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Preenie Senanayake, Judith Drazba, George Hoppe, Karen Shadrach, Sean Kessler, Ganes Sen, Joe G. Hollyfield; Angiotensin Converting Enzyme Dependent And Independent Synthesis Of Angiotensin II In Retina. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3338.
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Angiotensin II (Ang II) is increased in human diabetic retina and experimental diabetic retina. High glucose increases Ang II in cultured Müller cells.The aim of this study was to evaluate the synthesis of retinal Ang II in Angiotensin converting enzyme (ACE) null mice and in experimental diabetes under ACE inhibition.
ACE null C57BL/6 mice were generated by gene targeting. Genotype was confirmed by Southern analysis. Mice were sacrificed by carbon dioxide asphyxiation. The eyes were enucleated and fixed in PBS- paraformaldehyde. Diabetes was induced in 8 week old Sprague-Dawley rats (35 mg/kg streptozotocin-sodium citrate). Blood glucose >250 mg/dL confirmed the diabetic phenotype. Rats received ACE inhibitor captopril or saline. At 5 weeks post diabetes the rats were sacrificed. The eyes were enucleated and fixed with 4% paraformaldehyde-PBS. The eyes were then frozen in OCT and stored at -80º C. Ang II and Ang (1-7) was localized by immunohistochemistry and fluorescence imaging using anti- Ang II (Phoenix Pharmaceuticals,1:200) and anti-Ang (1-7) (in-house CCF Core 1, 1:200) antibodies. The immunoreactivity was resolved with anti-rabbit Alexa 488. Slides were mounted in anti-fade medium containing DAPI for labeling nuclei (Vectorshield Laboratories, Inc., Burlingame, CA). Images were taken with a Leica confocal laser-scanning microscope (Heidelberg, Germany).
Retinal tissue in both ACE+/+ and ACE-/- mice were histologically normal. In both models Ang II and Ang-(1-7) was detected throughout the retinal Müller cells. The two peptides appear to be co-localized. In the ACE-/- mice both peptides were present in higher amounts, Ang II by 1.7 fold and Ang-(1-7) by 2.4 fold. Increase in Ang II may be due to the stimulation of alternative pathways for the synthesis of Ang II. ACE gene deletion and ACE inhibition leads to the accumulation of Ang I, the precursor of Ang II. Ang I is converted to Ang-(1-9) and Ang-(1-7). Ang-(1-9) is also converted to Ang-(1-7). In addition, Ang II synthesized by the alternative pathways may also be converted to Ang-(1-7) by ACE 2. Furthermore, the lack of ACE inhibits the metabolism of Ang-(1-7) to Ang-(1-5). Thus, Ang-(1-7) is enhanced by multiple mechanisms.
Ang II in retinal Müller cells may be synthesized by the ACE pathway and alternate pathway(s) or may be independent of ACE.
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