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 2

Ndst1ECKO mice display disrupted vascularization in diaphragm.

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Ndst1ECKO mice display disrupted vascularization in diaphragm. (A) Wh...
(A) Whole-mount X-gal staining of E15.5 Tie2Cre+Rosa26LacZ diaphragm. The vascular plexus in diaphragm muscle and tendon was LacZ positive. (B) Costaining with anti-HS and anti–PECAM-1 antibodies showed that HS expressed by ECs was dramatically reduced in E15.5 Ndst1ECKO diaphragm. Cell nuclei were stained with Topro-3. (C) Whole-mount staining of E15.5–E16.5 diaphragm with anti–PECAM-1 antibody. Vascular density in Ndst1ECKO tendon was substantially reduced, with a larger avascular region (dotted outline). (D) Vascular length and branch point were both significantly decreased in E15.5–E16.5 Ndst1ECKO tendons (n = 5–7 per time point). (E) Whole-mount staining of E14.5–E15.5 diaphragm with anti–PECAM-1 antibody. Vascular density was significantly reduced in the Ndst1ECKO muscular region. Arrowheads, cord-like vessels. (F) Anti–PECAM-1 staining of E18.5 embryo showed reduced vascular density in Ndst1ECKO diaphragm (dashed outlines). (G) Microfill gel perfusion highlighted vasculature in adult diaphragm. The vasculature of Ndst1ECKO diaphragm lacked large vessels and was less branched in the anterior muscular region (see also Supplemental Figure 2A). Arrowhead, hernia sac. Scale bars: 0.5 mm (A and C, top); 10 μm (B); 0.2 mm (C, bottom; and E, top); 50 μm (E, bottom); 250 μm (F).