Heparan sulfate deficiency disrupts developmental angiogenesis and causes congenital diaphragmatic hernia
Bing Zhang, … , Jeffrey D. Esko, Lianchun Wang
Bing Zhang, … , Jeffrey D. Esko, Lianchun Wang
Published December 20, 2013
Citation Information: J Clin Invest. 2014;124(1):209-221. https://doi.org/10.1172/JCI71090.
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Research Article Vascular biology

Heparan sulfate deficiency disrupts developmental angiogenesis and causes congenital diaphragmatic hernia

Abstract

Congenital diaphragmatic hernia (CDH) is a common birth malformation with a heterogeneous etiology. In this study, we report that ablation of the heparan sulfate biosynthetic enzyme NDST1 in murine endothelium (Ndst1ECKO mice) disrupted vascular development in the diaphragm, which led to hypoxia as well as subsequent diaphragm hypoplasia and CDH. Intriguingly, the phenotypes displayed in Ndst1ECKO mice resembled the developmental defects observed in slit homolog 3 (Slit3) knockout mice. Furthermore, introduction of a heterozygous mutation in roundabout homolog 4 (Robo4), the gene encoding the cognate receptor of SLIT3, aggravated the defect in vascular development in the diaphragm and CDH. NDST1 deficiency diminished SLIT3, but not ROBO4, binding to endothelial heparan sulfate and attenuated EC migration and in vivo neovascularization normally elicited by SLIT3-ROBO4 signaling. Together, these data suggest that heparan sulfate presentation of SLIT3 to ROBO4 facilitates initiation of this signaling cascade. Thus, our results demonstrate that loss of NDST1 causes defective diaphragm vascular development and CDH and that heparan sulfate facilitates angiogenic SLIT3-ROBO4 signaling during vascular development.

Authors

Bing Zhang, Wenyuan Xiao, Hong Qiu, Fuming Zhang, Heather A. Moniz, Alexander Jaworski, Eduard Condac, Gerardo Gutierrez-Sanchez, Christian Heiss, Robin D. Clugston, Parastoo Azadi, John J. Greer, Carl Bergmann, Kelley W. Moremen, Dean Li, Robert J. Linhardt, Jeffrey D. Esko, Lianchun Wang

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Figure 3

Hypoxia, increased apoptosis, and attenuated proliferation of tendon cells in developing Ndst1ECKO diaphragm.

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Hypoxia, increased apoptosis, and attenuated proliferation of tendon cel...
(A) Vascular tip cells. Whole-mount costaining with anti–PECAM-1 antibody and Topro-3 showed tip cells (red arrows) and their characteristic structure as well as filopodia (yellow arrowheads), both of which were reduced in Ndst1ECKO diaphragms at E16.5 (n = 6). (B) EC proliferation. Diaphragms from BrdU-injected E16.5 embryos were costained for BrdU and PECAM-1. BrdU+ ECs (white outlines) were reduced in Ndst1ECKO diaphragms (n = 5). (C) Pericyte recruitment. E16.5 diaphragms were immunostained with anti–PECAM-1 and anti-NG2 antibodies. Pericyte coverage did not differ in the Ndst1ECKO and Ndst1f/f vasculature (n = 4–6). (D) Hypoxic tendon cells. E15.5 diaphragms were costained with anti-pimonidazole antibody and Topro-3 to visualize hypoxic cells. Hypoxic cells were much more prevalent in Ndst1ECKO than in Ndst1f/f diaphragm tendon (n = 5). (E) Apoptotic tendon cells. E15.5 diaphragms were costained with anti–caspase-3 and Topro-3 to visualize apoptotic cells. Apoptotic endocytes (yellow arrowheads) were more prevalent in Ndst1ECKO than in Ndst1f/f diaphragm (n = 4). (F) Proliferating tendon cells. Diaphragms from BrdU-injected E15.5 embryos were immunostained with anti-BrdU antibody. Red dashed outline, diaphragmatic tendon. BrdU+ tendon cells were significantly reduced in Ndst1ECKO diaphragms (n = 6). Scale bars: 40 μm (A, left; B; C; D, right; and E, right); 10 μm (A, right); 200 μm (D, left); 100 μm (E, left); 50 μm (F).