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Mutations in EPHB4 cause human venous valve aplasia
Oliver Lyons, … , Prakash Saha, Alberto Smith
Oliver Lyons, … , Prakash Saha, Alberto Smith
Published August 17, 2021
Citation Information: JCI Insight. 2021;6(18):e140952. https://doi.org/10.1172/jci.insight.140952.
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Research Article Angiogenesis Development

Mutations in EPHB4 cause human venous valve aplasia

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Abstract

Venous valve (VV) failure causes chronic venous insufficiency, but the molecular regulation of valve development is poorly understood. A primary lymphatic anomaly, caused by mutations in the receptor tyrosine kinase EPHB4, was recently described, with these patients also presenting with venous insufficiency. Whether the venous anomalies are the result of an effect on VVs is not known. VV formation requires complex “organization” of valve-forming endothelial cells, including their reorientation perpendicular to the direction of blood flow. Using quantitative ultrasound, we identified substantial VV aplasia and deep venous reflux in patients with mutations in EPHB4. We used a GFP reporter in mice to study expression of its ligand, ephrinB2, and analyzed developmental phenotypes after conditional deletion of floxed Ephb4 and Efnb2 alleles. EphB4 and ephrinB2 expression patterns were dynamically regulated around organizing valve-forming cells. Efnb2 deletion disrupted the normal endothelial expression patterns of the gap junction proteins connexin37 and connexin43 (both required for normal valve development) around reorientating valve-forming cells and produced deficient valve-forming cell elongation, reorientation, polarity, and proliferation. Ephb4 was also required for valve-forming cell organization and subsequent growth of the valve leaflets. These results uncover a potentially novel cause of primary human VV aplasia.

Authors

Oliver Lyons, James Walker, Christopher Seet, Mohammed Ikram, Adam Kuchta, Andrew Arnold, Magda Hernández-Vásquez, Maike Frye, Gema Vizcay-Barrena, Roland A. Fleck, Ashish S. Patel, Soundrie Padayachee, Peter Mortimer, Steve Jeffery, Siren Berland, Sahar Mansour, Pia Ostergaard, Taija Makinen, Bijan Modarai, Prakash Saha, Alberto Smith

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Figure 5

Effect of Efnb2 deletion on organization of VFCs.

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Effect of Efnb2 deletion on organization of VFCs.
(A and B) Localization...
(A and B) Localization of PECAM1 (blue), Prox1 (red), and SMA (white) in littermate controls and heterozygous (Efnb2lx/wt) and homozygous (Efnb2lx/lx) mice on P0, after tamoxifen induction of Efnb2 deletion on E15. In controls and Efnb2lx/wt mice, valves reached stage 1 of development, as normal. Homozygous deletion resulted in a failure to organize normally, with Prox1hi cells distributed over a wider upstream–downstream area of the vein and failure of VFCs to elongate and reorientate. (B) The bar chart shows the proportion of VVs identified at stage 0 (white) and stage 1 (grey) on P0 for the indicated genotypes, and the number of VVs analyzed for each condition is given above each bar. Ps derive from 2-sided Fisher’s exact test vs. control. (C) Hex-binned scatterplot of VFC elongation (length/width ratio) across the vein from superior to inferior. n = 2517 cells, ≥ 6 VVs. (D) Bar chart (± SEM) summarizing the results from C showing that both heterozygous and homozygous deletion resulted in significant reductions in VFC elongation. One-way ANOVA with Bonferroni’s post hoc test. For between groups 1-way ANOVA, F = 109 with 2 df, P = 3.2 × 10–46. (E) Hex-binned scatterplot of VFC reorientation (in VFCs with nuclear length/width ratio ≥ 2) across the vein from superior to inferior. n = 1226 cells, ≥ 6 VVs. After homozygous deletion, the VFCs with correctly reorientated nuclei were lost, particularly in the center of the vessel. (F) Bar chart (± SEM) summarizing the results from E. Homozygous deletion resulted in significantly reduced reorientation. One-way ANOVA with Bonferroni’s post hoc. For 1-way ANOVA, F = 7.1 with 2 df, P = 0.0009. *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.00005. Scale bars: 20 μm. VFCs, valve-forming cells; VVs, venous valves. SMA, smooth muscle α-actin.

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ISSN 2379-3708

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