3-nitrotyrosine and Retinal-Neovascularization

Hypertension and diabetes are known risk factors for retinal microvascular damage. However, the combined effects of diabetes with early and established stages of hypertension on retinal microvascular degeneration remain incompletely understood.. Male spontaneously hypertensive rats (SHR) were compared to SHR with streptozotocin-induced diabetes (SHR+D) for 6 or 10 weeks and Wistar rats as controls.. Hypertension alone (the SHR group) or in combination with diabetes (the SHR+D group) for 6 weeks induced additive increases in total retinal cell death, compared to the Wistar controls. This increase was associated with significant increases in phosphorylated-Jun N-terminal kinase (pJNK) activation, phosphorylated-Akt inhibition, plasma and retinal lipid peroxides, and soluble intracellular adhesion molecule-1 (sICAM-1) levels. After 10 weeks, a similar trend was still observed in retinal nitrotyrosine, nuclear factor kappaB p65, and tumor necrosis factor-α expression, associated with exacerbated pJNK activation and formation of acellular capillaries.. In conclusion, combining diabetes and hypertension-potentiated retinal oxidative/inflammatory stress promoted imbalance between the JNK stress and survival Akt pathways resulting in accelerated retinal cell death and acellular capillary formation.

Topics: Animals; Diabetes Mellitus, Experimental; Diabetic Retinopathy; Gene Expression; Hypertension; Inflammation; Intercellular Adhesion Molecule-1; Lipid Peroxidation; Male; MAP Kinase Kinase 4; NF-kappa B; Oxidative Stress; Phosphorylation; Proto-Oncogene Proteins c-akt; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Retina; Retinal Neovascularization; Tumor Necrosis Factor-alpha; Tyrosine

Retinal neovascularization is the major pathologic process in many ocular diseases and is associated with oxidative stress. Deficiency of aldose reductase (AR), the first enzyme in the polyol pathway for glucose metabolism, has been shown to reduce oxidative stress and blood vessel leakage. The present study aimed to investigate the effect of AR deficiency on retinal neovascularization in a murine oxygen-induced retinopathy (OIR) model.. Seven-day-old wild-type (WT) and AR-deficient (AR(-/-)) mice were exposed to 75% oxygen for 5 days and then returned to room air. Vascular obliteration, neovascularization, and blood vessel leakage were analyzed and compared. Immunohistochemistry for AR, nitrotyrosine (NT), poly(ADP-ribose) (PAR), glial fibrillary acidic protein (GFAP), and Iba-1, as well as Western blots for vascular endothelial growth factor (VEGF), phospho-Erk (p-Erk), phospho-Akt (p-Akt), and phospho-IκB (p-IκB) were performed.. Compared with WT OIR retinae, AR(-/-) OIR retinae displayed significantly smaller central retinal vaso-obliterated area, less neovascularization, and reduced blood vessel leakage. Significantly reduced oxidative stress and glial responses were also observed in AR(-/-) OIR retinae. Moreover, reduced microglial response in the avascular area but increased microglial responses in the neovascular area were found with AR deficiency. Furthermore, expression levels of VEGF, p-Erk, p-Akt, and p-IκB were significantly reduced in AR(-/-) OIR retinae.. Our observations indicated that AR deficiency reduced retinal vascular changes in the mouse model of OIR, indicating that AR can be a potential therapeutic target in ischemia-induced retinopathy.

Topics: Aldehyde Reductase; Animals; Animals, Newborn; Blotting, Western; Calcium-Binding Proteins; Capillary Permeability; Cell Movement; Cell Proliferation; Disease Models, Animal; Glial Fibrillary Acidic Protein; Humans; I-kappa B Proteins; Immunohistochemistry; Infant, Newborn; MAP Kinase Signaling System; Mice; Mice, Inbred C57BL; Mice, Knockout; Microfilament Proteins; Nerve Tissue Proteins; Oxidative Stress; Oxygen; Poly Adenosine Diphosphate Ribose; Proto-Oncogene Proteins c-akt; Retinal Neovascularization; Retinal Vessels; Retinopathy of Prematurity; Tyrosine; Vascular Endothelial Growth Factor A

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 vaso-obliteration. 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.

Topics: Acetylcysteine; Animals; Animals, Newborn; Apoptosis; Blotting, Western; Catechin; Cells, Cultured; Disease Models, Animal; Endothelial Cells; Glutathione; Hyperoxia; Hypoxia; Ischemia; Lipid Peroxidation; Metalloporphyrins; Mice; Mice, Inbred C57BL; Peroxynitrous Acid; Protective Agents; Retinal Neovascularization; Retinal Vessels; Tyrosine

Exposure of premature human infants to hyperoxia results in the obliteration of developing retina capillaries, leading to a vision-threatening retinopathy termed retinopathy of prematurity (ROP). The authors hypothesized that this process may be mediated in part by endothelial nitric oxide (NO)-derived oxidants such as peroxynitrite and tested this hypothesis in a mouse model of ROP.. Normal mice, mice treated with the nitric oxide synthase (NOS) inhibitor N:(G)-nitro-L-arginine (L-NNA), and knockout mice carrying a homozygous targeted disruption of the gene for endothelial NOS (eNOS) were studied in an experimental model of ROP. Retinas were compared for extent of capillary obliteration in hyperoxia, vascular endothelial growth factor (VEGF) expression, nitrotyrosine formation, and vitreous neovascularization.. Oxygen-induced retinal vaso-obliteration was significantly reduced by L-NNA treatment (43% decrease from controls). The eNOS-deficient mice showed a similar reduction in vaso-obliteration (46% decrease from controls), and vitreous neovascularization was also substantially reduced (threefold decrease). Retinal nitrotyrosine formation, a measure of in situ peroxynitrite modification of proteins, was significantly elevated in normal mice during hyperoxia, in a spatial and temporal pattern consistent with a role in oxygen-induced vaso-obliteration. This was not seen in eNOS-deficient mice. VEGF expression was similar in both groups of mice, although suppression in hyperoxia was slightly blunted in eNOS-deficient mice.. These data suggest a role for NO and peroxynitrite in the pathogenesis of ROP. Therapies aimed at modulation of eNOS activity may have therapeutic potential for preventing ROP.

Topics: Animals; Endothelial Growth Factors; Enzyme Inhibitors; Humans; Hyperoxia; Infant, Newborn; Lymphokines; Mice; Mice, Inbred C57BL; Mice, Knockout; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Nitroarginine; Retinal Neovascularization; Retinal Vessels; Retinopathy of Prematurity; Tyrosine; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors