A fragment of netrin-1 is implicated in the induction of permeability in diabetic retinopathy

Abstract

Purpose: Although elevated VEGF is frequently associated with breakdown in barrier function in diabetic retinopathy (DR), we have recently described roles for neuronal guidance cues in the pathogenesis of DR. Here we described a new role for a fragment of netrin-1, the VI-V peptide fragment of netrin-1, in the development of DR-associated breakdown of barrier function.

Methods: We first investigated retinal levels of full-length and fragmented netrin-1 by Western Blot in early stages of DR in a streptozotocin-induced model of diabetes. In a modified version of the in vivo Miles Assay, we i) subcutaneously and ii) intraviteously injected recombinant netrin-1 or the shorter VI-V netrin-1 peptide into wild type mice at 10 weeks of age. A blue dye (Evans blue (EB)), was later injected intravenously. Permeability was then evaluated by i) measuring dorsal spots of subcutaneous leaking EB, and ii) by quantifying EB in retinas after overnight extraction in formamide. To identify possible signaling pathways involved, human retinal microvascular endothelial cells were similarly stimulated in vitro.

Results: Our findings reveal a significant increase in the levels of fragmented netrin-1 at 4 and 8 weeks in diabetic retinas. Intravitreal injection of the netrin-1 VI-V peptide significantly increased vascular permeability in vivo both in retina and after subcutaneous injection, when compared to full-length netrin-1 or vehicle. In agreement, in vitro data confirmed that the VI-V fragment of netrin-1 provoked the phosphorylation of permeability-associated pathways such FAK, Src, and VE-Cadherin.

Conclusions: These results show a new mechanism implicating a wider spatial effect of netrin-1 via the VI-V peptide fragment. Current studies aim to elucidate the cellular and molecular mechanism by which this recently described fragment may be implicated in the pathogenesis of DR.

Keywords: 690 retina: neurochemistry • 499 diabetic retinopathy • 531 ganglion cells