Oxidative stress‐induced angiogenesis is mediated by miR‐205‐5p

Abstract miR‐205‐5p is known to be involved in VEGF‐related angiogenesis and seems to regulate associated cell signalling pathways, such as cell migration, proliferation and apoptosis. Therefore, several studies have focused on the potential role of miR‐205‐5p as an anti‐angiogenic factor. Vascular proliferation is observed in diabetic retinopathy and the ‘wet’ form of age‐related macular degeneration. Today, the most common treatments against these eye‐related diseases are anti‐VEGF therapies. In addition, both AMD and DR are typically associated with oxidative stress; hence, the use of antioxidant agents is accepted as a co‐adjuvant therapy for these patients. According to previous data, ARPE‐19 cells release pro‐angiogenic factors when exposed to oxidative insult, leading to angiogenesis. Matching these data, results reported here, indicate that miR‐205‐5p is modulated by oxidative stress and regulates VEGFA‐angiogenesis. Hence, miR‐205‐5p is proposed as a candidate against eye‐related proliferative diseases.

could inhibit the progression of tumour cells, targeting FOXO1 14 and CBX1, 15 among others, and promotes LRRK2-mediated apoptosis. 16 Enhancement of VEGFA and neovascular overgrowth are behind two associated proliferative retinal disorders: diabetic retinopathy (DR) 17 and the neovascular-aka 'wet' -age-related macular degeneration (AMD). 18,19 Even though the significant release of VEGF has been reported several times, [20][21][22] little is known about the role of miR-205-5p in these diseases. VEGFA is released by retinal cells, including the retinal pigment epithelium (RPE). 23,24 This layer constitutes the blood-retinal barrier, playing a pivotal role between the bloodstream and photoreceptors. 25 For several reasons (light, metabolic rate, cellular membrane interactions, etc), RPE is continually exposed to oxidative stress (OS) [26][27][28] ; hence, OS is considered a relevant risk factor for AMD. [27][28][29] Recent reports point out that OS in RPE cells intensifies the production and release of angiogenic factors, thus increasing the formation of new blood vessels. 20,30,31 Specifically, OS promotes VEGF production in RPE cells, leading to choroidal neovascularization. 18,32,33 In agreement with this, miR-205-5p overexpression also reduced reactive oxygen species (ROS) by increasing peroxiredoxin 2 levels, 34 suggesting that miR-205-5p could be sensitive to oxidative stimuli. Besides, OS is involved in eye diseases, such as AMD, and VEGFA-mediated angiogenesis plays a crucial role in the evolution of these diseases. 27,29,35,36 Indeed, VEGF inhibitors are widely used in both cancer and wet AMD, with beneficial results. 37,38 The present work focuses on the response of miR-205-5p-VEGFA in ARPE-19 cells to oxidative challenge as an approach to the potential therapeutic target of eye-related proliferative disorders.

| Cell culture
The ARPE-19 human cell line was obtained from American Type Culture Collection (ATCC). Cells were cultured in Dulbecco's modified Eagle's medium DMEM/F12 (Invitrogen), as previously described. 20 Cells from passage numbers 11-30 were used and cultured to 80%-90% confluence at a starting density of 1 × 10 6 cells/cm 2 on different plates depending on the technique. After 2 days, the cells were treated for 24 hours with 600 µmol/L H 2 O 2 (Scharlau) and 4 mM N-acetylcysteine (NAC) (Sigma-Aldrich) using media with 1% of fetal bovine serum (FBS). Cells and supernatant were collected and preserved for further experiments.
Human umbilical vein endothelial cells (HUVEC) were isolated from umbilical veins as previously described. 39 HUVEC were grown in Endothelial Cell Media supplemented with 20% FBS, penicillin/ streptomycin and amphotericin at 37°C and 5% CO 2 .

| Determination of intracellular ROS
Intracellular ROS levels were measured using dihydroethidium (DHE; Thermo Fisher Scientific), which is a superoxide indicator. This molecule has a blue fluorescence but, when oxidized to ethidium, it stains DNA red. ARPE-19 cells were seeded at 6 × 10 3 cells/ well in a 96 well plate. Cells were rinsed with PBS (phosphate-buffered saline) twice and incubated with 5 μmol/L DHE for 30 min at 37°C and 5% CO 2 . ROS levels were measured by a fluorescence multiplate reader (Victor X5; Perkin Elmer) excited at 518 nm and read at 605 nm.

| RNA isolation and miRNA expression analysis
The RNA was isolated from ARPE-19 cells using the miRNeasy Mini with the appropriate temperature cycles. Normalization was done with GAPDH. Relative expression was calculated as 2 -ΔΔCt .

| Analysis of miRNA target genes
In silico analysis of the putative targets of miR-205-5p was performed using Target Scan Human (http://www.targe tscan.org/ vert_72/). We analysed the pathways regulated by the putative targets of miR-205-5p with FUNRICH using the gene ontology database. With STRING (https ://string-db.org/) we performed the networking of the targets related to angiogenic pathways.

| Statistical analysis
Results of each experiment are presented as mean ± SEM. Statistical significance was set at 0.05 by using t test and 2-way-ANOVA.

| VEGFA mRNA is a direct target of miR-205-5p
Consistently with previous results, in silico analysis showed that VEGFA mRNA is a predicted target for miR-205-5p ( Figure 2B). Target Scan predicted a 3'-UTR binding site for VEGFA mRNA and miR-205-5p ( Figure 3A). In order to confirm the relation between VEGFA mRNA and miR-205-5p, VEGFA mRNA expression was analysed after H 2 O 2 treatment. We observed that VEGFA mRNA levels were up-regulated after H 2 O 2 exposure (Figure 3C), indicating a negative correlation with miR-205-5p levels ( Figure 3B and 3D).
Matching with the aforementioned results on miR-205-5p levels, VEGFA mRNA levels were also normalized by NAC exposure ( Figure 3E).

| D ISCUSS I ON
In a previous report, in which miR-205-5p was mapped in the mammalian eye, it was found in the epidermis and the anterior segmental epithelia and was not found in RPE. 42 Other studies have found that miR-125b 43 and miR-23a 44 are overexpressed in oxidative stressinduced RPE cells. 41 This current report is the first one indicating the presence of miR-205-5p in ARPE-19 cells as an RPE cell type. In contrast to the relevance of this miRNA on glioblastoma 13 or hepatocellular carcinoma, 45 little information is available about the presence of miR-205-5p in the eye.
In addition, miR-205-5p was identified as protective against OS via EGLN2 suppression. 46 Once considering this, reducing miR-205-5p levels after oxidative challenge is opposed to this proposed antioxidant mechanism. Perhaps excessive OS challenge (24 hours H 2 O 2 600 µmol/L) leads to a different scenario, far from the physiological conditions that occur in wet AMD or cancer. Future research must address the antioxidant role of miR-205-5p. Another relevant finding is that NAC normalized those decreased miR-205-5p levels, indicating that miR-205-5p is sensitive to ROS.

| VEGFA mRNA as a miR-205-5p target
According to Yue et al., VEGFA 3'UTR-mRNA is one of the putative targets of miR-205-5p. 7 In addition, we have identified some potential targets of miR-205-5p and their biological processes. Among the potential mRNAs regulated by miR-205-5p, 3.5% are angiogenic and 1.3% are related to endothelial cell migration (Figure 2). In fact, it F I G U R E 5 VEGFA-mediated Vasculogenesis is regulated by miR-205-5p. HUVEC cell tube formation under different ARPE-19 cell culture media conditions. ARPE-19 control medium (A), mimic(miR-205-5p)-treated ARPE-19 control (B), H 2 O 2 600 µmol/L treated ARPE-19 (C) and mimic-treated ARPE-19 + H 2 O 2 600 µmol/L medium (D). Scale bar 100 µmol/L. Total branch length (E) and total length (F) were calculated. Values are expressed as mean ± SEM (n = 3). Statistically significant differences were set at *P < .05 is well documented that miR-205-5p acts as a tumour suppressor by regulating cell migration and proliferation. 7,[11][12][13]47 Moreover, it has been suggested that miR-205-5p could interact indirectly via PI3K/AKT also involved in angiogenesis. 13 In the present report, we have observed that VEGFA and miR-205-5p are inversely expressed Anti-VEGF administration is currently used to control neovascular processes in both DR and wet AMD. 37 Similarly, anti-VEGF therapies are also helpful against different forms of cancer, such as glioblastoma and colorectal cancer. 55 In agreement with this, miR-205-5p suppresses VEGFA expression in glioblastoma 7 and also inhibits the PI3K/AKT pathway. 13 In accordance with this, reducing VEGF-angiogenesis and PI3K/AKT could decrease tumour cell viability and proliferation. Thus, miR-205-5p could be considered as a plausible target against local vascular proliferative retinal processes.
Moreover, increasing miR205-5p levels could be beneficial for these ocular proliferative disorders not only by the direct anti-VEGFA blockade but also by the PI3K/AKT pathway interaction.

ACK N OWLED G EM ENTS
The present work was funded by internal funds from

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.