Endothelial and non-endothelial responses to estrogen excess during development lead to vascular malformations

ABSTRACT Excess estrogen signaling is associated with vascular malformations and pathologic angiogenesis, as well as tumor progression and metastasis. Yet, how dysregulated estrogen signaling impacts vascular morphogenesis in vivo remains elusive. Here we use live imaging of zebrafish embryos to determine the effects of excess estrogen signaling on the developing vasculature. We find that excess estrogens during development induce intersegmental vessel defects, endothelial cell-cell disconnections, and a shortening of the circulatory loop due to arterial-venous segregation defects. Whole-mount in situ hybridization and qPCR analyses reveal that excess estrogens negatively regulate Sonic hedgehog (Hh)/Vegf/Notch signaling. Activation of Hh signaling with SAG partially rescues the estrogen-induced vascular defects. Similarly, increased vegfaa bioavailability, using flt1/vegfr1 mutants or embryos overexpressing vegfaa 165 , also partially rescues the estrogen-induced vascular defects. We further find that excess estrogens promote aberrant endothelial cell (EC) migration, possibly as a result of increased PI3K and Rho GTPase signaling. Using estrogen receptor mutants and pharmacological studies, we show that Esr1 and the G-protein coupled estrogen receptor (Gper1) are the main receptors driving the estrogen-induced vascular defects. Mosaic overexpression of gper1 in ECs promotes vascular disconnections and aberrant migration, whereas no overt vascular defects were observed in mosaic embryos overexpressing wild-type or constitutively active nuclear estrogen receptors in their ECs. In summary, developmental estrogen excess leads to a mispatterning of the forming vasculature. Gper1 can act cell-autonomously in ECs to cause disconnections and aberrant migration, whilst Esr signaling predominantly downregulates Hh/Vegf/Notch signaling leading to impaired angiogenesis and defective arterial-venous segregation. Subject codes : angiogenesis, animal models of human disease, mechanisms, vascular biology.