Semaphorin-4D Mediates Vascular Stabilization via Endothelial Plexin-B1 in Vivo

Abstract

<p>Objectives: To examine the role of Semaphorin-4D (Sema4D)-Plexin-B1 signaling in the recruitment of stem cells from human exfoliated deciduous teeth (SHED) as mural cells to stabilize the endothelial vessels in vivo.<br>Methods: Plexin-B1 in human umbilical vein endothelial cells (HUVECs) (ScienCell, CA, USA) was knocked down by target siRNA (Invitrogen) using Lipofectamine™ 3000 reagent (Invitrogen). Plexin-B1KD-HUVECs or Negative-HUVECs were suspended with (SHED) (AllCells, CA, USA) evenly with growth factor-reduced Matrigel (Corning#356231) with/without PDGFR-β inhibitor. The cell/Matrigel mixture was injected into the subcutaneous spaces of six to eight-week-old male severe combined immunodeficient (SCID/CB17) mice. Matrigel plugs were harvested on day 7 or day 14 after euthanization. H&E staining was performed to visualize the blood vessels within the Matrigel plugs. Immunofluorescence staining was conducted to examine SM22α, PDGFR-b, and NG2 expression patterns. The number of SM22α+SHED covered vessels (CD31+) was quantified using Image J software. Statistical analyses were conducted using GraphPad Prism 8 software (p=0.05) (GraphPad Software, Inc., San Diego, CA).<br>Results: Sema4D-treated HUVEC/SHED Matrigel plugs showed the highest levels of vascularization, as shown by the total number of perfused vessels at 7 and 14 days of implantation and a significantly higher number of vessels lined by SM22α positive SHED (SM22α+SHED) (p<0.05). Immunofluorescence staining for SM22α, PDGFR-b, and NG2 demonstrated the mural cell-like activity of SHED lining the endothelial vascular lumens. A significant reduction in mural cell recruitment was observed in the Sema4D treated group with Plexin-B1KD-HUVECs (P<0.05), confirming that Sema4D exerts its effects through endothelial Plexin-B1. When PDGFR-β was blocked, the effects of Sema4D on the recruitment of SHED as mural cells was significantly reduced (p<0.05), resulting in a significant reduction in perfused vessels (p<0.05).<br>Conclusions: Sema4D acts on endothelial Plexin-B1 to mediate the interaction between ECs and SHED and promotes the recruitment of SHED as mural cells via PDGF in vascular stabilization.<br></p>