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. 2012 Aug 3;111(4):426-36.
doi: 10.1161/CIRCRESAHA.112.269399. Epub 2012 Jun 20.

An unexpected role of semaphorin3a-neuropilin-1 signaling in lymphatic vessel maturation and valve formation

Giorgia Jurisic  1 , Hélène Maby-El Hajjami, Sinem Karaman, Alexandra M Ochsenbein, Annamari Alitalo, Shoib S Siddiqui, Carlos Ochoa Pereira, Tatiana V Petrova, Michael Detmar
Affiliations

An unexpected role of semaphorin3a-neuropilin-1 signaling in lymphatic vessel maturation and valve formation

Giorgia Jurisic et al. Circ Res. .

Abstract

Rationale: Lymphatic vasculature plays important roles in tissue fluid homeostasis maintenance and in the pathology of human diseases. Yet, the molecular mechanisms that control lymphatic vessel maturation remain largely unknown.

Objective: We analyzed the gene expression profiles of ex vivo isolated lymphatic endothelial cells to identify novel lymphatic vessel expressed genes and we investigated the role of semaphorin 3A (Sema3A) and neuropilin-1 (Nrp-1) in lymphatic vessel maturation and function.

Methods and results: Lymphatic and blood vascular endothelial cells from mouse intestine were isolated using fluorescence-activated cell sorting, and transcriptional profiling was performed. We found that the axonal guidance molecules Sema3A and Sema3D were highly expressed by lymphatic vessels. Importantly, we found that the semaphorin receptor Nrp-1 is expressed on the perivascular cells of the collecting lymphatic vessels. Treatment of mice in utero (E12.5-E16.5) with an antibody that blocks Sema3A binding to Nrp-1 but not with an antibody that blocks VEGF-A binding to Nrp-1 resulted in a complex phenotype of impaired lymphatic vessel function, enhanced perivascular cell coverage, and abnormal lymphatic vessel and valve morphology.

Conclusions: Together, these results reveal an unanticipated role of Sema3A-Nrp-1 signaling in the maturation of the lymphatic vascular network likely via regulating the perivascular cell coverage of the vessels thus affecting lymphatic vessel function and lymphatic valve development.

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Conflict of interest statement

Disclosures

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1. Ex vivo isolation of pure blood vascular and lymphatic endothelial cells from mouse colon tissue by high-speed cell sorting
(A) Colon single-cell suspensions were immunostained for the pan-endothelial marker CD31, the leukocyte marker CD45 and the lymphatic marker podoplanin. The CD31+CD45− population was further separated into podoplanin+ LECs and podoplanin− BECs. (B) qPCR for the blood vessel marker VEGFR1 and the lymphatic markers podoplanin and LYVE-1 was performed on sorted LECs and BECs. Four animal-matched pairs of LECs (gray bars) and BECs (white bars) showed consistent differential expression levels. Beta-actin was used for normalization of the expression levels. Error bars show standard deviation.
Figure 2
Figure 2. Semaphorin 3 family members are expressed specifically by lymphatic or blood vascular endothelium
(A) Specific expression of Sema3A and Sema3D by lymphatic endothelium, and of Sema3G by blood vascular endothelium, as assessed by qPCR analysis of ex vivo isolated mouse LECs and BECs. The semaphorin receptor Nrp-1 was expressed specifically by BECs and Nrp-2 levels were similar in LECs and BECs. (B) Higher expression of Sema3A and Sema3D by cultured human LECs than BECs and HCAECs. Beta-actin was used for normalization of the expression levels. Error bars show standard deviation. (C) Immunohistochemical analysis confirmed the expression of Sema3A in intestinal lymphatic vessels (arrowheads). Serial paraffin sections of mouse colon tissue were immunostained with antibodies for Sema3A or LYVE-1, or with control goat IgG. Asterisk depicts a LYVE-1-negative blood vessel that is also negative for Sema3A expression. Hematoxylin counterstain. Scale bars: 100 μm.
Figure 3
Figure 3. Nrp-1 is expressed on the developing collecting lymphatic vessel perivascular cells and on lymphatic valves
(A) Mouse embryonic mesenteries at E18.5, P3 and P6 were immunostained for Prox1 (magenta), CD31 (green), Nrp-1 (red) and DAPI (blue). The high-magnification single optical slice shows expression of Nrp-1 on lymphatic perivascular cells at E18.5, P3 and P6 (arrowheads). Additionally, at P6 there is an overlap of CD31 (green) and Nrp-1 on the lymphatic valve (arrows). Representative images of 4 mesenteries per stage are shown. MIP (maximum intensity projection) shows a 3D reconstruction of the image. Scale bars: 20 μm. (B) Immunofluorescence analyses of P7 mouse mesentery whole-mount preparations. The lymphatic markers VEGFR3 (magenta) and Prox1 (green) show stronger expression in the valve area (arrowheads) than in the remaining lymphatic vessel areas. Nrp-1 (red) signal was found on the artery (asterisks) and in the valve area. The lower panel represents a higher magnification of the upper panel. Images were obtained by confocal microscopy. Scale bars: 100 μm.
Figure 4
Figure 4. Blockade of Sema3A/Nrp-1 signaling leads to impaired lymphatic vessel function and abnormal morphology of developing collecting lymphatic vessels
(A) FITC-dextran (2000 kDa) was injected into the forelimb fat pad of control and of anti-Nrp-1A treated pups at day P5.5 to visualize the draining lymphatic vasculature. Low magnification images were taken through the skin and high magnification images were taken after removal of the skin to reveal the lymphatic vessels in more detail. Arrowheads point to the normal route of draining via the deep collecting lymphatic vessels. Arrows point to the alternative draining route via the superficial lymphatic vessel network. Scale bars: 500 μm. (B) Whole-mount preparations of mesenteries immunostained for CD31 (green) and VEGFR3 (magenta) were analyzed. When compared with control IgG-treated animals (first row) and anti-Nrp-1B-treated animals (second row), anti-Nrp-1A-treated animals had abnormally formed collecting lymphatic vessels (third row, arrows). Scale bars: 50 μm, n= 13–18 mice per group.
Figure 5
Figure 5. Smooth muscle cells (SMCs) aberrantly cover collecting lymphatic vessels after Sema3A/Nrp-1 signal blocking, and Sema3A inhibits migration of Nrp-1 expressing pericytes in vitro
(A) Alpha-smooth muscle actin (α-SMA, magenta) expressing SMCs normally sparsely cover lymphatic vessels and are absent from the valve areas that are visible by a stronger CD31 (green) staining (arrowheads). This phenotype was found in control and in anti-Nrp-1B antibody treated mice at P5.5. Anti-Nrp-1A treatment resulted in excessive coverage of collecting lymphatic vessels by SMCs in the valve areas (arrows). Scale bars: 50 μm. (B) Quantification of the valve areas covered by SMCs in P5.5 in utero treated mice. n= 2–3 mesenteries per group, bars show mean ± standard deviation. Cultured primary human pericytes express Nrp-1 as assessed by (C) immunofluorescence staining (scale bar: 10 μm), and by (D) qPCR where pericytes showed low CD31 levels and Nrp-1 levels comparable to those found in HUVEC (mean fold change ± standard deviation). (E) Sema3A prevents migration of pericytes to 1% FCS and fibronectin coating. This effect was blocked by incubation with the anti-Nrp-1A antibody but not with control IgG. A representative experiment (out of 3) is shown. Bars show mean values ± SEM, one-way ANOVA Tukey’s post test. **p<0.01.
Figure 6
Figure 6. Blocking Sema3A-Nrp-1 signal indirectly causes abnormal lymphatic valve morphology
(A) For lymphatic valve analysis, mesenteries were immunostained for integrin-alpha 9 (red) and fibronectin EIIIA (magenta). The white dotted line outlines the lymphatic vessels. Scale bars: 20 μm. (B) The total number of valves per mesentery and (C) the number of abnormal valves with excessive FNEIIIA deposition, integrin-alpha 9 mislocalization and ring-shaped leaflets per mesentery were quantified. n=2–3 mice per group, bars show mean values ± SD, one-way ANOVA Tukey’s post test. ***p < 0.001, ns=not significant.
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