Early Intervention of Tyrosine Nitration Prevents Vaso- Obliteration and Neovascularization in Ischemic Retinopathy Mohammed A. Abdelsaid, Bindu A. Pillai, Suraporn Matragoon, Roshini Prakash, Mohamed Al-Shabrawey, and Azza B. El-Remessy Program in Clinical and Experimental Therapeutics, University of Georgia, Augusta, Georgia (M.A.A., B.A.P., S.M., R.P., A.B.E.); Departments of Oral Biology (M.A.), Ophthalmology (M.A., A.B.E.), and Pharmacology and Toxicology (A.B.E.), Medical College of Georgia, Augusta, Georgia; and Veterans Affairs Medical Center, Augusta, Georgia (M.A.A., B.A.P., S.M., A.B.E.) Received June 19, 2009; accepted October 8, 2009 ABSTRACT Diabetic retinopathy and retinopathy of prematurity are blinding dis- orders that follow a pathological pattern of ischemic retinopathy and affect premature infants and working-age adults. Yet, the treatment options are limited to laser photocoagulation. The goal of this study is to elucidate the molecular mechanism and examine the therapeutic effects of inhibiting tyrosine nitration on protecting early retinal vas- cular cell death and late neovascularization in the ischemic retinop- athy model. Ischemic retinopathy was developed by exposing neo- natal mice to 75% oxygen [postnatal day (p) 7–p12] followed by normoxia (21% oxygen) (p12–p17). Peroxynitrite decomposition cat- alyst 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato iron III chlo- ride (FeTPPS) (1 mg/kg), the nitration inhibitor epicatechin (10 mg/kg) or the thiol donor N-acetylcysteine (NAC, 150 mg/kg) were adminis- tered (p7–p12) or (p7–p17). Vascular endothelial cells were incubated at hyperoxia (40% oxygen) or normoxia (21% oxygen) for 48 h. Vascular density was determined in retinal flat mounts labeled with isolectin B4. Expression of vascular endothelial growth factor, caspase-3, and poly(ADP ribose) polymerase (PARP), activation of Akt and p38 mitogen-activated protein kinase (MAPK), and tyrosine nitration of the phosphatidylinositol (PI) 3-kinase p85 subunit were analyzed by Western blot. Hyperoxia-induced peroxynitrite caused endothelial cell apoptosis as indicated by expression of cleaved caspase-3 and PARP leading to vaso- obliteration. These effects were associated with significant ty- rosine nitration of the p85 subunit of PI 3-kinase, decreased Akt activation, and enhanced p38 MAPK activation. Blocking ty- rosine nitration of PI 3-kinase with epicatechin or NAC restored Akt phosphorylation, and inhibited vaso-obliteration at p12 and neovascularization at p17 comparable with FeTPPS. Early in- hibition of tyrosine nitration with use of epicatechin or NAC can represent safe and effective vascular-protective agents in isch- emic retinopathy. Retinopathy of prematurity (ROP) and diabetic retinopa- thy (DR) are potentially blinding disorders that affect pre- mature infants and working-age adults, respectively, in the United States (Aiello et al., 1998; Chen and Smith, 2007). ROP and DR follow a pathological progression pattern char- acteristic of ischemic retinopathy, where the loss of retinal capillary is an early initiating event, leading to a poorly controlled process of retinal neovascularization and the de- velopment of proliferative retinopathy (for review see, Cald- well et al., 2003). So far, the standard treatment for retinal neovascularization is limited to laser photocoagulation. Al- though successful, this treatment is invasive and results in loss of peripheral vision (for review see, Ali and El-Remessy, 2009). The lack of approved pharmacological treatment for DR and ROP creates a great need for finding new effective therapeutic modalities to treat these devastating diseases. The mechanisms that control the process of retinal neovas- cularization are therefore of major clinical importance. Peroxynitrite formed by reaction of nitric oxide and super- oxide anion mediates a variety of biological processes includ- ing inhibition of key metabolic enzymes, lipid peroxidation, nitration of the protein tyrosine residue, and reduction of cellular antioxidant defenses by oxidation of thiol pools (Pacher et al., 2007). A critical role of increased oxidative stress and, in particular, the peroxynitrite is supported by previous studies showing that increases in peroxynitrite for- mation cause capillary endothelial cell apoptosis leading to This work was supported by the American Heart Association [Scientist Development Grant 0530170N] (to A.B.E.); the Juvenile Diabetes Research Foundation [Grant 1-2008-49] (to A.B.E.); and the University of Georgia Re- search Foundation (A.B.E.). Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.109.157941. ABBREVIATIONS: ROP, retinopathy of prematurity; DR, diabetic retinopathy; BRE, bovine retinal endothelial; VEGF, vascular endothelial growth factor; FeTPPS, 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato iron III chloride; PARP, poly(ADP ribose) polymerase; p, postnatal day; PI, phosphatidylinositol; NAC, N-acetylcysteine; PBS, phosphate-buffered saline; MAPK, mitogen-activated protein kinase; GSH, reduced glutathi- one; GSSG, oxidized glutathione. 0022-3565/10/3321-125–134 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 332, No. 1 U.S. Government work not protected by U.S. copyright 157941/3543259 JPET 332:125–134, 2010 Printed in U.S.A. 125