Adenoviral transduction of human E-selectin into isolated, perfused, rat aortic segments: an ex vivo model for studying leukocyte-endothelial interactions.
E-selectin, a member of the selectin family of adhesion molecules, is thought to play an important role in leukocyte-endothelial (EC) interactions during inflammation and atherosclerosis. To critically examine the role of E-selectin in leukocyte-EC interactions in the vascular system, we created a recombinant adenoviral vector containing a human E-selectin cDNA (AdRSVE-sel) and examined the effect of AdRSVE-sel in an ex vivo vascular model of a rat aortic segment. A segment of abdominal aorta was isolated from a male Sprague-Dawley rat transduced with AdRSVE-sel ex vivo. After 72 h, surface expression of transduced E-selectin in the segment was confirmed by Western blotting and immunohistochemistry using anti-E-selectin mAb. Aortic segments were connected to a perfusion system and the adhesion of human polymorphonuclear neutrophils (PMN), and a human monocytic cell line (THP-1) to the EC surface was studied in the presence of a physiological level of flow (0.85 ml/min, approximate luminal surface shear stress=1.76 dyn/cm2). Adhesion of PMN was assessed by scanning electron microscopy and quantified using fluorescently labeled PMN. AdRSVE-sel transduced aortic segments mediated significantly more PMN and THP-1 adhesion than control segments transduced with AdRSVLacZ. Pretreatment of AdRSVE-sel transduced aortic segments with anti-E-selectin mAb inhibited PMN adhesion significantly, as well as THP-1. These data indicate that human E-selectin expressed in rat aortic segments can support the adhesion of human PMN as well as THP-1 under physiological flow conditions. This genetically modified, excised, vascular-segment model provides a useful tool for the study of leukocyte recruitment in the vascular system.
Ishii
H
H
Department of Medicine, School of Medicine, Tokyo Medical and Dental University, Japan.
Yoshida
M
M
Rosenzweig
A
A
Gimbrone
M A
MA
Yasukochi
Y
Y
Numano
F
F
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D001012:Aorta, Abdominal / Q000378:metabolism; D002448:Cell Adhesion / Q000502:physiology; D002450:Cell Communication / Q000502:physiology*; D018076:DNA, Complementary / Q000235:genetics; D019040:E-Selectin / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D005822:Genetic Vectors; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D009504:Neutrophils / Q000166:cytology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D014161:Transduction, Genetic
D018076:DNA, Complementary; D019040:E-Selectin