Prostaglandin E² Stimulates Human Brain Endothelial Cell Migration Via a Signaling Pathway Involving Activation of Rho-Kinase II

Prostaglandin E₂ (PGE₂) plays a crucial 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. Exposure of human brain endothelial cells (HBECs) to PGE₂ elicited a chemotactic response in a time- and dose-dependent manner. The maximum migratory response was detected following 8 hours exposure of HBECs to PGE₂ (100 nM). Migration of HBECs in response to PGE₂ was accompanied by profound changes in the reorganization of actin filaments. Fluorescence microscopy examination of NBD-phallacidin-labeled endothelial cells showed increased formation of stress fibers, lamellipodia and podosomes after treatment with PGE₂ (100 nM) compared to control. Based on these results, we hypothesized that Rho-kinase (ROCK), an enzyme involved in regulation of actin dynamics and cell migration, mediated the effects of PGE₂ on HBEC migration. Western blot analyses revealed that ROCK II (type alpha), but not ROCK I (type beta), was expressed in HBECs. To examine ROCK II activation, we performed immunocomplex kinase assays using myosin light chain (MLC) as a substrate. PGE₂ (100 nM) induced a 2-fold increase of ³²P-incorporation into MLC indicating activation of ROCK II. Pretreatment of HBECs with the selective ROCK inhibitor, Y27632 (150 nM), blunted HBEC migration in response to PGE₂ but had no effect on migration induced by fetal bovine serum (10%). Knockdown of ROCK II by siRNA also abrogated the migratory response of HBECs to PGE₂. In contrast, similar treatment had no effect of HBEC migration stimulated by hepatocyte growth factor. Taken together, these results are consistent with the hypothesis that stimulation of HBECs with PGE₂ leads to activation of ROCK II, reorganization of the actin cytoskeleton and ultimately migration. A better characterization of the molecular events that regulate migration of brain endothelial cells is critical for the development of novel strategies to treat cerebrovascular diseases associated with deregulated angiogenesis.