Abstract
Aims/hypothesis Breakdown of the inner blood-retinal barrier (BRB) is an early event in the pathogenesis of diabetic macular oedema, that eventually leads to vision loss. We have previously shown that diabetes causes an imbalance of nerve growth factor (NGF) isoforms resulting in accumulation of its precursor proNGF and upregulation of the p75 neurotrophin receptor (p75(NTR)), with consequent increases in the activation of Ras homologue gene family, member A (RhoA). We also showed that genetic deletion of p75(NTR) in diabetes preserved the BRB and prevented inflammatory mediators in retinas. This study aims to examine the therapeutic potential of LM11A-31, a small-molecule p75(NTR) modulator and proNGF antagonist, in preventing diabetes-induced BRB breakdown. The study also examined the role of p75(NTR)/RhoA downstream signalling in mediating cell permeability.
Methods Male C57BL/6J mice were rendered diabetic using streptozotocin injection. After 2 weeks of diabetes, mice received oral gavage of LM11A-31 (50 mg kg(-1) day(-1)) or saline (NaCl 154 mmol/l) for an additional 4 weeks. BRB breakdown was assessed by extravasation of BSA-AlexaFluor-488. Direct effects of proNGF were examined in human retinal endothelial (HRE) cells in the presence or absence of LM11A-31 or the Rho kinase inhibitor Y-27632.
Results Diabetes triggered BRB breakdown and caused significant increases in circulatory and retinal TNF-alpha and IL-1 beta levels. These effects coincided with significant decreases in retinal NGF and increases in vascular endothelial growth factor and proNGF expression, as well as activation of RhoA. Interventional modulation of p75(NTR) activity through treatment of mouse models of diabetes with LM11A-31 significantly mitigated proNGF accumulation and preserved BRB integrity. In HRE cells, treatment with mutant proNGF (10 ng/ml) triggered increased cell permeability with marked reduction of expression of tight junction proteins, zona occludens-1 (ZO-1) and claudin-5, compared with control, independent of inflammatory mediators or cell death. Modulating p75(NTR) significantly inhibited proNGF-mediated RhoA activation, occludin phosphorylation (at serine 490) and cell permeability. ProNGF induced redistribution of ZO-1 in the cell wall and formation of F-actin stress fibres; these effects were mitigated by LM11A-31.
Conclusions/interpretation Targeting p75(NTR) signalling using LM11A-31, an orally bioavailable receptor modulator, may offer an effective, safe and non-invasive therapeutic strategy for treating macular oedema, a major cause of blindness in diabetes.