Semaphorin 3d and semaphorin 3e direct endothelial motility through distinct molecular signaling pathways

Abstract

Class 3 semaphorins were initially described as axonal growth cone guidance molecules that signal through plexin and neuropilin coreceptors and since then have been established to be regulators of vascular development. Semaphorin 3e (Sema3e) has been shown previously to repel endothelial cells and is the only class 3 semaphorin known to be capable of signaling via a plexin receptor without a neuropilin coreceptor. Sema3e signals through plexin D1 (Plxnd1) to regulate vascular patterning by modulating the cytoskeleton and focal adhesion structures. We showed recently that semaphorin 3d (Sema3d) mediates endothelial cell repulsion and pulmonary vein patterning during embryogenesis. Here we show that Sema3d and Sema3e affect human umbilical vein endothelial cells similarly but through distinct molecular signaling pathways. Time-lapse imaging studies show that both Sema3d and Sema3e can inhibit cell motility and migration, and tube formation assays indicate that both can impede tubulogenesis. Endothelial cells incubated with either Sema3d or Sema3e demonstrate a loss of actin stress fibers and focal adhesions. However, the addition of neuropilin 1 (Nrp1)-blocking antibody or siRNA knockdown of Nrp1 inhibits Sema3d-mediated, but not Sema3e-mediated, cytoskeletal reorganization, and siRNA knockdown of Nrp1 abrogates Sema3d-mediated, but not Sema3e-mediated, inhibition of tubulogenesis. On the other hand, endothelial cells deficient in Plxnd1 are resistant to endothelial repulsion mediated by Sema3e but not Sema3d. Unlike Sema3e, Sema3d incubation results in phosphorylation of Akt in human umbilical vein endothelial cells, and inhibition of the PI3K/Akt pathway blocks the endothelial guidance and cytoskeletal reorganization functions of Sema3d but not Sema3e.

Keywords: Actin; Akt; Cell Motility; Endothelial Cell; Neuropilin; PI3K; Plexin; Semaphorin.

FIGURE 1.

Sema3d and Sema3e inhibit endothelial cell motility and tubulogenesis. a, tracks representing the migration paths of individual HUVECs incubated with Sema3d, Sema3e, or a vehicle control for 3 h. b, quantification of total distance (in micrometers) and maximum displacement (in micrometers) of HUVECs in each condition. ***, p < 0.001 (one-way ANOVA between groups, post hoc multiple comparisons, Tukey's test). c, photomicrographs of HUVECs seeded in Matrigel and incubated with Sema3d, Sema3e, Cytocholasin D, or a vehicle control for 8 h. Cytochalasin D is used as a positive control for the inhibition of tubulogenesis. d, quantification of the number of tubules formed per high-power field. Cyt D, Cytochalasin D. **, p < 0.01; ***, p < 0.001 (one-way ANOVA between groups, post hoc multiple comparisons, Tukey's test, n = 9). Scale bars = 50 μm (a) and 1 mm (c).

FIGURE 2.

Sema3d inhibits endothelial migration independently of Plxnd1. a, graph representing the percentage of Plxnd1^+/− endothelial cells that migrated through a porous transwell insert in the presence of Sema3d or Sema3e compared with a vehicle control. ***, p < 0.001 (one-way ANOVA between groups, post hoc multiple comparisons, Tukey's test, n = 3). b, graph representing the percentage of Plxnd1^−/− endothelial cells that migrated through a porous transwell insert in the presence of Sema3d or Sema3e compared with a vehicle control. **, p < 0.01; ns, not significant (one-way ANOVA between groups, post hoc multiple comparisons, Tukey's test, n = 3). c, photomicrographs of stained endothelial cells that migrated through a transwell membrane in the presence of vehicle (left panel), Sema3d (center panel), or Sema3e (right panel). Scale bar = 100 μm.

FIGURE 3.

Sema3d and Sema3e induce loss of actin stress fibers and down-regulate focal adhesion complexes. a, HUVECs were incubated with alkaline phosphatase-tagged Sema3d, Sema3e, or alkaline phosphatase alone (AP) as a control for 0, 15, 30, 45, and 60 min and stained for F-actin (red) and vinculin (green). b, quantification of the percentage of HUVECs displaying loss of actin stress fibers at each time point (n = 60 cells/condition). c, quantification of the number of vinculin units per HUVEC at each time point (n = 10 cells/condition). d, percentage of HUVEC adhesion to collagen I after a 30-min incubation with Sema3e, Sema3d, or a vehicle control as quantified by a colorimetric assay. *, p < 0.05; ***, p < 0.001 (one-way ANOVA between groups, post hoc multiple comparisons, Tukey's test, n = 5). Scale bars = 50 μm (smaller bar) and 5 μm (larger bar).

FIGURE 4.

Sema3d-mediated, but not Sema3e-mediated, cytoskeletal reorganization and inhibition of tubulogenesis is dependent on Nrp1. a, HUVECs were exposed to Sema3d, Sema3e, or a vehicle control with or without an anti-NRP-1 blocking antibody for 60 min and subsequently stained for F-actin (red) and vinculin (green). b, quantification of the percentage of HUVECs displaying an absence of actin stress fibers under each condition. ***, p < 0.001 (one-way ANOVA between groups, post hoc multiple comparisons, Tukey's test, n = 60 cells/condition). Ab, antibody. c, quantification of the percentage of HUVECs with loss of actin stress fibers after Nrp1 or control siRNA transfection incubated with Sema3d, Sema3e, or vehicle control. ***, p < 0.001 (one-way ANOVA between groups, post hoc multiple comparisons, Tukey's test, n = 60 cells/condition). Also shown is a Western blot analysis of Nrp1 protein expression in HUVECs after siRNA-mediated knockdown. d, photomicrographs of HUVECs after Nrp1 or control siRNA-mediated knockdown, seeding in Matrigel, and incubation with Sema3d, Sema3e, or a vehicle control for 8 h. e, quantification of the number of tubules formed per high power field. ***, p < 0.001 (one-way ANOVA between groups, post hoc multiple comparisons, Tukey's test, n = 9). Scale bars = 5 μm (a) and 1 mm (d).

FIGURE 5.

Sema3d signals through PI3K/Akt to repel endothelial cells via actin cytoskeletal reorganization. a, Western blot analysis for phospho-Akt (Ser-473) of HUVECs treated with Sema3d, Sema3e, or vehicle for 5 min. b, quantification of Akt phosphorylation normalized to total Akt of HUVECs incubated with 10 nm of Sema3d, Sema3e, or a vehicle control for 5 min. **, p < 0.01; ns, not significant (one-way ANOVA between groups, post hoc multiple comparisons, Tukey's test, n = 3). c, Western blot for phospho-Akt (Ser-473) of HUVECs treated with increasing doses of Sema3d for 30 min. d, top panel, graph representing the percentage of HUVECs that migrated through a porous transwell insert in the presence of Sema3d or Sema3e compared with a vehicle control. ***, p < 0.001 (one-way ANOVA between groups, post-hoc multiple comparisons, Tukey's test, n = 3). Bottom panel, graph representing the percentage of HUVECs that migrated through a porous transwell insert toward medium containing wortmannin and either Sema3d or Sema3e compared with a vehicle control. *, p < 0.05; ns, not significant (one-way ANOVA between groups, post hoc multiple comparisons, Tukey's test, n = 3). e, HUVECs were incubated with Sema3d, Sema3e, or a vehicle control with or without wortmannin (1 μm) for 30 min and subsequently stained for F-actin (red) and vinculin (green). DMSO, dimethyl sulfoxide. f, quantification of the percentage of HUVECs displaying an absence of actin stress fibers under each condition. ***, p < 0.001 (one-way ANOVA between groups, post hoc multiple comparisons, Tukey's test, n = 60 cells/condition). Scale bar = 5 μm.