Abstract 823: Cooperation of the EP2, EP3 and EP4 Receptors in Regulation of Prostaglandin E2-induced Human Brain Microvascular Endothelial Cell Migration
Prostaglandin E2 (PGE2) plays a critical role in angiogenesis as well as in ischemic and inflammatory disorders of the brain associated with breakdown of the blood-brain barrier. However, the effects of PGE2 on brain endothelial cell migration, a key process in the angiogenic response and blood-brain barrier stability, are not well defined. Herein, we show that exposure of human brain microvascular endothelial cells (HBMECs) to PGE2 stimulated migration in a dose- and time-dependent manner. Conversely, HBMECs failed to migrate when exposed to prostaglandin D1 or prostaglandin A2. The effects of PGE2 on HBMEC migration were extracellular matrix-dependent. The maximum migratory response of HBMECs to PGE2 was detected in the presence of fibronectin. HBMECs expressed the EP1, EP2, EP3 and EP4 receptor subtypes. Exposure of HBMECs to butaprost (an EP2 receptor agonist), sulprostone (an EP3 receptor agonist) or PGE1-OH (an EP4 receptor agonist) also stimulated HBMEC motility, suggesting the involvement of these receptors in regulation of HBMEC motility. Knockdown of the EP2 receptor by siRNA completely blocked the migratory response of HBMECs to butaprost and decreased PGE2-induced migration by 43% over control. SiRNA-mediated knockdown of the EP4 and EP3 receptor subtypes also decreased the migratory response of HBMECs to PGE2 by 35% and 27% over control, respectively. Pharmacological treatment with the EP1 antagonist, AH8906, had no effect on PGE2-induced HBMEC migration. Challenge of HBMECs with PGE2 activated protein kinase A (PKA), whereas pretreatment of cells with the selective PKA inhibitors, H89 (0.1 μM) or PKAI (1 μM), completely abrogated their ability to migrate in response to PGE2 but not to hepatocyte growth factor. These results are consistent with the hypothesis that PGE2 regulates HBMEC migration via cooperation of the EP2, EP3 and EP4 receptors leading to PKA activation and suggest an important role for PGE2 in regulation of HBMEC angiogenic responses. Elucidation of the molecular events that regulate migration of brain endothelial cells is critical for the development of targeted strategies to treat cerebrovascular diseases associated with dysregulated angiogenesis.