D.H. Platt

Oxidative stress inactivates VEGF survival signaling in retinal endothelial cells via PI 3-kinase tyrosine nitration

In diabetic retinopathy, endothelial cell apoptosis is paradoxically increased despite upregulation of the potent pro-survival factor VEGF. We tested the hypothesis that elevated glucose levels disrupt VEGFs pro-survival function via peroxynitrite-mediated alteration of the Akt-1/p38 MAP kinase signaling pathway by studies of retinal endothelial cells in vitro. High glucose or exogenous peroxynitrite caused significant increases in apoptosis in the presence or absence of VEGF. Treatment with a peroxynitrite decomposition catalyst blocked these effects, implying a causal role of peroxynitrite. Peroxynitrite or high glucose treatment also increased phosphorylation of p38 MAP kinase, whereas phosphorylation of Akt-1 was significantly decreased in basal or VEGF-stimulated conditions. High glucose- or peroxynitrite-treated cells also showed significant increases in tyrosine nitration on the p85 subunit of PI 3-kinase that blocked PI 3-kinase and Akt-1 kinase activity. Inhibiting peroxynitrite formation or blocking tyrosine nitration of p85 restored the activity of PI 3-kinase and Akt-1 kinase, blocked phosphorylation of p38 MAP kinase and normalized pro-survival function. Transfecting the cells with constitutively active Akt-1 or inhibiting activity of p38 MAP kinase completely masked the pro-apoptotic effects of high glucose and exogenous peroxynitrite, suggesting an interaction between the Akt-1 and p38 MAP kinase pathways. In conclusion, high glucose treatment blocks the pro-survival effect of VEGF and causes accelerated endothelial cell apoptosis via the action of peroxynitrite in causing tyrosine nitration of PI 3-kinase, inhibiting activity of Akt-1 kinase and increasing the activity of p38 MAP kinase.

Cellular and Molecular Mechanisms of Retinal Angiogenesis

Angiogenesis is a multi-factorial process that involves different cell types and a number of cytokines and growth factors. Physiological angiogenesis is characterized by the existence of a delicate balance between pro-angiogenic and anti-angiogenic factors. In an in vivo setting, pro-angiogenic stimuli such as endothelial-specific mitogenic factors and extracellular matrix (ECM)- degrading enzymes must be tightly regulated and locally constrained. Differentiation factors, protease inhibitors, and the elements involved in reconstruction of ECM and recruitment of mural cells must be elicited in an appropriate temporal and spatial arrangement. Overexpression of angiogenesis-activating factors may cause hyper-vascularization. However, deficiency or disarray in expression of anti-angiogenic factors may result in leaky vessels, unstable capillaries, and formation of dysfunctional neovascular tufts as seen in retinopathy of prematurity, diabetic retinopathy, or other conditions of retinal neovascularization. In other words, pathological angiogenesis is characterized not only by excesses in pro-angiogenic factors but also an insufficiency in anti-angiogenic, pro-differentiation factors.