Aucuba japonica extract inhibits retinal neovascularization in a mouse model of oxygen‐induced retinopathy, with its bioactive components preventing VEGF‐induced retinal vascular hyperpermeability

Abstract Neovascularization in the retina is common pathophysiology of diabetic retinal microvasculopathy and exudative macular degeneration. Our study assessed the inhibitory activity of an ethanol‐based extract of Aucuba japonica (AJE) on abnormal angiogenesis in the retina with a hyperoxia‐induced neovascular retinopathy model. The inhibitory effects of aucubin, quercetin, and kaempferol, bioactive compounds, from A. japonica, on retinal vascular hyperpermeability were also examined. On the 7th postnatal day (P7), the C57BL/6 pups were exposed to a hyperoxic environment with 75% oxygen to develop the experimental angiogenesis in retinas. On the 12th postnatal day (P12), the pups were then returned to the normal atmospheric pressure of oxygen. From P12 to P16, the administration was intraperitoneal. The dose per day was 250 mg per kg weight. Retinal neovascularization was measured with retinal flat mounts prepared on P17. We also measured the vascular leakage mediated by the vascular endothelial growth factor (VEGF) in retinas. Mice treated with AJE had markedly smaller neovascular lesions, in comparison with vehicle‐administered mice. AJE downregulated the expression of both VEGF protein and mRNA. In addition, aucubin, quercetin, and kaempferol ameliorated VEGF‐induced retinal vascular leakage. The results of our study suggest that AJE is a potent antiangiogenic substance. AJE could also serve as a therapeutic agent for abnormal growth of vessels in the retina in patients with ischemic retinopathy. The bioactive compounds of AJE may be responsible for its antiangiogenic abilities.


| INTRODUC TI ON
Advanced retinal neovascularization causes visual impairment and a complete loss of sight in a significant proportion of the elderly over 65 years of age (Solomon, Lindsley, Vedula, Krzystolik, & Hawkins, 2019). Retinal neovascularization is also implicated in severe complications of retrolental fibroplasia, diabetic retinal microvasculopathy, and neovascular macular degeneration (Campochiaro, 2013).
A prominent proangiogenic and vascular permeability factor is the vascular endothelial growth factor (VEGF). VEGF also plays a crucial mediating role in the pathogenesis of these retinal diseases (Parikh et al., 2019). The use of anti-VEGF agents to inhibit the VEGF signaling pathway has recently successfully reduced retinal neovascularization in human subjects (Eyetech Study Group, 2003) and animal models (Muranaka et al., 2005).
Recently, we reported that A. japonica and its bioactive compound, aucubin, showed potent pharmacological effects on dry eye disease (Kang, Jung, & Kim, 2018).
The antiangiogenic abilities of A. japonica on the neovascular retinal diseases have not been described in reports, according to our research. To elucidate this, we examined the antiangiogenic activities of an ethanolic extract of A. japonica (AJE) in an oxygen-induced ischemic retinopathy (OIR) model. The ability of aucubin, kaempferol, and quercetin to inhibit retinal vascular hyperpermeability stimulated by administering exogenous VEGF intravitreally in rats was also assessed.

| AJE preparation
The leaves and stems of A. japonica were cultivated and collected in Geoje, Kyungsangnamdo, South Korea. Jeonbuk National University's (Jeonju, South Korea) herbarium has the voucher specimen (No. JBNU-AJE2018). The leaves (700 g) and stems (350 g) of A. japonica were extracted with 30% ethanol (10.5 L) at 85°C. The extraction took 3 hr with 175 g sample gotten by concentration and freeze-drying. AJE was qualitatively and quantitatively assessed with high-performance liquid chromatography (HPLC). AJE contained 59.7 ± 1.5 mg/g aucubin ( Figure 1).

| OIR model experimental neovascularization of the retina
As earlier described, the study was carried out C57BL/6 pups, with neovascularization of the retina stimulated in them (Lee et al., 2013). While with their nursing mothers, on the 7th postnatal day, the pups were exposed to 75% oxygen. The exposure lasted 5 days, and they were returned to normal oxygen exposure on P12. Four groups were then created, and the pups randomly allocated to these groups, with seven mice in each group. The groups are (a) OIR model mice; (b) OIR model mice administered F I G U R E 1 HPLC profile of an extract of Aucuba japonica 100 mg of AJE per kg body weight; and (c) OIR model mice administered 250 mg AJE per body weight. The intraperitoneal administration of AJE was carried out from P12 to P16. An equal volume of the vehicle was also administered to the mice for 5 days. The Institutional Animal Care and Use Committee approved the use of the animals as well as the care (Approval No. 19-015).

| Neovascular area analysis with isolectin staining and fluorescein-dextran angiography
The inhalation of isoflurane anesthetized the mice. This was carried out on the 17th postnatal day. The heart of the mice was also injected with 10 mg per body weight of fluorescein-dextran (FD40, Sigma). For 1.5 hr, the eyeballs were immersed in 4% paraformaldehyde after they had been enucleated. This process occurred 5 min after the cardiac injection. Mounts of whole retinas were made on microscopic slides after isolation. With a fluorescence microscope (BX51, Olympus), the mounts were viewed. With the ImageJ program (National Institutes of Health), we measured the part of the retina that had been vas-obliterated. The stain for the neurovascular area of the retina was rhodamine-conjugated isolectin B4 (Vector Laboratories Ltd.). Lecithin-based areas were identified with a fluorescence microscope. The ImageJ program was also applied in calculating the size of the neurovascular area.

| Real-time PCR
The weight of frozen samples of the retina was determined. TRIzol solution (Invitrogen Inc.) was applied for isolating the total RNA. An existing protocol was applied in the real-time PCR (Lee et al., 2016). Table 1 shows the primers for GAPDH and VEGF. The Bio-Rad iQ5 software (Bio-Rad Laboratories Inc.) was applied in determining the mRNA levels of VEGF.

| Immunohistochemical staining
At necropsy, with the eyes fixed and embedded in 4% paraformaldehyde and paraffin respectively, retina sections 4 μm thick were made. The sections were then deparaffinized, rehydrated, and treated. Xylene deparaffinized the subjects, and 1% H 2 O 2 in methanol was used for treating them. Incubation with anti-VEGF antibody (Catalog No. ab1316, Abcam) which lasted for 1 hr at 37°C followed.

| Vascular hyperpermeability of retina induced by VEGF
The induction followed a published protocol (Lara-Castillo et al., 2009). SD rats that are 7 weeks old were bought from Koatech

P R I M E R S E Q U E N C E S F O R R E A L-T I M E P C R A N A LY S I S Genes
Primers Sequences

| Statistical analysis
One-way analysis of variance then Tukey's multiple comparison test was applied for group data analysis. A statistically significant difference was indicated by a p-value <.05.

| AJE inhibits OIR model retinal neovascularization
Retinal degenerative diseases such as wet form macular degen-  (Stone et al., 1996). The upregulatory influence on VEGF was observed under normal conditions (Ashton, 1957(Ashton, , 1966. For ischemic retinopathy (P12-P17), the significant upregulation of proangiogenic VEGF mRNA translation triggered the development of abnormal neovascularization (Hoeben et al., 2004). VEGF has been well-recognized as a major regulator of vascular changes due to pathology, and its inhibition halts these changes (Aiello et al., 1994;Dorey, Aouididi, Reynaud, Dvorak, & Brown, 1996). As shown in Figure

| AJE downregulates VEGF expression
Vascular endothelial growth factor is well known for vascular permeability. VEGF is also known as a proangiogenic molecule for inducing proliferation of the endothelium, migration, and angiogenesis (Shweiki, Itin, Soffer, & Keshet, 1992). Several VEGF-inhibiting drugs have benefited patients with neovascular macular degeneration and proliferative diabetic retinopathy (Campochiaro, 2013;Dhoot & Avery, 2016). Real-time PCR and immunohistochemistry were applied in assessing the difference in VEGF expression in the retina. Figure 4a shows that the immunohistochemical staining of retinal sections revealed endothelial cells of neovessels on the surface of the retina staining intensely for VEGF (arrows) that may be contributing to the neovascularization. In the AJE-treated groups, VEGF-positive signals were mostly detected in blood vessels within the retina (arrowheads). As predicted, the VEGF mRNA levels were markedly decreased by the treatment of AJE during ischemic retinopathy compared to that of the OIR group (Figure 4b).
Regarding cellular mechanisms for suppressing retinal neovascularization by the treatment with AJE, the present data showed that the treatment led to significant suppression of VEGF expression. In

F I G U R E 4
The effect of AJE on VEGF expression in OIR mice. (a) Immunohistochemical staining for VEGF in the retinal section. Arrow indicates endothelial cells of neovessels on the surface of the retina stain intensely for VEGF. Arrowhead indicates blood vessels positively stained by VEGF within the retina. (b) Real-time PCR analysis of VEGF mRNA levels in OIR mice. The data are shown as the mean ± SEM, n = 7, *p < .05 versus OIR mice previous reports, macrophages, a rich source of VEGF, have been shown to facilitate the development of retinal neovascularization (Kvanta, Algvere, Berglin, & Seregard, 1996). Macrophages contribute to and participate in inflammatory processes that exacerbate new vessel formation. It has been reported that AJE has anti-inflammatory activity through inhibiting the overexpression of IL-1β, IL-8, and TNF-α in corneal epithelial cells (Kang et al., 2018). Collectively, the currently observed suppression of retinal neovascularization by the treatment with AJE is likely attributable to the inhibition of VEGF secretion and its anti-inflammatory activity. antiangiogenic activities by targeting VEGF in retinopathy of prematurity induced in rodents and human retinoblastoma cells (Song, Zhao, Xu, & Zhang, 2017 In traditional herbal medicines, herbal extracts were known to have various advantages of synergy and interactions among the various phytocompounds present in the herbs. Therefore, the focus of this study is to elucidate the molecular mechanism of AJE, not each single compound. Although the actual amount of active compound had no inhibitory activity against VEGF-induced vascular hyperpermeability, angio-supressive activity of AJE may be due to the synergistic effect among kaempferol and quercetin.

| CON CLUS ION
To the best of our knowledge, our work is the first to show that AJE significantly suppressed exudative and neovascular retinopathy in vivo. The effect of AJE on the translation of VEGF mRNA was also demonstrated using a mouse model of experimental OIR. In addition, AJE and its bioactive compounds, quercetin and kaempferol, also inhibit VEGF-mediated retinal vascular leakage in rats. Cumulatively, our findings indicate that the angio-suppressive activities of AJE in the retina are as a result of the potency of bioactive compounds quercetin and kaempferol. Therefore, this study proposes AJE as an antiangiogenic functional food for patients with abnormal retinal vessel growth.

ACK N OWLED G M ENTS
This research was supported by the R&D Program for Forest Science Technology (Project No. 2017040A00-1719-BA01) provided by Korea Forest Service (Korea Forestry Promotion Institute).

CO N FLI C T O F I NTE R E S T
The author declares that I do not have any conflict of interest.

E TH I C A L A PPROVA L
All applicable institutional guidelines for the care and use of animals were followed.