Inflammatory Role of ProNGF/p75NTR in Müller cells of the Diabetic Retina

Abstract

Purpose: : Diabetic retinopathy, the leading cause of blindness in U.S. working age adults, is characterized by neurodegeneration, inflammation, and vascular dysfunction. Our recent studies using diabetic animals show that neurodegeneration and vascular leakage are associated with an imbalance of nerve growth factor (NGF) leading to accumulation of its precursor (proNGF) and its receptor, p75^NTR. Here, we examine the role of proNGF/p75^NTR in mediating retinal inflammation and vascular dysfunction in the diabetic retina.

Methods: : STZ-diabetes was induced in wild type and p75^NTR knockout (KO) mice. After 4 weeks, vascular permeability was assayed by extravasation of BSA-conjugated fluorescein. Müller cells were cultured for 2 days in high glucose (25 mM), normal glucose (5 mM), or osmolar controls and stimulated with cleavage-resistant proNGF in the presence or absence of compound E (an inhibitor of p75^NTR intracellular domain cleavage) and epicatechin (a nitration inhibitor that reduces p75^NTR expression). Expression of p75^NTR, NGF, proNGF, and inflammatory mediators were determined in retinal and cell lysates by Western-Blot. Animals used for this research were handled in accord with ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

Results: : Deletion of p75^NTR prevented diabetes-induced vascular permeability detected in wild-type mice. In diabetic wild-type mice NGF protein levels decreased by 50% while NFkB and TNF-α levels increased by 1.8 fold and 6 fold, respectively (N=5-7, P < 0.01). These changes were not observed in diabetic p75^NTR KO mice. Interestingly, basal levels of NGF, NFkB, and TNF-α were altered in p75^NTR KO mice (N=5-7, P < 0.05). Müller cell studies showed that addition of exogenous, cleavage-resistant proNGF enhanced high glucose induced elevation of p75^NTR. Elevation of NFkB and TNF-α observed in high glucose, cleavage-resistant proNGF treated cells was reduced by co-treatment with compound E and epicatechin (N = 4-6, P < 0.05).

Conclusions: : Deletion of p75^NTR or inhibition of its cleavage reduced the diabetes induced elevation of inflammatory mediators that cause increased vascular permeability. Inhibition of the proNGF/p75^NTR inflammatory axis is a potential therapeutic target in the diabetic retina.

Keywords: diabetic retinopathy • inflammation • Muller cells