Truncated netrin-1 contributes to pathological vascular permeability in diabetic retinopathy
Khalil Miloudi, … , Timothy E. Kennedy, Przemyslaw Sapieha
Khalil Miloudi, … , Timothy E. Kennedy, Przemyslaw Sapieha
Published July 11, 2016
Citation Information: J Clin Invest. 2016;126(8):3006-3022. https://doi.org/10.1172/JCI84767.
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Research Article Angiogenesis

Truncated netrin-1 contributes to pathological vascular permeability in diabetic retinopathy

Abstract

Diabetic retinopathy (DR) is a major complication of diabetes and a leading cause of blindness in the working-age population. Impaired blood-retinal barrier function leads to macular edema that is closely associated with the deterioration of central vision. We previously demonstrated that the neuronal guidance cue netrin-1 activates a program of reparative angiogenesis in microglia within the ischemic retina. Here, we provide evidence in both vitreous humor of diabetic patients and in retina of a murine model of diabetes that netrin-1 is metabolized into a bioactive fragment corresponding to domains VI and V of the full-length molecule. In contrast to the protective effects of full-length netrin-1 on retinal microvasculature, the VI-V fragment promoted vascular permeability through the uncoordinated 5B (UNC5B) receptor. The collagenase matrix metalloprotease 9 (MMP-9), which is increased in patients with diabetic macular edema, was capable of cleaving netrin-1 into the VI-V fragment. Thus, MMP-9 may release netrin-1 fragments from the extracellular matrix and facilitate diffusion. Nonspecific inhibition of collagenases or selective inhibition of MMP-9 decreased pathological vascular permeability in a murine model of diabetic retinal edema. This study reveals that netrin-1 degradation products are capable of modulating vascular permeability, suggesting that these fragments are of potential therapeutic interest for the treatment of DR.

Authors

Khalil Miloudi, François Binet, Ariel Wilson, Agustin Cerani, Malika Oubaha, Catherine Menard, Sullivan Henriques, Gaelle Mawambo, Agnieszka Dejda, Phuong Trang Nguyen, Flavio A. Rezende, Steve Bourgault, Timothy E. Kennedy, Przemyslaw Sapieha

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Figure 9

The VI-V fragment of netrin-1 binds and signals through UNC5B.

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The VI-V fragment of netrin-1 binds and signals through UNC5B. (A) UNC5B...
(A) UNC5B, DCC, NEO1, and ADORA2B expression in endothelial cells (n = 3). (B) Representative Western blot showing loss of p–VE-cadherin, with silencing of the UNC5B receptor (n = 3). (C) p–VE-cadherin blot quantification (n = 3). (D) Paracellular resistance measured in real time by ECIS demonstrated that the VI-V fragment (3 nM) did not compromise endothelial barrier function in endothelial cells transfected with siUNC5B compared with those transfected with siNEG (1.8–6 hours, n = 4). (E) Representative confocal images of isolectin B4– (vessels), β-III tubulin– (retinal ganglion cells [RGCs]), DAPI- (nuclei), and UNC5B-stained retinal cross sections of adult mice revealed that retinal UNC5B was essentially vascular (n = 3). Scale bar: 20 μm. (F) LCM of the GCL, vessels, and INL, followed by qPCR of UNC5B (n = 3). (G) Binding strength of full-length netrin-1 versus that of the VI-V fragment to UNC5B via SPR (n = 4). FC, fragment crystallizable region. Data are expressed as the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, by 1-way ANOVA with Tukey’s post-hoc test (C) or 2-tailed Student’s t test (D and F).