Abstract 17980: Semaphorin3d and Semaphorin3e Signal Through Distinct Receptors to Modulate Motility of Endothelial Cells
Class 3 semaphorins were initially discovered to be axonal growth cone guidance molecules that signal through plexin and neuropilin coreceptors, and since have been established to be regulators of vascular development. Sema3e was previously shown to repel endothelial cells and is the only class 3 semaphorin capable of signaling via a plexin receptor without a neuropilin coreceptor. Sema3e signals through PlexinD1 to regulate vascular patterning by modulating the cytoskeleton and focal adhesion structures. Recently, we showed that Sema3d mediates endothelial cell repulsion and pulmonary vein patterning during embryogenesis. Here we show that both Sema3d and Sema3e signaling results in depolymerization of actin fibers and loss of focal adhesions when cultured with human umbilical vein endothelial cells (HUVECs), as measured by phalloidin and vinculin staining, respectively (n = 60 per group; Percentage of cells with loss of actin stress fibers and vinculin units per cell: control vs - Sema3d, P < 0.001; Sema3e, P < 0.001). Time lapse imaging of Sema3d or Sema3e exposed HUVECs reveals a statistically significant decrease in maximum displacement and total distance traveled by the cells (n=36 total number of cells; Total distance: control vs - Sema3d, P < 0.01; Sema3e, P < 0.001). Matrigel tube formation assays indicate that Sema3d and Sema3e decrease the total number of tubules formed in 8 hours (n=3 per group; tubules per HPF: control vs - Sema3d, P < 0.01; Sema3e, P < 0.001). However, the addition of Neuropilin-1 (Nrp-1) blocking antibody or siRNA knockdown of Nrp-1 inhibits Sema3d-mediated but not Sema3e-mediated depolymerization of actin (n=150 cells per group; Actin depolymerization with Nrp-1 siRNA knockdown or Nrp-1 antibody: control vs - Sema3d, NS; Sema3e, P < 0.001). In addition, siRNA knockdown of Nrp-1 eradicated Sema3d inhibition of tubulogenesis, but did not affect Sema3e-mediated inhibition of tubulogenesis. In conclusion, our study demonstrates that Sema3d and Sema3e can each mediate repellant guidance of endothelial cells and cytoskeletal reorganization, but they exert their effects via different receptors. In endothelial cells, Sema3e signals via PlexinD1 and is Nrp-1 independent, while Sema3d requires Nrp-1.
- © 2013 by American Heart Association, Inc.