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I. S. Ali Rahman, C.-M. Lai, N. Binz, E. P. Rakoczy; Assessment of Endothelin-2 Expression in Hyperglycemic and Retinal Neovascularisation Mouse Models. Invest. Ophthalmol. Vis. Sci. 2010;51(13):116.
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© ARVO (1962-2015); The Authors (2016-present)
To assess the influence of vascular endothelial growth factor (VEGF) and hyperglycemia on expression of vasoconstrictor, endothelin-2 (Edn2), in mouse models of hyperglycemia and retinal neovascularisation (RNV).
A diabetic mouse model, Akita, was crossed with a VEGF-driven RNV mouse model, Kimba, to produce the Akimba mouse, a model of both diabetes and RNV. Enucleated eyes from 8 and 24 weeks old male mice were collected with C57BL/6J mice (WT) used as controls. RNA was extracted from the posterior segment and amplified using whole transcriptome amplification method. Real-time PCR was performed on Edn2. Expression of Edn1, Edn3 and their receptors; EdnRa and EdnRb, were also assessed. Edn2 protein was quantitated in blood plasma and protein extracts from whole eyes using competitive ELISA and localised in paraffin sections using immunohistochemistry (IHC).
Edn2 mRNA expression in Kimba, Akita and Akimba was respectively 3.8-fold, 0.15-fold and 7.3-fold higher at 8 weeks postnatal and increased to 36-fold, 9-fold and 153-fold at 24 weeks postnatal when compared to aged-matched WT level.. Protein levels of Edn2 in 8 weeks old mouse eyes was 16±1 pg/ml in WT, 27±3 pg/ml in Kimba, 19.5±5 pg/ml in Akita and 32±7 pg/ml in Akimba mice. At 24 weeks, Edn2 protein levels significantly increased in WT and Akita to 30±6 pg/ml and 29±8 pg/ml respectively (p<0.01) while in Kimba and Akimba, protein levels remained unchanged at 35±7 pg/ml and 29±3 pg/ml respectively (p>0.05). There was no difference in circulating Edn2 plasma levels in all three genotypes at 8 and 24 weeks postnatal (p>0.05). Using IHC, Edn2 was localised in the ganglion cell layer, Müller cells and photoreceptor cell layer. We demonstrated that VEGF upregulation in the Kimba mouse eyes was sufficient to induce significant upregulation of Edn2 mRNA and protein expression. Prolonged diabetes alone also increased the expression of Edn2 in Akita mice. However, Edn2 expression was significantly upregulated when the upregulation of VEGF occurred in a prolonged hyperglycemic environment as in the Akimba mice.
Edn2 production in the Akimba retinae was significantly higher than in the Kimba or Akita mice suggesting that hyperglycemia enhances VEGF-induced expression of Edn2. VEGF-driven Edn2 may play a role in exacerbating the retinal damage in Akimba.
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