic maculopathy, such as female sex and older age, are not modifiable, any procedure preventing further axial elongation of highly myopic eyes might be useful to prevent vision impairment due to development or progression of myopic maculopathy. The results of the present study support the hypothesis that EGF and its family members are involved in the process of axial elongation. Future studies may therefore examine whether a blockade of the EGF system, by intravitreal application of antibodies to EGF or its family members, by intravitreal injection of antibodies to the EGF receptor, and by a knockdown of the expression of EGF and its family members may be useful to prevent further axial elongation in highly myopic adult patients with myopic maculopathy. 

Limitations of our study should be mentioned. First, we examined the influence of a knockdown of amphiregulin in the study, and it has remained open how far the results of the study can be extended to the other members of the EGF family. Second, future investigations may explore whether the findings made in our experimental investigation can be transferred to patients. Third, the study sample sizes were relatively small, so the statistical power was limited. This limitation, however, may serve to strengthen the conclusion that an shRNA-AAV–induced knockdown of amphiregulin expression reduced axial elongation in young guinea pigs with LIM. Fourth, the histomorphometric measurements of the thickness of the retina and choroid might have been influenced by alterations in the filling of the blood vessels shortly before and after enucleation and by postenucleation tissue swelling, fixation-related tissue shrinkage, and other artifacts caused by the preparation of the histologic slides. Fifth, the process of axial elongation may be influenced by the type of goggle used to induce myopization. The –10 diopter goggles applied in our study may differ in their effect from −4.0 diopter goggles or diffuser lenses. Sixth, although we assumed that the RPE is the main target of amphiregulin (and other EGF family members) in the process of axial elongation, we did not provide experimental data on sequels of the knockdown of amphiregulin on the molecular biological aspects of the RPE cells. Future studies may explore those aspects. Seventh, axial elongation in the present study as compared to a previous study by a similar group of authors was lower for the bilateral LIM group (0.70 ± 0.04 mm/0.68 ± 0.02 [right eyes/left eyes] vs. 0.87 ± 0.08 mm/0.89 ± 0.08 mm [right eyes/left eyes]) and for the guinea pig group without LIM (0.50 ± 0.03 mm and 0.49 ± 0.07 mm [right eyes/left eyes] vs. 0.71 ± 0.12 mm and 0.71 ± 0.09 mm [right eyes/left eyes]) (Supplementary Table S3).¹¹ The reason for the discrepancy may be the difference in the total study duration, with 28 days in the present study and 36 days in the previous investigation.¹¹ 

In conclusion, shRNA-AAV induced knockdown of amphiregulin expression in association with suppression of EGFR signaling and attenuated axial elongation in guinea pigs with LIM. This finding supports the notion that EGF plays a role in the process of myopic axial elongation. 

Acknowledgments

Supported by the National Natural Science Foundation of China (82220108017, 82141128); The Capital Health Research and Development of Special (2020-1-2052); Science & Technology Project of Beijing Municipal Science & Technology Commission (Z201100005520045, Z181100001818003) to WBW. 

Disclosure: L. Dong, None; R.-H. Zhang, None; H.-T. Wu, None; H.-Y. Li, None; W.-D. Zhou, None; X.-H. Shi, None; C.-Y. Yu, None; Y.-T. Li, None; Y.-F. Li, None; J.B. Jonas, European patent EP 3 271 392, JP 2021-119187, and US 2021 0340237 A1 (P); W.-B. Wei, None