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X.-Q. Song, S.-Y. Park, Q. Zhang, Y. A. Lussier, R. H. Xing, M. A. Grassi; Wnt/β-Catenin Signaling in Diabetic Retinopathy. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2910.
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Recent genetic and biochemical evidence suggests that the Wnt/ß-catenin pathway may be involved in the pathogenesis of diabetic retinopathy, which is a leading cause of blindness. Here we report Wnt/β-catenin alterations in high glucose cell assays.
Cell cultures: TR-iBRB (rat retinal endothelial) cells were cultured, respectively, in 5mM glucose/DMEM/ECGF/10% FBS or 25mM glucose/DMEM/ECGF/0.5% human albumin for control and experimental treatments. For 3D cultures, 104 cells were cultured in Matrigel in a 4-well chamber slide under 5mM or 25mM glucose/DMEM, 0.5% human albumin for 2 weeks.Western blot: Western blots using antibodies against GSK-3β, phospho-Ser9-GSK-3β, β-catenin, cyclin D1, and GAPDH were performed. Real-time PCR: Total RNA was isolated with a Qiagen RNeasy kit. For cDNA synthesis, 1µg RNA was used in 20µl. Real-time PCR was performed in 25µl with ABI 7900 Real-time PCR System.
High glucose reduces tube formation: In 3D, cells in the 25mM glucose medium revealed less sprouting, branching, tubulogenesis and capillary surface area compared with the 5mM glucose medium after 7 days.Biphasic response: Western blot data showed that the β-catenin protein level was elevated initially after being treated with 25mM glucose for 1 to 4 hrs; 18 hrs later the β-catenin levels declined below baseline. Levels of GSK-3β, an upstream regulator of β-catenin, were compatible with this biphasic response. High glucose affects target gene transcription of β-catenin: Real-time PCR demonstrated that the mRNA of two downstream products of β-catenin signaling, cyclin D1 and VEGFa, are increased initially after treatment with 25mM glucose; 24 hours later, mRNA levels returned to baseline.
High glucose exposure results in a biphasic response in both β-catenin and GSK3β signaling in TRiBRB cells. Further, high glucose treatment inhibited TRiBRB cell sprouting and forming tube-like structures in Matrigel 3-D culture. These findings suggest that Wnt/β-catenin signaling is likely an important player in regulating retinal angiogenesis in response to high glucose insult frequently seen in diabetic patients.
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