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Barbara A Mysona, Dorothy Rodenbeck, Ahmed Y Shanab, Suraporn Matragoon, Azza B El-Remessy; ProNGF causes retinal endothelial cell permeability via activation of p75NTR/RhoA pathway. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5827. doi: https://doi.org/.
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Our aim is to understand the molecular mechanisms contributing to breakdown of the inner blood-retinal barrier (BRB), an event which leads to macular edema, a major cause of diabetic blindness. Previously, our lab has shown that p75NTR deletion or inhibition blocks diabetes and proNGF-induced inflammation and BRB breakdown. Here, we test the hypothesis that proNGF acts directly on endothelial cells to stimulate p75NTR mediated activation of RhoA leading to barrier dysfunction.
Human retinal endothelial (HRE) cell permeability was assayed by measuring extravasation of FITC-dextran (70 kD) through HRE monolayers grown on collagen/fibronectin coated transwells. Cells were treated in the presence or absence of human cleavage-resistant mutant proNGF, (hm-proNGF, 1 to 50 ng/ml) with or without pretreatment with p75NTR inhibitors compound A (Uri Saragovi, McGill University) or LM11A-31 (Dr. Frank Longo, Stanford University) and Rho-kinase inhibitor Y-27632, 10 μM. Cells were also grown in high (25 mM) compared to normal (5 mM) glucose in the presence or absence of hm-proNGF (10 ng/ml). Barrier function was measured by monitoring accumulation of fluorescence in the abluminal compartment. Lysates from confluent HRE cells were analyzed by Western blot for RhoA activation, VEGF, and TNF-α.
Treatment with hm-proNGF yielded a maximal 2-fold increase in HRE cell permeability at 10 ng/ml (n=6, P < 0.05) that was blocked by p75NTR and Rho-kinase inhibition (n=6-8, P < 0.05). Analysis of HRE lysates revealed that cells treated with hm-proNGF exhibited a 50% increase in active RhoA that was blocked by inhibition of p75NTR. In HRE cells, treatment with proNGF did not stimulate increased expression of VEGF or TNF-α.
Our data provide evidence that proNGF/p75NTR can directly mediate RhoA activation and increased HRE cell permeability independent of VEGF or TNF-α. The p75NTR/Rho kinase pathway may be a potential therapeutic target for treating macular edema, a major cause of diabetic blindness.
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