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

Abstract

16 17 Word count: 7849 18 19 Subject codes: angiogenesis, animal models of human disease, mechanisms, 20 vascular biology. 21 22 23 24 25 26 27 2 ABSTRACT 28 29 Excess estrogen signaling is associated with vascular malformations and pathologic 30 angiogenesis, as well as tumor progression and metastasis. Yet, how dysregulated 31 estrogen signaling impacts vascular morphogenesis in vivo remains elusive. Here 32 we use live imaging of zebrafish embryos to determine the effects of excess estrogen 33 signaling on the developing vasculature. We find that excess estrogens during 34 development induce intersegmental vessel defects, endothelial cell-cell 35 disconnections, and a shortening of the circulatory loop due to arterial-venous 36 segregation defects. Whole-mount in situ hybridization and qPCR analyses reveal 37 that excess estrogens negatively regulate Sonic hedgehog (Hh)/Vegf/Notch 38 signaling. Activation of Hh signaling with SAG partially rescues the estrogen-induced 39 vascular defects. Similarly, increased vegfaa bioavailability, using flt1/vegfr1 mutants 40 or embryos overexpressing vegfaa 165 , also partially rescues the estrogen-induced 41 vascular defects. We further find that excess estrogens promote aberrant endothelial 42 cell (EC) migration, possibly as a result of increased PI3K and Rho GTPase 43 signaling. Using estrogen receptor mutants and pharmacological studies, we show 44 that Esr1 and the G-protein coupled estrogen receptor (Gper1) are the main 45 receptors driving the estrogen-induced vascular defects. Mosaic overexpression of 46 gper1 in ECs promotes vascular disconnections and aberrant migration, whereas no 47 overt vascular defects were observed in mosaic embryos overexpressing wild-type or 48 constitutively active nuclear estrogen receptors in their ECs. In summary, 49 developmental estrogen excess leads to a mispatterning of the forming vasculature.50 Gper1 can act cell-autonomously in ECs to cause disconnections and aberrant 51 migration, whilst Esr signaling predominantly downregulates Hh/Vegf/Notch signaling 52 leading to impaired angiogenesis and defective arterial-venous segregation. 53 54 55 3 56 vascular malformations 57 58 4 INTRODUCTION 59 Vascular development is largely conserved across species, and studies in fish, birds 60 and mammals have brought significant understanding into the main molecular 61 mechanisms orchestrating vascular morphogenesis 1 . Notochord-derived sonic 62 hedgehog (HH) induces vascular endothelial growth factor A (Vegfa) expression in 63 the ventral somites. VEGFA, via activation of kinase insert domain receptor 64 (KDR/VEGFR2), orchestrates angioblast differentiation and proliferation, as well as 65 vasculogenesis and angiogenesis during development and disease 1,2 . VEGFA-66 dependent activation of phosphatidylinositol 3-kinase (PI3K)/AKT and the mitogen-67 activated kinase (MAPK) pathways activates the Rho family of small GTPases Rac1, 68 Cdc42 and RhoA, to promote directional migration 3,4 . Furthermore, VEGF signaling 69 promotes arterial specific...