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A. B. El-Remessy, M. M. Al-Gayyar, M. A. Abdelsaid, S. Matragoon, B. A. Pillai, J. J. Nussbaum; Impaired Balance of NGF/proNGF Causes Retinal Neuronal/Vascular Injury via Activation of RhoA. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3823.
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We have previously shown positive correlation between breakdown of blood retina barrier (BRB), neuronal death and accumulation of the proform of nerve growth factor (proNGF) in diabetic rat retinas. The purpose of this study is to elucidate the causal role of proNGF in mediating glial activation, BRB breakdown and neuronal death and to characterize its downstream signal. In addition, levels of NGF and proNGF were assessed in human samples from non-diabetic, diabetic patients (DR) or proliferative diabetic retinopathy (PDR) patients.
Overexpression of the proNGF in SD rats was achieved by intravitreal injection of the GFP-conjugated plasmid of cleavage resistant proNGF construct (5µg) that was successfully incorporated into rat retina by electroporation. Additional groups received either proNGF/the specific Rho kinase inhibitor Y27632 (100 nmoles/eye) or GFP-conjugated plasmid only. Neuronal cell death was determined by TUNEL assay and ganglion cell (GC) count. BRB was assessed by extravasation of BSA-fluoresciene. Expression of NGF, proNGF and P38MAPK were determined by Western-Blot. Activation of RhoA was determined by pull down assay.
Significant accumulation of proNGF (3- and 5-fold) and reduction of NGF levels (35% or 65%) were observed in samples from DR and PDR-patients, respectively, compared to samples from non-diabetic. Overexpression of proNGF in rat retina mimicked diabetes action by inducing BRB breakdown (2-fold), retinal neuronal cell death (5-fold) and reducing GC count (25%). ProNGF caused glial activation as indicated by GFAP staining and activation of RhoA/p38 MAPK pathway. Treatment of proNGF expressing animals with Y27632 blocked activation of RhoA/p38MAPK pathway and preserved BRB and restored GC in rat retina.
Diabetes-induced peroxynitrite alters the homeostasis of NGF in the retina by decreasing NGF maturation, increasing proNGF accumulation which activates the common pathway RhoA/p38MAPK leading to retinal neurodegeneration and vascular permeability. Similar to diabetes, overexpression of proNGF in normal rat retina caused vascular and neuronal injury that was reversed by Rho kinase inhibitor. Thus, inhibiting RhoA may be effective therapeutic targets in DR.
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