Abstract P098: G-protein Coupled Estrogen Receptor Stimulates Capillary Formation by Human Umbilical Vein Endothelial Cells via ALK1-SMAD 1/5/8 Pathway Activation

Abstract

Estradiol (E2) induces vascular repair by promoting endothelial growth and capillary formation. Based on our previous findings that the capillary stimulating effects of E2 are mimicked by its non-permeable analog, BSA-tagged E2, we hypothesize that the stimulatory effects are potentially mediated via the newly discovered membrane bound G-protein coupled estrogen receptor (GPER). To investigate this, we assessed capillary formation by endothelial cells in response to E2, and GPER agonist (G1)and antagonist (G15) in a 2-D matrigel based assay. Capillary formation was assessed microscopically by quantifying junction/sprout formation and capillary length. E2 (10nM) increased capillary formation and this effect was mimicked by G1 (10nM; stimulated from 100% to 517±46% and 210±41%, respectively; p<.05 vs control). The effects of E2 and G1 were significantly abrogated by G15 (100nM; p<.05), suggesting a role for GPER in mediating the capillary stimulatory effects of E2. Because G-protein coupled mechanisms, Akt, ALK1 and SMAD1/5/8 are involved in capillary formation, we investigated their role in GPER induced capillary formation. ECs expressed GPER, ALK1 and SMAD1/5/8. Treatment with G1 (10-100nM) up-regulated the expression of ALK1 from 100% to 168±21% (p<.05 vs control) and phosphorylated SMAD1/5/8 from 100% to 208±36% (p<.05 vs control). Similar to capillary formation the stimulatory effects of G1 on SMAD1/5/8 were blocked significantly (p<.05) by G15 (100nM), Pertussis Toxin (0,1ng/ml; G protein pathway inhibitor), LY294002 (5μM; Akt/PI3K inhibitor) and ALK1Fc (100ng/ml; specific ALK-1 antagonist). Silencing of GPER and SMAD1 by siRNA (50nM) abrogated the stimulatory effects of E2 and G1 on capillary formation, and SMAD1/5/8 and Akt phosphorylation. Moreover, treatment with G1(100nM) upregulated ID-1 expression from 100% to 179±26% (p<.05 vs control), a downstream target of ALK1/pSMAD1/5/8. We conclude that E2 via GPER promotes EC-mediated capillary formation by a mechanism that involves non-genomic activation of ALK1→ pSMAD1/5/8 ↔ pAkt → ID-1. GPER agonists in general may promote healing of injured vascular beds by promoting EC activity leading to more rapid endothelial recovery and capillary formation following injury.