title: Cilostazol strengthens barrier integrity in brain endothelial cells.
abstract: We studied the effect of cilostazol, a selective inhibitor of phosphodiesterase 3, on barrier functions of blood-brain barrier (BBB)-related endothelial cells, primary rat brain capillary endothelial cells (RBEC), and the immortalized human brain endothelial cell line hCMEC/D3. The pharmacological potency of cilostazol was also evaluated on ischemia-related BBB dysfunction using a triple co-culture BBB model (BBB Kit™) subjected to 6-h oxygen glucose deprivation (OGD) and 3-h reoxygenation. There was expression of phosphodiesterase 3B mRNA in RBEC, and a significant increase in intracellular cyclic AMP (cAMP) content was detected in RBEC treated with both 1 and 10 μM cilostazol. Cilostazol increased the transendothelial electrical resistance (TEER), an index of barrier tightness of interendothelial tight junctions (TJs), and decreased the endothelial permeability of sodium fluorescein through the RBEC monolayer. The effects on these barrier functions were significantly reduced in the presence of protein kinase A (PKA) inhibitor H-89. Microscopic observation revealed smooth and even localization of occludin immunostaining at TJs and F-actin fibers at the cell borders in cilostazol-treated RBEC. In hCMEC/D3 cells treated with 1 and 10 μM cilostazol for 24 and 96 h, P-glycoprotein transporter activity was increased, as assessed by rhodamine 123 accumulation. Cilostazol improved the TEER in our triple co-culture BBB model with 6-h OGD and 3-h reoxygenation. As cilostazol stabilized barrier integrity in BBB-related endothelial cells, probably via cAMP/PKA signaling, the possibility that cilostazol acts as a BBB-protective drug against cerebral ischemic insults to neurons has to be considered.
authors.affiliation: Department of Neuropsychiatry, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki, Japan.
authors.forename: Shoji
authors.initials: S
authors.lastname: Horai
authors.forename: Shinsuke
authors.initials: S
authors.lastname: Nakagawa
authors.forename: Kunihiko
authors.initials: K
authors.lastname: Tanaka
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Morofuji
authors.forename: Pierre-Oliver
authors.initials: PO
authors.lastname: Couraud
authors.forename: Maria A
authors.initials: MA
authors.lastname: Deli
authors.forename: Masaki
authors.initials: M
authors.lastname: Ozawa
authors.forename: Masami
authors.initials: M
authors.lastname: Niwa
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism; D000818:Animals; D001681:Biological Assay; D001812:Blood-Brain Barrier / Q000166:cytology* / Q000187:drug effects / Q000502:physiology*; D002196:Capillaries / Q000166:cytology; D002460:Cell Line; D000077407:Cilostazol; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3 / Q000235:genetics / Q000378:metabolism; D016572:Cyclosporine / Q000494:pharmacology; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D005947:Glucose / Q000172:deficiency; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology; D010100:Oxygen / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D013449:Sulfonamides / Q000494:pharmacology; D013777:Tetrazoles / Q000494:pharmacology*; D019108:Tight Junctions / Q000187:drug effects / Q000378:metabolism
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D007546:Isoquinolines; D012333:RNA, Messenger; D013449:Sulfonamides; D013777:Tetrazoles; D016572:Cyclosporine; D017868:Cyclic AMP-Dependent Protein Kinases; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; D005947:Glucose; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide; D000077407:Cilostazol; D010100:Oxygen