Flow cytometric study of injuries in cultured endothelial cells by neutrophils of the inherited cataract rats.
We examined the mechanism of endothelial injuries in the inherited cataract rats (ICR), which have a number of age-associated spontaneous injuries in the aortic endothelium. Cell cycle traverse rate of endothelial cells of ICR was shorter than that of Wistar rats. The rate was estimated from bromodeoxyuridine (BrdU) incorporation into cell nuclei measured periodically after BrdU pulse labeling. Next we established the method for measurement of cultured endothelial cell injury by neutrophils with flow cytometry by assessing the regeneration of injured endothelial cells. By the use of the gate analysis method, contaminated neutrophils were excluded from the analysis. Endothelial cell injury by neutrophils of Wistar rats was detectable at 1 x 10(5) neutrophils (1 neutrophil to 1 endothelial cell) when stimulated with 10 ng/ml phorbol myristate acetate (PMA). Extent of injury increased with an increasing number of neutrophils and the concentration of a stimulator, PMA. We detected endothelial cell injury by ICR neutrophils not only when they were stimulated but also in a resting condition, and ICR neutrophils yielded more injury than Wistar rat neutrophils. Number of adhered neutrophils to endothelial cells and effects of plasma or lymphocytes were the same between two strain rats. Scavengers of hydrogen peroxide and singlet oxygen inhibited the ICR neutrophil-induced endothelial cell injury. These findings indicate that an increase of generation of excited oxygen species from neutrophils, particularly of singlet oxygen, may cause injury of endothelial cells in this specific strain of rats.
Masuda
M
M
Department of Clinico-laboratory Medicine, Kansai Medical University, Osaka, Japan.
Ihara
N
N
Komiyama
Y
Y
Murakami
T
T
Murata
K
K
D000818:Animals; D001011:Aorta / Q000473:pathology; D002084:Butylated Hydroxytoluene / Q000494:pharmacology; D002386:Cataract; D002453:Cell Cycle; D002478:Cells, Cultured; D003574:Cytochrome c Group / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000378:metabolism / Q000473:pathology*; D005434:Flow Cytometry; D016166:Free Radical Scavengers; D008297:Male; D009504:Neutrophils / Q000502:physiology*; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D013482:Superoxide Dismutase / Q000494:pharmacology
D003574:Cytochrome c Group; D016166:Free Radical Scavengers; D011480:Protease Inhibitors; D002084:Butylated Hydroxytoluene; D013482:Superoxide Dismutase