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Barbara Mysona, Suraporn Matragoon, Azza El-Remessy; Role of proNGF/p75NTR mediated RhoA activation in diabetes-induced BRB breakdown. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2699.
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Diabetic macular edema is characterized by early breakdown of the inner blood-retinal barrier (BRB). Understanding the mechanism of this breakdown is essential for developing alternative therapies to laser photocoagulation and anti-VEGF injections. Previously, we have shown that modulation of p75NTR expression prevents diabetes- and proNGF-induced retinal inflammation and BRB breakdown. Increased activation of RhoA has also been demonstrated in diabetic and proNGF overexpressing retinas. We hypothesize that diabetes-induced proNGF stimulates p75NTR dependent activation of RhoA in endothelial cells leading to barrier dysfunction.
Inhibition of Rho-kinase using intravitreal injection of Y-27632 (100 nmole/eye) was employed in streptozotocin (STZ) induced diabetic male Sprague-Dawley rats and in proNGF overexpressing rats. BRB breakdown was assayed by extravasation of BSA-conjugated fluorescein. Barrier function of confluent human retinal endothelial (HRE) cells treated with human mutant proNGF (10, 20, 50 ng/ml) was assessed by electrical cell-substrate impedance sensing machine (ECIS) in presence or absence of Y-27632 (10 μM) or gamma-secretase inhibitor (compound E, 10 μM). Protein expression was analyzed by western blot.
Inhibition of Rho kinase significantly decreased BRB breakdown in retinas after 5 weeks STZ-diabetes and after 1-week proNGF overexpression. In vitro, mutant proNGF induced dose-dependent HRE cell permeability which was blocked by inhibition of Rho-kinase or p75NTR cleavage. ProNGF treatment was accompanied by decreased expression of tight junction proteins ZO-1 and claudin-5 compared to controls.
Our data support a novel role for proNGF/p75NTR in activating RhoA mediated endothelial cell permeability and BRB breakdown in early diabetes. The p75NTR/Rho kinase pathway may be a potential therapeutic target in the diabetic retina.
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