SUPPLEMENT
ACE2 and Ang-(1-7) Confer Protection Against Development of Diabetic Retinopathy
Amrisha Verma1, Zhiying Shan2, Lihui Yuan2, Bo Lei3, Takahiko Nakagawa4, Maria B Grant5,
Alfred S Lewin6, and William W Hauswirth1, Mohan K Raizada2, and Qiuhong Li1
Departments of 1Ophthalmology, 2Physiology & Functional Genomics, 5Pharmacology and
Therapeutics, 6Molecular Genet. & Microbiology, University of Florida, Gainesville, FL
3
The First Affiliated Hospital of Chongqing Medical University, Chongqing Key Laboratory of
Ophthalmology, Chongqing Eye Institute, Chongqing 400016, China.
4
Division of Renal Disease and Hypertension, University of Colorado Denver, Aurora, CO
Correspondence Author:
Qiuhong Li, Ph. D.
Departments of Ophthalmology
University of Florida,
Gainesville, FL 32610-0284
Email: qli@ufl.edu
Short title: ACE2/Ang-(1-7) protects retina from diabetes-induced vascular damage
Key words: ACE2, Angiotensin-(1-7), renin angiotensin system (RAS), diabetes, diabetic
retinopathy, eNOS, animal model
References:
1.
2.
3.
4.
5.
6.
7.
8.
Huentelman MJ, Zubcevic J, Katovich MJ, Raizada MK. Cloning and characterization of
a secreted form of angiotensin-converting enzyme 2. Regul Pept. 2004;122(2):61-67.
Hauswirth WW, Lewin AS, Zolotukhin S, Muzyczka N. Production and purification of
recombinant adeno-associated virus. Methods Enzymol. 2000;316:743-761.
Zolotukhin S, Potter M, Hauswirth WW, Guy J, Muzyczka N. A "humanized" green
fluorescent protein cDNA adapted for high-level expression in mammalian cells. J Virol.
1996;70(7):4646-4654.
Kuwabara T, Cogan DG. Studies of retinal vascular patterns. I. Normal architecture. Arch
Ophthalmol. 1960;64:904-911.
Alves MF, Araujo MC, Juliano MA, Oliveira EM, Krieger JE, Casarini DE, Juliano L,
Carmona AK. A continuous fluorescent assay for the determination of plasma and tissue
angiotensin I-converting enzyme activity. Braz J Med Biol Res. 2005;38(6):861-868.
Yan ZH, Ren KJ, Wang Y, Chen S, Brock TA, Rege AA. Development of
intramolecularly quenched fluorescent peptides as substrates of angiotensin-converting
enzyme 2. Anal Biochem. 2003;312(2):141-147.
Meagher EA, FitzGerald GA. Indices of lipid peroxidation in vivo: strengths and
limitations. Free Radic Biol Med. 2000;28(12):1745-1750.
Dawn-Linsley M, Ekinci FJ, Ortiz D, Rogers E, Shea TB. Monitoring thiobarbituric acidreactive substances (TBARs) as an assay for oxidative damage in neuronal cultures and
central nervous system. J Neurosci Methods. 2005;141(2):219-222.
Figure Legend:
Supplement Figure 1: Transmission electron micrographs of retinal capillaries from a untreated
2 month diabetic eNOS-/- mouse eye (A), and an eye that received AAV-ACE2 treatment 2
weeks before STZ-induction of diabetes (B). CL: capillary lumen; En: endothelial cell; P:
pericyte; * indicates the capillary basement membrane. The scale bar = 500nm. We have
previously shown that the basement membranes of retinal capillaries from the diabetic eNOS-/animals at two month after STZ induction of diabetes was significantly thicker than those from
agematched, non-diabetic animals4. The thickening of the basement membrane was prevented in
the AAV-ACE2 treated eyes (73.81+17nm, versus 95.72+20 nm in untreated DM eye).