Abstract 14530: The ADP Receptor P2Y12 Regulates Endothelial Tip Cell Behaviors Through Suppression of Notch-Flt1 Signaling During Vascular Development
Purinergic signaling is vasoactive and involved in some vascular pathological conditions. Deficiency of extracellular nucleotide phosphohydrolysis causes severe defect of cell infiltration in new vessel growth. Previous in vitro experiments on endothelial cells demonstrated that extracellular nucleotides could regulate their mitogenic and chemotactic behaviors; however, whether purinergic signaling in endothelial cells plays a role in angiogenesis and vascular development is not known.
We cloned zebrafish purinergic receptor P2Y, G-protein coupled, 12 (P2Y12) and identified it was specifically expressed in vascular endothelial cells with whole-mount in situ hybridization. Inhibition of P2Y12 signaling in Tg(fli1:egfp) embryos through knocking down of P2Y12 expression with morpholino resulted in defect in angiogenesis of intersegmental vessel (ISV) in 90.1% ± 2.7% of the embryos (n = 137). Blocking ADP-P2Y12 signaling with MRS2395 (P2Y12 antagonist) also led to ISV growth defect in 92.3% ± 2.8% of the embryos (n = 127). Long-term time-lapse confocal analysis of Tg(fli1a:negfp)y7;(kdrl:ras-cherry)s896 embryos showed this vessel deficiency was due to defect of tip cell proliferation and migration. In the embryos inhibition of ADP-P2Y12 signaling significantly upregulated the expression of both Notch ligand dll4 and Notch-induced vascular endothelial growth factor receptor flt1, demonstrating that Notch signaling was activated. Remarkably, knockdown of dll4 in P2Y12 morpholino injected embryos could rescue both the decreased ISV endothelial cell number and length. Moreover, knockdown of flt1 also effectively restored ISV growth in P2Y12 morphants.
In conclusion, the present study demonstrates the essential role of purinergic signaling in vascular development, and establishes a link between ADP-P2Y12 pathway and Notch-flt1 signaling in control of tip cell behaviors during angiogenesis.
- © 2013 by American Heart Association, Inc.