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
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 (p75NTR), with consequent increases in the activation of Ras homologue gene family, member A (RhoA). We also showed that genetic deletion of p75NTR 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 p75NTR modulator and proNGF antagonist, in preventing diabetes-induced BRB breakdown. The study also examined the role of p75NTR/RhoA downstream signalling in mediating cell permeability.
Male C57BL/6 J 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.
Diabetes triggered BRB breakdown and caused significant increases in circulatory and retinal TNF-α and IL-1β 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 p75NTR 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 p75NTR 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.
Targeting p75NTR 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.
KeywordsCytoskeleton rearrangement Diabetic retinopathy Occludin p75NTR receptor proNGF RhoA Vascular permeability
Diabetic macular oedema
Human mutant proNGF
Human retinal endothelial (cells)
Nerve growth factor
Proliferative diabetic retinopathy
p75 neurotrophin receptor
Ras-related C3 botulinum toxin substrate 1
Ras homologue gene family, member A
Vascular endothelial growth factor
We are grateful to D. Antonetti (Kellogg Eye Center, University of Michigan, MI, USA) for providing the antibody to p-occludinS490. Part of the data was presented as an abstract at the 75th Annual Meeting of the American Diabetes Association in 2015.
SLE, AA, RM, TL and MC contributed to the design, acquisition and assembly of data, data analysis and interpretation and drafting the manuscript. FL contributed to the conceptual design of experiments and to manuscript writing and communication. ABE conceived the idea, contributed to the overall experimental design, data analysis and interpretation and writing and critical revision of the manuscript, as well as manuscript correspondence. All authors read and approved the final manuscript. ABE is responsible for the integrity of the work as a whole.
This work was supported by RO-1-EY-022408 (ABE), a pre-doctoral fellowship from the American Heart Association (SLE) and by the Jean Perkins Foundation (FML).
Duality of interest
FML is listed as an inventor on patents relating to LM11A-31, which are assigned to UNC and UCSF, and is eligible for royalties distributed by the assigned universities. FML has a financial interest in PharmatrophiX, a company focused on the development of small-molecule ligands for neurotrophin receptors, which has licensed several of these patents. All other authors declare that there is no duality of interest associated with their contribution to this manuscript.
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