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Cilostazol Strengthens Barrier Integrity in Brain Endothelial Cells

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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.

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Acknowledgments

Cilostazol was kindly provided by Otsuka Pharmaceutical Co., Ltd., Japan. This work was supported in part by a Grant-in-Aid for Scientific Research (C) (22590243) and a Grant-in-Aid for Young Scientists (B) (24790258) from the Japan Society for the Promotion of Science (JSPS), Japan.

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Authors and Affiliations

  1. Department of Neuropsychiatry, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki, 852-8501, Japan

    Shoji Horai & Masaki Ozawa

  2. Department of Pharmacology, Nagasaki University Graduate School of Biomedical Sciences, 1-12-4 Sakamoto, Nagasaki, 852-8523, Japan

    Shinsuke Nakagawa, Kunihiko Tanaka, Yoichi Morofuji & Masami Niwa

  3. BBB Laboratory, PharmaCo-Cell Company Ltd., 1-43 Dejima, Nagasaki, 850-0862, Japan

    Shinsuke Nakagawa, Maria A. Deli & Masami Niwa

  4. Inserm U1016 Institute Cochin, CNRS UMR8104, Université Paris Descartes, Sorbonne Paris Cité, 22 rue Méchain, Paris, 75014, France

    Pierre-Oliver Couraud

  5. Laboratory of Molecular Neurobiology, Institute of Biophysics, Biological Research Center, Hungarian Academy of Sciences, Temesvári körút 62, Szeged, 6726, Hungary

    Maria A. Deli

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  1. Shoji Horai
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  2. Shinsuke Nakagawa
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  3. Kunihiko Tanaka
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Correspondence to Shinsuke Nakagawa.

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Horai, S., Nakagawa, S., Tanaka, K. et al. Cilostazol Strengthens Barrier Integrity in Brain Endothelial Cells. Cell Mol Neurobiol 33, 291–307 (2013). https://doi.org/10.1007/s10571-012-9896-1

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  • DOI: https://doi.org/10.1007/s10571-012-9896-1

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