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K. G. Shadrach, G. Hoppe, J. Drazba, J. Dudley, V. Sarthy, J. G. Hollyfield, P. D. Senanayake; High Glucose Increases Angiotensin II in Müller Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1390.
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The objective of this study was to examine whether high glucose has any effect on the content and distribution of angiotensin II (Ang II) in retinal Müller cells
The rat Müller cell line, rMC1, was grown in DMEM containing 5.5 mM glucose, 10%FBS and 1% penicillin/streptomycin/glutamine in 5% CO2 at 37OC. When the cells reached 70% confluence, they were cultured in 5.5 mM or 25 mM glucose with 1% FBS and 1% penicillin/streptomycin/glutamine. The medium was changed every day. On the third day, the medium was removed and cells to be used for immunohistochemistry were fixed with 4% paraformaldehyde-PBS, whereas cells for radioimmunoassay were frozen at -80O C. Ang II distribution was determined using a polyclonal antibody for Ang II (Phoenix H-002-12, 1:200) and immunoreactivity was detected with anti-rabbit Alexa 488 secondary antiserum. Slides were mounted in anti-fade medium containing DAPI. Confocal laser-scanning microscopy was used to examine Ang II distribution on Müller cells.
The radioimmunoassay showed that Ang II levels were 2.4 ± 0.1 pg/culture when Müller cells were grown in normal glucose. In medium with high glucose, however, Ang II levels were found to be 23.3 ± 2.9 pg/culture (mean ± SEM, n=6, p< 0.001), indicating that high glucose dramatically increases in Müller cells. Immunocytochemical studies showed that there was a low level expression of Ang II in Müller cells grown in normal glucose while the intensity of immunostaining was significantly enhanced in the presence of high glucose
These data suggest that significant changes in Ang II levels occur in Müller cells under conditions of glycemic stress. These observations are important for understanding the pathophysiology of diabetic retinopathy.
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