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The role of endothelial cell on neutrophil transendothelial migration

Research Project

Project/Area Number 08671507
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Thoracic surgery
Research InstitutionAkita University

Principal Investigator

MINAMIYA Yoshihiro  Akita University School of Medicine Assistant Professor, 医学部, 講師 (30239321)

Co-Investigator(Kenkyū-buntansha) KITAMURA Michihiko  Akita University School of Medicine Associate Professor, 医学部, 助教授 (10153131)
ENOMOTO Katsuhiko  Akita University School of Medicine Professor, 医学部, 教授 (20151988)
泉 啓一  秋田大学, 医学部, 講師 (60176237)
Project Period (FY) 1996 – 1997
Project Status Completed (Fiscal Year 1997)
Budget Amount *help
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1997: ¥200,000 (Direct Cost: ¥200,000)
Fiscal Year 1996: ¥2,000,000 (Direct Cost: ¥2,000,000)
Keywordsneutrphil / endothelial cell / transendothelial migration / myosin light chain / myosin light chain kinase / ミオシン軽鎖中ナーゼ / ミオシン軽鎖 / ミオシン軽鎖キナーゼ / アクチン
Research Abstract

Although extravasation of neutrophils is a critical step in acute inflammation, the role of the endothelial cytoskeleton in neutrophil transmigration has not been fully investigated. We used an in vitro model of neutrophil transmigration across a monolayr of human umbilical endothelial cells (HUVEC) cultured on amniotic membrane. Human neutrophils were allowed to migrate across the HUVEC monolayr in response to a gradient leukotriene B_4 and then the number of migrated neutrophils were counted microscopically. We also followed endothelial F-actin and myosin filament formation using rhodamine-phalloidin and anti-myosin antibody staining. Myosin light chain (MLC) phosphorylation in endothelial cells was determined by immunoprecipitation of [^<32>P] labeled HUVEC with anti-myosin polyclnal antibody. Normally, neutrophil migration induced F-actin formation, myosin filament formation and MLC phosphorylation in HUVEC.When HUVEC was pretreated with the myosin light chain kinase (MLCK) inhibitor, ML-9, neutrophil migration was diminished and F-actin formation, myosin filament formation and MLC phosphorylation were inhibited. Pretreatments of HUVEC with the intracellular calcium ion chelator, bis-(O-aminophenoxyl) ethane-N,N,N', N'-tetraacetic acid acetoxymethyl ester (BAPTA/AM), and the calmodulin antagonist, trifluoperazine, had similar effects. These results indicate that a calcium/calmodulin-dependent MLCK in endothelial cells regulates neutrophil transendothelial migration.

Report

(3 results)
  • 1997 Annual Research Report   Final Research Report Summary
  • 1996 Annual Research Report

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Published: 1996-04-01   Modified: 2016-04-21  

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