Activation of the Wnt Pathway Plays a Pathogenic Role in Diabetic Retinopathy in Humans and Animal Models
2009; Elsevier BV; Volume: 175; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2009.080945
ISSN1525-2191
AutoresYing Chen, Yang Hu, Ti Zhou, Kelu Zhou, Robert Mott, Mingyuan Wu, Michael E. Boulton, Timothy J. Lyons, Guoquan Gao, Jian‐xing Ma,
Tópico(s)Nerve injury and regeneration
ResumoAlthough Wnt signaling is known to mediate multiple biological and pathological processes, its association with diabetic retinopathy (DR) has not been established. Here we show that retinal levels and nuclear translocation of β-catenin, a key effector in the canonical Wnt pathway, were increased in humans with DR and in three DR models. Retinal levels of low-density lipoprotein receptor-related proteins 5 and 6, coreceptors of Wnts, were also elevated in the DR models. The high glucose-induced activation of β-catenin was attenuated by aminoguanidine, suggesting that oxidative stress is a direct cause for the Wnt pathway activation in diabetes. Indeed, Dickkopf homolog 1, a specific inhibitor of the Wnt pathway, ameliorated retinal inflammation, vascular leakage, and retinal neovascularization in the DR models. Dickkopf homolog 1 also blocked the generation of reactive oxygen species induced by high glucose, suggesting that Wnt signaling contributes to the oxidative stress in diabetes. These observations indicate that the Wnt pathway plays a pathogenic role in DR and represents a novel therapeutic target. Although Wnt signaling is known to mediate multiple biological and pathological processes, its association with diabetic retinopathy (DR) has not been established. Here we show that retinal levels and nuclear translocation of β-catenin, a key effector in the canonical Wnt pathway, were increased in humans with DR and in three DR models. Retinal levels of low-density lipoprotein receptor-related proteins 5 and 6, coreceptors of Wnts, were also elevated in the DR models. The high glucose-induced activation of β-catenin was attenuated by aminoguanidine, suggesting that oxidative stress is a direct cause for the Wnt pathway activation in diabetes. Indeed, Dickkopf homolog 1, a specific inhibitor of the Wnt pathway, ameliorated retinal inflammation, vascular leakage, and retinal neovascularization in the DR models. Dickkopf homolog 1 also blocked the generation of reactive oxygen species induced by high glucose, suggesting that Wnt signaling contributes to the oxidative stress in diabetes. These observations indicate that the Wnt pathway plays a pathogenic role in DR and represents a novel therapeutic target. Diabetic retinopathy (DR), the leading cause of blindness in the working age population, represents a common concern in types 1 and 2 of diabetes mellitus (DM).1Wild S Roglic G Green A Sicree R King H Global prevalence of diabetes: estimates for the year 2000 and projections for 2030.Diabetes Care. 2004; 27: 1047-1053Crossref PubMed Scopus (11797) Google Scholar Accumulating evidence suggests that DR is a chronic inflammatory disorder.2Joussen AM Poulaki V Le ML Koizumi K Esser C Janicki H Schraermeyer U Kociok N Fauser S Kirchhof B Kern TS Adamis AP A central role for inflammation in the pathogenesis of diabetic retinopathy.FASEB J. 2004; 18: 1450-1452Crossref PubMed Scopus (951) Google Scholar Retinal inflammation is believed to play a causative role in vascular leakage, which can lead to diabetic macular edema, and in retinal neovascularization (NV). It has been shown that levels of soluble intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 are significantly higher in the vitreous from patients with proliferative diabetic retinopathy than in nondiabetic vitreous.3Funatsu H Noma H Mimura T Eguchi S Hori S Association of vitreous inflammatory factors with diabetic macular edema.Ophthalmology. 2009; 116: 73-79Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar, 4Limb GA Hickman-Casey J Hollifield RD Chignell AH Vascular adhesion molecules in vitreous from eyes with proliferative diabetic retinopathy.Invest Ophthalmol Vis Sci. 1999; 40: 2453-2457PubMed Google Scholar Increased ICAM-1, vascular cell adhesion molecule-1, and e-selectin levels were found in the serum from patients with diabetic microangiopathy.5Khalfaoui T Lizard G Ouertani-Meddeb A Adhesion molecules (ICAM-1 and VCAM-1) and diabetic retinopathy in type 2 diabetes.J Mol Histol. 2008; 39: 243-249Crossref PubMed Scopus (54) Google Scholar, 6Adamiec-Mroczek J Oficjalska-Mlynczak J Misiuk-Hojlo M Proliferative diabetic retinopathy: the influence of diabetes control on the activation of the intraocular molecule system.Diabetes Res Clin Pract. 2009; 84: 46-50Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 7Kado S Nagata N Circulating intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin in patients with type 2 diabetes mellitus.Diabetes Res Clin Pract. 1999; 46: 143-148Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar In diabetic animal models, increased retinal ICAM-1 expression is believed to be responsible for leukocyte adhesion or leukostasis and increased vascular permeability. Leukostasis is believed to contribute to capillary nonperfusion and local ischemia, which subsequently induces the overexpression of vascular endothelial growth factor (VEGF).8Zhang XL Wen L Chen YJ Zhu Y Vascular endothelial growth factor up-regulates the expression of intracellular adhesion molecule-1 in retinal endothelial cells via reactive oxygen species, but not nitric oxide.Chin Med J (Engl). 2009; 122: 338-343PubMed Google Scholar, 9Olson JA Whitelaw CM McHardy KC Pearson DW Forrester JV Soluble leucocyte adhesion molecules in diabetic retinopathy stimulate retinal capillary endothelial cell migration.Diabetologia. 1997; 40: 1166-1171Crossref PubMed Scopus (90) Google Scholar, 10Antonetti DA Barber AJ Khin S Lieth E Tarbell JM Gardner TW Vascular permeability in experimental diabetes is associated with reduced endothelial occludin content: vascular endothelial growth factor decreases occludin in retinal endothelial cells. Penn State Retina Research Group.Diabetes. 1998; 47: 1953-1959Crossref PubMed Scopus (510) Google Scholar, 11Antonetti DA Lieth E Barber AJ Gardner TW Molecular mechanisms of vascular permeability in diabetic retinopathy.Semin Ophthalmol. 1999; 14: 240-248Crossref PubMed Scopus (172) Google Scholar Increased VEGF levels are responsible for the retinal vascular leakage and retinal NV.12Ishida S Usui T Yamashiro K Kaji Y Ahmed E Carrasquillo KG Amano S Hida T Oguchi Y Adamis AP VEGF164 is proinflammatory in the diabetic retina.Invest Ophthalmol Vis Sci. 2003; 44: 2155-2162Crossref PubMed Scopus (342) Google Scholar, 13Aiello LP Gardner TW King GL Blankenship G Cavallerano JD Ferris 3rd, FL Klein R Diabetic retinopathy.Diabetes Care. 1998; 21: 143-156PubMed Google Scholar Recent studies have indicated that oxidative stress, induced by hyperglycemia, contributes to retinal inflammation in diabetes.14Ali TK El-Remessy AB Diabetic retinopathy: current management and experimental therapeutic targets.Pharmacotherapy. 2009; 29: 182-192Crossref PubMed Scopus (43) Google Scholar, 15Piconi L Quagliaro L Ceriello A Oxidative stress in diabetes.Clin Chem Lab Med. 2003; 41: 1144-1149Crossref PubMed Scopus (176) Google Scholar However, the pathogenic mechanisms by which diabetes and oxidative stress induce inflammation are not certain at the present time.Wnts are a group of secreted, cysteine-rich glycoproteins, which bind to a coreceptor complex of frizzled (Fz) receptors and low-density lipoprotein receptor-related protein 5 or 6 (LRP5/6) and regulate expression of a number of target genes through an intracellular signaling pathway, namely the Wnt pathway.16He X Semenov M Tamai K Zeng X LDL receptor-related proteins 5 and 6 in Wnt/beta-catenin signaling: arrows point the way.Development. 2004; 131: 1663-1677Crossref PubMed Scopus (847) Google Scholar In the absence of Wnt ligands, β-catenin, a down-stream effector of the canonical Wnt pathway, is phosphorylated by a protein complex containing glycogen synthase kinase-3β. The phosphorylated β-catenin is constantly degraded, to prevent its accumulation. On binding of certain Wnts to the Fz-LRP5/6 coreceptors, phosphorylation of β-catenin is inhibited, which prevents the degradation of β-catenin and results in its accumulation.17Dale TC Signal transduction by the Wnt family of ligands.Biochem J. 1998; 329: 209-223Crossref PubMed Scopus (435) Google Scholar β-catenin is then translocated into the nucleus, associates with T-cell factor for DNA binding, and regulates expression of target genes including VEGF.18Phng LK Potente M Leslie JD Babbage J Nyqvist D Lobov I Ondr JK Rao S Lang RA Thurston G Gerhardt H Nrarp coordinates endothelial Notch and Wnt signaling to control vessel density in angiogenesis.Dev Cell. 2009; 16: 70-82Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 19Tachikawa K Schroder O Frey G Briggs SP Sera T Regulation of the endogenous VEGF-A gene by exogenous designed regulatory proteins.Proc Natl Acad Sci USA. 2004; 101: 15225-15230Crossref PubMed Scopus (46) Google Scholar, 20Easwaran V Lee SH Inge L Guo L Goldbeck C Garrett E Wiesmann M Garcia PD Fuller JH Chan V Randazzo F Gundel R Warren RS Escobedo J Aukerman SL Taylor RN Fantl WJ beta-Catenin regulates vascular endothelial growth factor expression in colon cancer.Cancer Res. 2003; 63: 3145-3153PubMed Google ScholarLRP5/6 are known to play a critical role in Wnt/β-catenin signaling.21MacDonald BT Yokota C Tamai K Zeng X He X Wnt signal amplification via activity, cooperativity, and regulation of multiple intracellular PPPSP motifs in the Wnt co-receptor LRP6.J Biol Chem. 2008; 283: 16115-16123Crossref PubMed Scopus (72) Google Scholar, 22Mao B Wu W Li Y Hoppe D Stannek P Glinka A Niehrs C LDL-receptor-related protein 6 is a receptor for Dickkopf proteins.Nature. 2001; 411: 321-325Crossref PubMed Scopus (883) Google Scholar, 23Mao J Wang J Liu B Pan W Farr 3rd, GH Flynn C Yuan H Takada S Kimelman D Li L Wu D Low-density lipoprotein receptor-related protein-5 binds to Axin and regulates the canonical Wnt signaling pathway.Mol Cell. 2001; 7: 801-809Abstract Full Text Full Text PDF PubMed Scopus (680) Google Scholar On binding with Wnt ligands, LRP6 dimerizes with Fz receptor, which is the first and essential step in activation of the Wnt pathway. The cytoplasmic domain of LRP6 has multiple modular phosphorylation sites, and phosphorylation of LRP6 is an essential event for activation of the canonical Wnt pathway, as the phosphorylation of LRPE6 promotes the recruitment of the scaffold protein Axin, and thus, activates the canonical Wnt signaling pathway.24Davidson G Wu W Shen J Bilic J Fenger U Stannek P Glinka A Niehrs C Casein kinase 1 gamma couples Wnt receptor activation to cytoplasmic signal transduction.Nature. 2005; 438: 867-872Crossref PubMed Scopus (460) Google Scholar, 25Zeng X Tamai K Doble B Li S Huang H Habas R Okamura H Woodgett J He X A dual-kinase mechanism for Wnt co-receptor phosphorylation and activation.Nature. 2005; 438: 873-877Crossref PubMed Scopus (647) Google ScholarRecent evidence indicates that the canonical Wnt pathway plays a role in angiogenesis.26Goodwin AM D'Amore PA Wnt signaling in the vasculature.Angiogenesis. 2002; 5: 1-9Crossref PubMed Scopus (152) Google Scholar, 27Masckauchan TN Agalliu D Vorontchikhina M Ahn A Parmalee NL Li CM Khoo A Tycko B Brown AM Kitajewski J Wnt5a signaling induces proliferation and survival of endothelial cells in vitro and expression of MMP-1 and Tie-2.Mol Biol Cell. 2006; 17: 5163-5172Crossref PubMed Scopus (177) Google Scholar Extensive studies have shown that the Wnt pathway up-regulates nuclear factor κB, signal transducer and activator of transcription 3 and a number of inflammatory factors, and thus, plays a key role in inflammation.28Yan S Zhou C Zhang W Zhang G Zhao X Yang S Wang Y Lu N Zhu H Xu N beta-Catenin/TCF pathway upregulates STAT3 expression in human esophageal squamous cell carcinoma.Cancer Lett. 2008; 271: 85-97Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 29Yamashina K Yamamoto H Chayama K Nakajima K Kikuchi A Suppression of STAT3 activity by Duplin, which is a negative regulator of the Wnt signal.J Biochem. 2006; 139: 305-314Crossref PubMed Scopus (21) Google Scholar The present study investigated the possible role of the Wnt signaling pathway in DR by using human donor eyes, diabetic animal models, and cultured cells.Materials and MethodsHuman TissueNormal and diabetic eyes fixed in 10% neutral buffered formalin (NBF) within 12 hours postmortem and were obtained from National Diseases Research Interchange (Philadelphia, PA) with full ethical approval for use in research. Diabetic eyes were categorized according to a standardized protocol.30Khaliq A Foreman D Ahmed A Weich H Gregor Z McLeod D Boulton M Increased expression of placenta growth factor in proliferative diabetic retinopathy.Lab Invest. 1998; 78: 109-116PubMed Google ScholarAnimalsAkita mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and Brown Norway rats were purchased from Charles River (Wilmington, MA). Care, use, and treatment of all animals in this study were in strict agreement with the Statement for the Use of Animals in Ophthalmic and Vision Research from the Association for Research in Vision and Ophthalmology.Isolation and Culture of Bovine Retinal Capillary Endothelial Cells and PericytesBovine retinal capillary endothelial cells (RCEC) and pericytes were isolated from bovine eyes, as described by Grant and Guay31Grant MB Guay C Plasminogen activator production by human retinal endothelial cells of nondiabetic and diabetic origin.Invest Ophthalmol Vis Sci. 1991; 32: 53-64PubMed Google Scholar with some modifications. At passage 3 or 4, the purity of the cells in culture was determined. The identity of RCEC was confirmed by a characteristic cobblestone morphology and the incorporation of acetylated low-density lipoprotein labeled with a fluorescent probe, DiI (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate) (Biomedical Technologies, Inc; Stoughton, MA). Purity of the pericyte culture was determined by immunostaining using a fluorescein isothiocyanate-conjugated antibody specific to α-smooth muscle actin (Sigma; St. Louis, MO).Induction of Diabetes in RatsExperimental diabetes was induced by an intraperitoneal injection of streptozotocin (STZ) (50 mg/kg in 10 mmol/L of citrate buffer; pH 4.5) into anesthetized Brown Norway rats (8 weeks of age) after an overnight fast. Age-matched control rats received an injection of citrate buffer alone for nondiabetic control. Blood glucose levels were measured 48 hours after the STZ injection and monitored weekly thereafter. Only the animals with glucose levels >350 mg/dl were considered diabetic.The Oxygen-Induced Retinopathy Model and Analysis of Retinal NVThe oxygen-induced retinopathy (OIR) model was induced in Brown Norway rats as described previously.32Ricci B Oxygen-induced retinopathy in the rat model.Doc Ophthalmol. 1990; 74: 171-177Crossref PubMed Scopus (36) Google Scholar Quantification of preretinal vascular cells was described by Smith et al.33Smith LE Wesolowski E McLellan A Kostyk SK D'Amato R Sullivan R D'Amore PA Oxygen-induced retinopathy in the mouse.Invest Ophthalmol Vis Sci. 1994; 35: 101-111PubMed Google Scholar Briefly, the eyes of eight rats from each group at postnatal day 18 (P18) were enucleated, fixed with 10% formaldehyde, sectioned, and then stained with H&E. The nuclei of vascular cells on the vitreal side of the retina were counted under a light microscope in a double-blind study. Ten sagittal sections from each eye were examined, and cell numbers were averaged in each group of animals. The average number of preretinal vascular nuclei was compared with that in the control group by using Student’s t-test.Retinal Angiography with High-Molecular-Weight FluoresceinRats at P18 were anesthetized with 10 mg/kg xylazine plus 75 mg/kg ketamine i.p. and perfused with 50 mg/ml high molecular weight fluorescein isothiocyanate-dextran (molecular weight 2 × 106; Sigma) via intraventricle injection as described by Smith et al.33Smith LE Wesolowski E McLellan A Kostyk SK D'Amato R Sullivan R D'Amore PA Oxygen-induced retinopathy in the mouse.Invest Ophthalmol Vis Sci. 1994; 35: 101-111PubMed Google Scholar The animals were immediately euthanized. The eyes were enucleated and fixed with 4% paraformaldehyde in PBS for 10 minutes. The retina was then separated from the eyecup and fixed with 4% paraformaldehyde for 3 hours. Several incisions were made to the retina, which was flat-mounted on a gelatin-coated slide. The vasculature was then examined under a fluorescence microscope (Axioplan2 Imaging; Carl Zeiss; Jena, Germany).ImmunohistochemistryImmunohistochemistry was performed as described.34Chen Y Moiseyev G Takahashi Y Ma JX RPE65 gene delivery restores isomerohydrolase activity and prevents early cone loss in Rpe65−/− mice.Invest Ophthalmol Vis Sci. 2006; 47: 1177-1184Crossref PubMed Scopus (77) Google Scholar The primary antibodies specific for LRP5/6 (Abcam; Cambridge, MA) and hypoxia-inducible factor-1α (HIF-1α) (Santa Cruz Biotechnology; Santa Cruz, CA) were used at a dilution of 1:200, and antibody for β-catenin (Cell Signaling Technology, Danvers, MA) at a dilution of 1:300. The secondary antibodies were fluorescein isothiocyanate-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratory, Inc; West Grove, PA) and Texas Red-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratory) at a dilution of 1:200.Measurement of Reactive Oxygen Species GenerationCellular oxidative stress was determined by measuring intracellular reactive oxygen species (ROS) generation.35Degli Esposti M Measuring mitochondrial reactive oxygen species.Methods. 2002; 26: 335-340Crossref PubMed Scopus (143) Google Scholar, 36Amer J Goldfarb A Fibach E Flow cytometric measurement of reactive oxygen species production by normal and thalassaemic red blood cells.Eur J Haematol. 2003; 70: 84-90Crossref PubMed Scopus (126) Google Scholar Briefly, the treated and untreated cells at a density of 2 × 106 cells/ml were incubated with freshly prepared 5-(and 6-)chloromethyl-2′, 7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2-DCF-DA) at 37°C in the dark. The CM-H2-DCF-DA–loaded cells were rinsed twice in PBS and analyzed immediately by fluorometer at 488 nm excitation and 530 nm emission. Data were expressed as fluorescence intensity in arbitrary units from the average of three separate experiments.Intravitreal Injection of Dickkopf Homolog 1Briefly, animals were anesthetized with a 50:50 mix of ketamine (100 mg/ml) and xylazine (20 mg/ml), and their pupils were dilated with topical application of phenylephrine (2.5%) and tropicamide (1%). A sclerotomy was created approximately 0.5 mm posterior to the limbus with a blade, and a glass injector (∼33 gauge) connected to a syringe filled with 3 μl of Dickkopf homolog 1 (DKK1) with desired concentrations or bovine serum albumin (BSA) was introduced through the sclerotomy into the vitreous cavity.Soluble ICAM-1 Enzyme-Linked Immunosorbent AssayA commercial soluble ICAM-1 (sICAM-1) enzyme-linked immunosorbent assay kit (R&D Systems, Inc; Minneapolis, MN) was used to measure sICAM-1 levels in mouse retinal tissues, following the manufacturer’s protocol. The samples from mouse tissues were diluted 10 times to ensure that the sICAM-1 concentration fell within the range of the sICAM-1 standard curves.Western Blot AnalysisThe same amounts of retinal proteins from each rat or mouse were used for Western blot analysis using specific primary antibodies for each protein and blotted with a horseradish peroxidase-conjugated secondary antibody.34Chen Y Moiseyev G Takahashi Y Ma JX RPE65 gene delivery restores isomerohydrolase activity and prevents early cone loss in Rpe65−/− mice.Invest Ophthalmol Vis Sci. 2006; 47: 1177-1184Crossref PubMed Scopus (77) Google Scholar The signal was developed with a chemiluminescence detection kit (ECL; Amersham International; Piscataway, NJ). Blots were then stripped and reblotted with an antibody specific for β-actin.The primary antibodies used are specific for LRP5/6 (Santa Cruz Biotechnology) and HIF-1α (Santa Cruz Biotechnology) at a dilution of 1:500, and the anti-β-catenin antibody (Cell Signaling Technology) at a dilution of 1:3000.Vascular Permeability AssayVascular permeability was quantified by using Evans blue as a tracer as described previously.37Gao G Shao C Zhang SX Dudley A Fant J Ma JX Kallikrein-binding protein inhibits retinal neovascularization and decreases vascular leakage.Diabetologia. 2003; 46: 689-698PubMed Google ScholarResultsThe Wnt Pathway Is Activated in the Retina of Human Patients with DRTo evaluate the activation status of the Wnt pathway in the retina of diabetic patients, we have measured retinal levels of total β-catenin, an essential effector of the canonical Wnt pathway. Ocular sections from six diabetic donors with nonproliferative diabetic retinopathy (NPDR) and those from five nondiabetic donors were stained for β-catenin by using immunohistochemistry. Under the same development intensity, there was a statistically significant increase in β-catenin staining intensity in the inner retina from the donors with NPDR, while there were only basal levels of β-catenin signal in the retina from the nondiabetic donors (Figure 1, A–E). Moreover, immunohistochemistry showed that the donors with NPDR had more intensive β-catenin signals (brown color) in the nuclei of the retinal cells, compared with that in the nondiabetic donors (Figure 1), suggesting increased nuclear translocation of β-catenin in the retinas from patients with NPDR.Activation of the Wnt Pathway in the Retinas of Akita Mice, STZ-Induced Diabetic Rats, and Rats with OIRTo confirm the activation of the Wnt pathway in the retina of DR animal models, we have measured β-catenin levels in the retinas from Akita mice, a genetic model of type-1 diabetes, STZ-induced diabetic rats, and in OIR rats, a model of ischemia-induced retinal NV. As shown by Western blot analysis, β-catenin levels were elevated in the retinas from Akita mice at the age of 16 weeks, compared with that in their nondiabetic littermates (Figure 2A). Similarly, retinal β-catenin levels were also elevated in STZ-diabetic rats at 16 weeks after the induction of diabetes, compared with age-matched nondiabetic rats (Figure 2B). In rats at the age of postnatal day 16 (P16) under constant normoxia, low levels of β-catenin were detected in the retina, while the OIR rats at the same age showed dramatically increased β-catenin levels in the retina (Figure 2C).Figure 2Increased β-catenin levels in the retinas of Akita mice, STZ-induced diabetic rats, and OIR rats. The retinas from Akita mice at 16 weeks of age, STZ-DM rats at 16 weeks following the STZ injection, OIR rats at the age of P16, and age-matched nondiabetic or normoxic controls were used for Western blot (A–C) and immunohistochemistry (D–Q) analyses of β-catenin. A–C: The same amount (50 μg) of retinal proteins from each animal was blotted with an antibody specific for β-catenin. The membranes were stripped and reblotted with an antibody for β-actin. Each lane represents an individual animal. D–Q: Representative retinal sections from Akita mice (G–I) and their nondiabetic littermates (D–F), STZ-DM rats (M–O) and non-DM rats (J–L), OIR rats (Q), and age-matched normal rats maintained under constant normoxia (P) were immunostained with an antibody for β-catenin. F, I, L, and O: The nucleus was counterstained with 4’, 6-diamidino-2-phenylindole (DAPI) (colored red) and merged with β-catenin signal. Red arrows (in I and O) indicate the nuclei showing green or orange color as a result of increased β-catenin signal in the nuclei of diabetic retinas, while the white arrows (in F and L) point to nuclei (red color) in nondiabetic retinas. Scale bar = 20 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To identify the cellular location of the β-catenin accumulation, ocular sections from the eyes of Akita mice, STZ-diabetic rats, OIR rats, and their respective controls were stained with an antibody specific for β-catenin by using immunohistochemistry. More intensive β-catenin signals (green color) were detected in the inner retinas of the Akita mice, STZ-diabetic rats, and OIR rats, when compared with their respective controls (Figure 2, D–Q). Increased β-catenin signals in the nuclei of retinal cells were also observed in the diabetic animals and OIR rats (Figure 2, D–Q).Increased Retinal Levels of LRP5/6 in Diabetic and OIR RatsTo further assess the activation status of the Wnt pathway, retinal levels of LRP5/6, coreceptors in the Wnt pathway, were measured by Western blot analysis. The results showed that retinal levels of LRP6 were higher in the retinas from STZ-induced diabetic rats at 16 weeks following the onset of diabetes than that in nondiabetic controls (Figure 3A). Similarly, retinal LRP6 levels were also elevated in the retinas from OIR rats at the age of P16, compared with age-matched normoxia controls (Figure 3B).Figure 3Up-regulated expression of LRP5/6 in the retinas of STZ-diabetic and OIR rats. A and B: The same amount of retinal proteins (100 μg) from three STZ-induced diabetic rats 16 weeks after the onset of diabetes and age-matched nondiabetic rats (A), and four OIR rats and normal rats at age of P16 (B) was used for Western blot analysis using an antibody specific for LRP5/6 (Santa Cruz Biotechnology). The same membranes were stripped and reblotted with an antibody for β-actin. C–F: Retinal sections from STZ-diabetic rats (D) and non-DM controls (C), and those from OIR rats (F) and their normoxic controls (E) were immunostained with the antibody against LRP5/6 (green). The nuclei were counterstained with DAPI (red). Original magnification, ×400.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Immunohistochemical analysis in ocular sections showed increased LRP5/6 signals in the inner retina of STZ-induced diabetic rats (green color in Figure 3, C and D), compared with nondiabetic controls. In OIR rats, the more intensive LRP5/6 signals were detected primarily in the retinal vasculature (Figure 3, E and F).Hypoxia and Oxidative Stress Are Responsible for the Wnt Pathway Activation in DiabetesTo identify the cause for the Wnt pathway activation in diabetes, we evaluated the effects of hypoxia and hyperglycemia, known pathogenic factors of DR, on Wnt signaling in vitro. As shown by Western blot analysis, exposure of primary RCEC to hypoxia (2% oxygen) for 14 hours increased the total β-catenin levels (Figure 4A), indicating that hypoxia is a causative factor for the Wnt pathway activation in the retina of the diabetic and OIR models.Figure 4Induction of Wnt signaling by hypoxia and oxidative stress. A: RCEC were exposed to 2% oxygen and normoxia for 14 hours. Levels of total β-catenin were determined by Western blot analysis using the same amount (50 μg) of total proteins from each sample and normalized to β-actin levels. Note that the blot represents two independent experiments. B–D: RCEC were treated with low glucose (LG; 5 mmol/L glucose and 25 mmol/L mannitol, B), high glucose (HG, 30 mmol/L glucose, C), and high glucose plus 10 μmol/L aminoguanidine (HG+AG, D) for 24 hours. The subcellular distribution of β-catenin was revealed by immunocytochemistry by using the antibody for β-catenin. E: The same amount of isolated nuclear proteins (50 μg) from each of the above groups was blotted with an antibody for β-catenin and normalized to TATA box-binding protein (TBP) levels.View Large Image Figure ViewerDownload Hi-res image Download (PPT)RCEC were also exposed to 30 mmol/L glucose for 24 hours, in the presence and absence of 10 μmol/L aminoguanidine, which is known to have anti-oxidant activities. The subcellular distribution of β-catenin in RCEC was determined by using immunocytochemistry. In the cells cultured under the low glucose medium (5 mmol/L glucose and 25 mmol/L mannitol), β-catenin was distributed primarily in the cytosol and membrane, and was undetectable in the nuclei (green color in Figure 4B). The high glucose medium induced β-catenin nuclear translocation (Figure 4C), suggesting that high glucose alone is sufficient to activate the Wnt pathway. Under the same condition, aminoguanidine inhibited the nuclear translocation of β-catenin induced by high glucose (Figure 4D). Consistently, Western blot analysis using isolated nuclear proteins showed that nuclear levels of β-catenin were elevated in the RCEC exposed to the high glucose medium, compared with those in the low glucose medium. Aminoguanidine blocked the high glucose-induced increase of nuclear β-catenin levels, suggesting that oxidative stress is responsible for the high glucose-induced activation of the Wnt pathway (Figure 4E).Blockade of the Wnt Pathway Ameliorates Retinal Inflammation, Vascular Leakage, and NV in DR ModelsTo further establish the causative role of the Wnt pathway activation in DR, we blocked the Wnt pathway activation in the retinas of the DR models by using DKK1, a specific inhibitor of the Wnt pathway. An intravitreal injection of different doses of purified DKK1 into STZ-diabetic rats reduced retinal soluble ICAM-1 levels in a dose-dependent manner, when compared with that in the contralateral eyes injected with the same amounts of BSA, suggesting that Wnt signaling is responsible for retinal inflammation in diabetic rats (Figure 5A). To evalu
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