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 Vascular Endothelial Growth Factor C (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 1phosphate (S1P) 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 promoting membrane localization of the tight junction molecule Claudin-5. Our findings suggest a 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.

Unique functions for Notch4 in murine embryonic lymphangiogenesis

“…Lymphangiographies were performed as described on E17.5 embryos [15]. Briefly, 2 µL of 0.4% Trypan blue solution (Sigma) was injected into the dermis in periorbital region with a 36G beveled needle attached to a Nano l syringe (WPI).…”

“…In embryonic dermal lymphangiogenesis, Notch1 deletion in LECs did not affect lymphatic branching, but increased lymphatic vessel caliber which was proposed to be secondary to an increase in LEC proliferation and decreased LEC apoptosis [14]. More recently, it was shown that loss of one copy of Dll4 was associated with reduced embryonic dermal lymphangiogenesis in mice [15], a phenotype opposite to that seen in retinal angiogenesis [7,8]. Additional studies are needed to clarify the differences in the lymphangiogenic phenotypes observed upon disruption of lymphatic endothelial Notch signaling.…”

AIBP-CAV1-VEGFR3 axis dictates lymphatic cell fate and controls lymphangiogenesis

“…Although Notch signaling limits angiogenesis [69][70][71] , Notch has been reported to inhibit or augment lymphangiogenesis in a contextdependent fashion 38,39,72,73 . In addition, a recent study also documents the contrary role of two angiostatic components, DLL4 and S1PR1, in lymphangiogenesis 74 .…”

Homeostatic maintenance of the lymphatic vasculature

“…Conversely, silencing or pharmacological blockade of S1PR1 resulted in increased RHOA-ROCK activity, junctional destabilization, and transient loss of junctional CLDN5. Consequently, junctional architecture and CLDN5 localization in S1PR1-silenced or inhibitor-treated LECs was restored by pharmacological inhibition of ROCK [54]. The peptide hormone adrenomedullin has also been implicated as an inhibitor of RHOA-ROCK activity under inflammatory conditions through protein kinase A (PKA) and Ras-related protein 1 (RAP1) signaling, thereby mediating junctional stabilization of dermal LECs [55].…”

“…Laminar shear enhances proliferation and sprouting in cultured LECs via Notch inhibition as well as by activating Ca 2+ signaling and the flow-sensitive transcription factors Krüppel-like factor 2/4 (KLF2/4) [87,88]. Laminar shear also induces VEGF-C-independent activation of VEGFR3 [86], which is, however, antagonized by S1PR1 activity to promote LEC quiescence in lumenized vessels in vivo [54]. By contrast, OSS induces LEC quiescence through upregulation of FOXC2 [40,47], which promotes cell-cell junction integrity and quiescence by restricting TAZ-mediated proliferation [47] as well as MAPK activation [48].…”