Study on Modulation of the p75 Neurotrophin Receptor Using LM11A-31 Prevents Diabetes-induced Retinal Vascular Permeability in Mice via Inhibition of Inflammation and the RhoA Kinase Pathway

Abstract

Aims/hypothesis: The breakdown of the inner blood–retinal barrier (BRB) is a key event in the pathogenesis of diabetic macular oedema, which leads to vision loss. We previously demonstrated that diabetes produces an imbalance of nerve growth factor (NGF) isoforms, leading in the buildup of proNGF and overexpression of the p75 neurotrophin receptor (p75NTR), with subsequent increases in Ras homologue gene family, member A activation (RhoA). We also discovered that in diabetes, genetic deletion of the p75NTR gene retained the BRB and inhibited the production of inflammatory mediators in the retina. The goal of this study is to see if LM11A-31, a small-molecule p75NTR modulator and proNGF antagonist, can help prevent BRB breakdown caused by diabetes. The role of p75NTR/RhoA downstream signalling in mediating cell permeability was also investigated. Methods: Streptozotocin injection was used to make male C57BL/6 J mice diabetic. Mice were given oral gavage with LM11A-31 (50 mg kg1 day1) or saline (NaCl 154 mmol/l) for an additional 4 weeks after 2 weeks of diabetes.Extravasation of BSA–AlexaFluor-488 was used to measure BRB breakdown. In the presence or absence of LM11A-31 or the Rho kinase inhibitor Y-27632, the direct effects of proNGF were investigated in human retinal endothelial (HRE) cells. Results: BRB breakdown was induced by diabetes, which resulted in large increases in TNF- and IL-1 levels in the circulatory and retinal systems. Significant decreases in retinal NGF and elevations in vascular endothelial growth factor and proNGF expression, as well as RhoA activation, accompanied these effects. ProNGF accumulation was significantly reduced and BRB integrity was preserved after interventional modulation of p75NTR activity in diabetes animal models using LM11A-31. Treatment with mutant proNGF (10 ng/ml) increased cell permeability and reduced expression of tight junction proteins, zona occludens-1 (ZO-1) and claudin-5, in HRE cells, independent of inflammatory mediators or cell death, compared to control. ProNGF-mediated RhoA activation, occludin phosphorylation (at serine 490), and cell permeability were all considerably reduced when p75NTR was modulated.The effects of ProNGF were moderated by LM11A-31, which caused redistribution of ZO-1 in the cell wall and the development of F-actin stress fibres. Conclusions/interpretation: Using LM11A-31, an orally accessible receptor modulator, to target p75NTR signalling may provide an effective, safe, and non-invasive therapeutic method for treating macular oedema, a leading cause of blindness in diabetics.