Introduction of short interfering RNA to silence endogenous E-selectin in vascular endothelium leads to successful inhibition of leukocyte adhesion.
Short interfering RNAs (siRNAs) are powerful sequence-specific reagents that suppress gene expression in mammalian cells. We report for the first time that gene silencing of endothelial E-selectin by siRNAs leads to successful inhibition of leukocyte-endothelial interaction under flow. siRNAs designed to target human E-selectin were tranfected into human umbilical vein endothelial cells (HUVEC). Western blotting analysis revealed that transfection of these siRNAs, but not the scrambled control siRNA (100nM each), attenuated E-selectin expression in HUVEC activated with TNF-alpha (10ng/ml, 4h) without affecting expression of ICAM-1. Moreover, a leukocyte adhesion assay under flow (shear stress=1.0dyne/cm(2)) demonstrated that HUVEC transfected with a siRNA against E-selectin (siE-01) supported significantly less HL60 adhesion as compared to those transfected with the control siRNA (scE-01) after activation (p<0.03). This technique provides a powerful strategy to dissect a specific function of a given molecule in leukocyte-endothelial interaction.
Nishiwaki
Yasunobu
Y
Department of Medical Biochemistry, Tokyo Medical and Dental University, Tokyo, Japan.
Yokota
Takanori
T
Hiraoka
Megumi
M
Miyagishi
Makoto
M
Taira
Kazunari
K
Isobe
Mitsuaki
M
Mizusawa
Hidehiro
H
Yoshida
Masayuki
M
D002448:Cell Adhesion; D002460:Cell Line; D019040:E-Selectin / Q000235:genetics*; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D020868:Gene Silencing; D006801:Humans; D007962:Leukocytes / Q000166:cytology*; D034741:RNA, Small Interfering / Q000502:physiology*
D019040:E-Selectin; D034741:RNA, Small Interfering