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 2

Truncated netrin-1 is highly expressed in early phases of diabetes in a mouse STZ model.

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Truncated netrin-1 is highly expressed in early phases of diabetes in a ...
(A) STZ or citrate vehicle was administered to 6-week-old C57BL/6J mice. Retinae were then extracted at 4, 6, and 8 weeks of diabetes (db). (B) Netrin-1 localization in the GCL in adult murine retina. Representative confocal images of isolectin B4– (vessel stain), DAPI- (nuclei stain), and netrin-1–immunostained retinal cross sections (20-μm), reconstructed in 3D (n = 3). (C) LCM of the GCL, INL, and ONL, followed by qPCR confirmed expression of netrin-1 in the GCL (n = 3). (D) Representative Western blot analyses of retinae confirmed increased levels of the VI-V fragment at 4, 6, and 8 weeks of diabetes (n = 3). VI-V fragment corresponds to the upper band indicated by the arrow. (E) Quantification of full-length netrin-1 signal density at 4 (n = 7), 6 (n = 3), and 8 weeks (n = 7). (F) Quantification of VI-V fragment signal density at 4 (n = 7), 6 (n = 3), and 8 weeks (n = 7). (G) Netrin-1 Ab targeting the aa sequence of domain VI of netrin-1 (clone NORA-1). (H) Gel electrophoresis of immunoprecipitated netrin-1 (n = 10 diabetic retinae). Data are expressed as the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, by 2-tailed Student’s t test (E and F) and 1-way ANOVA with Tukey’s post-hoc test (C). fr, fragment.