Experimental Diabetes Causes Breakdown of the Blood-Retina Barrier by a Mechanism Involving Tyrosine Nitration and Increases in Expression of Vascular Endothelial Growth Factor and Urokinase Plasminogen Activator Receptor

Abstract

The purpose of these experiments was to determine the specific role of reactive oxygen species (ROS) in the blood-retinal barrier (BRB) breakdown that characterizes the early stages of vascular dysfunction in diabetes. Based on our data showing that high glucose increases nitric oxide, superoxide, and nitrotyrosine formation in retinal endothelial cells, we hypothesized that excess formation of ROS causes BRB breakdown in diabetes. Because ROS are known to induce increases in expression of the well-known endothelial mitogen and permeability factor vascular endothelial growth factor (VEGF) we also examined their influence on the expression of VEGF and its downstream target urokinase plasminogen activator receptor (uPAR). After 2 weeks of streptozotocin-induced diabetes, analysis of albumin leakage confirmed a prominent breakdown of the BRB. This permeability defect was correlated with significant increases in the formation of nitric oxide, lipid peroxides, and the peroxynitrite biomarker nitrotyrosine as well as with increases in the expression of VEGF and uPAR. Treatment with a nitric oxide synthase inhibitor (N--nitro-L-arginine methyl ester, 50 mg/kg/day) or peroxynitrite scavenger (uric acid, 160 mg/kg/day) blocked the breakdown in the BRB and prevented the increases in formation of lipid peroxides and tyrosine nitration as well as the increases in expression of VEGF and uPAR. Taken together, these data indicate that early diabetes causes breakdown of the BRB by a mechanism involving the action of reactive nitrogen species in promoting expression of VEGF and uPAR.

Diabetes-induced peroxynitrite impairs the balance of pro-nerve growth factor and nerve growth factor, and causes neurovascular injury

“…In parallel, analysis of oxidative stress and peroxynitrite markers showed significant increases in 4-HNE adducts and nitrotyrosine formation in aqueous humour samples from diabetic patients and diabetic rat retinas compared with non-diabetic controls. These results lend further support to previous studies showing enhanced peroxynitrite formation in clinical and experimental diabetes [1,3,12,22]. Treatment of our diabetic animals with atorvastatin exerted similar protective effects to FeTPPs in reducing peroxynitrite and 4-HNE adducts, as well restoring the balance between NGF and proNGF to normal levels.…”

“…A growing body of evidence supports the concept that diabetes disturbs homeostasis in the retina by activating glial cells, reducing neurotrophic and prosurvival inputs, and increasing proinflammatory cytokines, leading to accelerated cell death and vascular permeability, thus impairing vision, notions which have been reviewed previously [18][19][20]. In agreement, we and others have reported that increases in proinflammatory cytokines, including vascular endothelial growth factor (VEGF), NGF, TNF-α, intercellular adhesion molecule-1 and inducible nitric oxide synthase, in diabetic retinas were correlated with neuronal cell death and vascular permeability [1,3,12,13,[21][22][23]. However, the role of neurotrophins in the diabetic eye remains unknown.…”

“…Blood-retinal barrier function Integrity of the BRB was measured as previously described by our group [3,12]. Plasma was assayed for fluoresciene concentration using a plate reader (excitation 370 nm, emission 460 nm) (BioTek).…”

“…Diabetes-induced peroxynitrite formation causes BRB breakdown We have previously shown that diabetes causes BRB breakdown in streptozotocin-induced animal models of diabetes [3,12,13]. However, the causal role of peroxynitrite has not been investigated.…”

“…The molecular link between two well-documented early hallmarks of diabetic retinopathy, i.e. accelerated neuronal death and vascular permeability, remains obscure [1][2][3][10][11][12][13]. While proinflammatory cytokines are known to activate Rho family kinases leading to actin cytoskeleton rearrangement and cell permeability in vasculature, Rho family members are also essential regulators of neuronal survival in the nervous system, as reviewed by Linseman and Loucks [14].…”