Abstract
Purpose:
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.
Methods:
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.
Results:
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.
Conclusions:
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.
Keywords: 499 diabetic retinopathy •
505 edema •
688 retina