Retina d Opioid Receptor Agonism Preserves the Retinal Pigmented Epithelial Cell Tight Junctions and Ameliorates the Retinopathy in Experimental Diabetes Jacqueline M. Lopes de Faria,1 Diego A. Duarte,1 Rafael Simó,2 Marta Garcı́a-Ramirez,2 Marcella N. Dátilo,1 Francieli C. Pasqualetto,1 and José B. Lopes de Faria1 1 Renal Pathophysiology Laboratory, Investigation on Diabetes Complications, Faculty of Medical Sciences, State University of Campinas (UNICAMP), Campinas, Brazil 2 Vall d’Hebron Research Institute (VHIR) and CIBERDEM, Barcelona, Spain Correspondence: Jacqueline M. PURPOSE. Outer blood retinal barrier breakdown is a neglected feature of diabetic retinopathy Lopes de Faria, Faculty of Medical (DR). We demonstrated that the agonism of the d opioid receptor (DOR) by epicatechin Sciences, University of Campinas preserves the tight junction proteins in ARPE-19 cells under diabetic conditions. Presently, we (Unicamp), Campinas, SP 13083-970, aimed to evaluate the possible role of the DOR on the maintenance of tight junction of RPE Brazil; lopesdefariajm@gmail.com. layer and on the early markers of experimental DR. Submitted: January 31, 2019 METHODS. DR markers and external retinal tight junction proteins were evaluated in CL57B Accepted: August 5, 2019 diabetic mice submitted to intravitreous injection of short hairpin RNA (shRNA)-DOR (108 transducing units [TU]/mL) treated or not with DOR agonist (0.05 g/animal/d of epicatechin Citation: Lopes de Faria JM, Duarte DA, Simó R, et al. d opioid receptor in drinking water) for 16 weeks. The presence of DOR in human retina from postmortem eyes agonism preserves the retinal pig- from diabetic and nondiabetic donors were also performed. mented epithelial cell tight junctions RESULTS. DOR is present in RPE layer and in neuro retina. The treatment with DOR agonist and ameliorates the retinopathy in prevented the upregulation of the early markers of retinopathy (glial fibrillary acidic protein, experimental diabetes. Invest Oph- VEGF) and the downregulation of pigment epithelium-derived factor, occludin, claudin-1, and thalmol Vis Sci. 2019;60:3842–3853. https://doi.org/10.1167/iovs.19-26761 zonula occludens-1 tight junction expressions. The silencing of DOR in retina of diabetic mice partially abolished the protective effects of epicatechin. In human retina specimens, DOR is present throughout the retina, similarly in nondiabetic and diabetic donors. CONCLUSIONS. This set of experiments strongly indicates that the DOR agonism preserves RPE tight junctions and reduces the early markers of retinopathy in model of diabetes. These novel findings designate DOR as a potential therapeutic tool to treat DR with preservation of the RPE tight junction proteins. Keywords: diabetic retinopathy, diabetes, d opioid receptor, retinal pigmented epithelial D iabetic retinopathy (DR) is one of the most common causes of irreversible blindness worldwide. In the United States, the prevalence of DR is expected to increase substan- Moreover, the RPE plays a major role in the absorption of light; protection against photo-oxidation; regulation of nutrients, ions, and water between the neural retina (apical side) and the tially by 2020 due to the increasing rates of diabetes over time choriocapillaris (basal side); exchange of 11-cis retinal and all- with the aging of the population.1 DR often causes visual trans-retinol between the photoreceptors and RPE in the visual disabilities during the working ages, thus incurring significant cycle; phagocytosis of shed photoreceptor outer segments; and human, social, and economic costs on countries around the secretion of growth factors such as the pigment epithelium- world.2 The major outcomes of DR, including visual impair- derived factor (PEDF),6 vascular endothelial growth factor ments, consist of advanced forms of proliferative DR and (VEGF),7 transforming growth factor-b (TGF-b),8 insulin-like clinically significant macular edema, which can occur together growth factor-I (IGF-I),9 brain-derived growth factor (BDNF),10 or not. Data show that 30% (confidence interval: 25.9%–35.5%) and connective tissue growth factor (CTGF).11,12 More recently, of type 2 diabetes patients with nonproliferative retinopathy the role of the outer BRB in the pathogenesis of DR, specifically experience visual threatening due to diabetic maculopathy.3 diabetic macular edema, has gained more attention. Proteomics The RPE is a monolayer of polarized, pigmented cells of of diabetic human donor eyes with no retinopathy has revealed neuroectodermal origin located between the neural retina and alterations in the proteins involved in membrane dynamics, the choroid and helps in maintaining the structural and metabolic events, and cytoskeletal structure.13 A previous functional integrity of the retina.4 The RPE constitutes the ARPE-19 cell-based proteomic study showed different profile of outer blood retinal barrier (BRB), wherein the apical side is the secreted proteins from the RPE cells that are functionally directed toward the neural retina and the basal side is directed associated with cytoskeleton adhesion/junction when exposed toward the Bruch’s membrane, followed by choroid. The to high glucose (HG) conditions.14 functionality of RPE, apart from imparting a physical barrier, is Recently, we have demonstrated that the process by which to maintain retinal homeostasis by a series of secretory factors.5 tight junctions claudin-1 and occludin decrease in ARPE-19 cell Copyright 2019 The Authors iovs.arvojournals.org j ISSN: 1552-5783 3842 This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. Downloaded from iovs.arvojournals.org on 04/26/2021 d Opioid Receptor Protects the Retina Against Diabetes IOVS j September 2019 j Vol. 60 j No. 12 j 3843 and primary porcine RPE monolayers exposed to diabetes solution containing 108 transducing units (TU)/mL virus milieu conditions occurs through caveolin 1 (CAV-1) endocy- particles were intravitreally injected in using the Hamilton tosis.15 The HG condition leads to a profound imbalance in syringe with a 30G needle. Eight-week-old C57BL/6JUnib mice paracellular resistance and permeability, thus mimicking the were randomized to receive an intravitreal injection of one of outer BRB breakdown in vivo.15 The CAV-1 endocytosis the three different DOR shRNA construct (TRCN0000027801, process depends on the upregulation of inducible nitric oxide TRCN0000027842, and TRCN0000027797) and a nontarget synthase (iNOS) expression by TNF-a. The increased iNOS genes shRNA construct (Empty vector) as a negative control generates massive nitric oxide production, which results in S- (Sigma-Aldrich Corp.). After 1 month, the animals were nitrosylation of CAV-1.15 Treatment with epicatechin abolished submitted to electroretinogram (ERG) to test the possible these translational modifications of CAV-1 through direct retinal toxicity and then euthanized, and retinas were collected interaction with the d-opioid receptor (DOR) in the RPE to test the efficiency of DOR silencing. cells.15 These are the first observations that epicatechin Protocol 3. Treatment With DOR-Agonist in Drinking protects the integrity of the RPE barrier through DOR agonism, Water in Mice Submitted to Intravitreous Injection of thus preserving the retina from the deleterious effects of HG- shRNA to the Retinal DOR Gene. The diabetic mice received mimics diabetes. an intravitreous injection of empty vector in the left eye and The anti-angiogenic PEDF is a member of the serine protease DOR shRNA construct (TRCN0000027797) in the right eye, in inhibitor family, which is characterized by anti-angiogenic, the presence or absence of the DOR agonist in drinking water neuroprotective, and anti-inflammatory properties. In a healthy (0.05 g/animal/d). After 12 weeks of treatment, the animals eye, PEDF is secreted from the RPE and maintains the retinal as were submitted to ERG and then euthanized; the eye globes well as the choriocapillaris structure by inhibiting endothelial cell and retinas were collected for subsequent experiments. proliferation.16 PEDF is predominantly secreted from the apical surface of the RPE. In the postnatal period, PEDF-deficient mice Full-Flash ERG have shown enhanced retinal vascularization; this implies the modulating role of PEDF in the early stage of postnatal retinal Retinal function was measured using the UTAS-E3000 system vascularization.17,18 PEDF expression is downregulated in human (LKC Technologies, Inc., Gaithersburg, MD, USA), as previously RPE cells cultured in HG levels, as well as in the vitreous of described.24–27 The measurements were taken after overnight proliferative DR and diabetic macular edema patients.19 Studies dark adaptation. An intensity-response was recorded using a have also shown that PEDF-34 has neuroprotective and ganzfeld flash with an intensity of 0.25 cd.s/m2 at 0 to 250 or 0 regenerative capacity in optic nerve crush rat models.20 to 4000 milliseconds. Recordings were amplified and digitized Therefore, more studies are needed to explore the role of PEDF using a 24-bit A/D converter band passed from 0.3 to 300 Hz as a potential therapeutic tool to treat diabetic macular edema. with a 50-Hz notch filter. The quantitative analyses were The aim of the present study was to investigate whether the performed using the b-waves and oscillatory potential ampli- DOR agonist protects the retina, specially the outer tight junction tude expressed in microvolts (lV) and c-waves deflection proteins in an experimental model of DR. This study is the first to expressed in area under the curve. demonstrate that DOR agonism protects the RPE maintaining the expression of tight junctions accompanied by a decrease in VEGF In Vitro Assay and an increase in PEDF retinal levels. Collectively, these observations provide a new approach to treat DR. The human retinal pigment epithelial cell line (ARPE-19) was cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F-12 medium, 10% of fetal bovine serum (FBS; Cultlab, Campinas, MATERIALS AND METHODS Brazil), and 1% of penicillin and streptomycin. ARPE-19 cells at passage 15 to 17 were used for this study. Cell culture at 70% Animal Model and Experimental Design confluence were serum starved with 1% of FBS for 15 hours and exposed to normal glucose (NG; 5.5 mM) or HG (30 mM), The study was conducted in accordance with the ARVO or HG plus D-Ala(2)-Deltorphin II (HGþD2; 25 nM; HY-P1013, Statement for the Use of Animals in Ophthalmic and Vision MedChemExpress, Monmouth Junction, NJ, USA), or HG plus Research, and all procedures involving animals were approved D-Ala(2),D-Leu(5)]enkephalin (HGþDADLE; 50 nM; #E7131, by the Ethics Committee on the Use of Animals for the State Sigma-Aldrich, Corp.) for 24 hours in the presence or absence University of Campinas under regulation number 3986-1. The of the DOR antagonist Naltrindole (Nalt; 10 lM; 111469-81-9, mice were housed in a temperature-controlled room (228C) Cayman Chemical, Ann Arbor, MI, USA). with a 12-hour:12-hour light to dark cycle and had free access to food and water. Transepithelial Electrical Resistance (TER) Protocol 1. Treatment With DOR-Agonist in Drinking Water. Experimental diabetes was induced in 8-week-old Measurements C57BL/6JUnib male mice from the Multidisciplinary Center ARPE-19 cells were cultured in a Transwell-Clear Polyester for Biological Research by five consecutive daily intraperito- Membrane Insert (HTS, Costar; Corning, Inc., Corning, NY, neal injections of streptozotocin (50 mg/kg; Sigma-Aldrich USA). At day 22, the respective treatments were performed as Corp., St. Louis, MO, USA); the control animals received described above. After 24, 48, 120, and 196 hours, the vehicle alone. Thirty days after, the animals presenting a voltmeter (Millicell-ERS; Millipore, Burlington, MA, USA) was blood glucose of ‡15 mmol/L, measured by Accu-Chek used to evaluate the TER expressed in ohms and corrected by (Roche, Mississauga, ON, Canada), were considered diabetic area of the polyester membrane (ohms/cm2) subtracted from and randomized to receive or not the DOR agonist (high- the blank (polyester membrane only).14 purity ‡98% ()-epicatechin; Sigma-Aldrich Corp.) in drinking water. Based on previous studies,21,22 the oral dose of Western Blot Analysis epicatechin used in this study was 0.2% (0.2 g/100 mL) in drinking water (0.05 g/animal/d). The protein extracts obtained from total cell lysate were Protocol 2. Efficiency of the Different Short Hairpin subjected to SDS-PAGE in a Bio-Rad slab gel apparatus (Mini- RNA (shRNA) Constructs. As previously described,23 1 lL PROTEAN Tetra cell; Bio-Rad, Hercules, CA, USA) and Downloaded from iovs.arvojournals.org on 04/26/2021 d Opioid Receptor Protects the Retina Against Diabetes IOVS j September 2019 j Vol. 60 j No. 12 j 3844 electrophoretically transferred to a nitrocellulose membrane. Suite (Wetzlar, Germany) using two images per retinal section The membranes were incubated with primary antibodies for of at least three nonconsecutive retinal slides (100-lm apart) phospho-NF-jB (Ser536) 1:1000 (Cell Signaling Technology, per animal and three to five animals per group. At the end, at Danvers, MA, USA), NF-jB 1:250 (Santa Cruz Biotechnology, minimum six retinal images/animal were considered for the Dallas, TX, USA) followed by the appropriated secondary analyzes. antibodies. As internal control for protein loadings, the membranes were hybridized against vinculin 1:1000 (Cell Immunofluorescence Signaling Technology). Immunoreactive bands were visualized using the enhanced chemiluminescence method (Super Signal The immunofluorescence was performed as previously de- CL-HRP Substrate System; Pierce, Rockford, IL, USA). The scribed.25 The tissues were incubated with anti-claudina-1, membranes were scanned with a digital photo documentator occludin, and zonula occludens-1 (ZO-1; Invitrogen, Carlsbad, (ImageQuant LAS 500, GE, Boston, MA, USA) and analyzed CA, USA) and anti-human DOR (Merck) followed by the quantitatively with ImageJ Software (National Institutes of appropriate secondary antibody. The slides were examined Health, Bethesda, MD, USA). At least three independent under a scanning microscope (LSM510 Zeiss, Jena, Thuringia, experiments were carried out. Germany). The negative controls consisted in omitting the primary antibody. For the semi-quantitative analyses, at least Human Samples two images of each retinal section and three nonconsecutive retinal sections (100-lm apart) per animal and three to five Human postmortem eyes were obtained from diabetic (n ¼ 7) animals per group were included into the analyses using the and nondiabetic donors (n ¼ 7) matched by age). To be Image J Software (National Institutes of Health). included in this study, an ophthalmoscopic examination documenting the absence of microvascular abnormalities associated with DR or only mild nonproliferative DR must RNA Extraction and Quantitative RT-PCR have been performed within 2 years prior to the individual’s Real-time PCR was performed as previously described.26 death. The demographic characteristics of donors are Briefly, the retina extraction was transferred to 300-lL tubes presented in the Supplementary Table S1. The study was containing Trizol (Invitrogen). The concentration of RNA was performed following the tenets of the Declaration of Helsinki determined using a NanoVue (GE). For cDNA synthesis, we for research involving human tissue. The time elapsed from used random primers and a dNTP mix. Subsequently, 4 lL 5X- death to eye enucleation was 3.4 6 1.9 hours. After concentrated enzyme buffer, 1 lL 0.1 M dithiothreitol, and 1 lL enucleation, one eye from each donor was snap-frozen in Superscript III reverse transcriptase enzyme (Thermo Fisher liquid nitrogen and stored at 808C in the Blood and Tissue Scientific) were added. Primers for the DOR (Prrd1, Bank of Vall d’Hebron University Hospital (Barcelona, Spain) Mm01180757_m1, and NM_000911.3), Occludin (Oclin, until the eye was assayed. The other eye was fixed in 4% Mm00500911_m1), and GFAP (Mm01253033_m1) genes were paraformaldehyde and embedded in paraffin for the immu- nofluorescence study. The neuroretina and RPE were obtained from Applied Biosystems (Foster City, CA, USA). b- harvested before mRNA or protein extracts were obtained. actin gene expression was used as a control (Actb, Briefly, the vitreous and neuroretina were removed, and the Mm02619580_g1). The RT-PCR was performed using primers, RPE layer was then carefully peeled off from Bruch’s and b-Actin was used as a constitutive gene. membrane using forceps (Dumont No 5, Sigma-Aldrich Corp., Madrid, Spain) under the dissecting microscope (Olympus Statistical Analysis SZ61, Barcelona, Spain). The results were expressed as the mean 6 SD. Comparisons between groups were done using ANOVA followed by Fisher’s Human Protein Extraction and Western Blotting protected least-significant difference test. The analyses were Protein extracts from isolated RPE and neuroretina samples performed using StatView software (SAS Institute, Inc., Cary, were prepared by homogenization with radio immunoprecip- NC, USA). P < 0.05 was considered significant. itation assay (RIPA) buffer, and protein concentrations were determined using a bicinchoninic acid assay (BCA) kit (Thermo Fisher Scientific, Rockford, IL, USA). Twenty micrograms of RESULTS protein was separated by SDS-PAGE acrylamide gels and transferred to nitrocellulose membranes (GE Healthcare, Physiological Parameters Piscataway, NJ, USA). The membranes were incubated in As expected, the final body weights were lower and the blood primary antibody against DOR (1:250 Merck, Darmstadt, glucose levels were higher in the diabetic group compared Germany), and immunoreactive bands were visualized using with the nondiabetic group (P < 0.0001) (Table). The water enhanced chemiluminescence (Millipore, Madrid, Spain). For consumption was higher in the diabetic group compared with densitometric analysis, we used a GS-800 calibrated densitom- the nondiabetic group (P < 0.001). None of the treatment eter (Bio-Rad Laboratories, Madrid, Spain) and Quantity One regimens altered body weight, blood glucose levels, or water software version 4.6.2 (Bio-Rad Laboratories). consumption among the diabetic animals. Immunohistochemistry The Expression of the DOR Is Not Altered Either by The immunohistochemistry was performed as previously the Presence of Elevated Blood Glucose or by described.24 The retinal sections were incubated with anti- Treatment With the DOR Agonist GFAP and anti-VEGF (1:250, Santa Cruz Biotechnology) overnight at 48C. The slides were then incubated with the DOR is expressed in the RPE and outer and inner plexiform appropriate secondary antibodies. The negative controls layers. We found that both DOR protein expression and mRNA consisted in omitting the primary antibody. The semi-quanti- levels did not change in the presence of elevated blood glucose tative analyses were performed using the Leica Application treated or not with DOR agonist (Figs. 1B, 1C, respectively). Downloaded from iovs.arvojournals.org on 04/26/2021 d Opioid Receptor Protects the Retina Against Diabetes IOVS j September 2019 j Vol. 60 j No. 12 j 3845 TABLE. Physiological Parameters of Study Group Groups Blood Glucose, mg/dL Body Weight, g Water Intake, mL/d No diabetic, n ¼ 6 154.0 6 18.3 30.5 6 2.7 9.9 6 3.0 Diabetic, n ¼ 7 539.8 6 29.6* 21.1 6 2.8* 28.3 6 2.4† Diabetic þ DOR agonist, n ¼ 6 552.7 6 74.2* 20.5 6 2.8* 27.0 6 1.6† Nondiabetic-shRNA, n ¼ 9 152.6 6 38.5 31.4 6 1.8 8.0 6 2.0 Diabetic-shRNA, n ¼ 7 558.8 6 39.3* 21.0 6 1.7* 27.6 6 2.1† Diabetic-shRNA þ DOR agonist, n ¼ 6 594.5 6 9.4* 20.1 6 2.3* 27.1 6 1.5† * P < 0.0001. † P < 0.001. The DOR Agonist Epicatechin Ameliorates the Efficiency and Toxicity of the shRNA Constructs Early Markers of DR and Protects the RPE Tight To verify the possible role of the DOR in the beneficial Junctions in Experimental Diabetic Mice effects of the epicatechin treatment (Figs. 1, 2), mice received an intravitreous injection of shRNA-DOR (108 TU/ The presence of elevated blood glucose exacerbates GFAP and mL, 1lL) with three different shRNA constructs for the DOR VEGF immunolabeling (Figs. 2A, 2B; P < 0.0001) and increases gene to determine the most efficient one. None of the GFAP mRNA levels in retinal tissue (Fig. 2C; P ¼ 0.007). constructs altered the a, b, or c-waves of full-flash ERG, Treatment with the DOR-agonist prevents glial reaction and indicating the absence of retinal toxicity (Supplementary reduces the levels of VEGF in diabetic retina (P < 0.0001). Figs. S1D–F); the best efficiency was found in the PEDF expression, used as a marker of RPE function, was TRCN0000027797 construct, with an approximately 32% markedly reduced in diabetic retina (Figs. 2A, 2B; P < 0.0001). reduction in DOR mRNA levels (Supplementary Fig. S1A) The DOR agonism completely restored retinal PEDF expression and a 29% reduction in retinal protein expression (Supple- to nondiabetic levels (P < 0.0001). mentary Figs. S1B, S1C). The presence of elevated blood glucose increased claudin-1, occludin, and ZO-1 tight junction proteins immunolabeling Silencing of DOR Partially Reversed the Protective (Figs. 3A, 3B; P < 0.03) and occludin mRNA levels in the RPE layer (Fig. 3C; P ¼ 0.01), and the treatment with epicatechin Effects of Epicatechin on the Early Markers of DR prevented these increments in all tight junctions to nondia- Diabetic mice that received an intravitreous injection of betic levels (P < 0.01). shRNA-DOR (TRCN0000027797) and were treated with DOR Retinal function was estimated through full-flash ERG. agonist in drinking water (0.05 g epicatechin/d/animal) for 12 Diabetic mice showed a significant decrease in the amplitudes weeks showed partial reversal of the expression of GFAP (P ¼ of the b- and c-waves (Fig. 4; P < 0.02). However, the oral 0.001), VEGF (P ¼ 0.04), PEDF (P ¼ 0.02) (Fig. 5), claudin-1 (P treatment with epicatechin was not able to improve these ¼ 0.003), occludin (P ¼ 0.03), and ZO-1 (P ¼ 0.003) (Fig. 6) parameters. compared with the contralateral eyes (intraocularly injected FIGURE 1. DOR agonist does not affect the protein levels of the DOR receptor. (A) Representative photomicrograph of DOR in retinal tissue. DOR immunostaining (green) counterstained with the nuclei marker DAPI (blue). Magnification: 4003. Scale bars: 50 lm. (B) Semiquantitative analyses of DOR immunofluorescence. The bars represent the mean 6 SD for the percentage of DOR-positive per retinal area expressed as a percentage of variation relative to control. (C) Relative DOR mRNA expression in retinal tissue. The bars represent mean 6 SD for the relative expression to control. DM, diabetic; DMþDOR-A, diabetic treated with DOR agonist; ND, no diabetic; NS, no significant statistical difference. Downloaded from iovs.arvojournals.org on 04/26/2021 d Opioid Receptor Protects the Retina Against Diabetes IOVS j September 2019 j Vol. 60 j No. 12 j 3846 FIGURE 2. DOR agonist ameliorates early markers of DR and improves PEDF expression in an experimental model of diabetes using mice. (A) Representative photomicrograph of glial reactivity revealed by GFAP and retinal dysfunction represented by VEGF and PEDF in retinal tissue. PEDF immunostaining (green) counterstained with the nuclei marker DAPI (blue). Magnification: 2003. (B) Semiquantitative analyses of the GFAP, VEDF, and PEDF immunoreaction. The bars represent the mean 6 SD for the percentage of GFAP, VEGF, or PEDF-positive per retinal area expressed as a percentage of variation relative to control. (C) Relative GFAP mRNA expression in retinal tissue. The bars represent the mean 6 SD for the relative expression to control. DM, diabetic; DMþDOR-A, diabetic treated with DOR agonist; ND, no diabetic; NS, no significant statistical difference. with empty lentivirus-green fluorescent protein labeled), normal levels. The co-treatment with naltrindole abolished which served as a control. These results indicate that the effect of DOR-1 activation (P ¼ 0.04). The anti- epicatechin prevented the early markers of DR and protected inflammatory effect of DOR-1 activation in ARPE-19 also the RPE tight junctions in diabetic mice, partially through DOR improved the TER feature of ARPE-19 cells barrier. At 48 agonism. hours’ time point, there was a significant increase in TER in HG conditions (P  0.008), which was prevented by the The Activation of DOR-1 But Not 2 Inhibits presence of DOR-1 activator up to 196 hours of observation (P ¼ 0.02 and P  0.05 for 48, 120, and 196 hours, Inflammatory Signaling in ARPE-19 Cells Exposed respectively); the presence of naltrindole blocker did not to HG Condition Restoring a Barrier Property: a alter the protective effect of DOR-1 activator (P ‡ 0.05). This Proof of Concept last observation could be explained by the nonspecificity of the naltrindole in presence of D-Ala(2),D-Leu(5)]enkepha- In order to confirm whether the stimulation of DOR in RPE lin), a highly specific DOR-1 activator within this period of could be protective as a possible tool to maintain the barrier time. property under diabetic milieu conditions, ARPE-19 cells To verify whether DOR is present in the human retina, we cultured under HG conditions and submitted to treatment immunolabeled and quantified the protein and gene expres- with both activators of DOR, type 1 and type 2, in presence sions of DOR from isolated RPE and neuroretina samples from or not of unspecific blocker naltrindole for 24 hours. The nondiabetic and diabetic donors. The immunofluorescence inflammatory signaling was evaluated through western blot images revealed that DOR is widely distributed in RPE and assay and the ARPE-19 barrier function by TER test. As neuro retina (Fig. 8A). We did not find any significant shown (Fig. 7A), the ARPE-19 cells exposed to HG showed a difference between diabetic and nondiabetic donors in terms significant increasing in p65-NFkB expression (P < 0.008) of protein content (1.2 6 0.2 vs. 1.4 6 0.5 arbitrary units [AU] but the treatment with DOR-2 activator (D-Ala(2)-Deltorphin for neuroretina, and 0.8 6 0.4 vs. 1.0 6 0.7 AU for RPE) (Fig. II) did not interfere in the expression of p65-NFkB, as well as 8B) and mRNA levels (1.35 6 1.27 vs. 2.12 6 1.44 AU, and in presence of naltrindole (P > 0.05). By the other hand, 1.98 6 2.77 vs. 3.23 6 5.77 AU for RPE) of DOR (Fig. 8C). ARPE-19 under HG and treated with DOR-1 activator (D- These observations strengthen the potential translational Ala(2),D-Leu(5)]enkephalin) (Fig. 7B) showed a significant importance of the DOR agonism as a new therapeutic tool to reduction in p65-NFkB expression (P ¼ 0.02) achieving the treat retinopathy in diabetic patients. Downloaded from iovs.arvojournals.org on 04/26/2021 d Opioid Receptor Protects the Retina Against Diabetes IOVS j September 2019 j Vol. 60 j No. 12 j 3847 FIGURE 3. DOR agonist prevents external retinal barrier dysfunction in an experimental model of diabetes using mice. (A) Representative photomicrograph of claudin-1, occludin, and ZO-1 in the RPE layer. Magnification: 2003. Scale bars: 50 lm. (B) Semiquantitative analyses of the claudin-1, occludin, and ZO-1 immunofluorescence. The bars represent the mean 6 SD for the percentage of claudin-1, occludin, and ZO-1-positive per RPE layer expressed as a percentage of variation relative to control. (C) Relative occludin mRNA expression in retinal tissue. The bars represent the mean 6 SD of the relative expression to control. DM, diabetic; DMþDOR-A, diabetic treated with DOR agonist; ND, no diabetic; NS, no significant statistical difference. DISCUSSION direction. The glucose depends upon the transporters (GLUT1 and GLUT3), which are diminished in high-glucose condi- This study presents DOR agonism as a potential novel target for tions.29 This deleterious effect alters the neuron cell activity, a the treatment of DR in experimental model of diabetes. We highly metabolic cell. However, due to the metabolism of provided evidence that using epicatechin as a DOR agonist neurons, including photoreceptors, a large quantity of water is efficiently prevents the early diabetic changes present in retinal produced, and it is actively transported by the Naþ-Kþ-ATPase, tissue, such as upregulation of GFAP and VEGF followed by located in the apical membrane, as active transport.30 The downregulation of PEDF. In addition to those observed constant elimination of the water from the subretinal space changes, diabetic mice treated with epicatechin showed produces an adhesive force between the retina and the RPE preservation of the outer tight junction proteins claudin-1, that is lost due to the inhibition of Naþ-Kþ-ATPase. Studies with occludin, and ZO-1. Epicatechin was shown to be a potential cultured bovine RPE cells in high-glucose conditions have scaffold for the opioid receptor ligands of the C2 and C3 demonstrated a loss of Naþ/Kþ ATPase function, which in turn positions of the epicatechin molecule.28,29 Corroborating with decreases permeability.31 The impairment of glucose trans- possible role of DOR in these effects, diabetic mice treated porter and of Naþ/Kþ ATPase decreases the glucose offered to with oral epicatechin but submitted to intravitreous injection the neuroretina and weakens the adhesive forces between the of shRNA-DOR in its right eye (with an approximately 30% retina and the RPE, reducing the permeability. 31 The reduction in DOR protein expression), had the beneficial importance of RPE to DR development depends on the effects partly diminished. These compelling results point out photoreceptor abnormalities present early in the DR patho- DOR agonism as a possible mechanism by which epicatechin genesis.32–35 Another important feature of increasing outer exerts the effects. In this present study, although the DOR retinal barrier resistance is the fluid accumulation in the agonism protected the retina from the ultrastructural changes subretinal space, particularly in the macular area,36–39 which related to diabetes, the ERG traces did not reflect this is a hallmark of diabetic macular edema and has important improvement. This observation might be due to relatively clinical relevance. Thus, the RPE role in DR deserves further short time duration or dose dependent of the oral treatment investigation and could represent a possible site for novel with epicatechin. Further studies with longer duration or even therapeutic approaches. with different doses are needed in order to understand this In our previous in vitro study with ARPE-19, the cells finding. exposed to HG, a condition that mimics diabetes, showed a The role of RPE as the outer retinal barrier and its secretory significant increase in transepithelial resistance and reduced activity in a DR experimental model require further study. The cell permeability accompanied by increased expression of outer barrier transports water and electrolytes in one direction ZO-1 and decrease of claudin-1 and occludin; the presence of (from the neuroretina to the choriocapillaris) and retinol, epicatechin abrogated these effects.14 Differently from the in glucose, ascorbic acid, and fatty acids in the opposite vitro study using human immortalized cells, the expressions Downloaded from iovs.arvojournals.org on 04/26/2021 d Opioid Receptor Protects the Retina Against Diabetes IOVS j September 2019 j Vol. 60 j No. 12 j 3848 FIGURE 4. DOR agonist does not improve retinal function in an experimental model of diabetes using mice. (A) Representative full-flash ERG waveforms at 0.25 cd.s/m2 in studied mice. b-wave positive deflection generated in part by the Müller and mainly by the bipolar cell potentials. (B) Representative waveforms of c-waves at a light stimulus intensity of 0 dB; c-wave positive deflection generated by the potentials of the RPE cells. (C– E) The bars represent the mean 6 SD for the b-waves amplitude and oscillatory potential amplitudes expressed in microvolts (lV) and c-waves amplitude (lV) expressed in area under the curve (arbitrary units). DM, diabetic; DMþDOR-A, diabetic treated with DOR agonist; ND, no diabetic; NS, no significant statistical difference. of claudin-1 and occludin were diminished under HG anti-inflammatory and anti-oxidative protective effects of conditions in tissue of diabetic mice. Previous studies have activation of DOR.46–49 A study by Husain et al.50 showed yielded controversial findings regarding the transepithelial that in a model of retinal ischemia, TNF-a production was resistance and permeability of the ARPE-19 monolayer significantly inhibited by morphine, and the combination exposed to HG in vitro.15,40,41 Villarroel et al.40 verified the with naloxone reversed the morphine-induced suppression of increase of claudin-1 in ARPE-19 cells exposed to HG; TNF-a production in vivo and in vitro. In the present study, however, at that time, the gene silencing of claudin-1, the the increased expression of the ZO-1, occludin, and claudin-1 status of cell permeability, and resistance were not altered, tight junction proteins represent one function of the RPE demonstrating how complex is the functioning of the barrier, that is, paracellular diffusion. Besides the paracellular epithelial cell barrier. Studies with other types of epithelial diffusion, other mechanisms are operating to maintain the cells show that stress conditions are mediated by TNFa selectivity and efficiency of the RPE barrier, such as facilitated signaling via NF-jB.42–45 Other studies have demonstrated diffusion and active transport of CL and transcytosis. Downloaded from iovs.arvojournals.org on 04/26/2021 d Opioid Receptor Protects the Retina Against Diabetes IOVS j September 2019 j Vol. 60 j No. 12 j 3849 FIGURE 5. Silencing of DOR partially reverses the effect of its agonist in the improvement of retinal dysfunction in an experimental model of diabetes using mice. (A) Representative photomicrograph of glial reactivity revealed by GFAP and retinal dysfunction represented by VEGF and PEDF in retinal tissue. PEDF immunostaining (green) counterstained with the nuclei marker DAPI (blue). Magnification: 2003. Scale bars: 50 lm. (B) Semiquantitative analyses of the GFAP, VEDF, and PEDF immunoreaction. The bars represent the mean 6 SD for the percentage of GFAP, VEGF, or PEDF-positive per retinal area expressed as a percentage of variation relative to control. DM-Empty, diabetic injected with empty virus; DM- EmptyþDOR-A, diabetic injected with empty virus and treated with DOR agonist; DM-SiþDOR-A, diabetic injected with DOR silencing and treated with DOR agonist; ND-Empty, no diabetic injected with empty virus; NS, no significant statistical difference. The profound augment of the tight junctions observed in studies showed the binding of flavonoids with various this study may represent a late RPE barrier dysfunction and receptors: androgen and/or estrogen, IGF-1, epidermal growth possibly represent a compensatory mechanism to rescue the factor, aryl hydrocarbon, Fas, VEGF, and laminin.55 Epicatechin, barrier properties under the metabolic condition induced by as well as many other flavonoids, has been identified as a new diabetes. The treatment with epicatechin as a DOR agonist was scaffold for the development of opioid receptor ligands. The able to protect the RPE tight junctions in diabetic mice and in biological results suggest that the stereochemistry of the C2 presence of DOR silencing, this beneficial effect was mitigated. and C3 positions of the flavonoid molecule, including DOR is widely expressed in peripheral and central systems, epicatechin, is important for the selectivity of DOR.27,28 In including the brain, heart, liver, and kidney.51,52 The concept this study, we showed that intravitreal injection of shRNA-DOR that DOR is a cardio53–55 and neuro protector56–58 has been diminished the protective effects of epicatechin on diabetic demonstrated and strengthened in recent years. However, the retinal tissue, thus providing evidence that its protective effect underlying mechanisms of DOR protection remain unclear. We depends on the DOR receptor, at least partly. previously demonstrated that the agonism of DOR in in vitro In presence of compromised RPE, the monolayer of these conditions that mimic diabetes preserved the tight junction’s polarized cells fails in its directional transport by altering and maintenance in ARPE-19 cells as a negative regulator of the accumulating subretinal fluid and thus disturbing the photo- TNF-a pathway; however, the presence of the DOR blocker naltrindole abolished this effect.14 In this study, we moved receptor segments and neuro retina. Impairment of water onward and demonstrated that the expression of DOR is transport from the subretinal space to choriocapillaris leads to present in retinal tissue, including the RPE and neuro retina, the accumulation of water in the subretinal space. and does not change in the presence of elevated blood glucose The agonism of the DOR using ()-epicatechin protects or with treatment with epicatechin; however, under DOR the tight junction integrity of the RPE and prevents the early silencing, the oral treatment with epicatechin was weakened markers of DR. The preservation of RPE function leads to an in diabetic retina, suggesting that epicatechin operates partly increase in the endogenous anti-angiogenic factor PEDF, thus via DOR agonism. counter-balancing the increasing of VEGF levels early on in The epicatechin antioxidant properties are well known. the pathogenesis of DR. DOR agonism may be used as a new Several studies have shown the protective role of flavonoids on pharmacological therapy for retinal metabolic disorders as the membrane surface through interactions with the lipid and DR and disorders in which RPE tight junctions are disar- protein components of these membranes.28,58,59 Previous ranged. Downloaded from iovs.arvojournals.org on 04/26/2021 d Opioid Receptor Protects the Retina Against Diabetes IOVS j September 2019 j Vol. 60 j No. 12 j 3850 FIGURE 6. Silencing of DOR partially reverses the effect of its agonist in the improvement of external retinal barrier dysfunction in an experimental model of diabetes using mice. (A) Representative photomicrograph of claudin-1, occludin, and ZO-1 in the RPE layer. Magnification: 3200. Scale bars: 50 lm. (B) Semiquantitative analyses of the claudin-1, occludin, and ZO-1 immunofluorescence. The bars represent the mean 6 SD for the percentage of claudin-1, occludin, or ZO-1-positive per RPE layer expressed relative to control. DM-Empty, diabetic injected with empty virus; DM- EmptyþDOR-A, diabetic injected with empty virus and treated with DOR agonist; DM-SiþDOR-A, diabetic injected with DOR silencing and treated with DOR agonist; ND-Empty, no diabetic injected with empty virus; NS, no significant statistical difference. FIGURE 7. DOR 1 but not DOR 2 is involved in inflammatory pathway in ARPE-19. The phosphorylation of NFjB (p65-NFjB) in total cell lysate was evaluated after 24 hours in ARPE-19 cells exposed to NG, HG, HGþD2, HGþNaltþD2 in (A) or to NG, HG, HGþD1, HGþNaltþD1, *P ¼ 0.0008. (B) Protein loading was evaluated re-probing the membranes against vinculin. The bars represent mean 6 SD. n ¼ 6 per group, †P ¼ 0.05, ‡P ¼ 0.02 versus NG, §P ¼ 0.04 vs. HGþD1. (C) TER assay. The electrical resistances were measured in ARPE-19 cells exposed to NG, HG, HGþD1, and HGþNaltþD1 at 0, 24, 48, 120, and 196 hours. The bars represent the TER expressed in Ohms/cm2 (mean 6 SD) in each group. n ‡ 5 per group. *P  0.05; ‡P ¼ 0.02 versus HG; #P ¼ 0.02 versus NG; **P  0.05 versus HG. D1, DOR 1 activator, D-Ala(2),D-Leu(5)]enkephalin; D2, DOR 2 activator, D-Ala(2),Deltorphin II; Nalt, Naltrindole (a nonspecific DOR blocker); NG, normal. Downloaded from iovs.arvojournals.org on 04/26/2021 d Opioid Receptor Protects the Retina Against Diabetes IOVS j September 2019 j Vol. 60 j No. 12 j 3851 FIGURE 8. DOR expression in the RPE and neuroretina from human retinal donors. (A) DOR expression in human retinas from a representative nondiabetic donor. DOR immunostaining (green) in retinal paraffined sections counterstained with DAPI (blue). Confocal microscopy (oil immersion, 603). Scale bars: 40 lm. (B) DOR representative images of Western blotting. DOR band values were obtained by densitometry and intensities normalized by b-actin bands. Bars represent the mean 6 SD of the intensity ratio. (C) Real-time quantitative PCR of DOR mRNA. Bars represent the mean 6 SD of the values obtained in the control versus diabetic human eye donors studied. DOR mRNA levels were normalized with b-actin. N ¼ 14. GCL, ganglion cell layer; INL, inner nuclear layer; NR, neuroretina; NS, no significant statistical difference; ONL, outer nuclear layer; OPL, outer plexiform layer. Acknowledgments in the Liverpool Diabetic Eye Study: a cohort study. Liverpool Diabetic Eye Study. Lancet. 2003;361:195–200. The authors thank individuals from the Renal Pathophysiology 4. Campbell M, Humphries P. The blood-retina barrier: tight Laboratory, Investigation on Diabetes Complications, Faculty of junctions and barrier modulation. Adv Exp Med Biol. 2012; Medical Sciences (FCM), and UNICAMP: Antônio R. Calixto for the 763:70–84. RT-PCR experiments; Daniele C. 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