| Research backgroundDiabetic retinopathy is characterized by an increased retinal neovascularization mainly due to the action of angiogenic factor VEGF. Previous reports indicate that VEGF can induce the expression of Ets-1 and the phosphorylation of ERK1/2 in vivo and in vitro, and in turn, Ets-1 and ERK1/2 can regulate the expression of VEGF in some cultured cells and animal models. However, the role played by Ets-1 and ERK1/2 in the expression of VEGF, and the interactions of Ets-1 and ERK1/2 in the retina of diabetic animal have not been determined. Thus, the purpose of this study was to determine the relationship of Ets-1, ERK1/2, and VEGF in the retina of streptozotocin (STZ)-induced diabetic rats. Objectives1. To investigate the role of transcription factor Ets-1 and ERK1/2 in VEGF expression and the interactions of Ets-1 and ERK1/2 in the retina of diabetic rats.2. To detect the localization of Ets-1 and ERK1/2 and the involvement of VEGF receptors (Flt-1 and KDR/Flk-1) in the experimental diabetic rat retina.Methods1. Eight-week-old female Sprague-Dawley rats (glucose concentrations 4-10 mmol/L) were housed under a 12-h light/dark cycle with free access to rat food and water. Diabetes was induced by an intraperitoneal injection of 65 mg/kg STZ dissolved in 0.05 mol/L sodium citrate buffer, pH 4.5, and control rats received equivalent volume of buffer. Food was withdrawn 16 hours before the injection. Blood glucose concentrations of the injected animals were measured 24 hours after induction, weekly during the first month and monthly afterwards. Rats were kept up to 3 months post induction and were inspected daily for general appearance and behavior and weekly for weight. The rats were considered diabetic if their blood glucose levels were >13.9 mmol/l (250 mg/dl) within 24 hours after the injection of STZ.2. To follow the time course in the expression of Ets-1, phosphorylated ERK1/2 (pERK1/2), and VEGF, rats were killed at 1, 2, 4, and 8 weeks after the injection of STZ, and total proteins were extracted from the isolated retinas. To investigate the effects of Ets-1 blockade and ERK1/2 inhibition on the expression of VEGF, an adenovirus vector encoding dominant-negative Ets-1 (1ul) and PD98059 (2ul) were injected intravitreally 1 day after the injection of STZ, respectively. For control, an equivalent amount of null adenovirus or an equivalent amount of DMSO (0.1%, v/v) diluted in saline were injected into the contralateral eye, respectively. At 4 weeks after first intravitreal injection, total protein and total RNA were obtained.3. Animals were sacrificed at 4 weeks after STZ-injection. Frozen cross sections of eyeballs with 14 um thickness were used to perform double immunofluorescence staining with anti-GFAP (1:400) and anti-Ets-1 (1:100) or anti-pERK1/2 (1:1000) antibodies.Total proteins were isolated to detect the expression of Flt-1 and Flk-1 in the diabetic rat retina.4. Western blot and Northern blot analyses were used to detect protein and RNA expression, respectively.Results1. All diabetic rats gained significantly less weight than their age-matched control groups. The blood glucose values of the diabetic rats were significantly higher than control values for the entire duration of the experiment (P< 0.01).2. The levels of both Ets-1 and VEGF proteins were increased in a time-dependent manner, reaching a 4-fold increase and a 3.5-fold increase of control at 8 weeks, respectively (P<0.001). However, the protein level of Ets-1 (1.3±0.2-fold at 1 week, 2.0±0.3-fold at 2 weeks) increased more rapidly than that of VEGF (no significant increase at 1 week, 1.7±0.2-fold at 2 weeks). The phosphorylation of ERK1/2 also increased in a similar time-dependent manner with the expression Ets-1,reaching a 4.5-fold increase of that in the controls at 8 weeks (P<0.001). 3. Four weeks after the intraocular injection of adenovirus vector encoding dominant-negative Ets-1, the mRNA level and protein level of Ets-1 in the experimental eyes were much lower than that in the control eyes at 4 weeks (P<0.001). At the same time, the treatment with dominant-negative Ets-1 resulted in a significant reduction of both ERK1/2 phosphorylation and VEGF protein level compared with the eyes from rats injected with the control null adenovirus vector. ERK1/2 phosphorylation and VEGF protein level were decreased by 60.2±4.0% (P<0.001) and 58.3±6.0% (P<0.001), respectively.4. Immunostaining of Ets-1 and pERK1/2 exhibited identical laminar distribution and both Ets-1 and pERK1/2 immunoreactivities displayed typical Muller cell morphology. Furthermore, confocal microscopy study showed the extensive colocalization of immunohistochemical expression of pERK1/2 and Ets-1 with GFAP.5. Both Flt-1 and Flk-1 expression were up-regulated in the diabetic rat retina at 4 weeks after diabetes induction, reaching a 4.2- and 3-fold increase of control at 4 weeks after STZ injcetion (P<0.01), respectively..Conclusions1. Our results indicated the followings in the retina of diabetic rats: (1) the alteration of Ets-1, pERK1/2, and VEGF synchronized approximately with each other; (2) the phosphorylation of ERK1/2 was regulated by the expression of Ets-1; (3) Ets-1 protein expression was ERK1/2 pathway dependent; (4) the protein level of VEGF was regulated by both Ets-1 expression and ERK1/2 phosphorylation; (5) increased VEGF expression is accompanied by up-regulation of both Flt-1 and Flk-1.2. According to our results, we propose that VEGF, Ets-1, and ERK1/2 may act synergistically in the pathophysiology of diabetic retinopathy. VEGF binds to its receptors and activates VEGF-VEGFR-ERK1/2-Ets-1-VEGF cascades, and this VEGF-induced auto-loop keeps repeating until retinal neovascularization develops.
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