S1PR1 regulates the quiescence of lymphatic vessels by inhibiting laminar shear stress–dependent VEGF-C signaling
Xin Geng, Keisuke Yanagida, Racheal G. Akwii, Dongwon Choi, Lijuan Chen, YenChun Ho, Boksik Cha, Md. Riaj Mahamud, Karen Berman de Ruiz, Hirotake Ichise, Hong Chen, Joshua D. Wythe, Constantinos M. Mikelis, Timothy Hla, R. Sathish Srinivasan
Xin Geng, Keisuke Yanagida, Racheal G. Akwii, Dongwon Choi, Lijuan Chen, YenChun Ho, Boksik Cha, Md. Riaj Mahamud, Karen Berman de Ruiz, Hirotake Ichise, Hong Chen, Joshua D. Wythe, Constantinos M. Mikelis, Timothy Hla, R. Sathish Srinivasan
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Research Article Development Vascular biology

S1PR1 regulates the quiescence of lymphatic vessels by inhibiting laminar shear stress–dependent VEGF-C signaling

Abstract

During the growth of lymphatic vessels (lymphangiogenesis), lymphatic endothelial cells (LECs) at the growing front sprout by forming filopodia. Those tip cells are not exposed to circulating lymph, as they are not lumenized. In contrast, LECs that trail the growing front are exposed to shear stress, become quiescent, and remodel into stable vessels. The mechanisms that coordinate the opposed activities of lymphatic sprouting and maturation remain poorly understood. Here, we show that the canonical tip cell marker Delta-like 4 (DLL4) promotes sprouting lymphangiogenesis by enhancing VEGF-C/VEGF receptor 3 (VEGFR3) signaling. However, in lumenized lymphatic vessels, laminar shear stress (LSS) inhibits the expression of DLL4, as well as additional tip cell markers. Paradoxically, LSS also upregulates VEGF-C/VEGFR3 signaling in LECs, but sphingosine 1-phosphate receptor 1 (S1PR1) activity antagonizes LSS-mediated VEGF-C signaling to promote lymphatic vascular quiescence. Correspondingly, S1pr1 loss in LECs induced lymphatic vascular hypersprouting and hyperbranching, which could be rescued by reducing Vegfr3 gene dosage in vivo. In addition, S1PR1 regulates lymphatic vessel maturation by inhibiting RhoA activity to promote membrane localization of the tight junction molecule claudin-5. Our findings suggest a potentially new paradigm in which LSS induces quiescence and promotes the survival of LECs by downregulating DLL4 and enhancing VEGF-C signaling, respectively. S1PR1 dampens LSS/VEGF-C signaling, thereby preventing sprouting from quiescent lymphatic vessels. These results also highlight the distinct roles that S1PR1 and DLL4 play in LECs when compared with their known roles in the blood vasculature.

Authors

Xin Geng, Keisuke Yanagida, Racheal G. Akwii, Dongwon Choi, Lijuan Chen, YenChun Ho, Boksik Cha, Md. Riaj Mahamud, Karen Berman de Ruiz, Hirotake Ichise, Hong Chen, Joshua D. Wythe, Constantinos M. Mikelis, Timothy Hla, R. Sathish Srinivasan

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Figure 4

S1PR1 does not inhibit VEGF-C signaling in statically cultured HLECs, and it is not necessary for canonical LSS response.

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S1PR1 does not inhibit VEGF-C signaling in statically cultured HLECs, an...
(A and B) S1PR1 does not regulate VEGF-C signaling in statically cultured HLECs. (A) HLECs were treated with 100 ng/mL VEGF-C in the presence or absence of S1PR1 agonist SEW2871 or antagonist W146. SEW2871 did not cause any obvious differences in VEGF-C signaling (phosphorylation of ERK and AKT). W146 slightly reduced the VEGF-C signaling. (B) HLECs were transfected with control siRNA or siS1P1 and then treated with the indicated concentrations of VEGF-C. No obvious differences were observed in VEGF-C signaling between control and siS1PR1-treated HLECs. n = 3 for A; n = 4 for B. (C–F) S1PR1 does not regulate canonical LSS-responses in HLECs. (C) Exposure of HLECs to LSS (5 dynes/cm2) for 10 minutes activated the phosphorylation of ERK and AKT. Prolonged exposure (30–60 minutes) to LSS caused the phosphorylation of ERK and AKT to return to background levels. (D) LSS-induced phosphorylation of ERK, AKT, P38, and eNOS was not affected by siS1PR1. (E) Shear stress responsive genes KLF2 and KLF4 were mildly reduced in siS1PR1-treated HLECs cultured under static conditions. Both KLF2 and KLF4 were upregulated in both control and siS1P1-treated HLECs that were cultured with LSS for 24 hours. A slight reduction in KLF4 expression was observed in siS1PR1-treated HLECs compared with controls. (F) siS1PR1 treatment caused the elongation of statically cultured HLECs. However, both control and siS1PR1-transfected HLECs align normally in the direction of LSS. n = 3 for C, E, and F; n = 4 for D. *P < 0.05. Data are presented as mean ± SEM. Scale bar: 100 μM.