Early intervention of tyrosine nitration prevents vaso-obliteration and neovascularization in ischemic retinopathy

Diabetic retinopathy and retinopathy of prematurity are blinding disorders 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 vascular cell death and late neovascularization in the ischemic retinopathy model. Ischemic retinopathy was developed by exposing neonatal mice to 75% oxygen [postnatal day (p) 7-p12] followed by normoxia (21% oxygen) (p12-p17). Peroxynitrite decomposition catalyst 5,10,15,20-tetrakis(4- sulfonatophenyl)porphyrinato iron III chloride (FeTPPS) (1 mg/kg), the nitration inhibitor epicatechin (10 mg/kg) or the thiol donor N-acetylcysteine (NAC, 150 mg/kg) were administered (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 vasoobliteration. These effects were associated with significant tyrosine nitration of the p85 subunit of PI 3-kinase, decreased Akt activation, and enhanced p38 MAPK activation. Blocking tyrosine 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 inhibition of tyrosine nitration with use of epicatechin or NAC can represent safe and effective vascular-protective agents in ischemic retinopathy.