（論文

要様式３）

旨C-type natriuretic peptide modulates permeability of theblood-brain barrier

血液脳関門の透過性に対するC 型ナトリウム利尿ペプチドの関与
Manoj Bohara(ボハラマノズ)【序論および目的】

Blood-brain barrier (BBB) functions for transport of molecules into/out of brain and is contributedby its tight junction proteins Zonula Occludens-1 (ZO-1), Claudin-5 and Occludin-1. C-typenatriuretic peptide (CNP) belongs to the natriuretic peptide family, together with atrial natriuretic
peptide (ANP) and brain natriuretic peptide (BNP). CNP potently stimulates guanylate cyclase
activity by binding to guanylate cyclase-B (GC-B) receptor which enhances the production of
cyclic GMP (cGMP). Despite the abundance of CNP in brain and regulatory function of CNP on
vascular cells, the effect of CNP on BBB permeability has not been clarified yet, which led us toundertake this study.
【材料および方法】

Three systems were used to evaluate the BBB permeability: i) in-vitro BBB constructed byco-culture of primary bovine brain microvascular endothelial cells (BBMEC) and primary
astrocytes, ii) commercially available in-vitro BBB kitTM composed of endothelial cells, astrocytes
and pericytes, and iii) in-vivo permeability using mice. Measurement of transendothelial electricalresistance (TEER), western blotting, RT-PCR, immunocytochemical analyses and siRNAtransfection were performed to check the effect of CNP on BBB and the underlying mechanism.
Fluorimetric analysis using sodium fluorescin and FITC-dextran was performed in mice toevaluate change of in-vivo BBB permeability. All procedures were approved by the Institutional
Animal Care and Use Committee of Kagoshima University (ID: MD11037) and were inaccordance with its guidelines.
【結果】

Transendothelial electrical resistance (TEER) of in-vitro BBB model, composed of bovine brain
microvascular endothelial cells and astrocytes, was significantly dose-dependently decreased by

CNP (1, 10 and 100 nM). CNP treatment reduced both the mRNA and protein expressions of tightjunction (TJ) protein zonula occludens-1 (ZO-1). The effects on TEER, mRNA and protein
expressions of ZO-1 were mimicked by cGMP analogue 8-bromo-cGMP (1 µM) and reversed byprotein kinase G (PKG) inhibitor Rp-8-CPT-cGMPS (100 µM), thus implying the role of PKG
and cGMP signaling in BBB function. Transcription factor JunD knockdown by small interfering
RNA resulted in no change of permeability by CNP. In-vivo study of mouse brain by fluorimetricanalysis with intravenous (i.v.) administration of sodium fluorescein (40 mg/kg) also showed asignificant increase in BBB permeability by CNP (10 nmol/kg, i.v.).
【結論及び考察】

Our findings in this study represent that CNP can effectively enhance the permeability of BBB
both in-vitro and in-vivo via cGMP/PKG and JunD dependent mechanism. The significant
enhancement of BBB permeability to sodium fluorescein can be considered to be of functionalsignificance as it mimics the permeation of various small molecular weight drugs across BBB.
Indeed, further experiments should be required to refine the system for delivery of therapeutic
compounds into the CNS. Nevertheless, the results we presented demonstrate the pivotal role of
CNP in modulating BBB permeability by altering ZO-1 expression and might form a basis fordeveloping a new drug-delivery system into brain.
(Journal of Cerebral Blood Flow and Metabolism, 2013 Dec 2 Accepted)