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Rashidul Haque, P Iuvone, Kimberly Choi, Daniel Park, Annie Farrell, Elizabeth Hur; Role of Prorenin Receptor (PRR) and NADPH Oxidase (NOX) in the Regulation of VEGF in ARPE-19 Cells under Hyperglycemic Conditions. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5567.
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Hyperglycemia-induced increase of reactive oxygen species (ROS) is thought to be a key event in the pathogenesis of diabetic retinopathy. Diabetes-induced increase in vascular endothelial growth factor (VEGF) expression, leukocyte adhesion, and breakdown of the blood-retinal barrier are all reported to be associated with increase in ROS. Also, the stimulation of angiotensin II (Ang II), the effector peptide of renin-angiotensin system, has been reported to increase the expression of VEGF through the activation of the Ang II type 1 receptor (AT1R). The objective of this study was to investigate whether hyperglycemia in ARPE-19 cells could promote the expression of VEGF independently of Ang II through PRR, via a NOX-dependent mechanism.
ARPE-19 cells were cultured in DMEM:F12 medium with 5.5 mM glucose (normal physiological condition), supplemented with 10% FBS and penicillin/streptomycin (LONZA), at 37°C in 5% CO2 and 90% relative humidity. Hyperglycemia-mediated induction of VEGF independently of Ang II was investigated using the ACE inhibitor perindopril (10 µmol/L, Sigma), which blocks the synthesis of Ang II, and siRNA-mediated down-regulation of PRR and Nox4. To determine the signaling role of NADPH-derived superoxide (O2-) and hydrogen peroxide (H2O2) in the regulation of VEGF expression, cells were incubated with 100 U/ml of PEG-SOD (superoxide dismutase) and PEG-CAT (catalase), respectively. Cells treated with 5.5 mM glucose, scramble siRNAs, and PEG alone were used as controls. The expression of mRNAs was measured by quantitative real-time PCR (qRT-PCR).
In perindopril-treated cells, hyperglycemia (33 mM glucose) significantly induced the expression of PRR (p<0.001), Nox4 (p<0.01), and VEGF (p<0.01), but not that of AT1R, Nox1, and Nox2, when compared with controls. Also, in perindopril-treated cells, PRR and Nox4 siRNAs, but not the control siRNA, significantly reduced the hyperglycemia-induced expression of VEGF (p<0.001). Both PEG-CAT and PEG-SOD, but not the PEG alone, significantly attenuated the hyperglycemia-mediated induction of VEGF (p<0.01), indicative of the involvement of NOX-derived O2- and H2O2 signaling in the regulation of VEGF.
Our results support the hypothesis that NOX4-derived ROS signaling is implicated in the hyperglycemia-induced increase of VEGF expression through PRR in ARPE-19 cells.
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