Establishment and characterization of an immortalized bovine glomerular endothelial cell line.
Bovine subcultures (second passage) of glomerular endothelial cells (GEN) isolated from one-year-old kidney were successfully transfected by recombinant plasmids containing the simian virus (SV)-40 T antigen (Tag) using a lipofectin-mediated procedure. One cell clone was selected, propagated and characterized. This clone can be grown in RPMI 1640 medium supplemented with 10% fetal calf serum. The advantage of this cell line is the cultivation of bovine GEN without the addition of fibroblast growth factor or a coating of fibronectin or gelatin on the culture plate. More than 80 passages were achieved and the doubling time was 32 h. The Tag was easily identified in transfected-GEN by indirect immunofluorescence. These cells weakly expressed factor VIII-related antigen, slightly took up acetylated-low density lipoprotein and secreted a detectable amount of angiotensin-converting enzyme. Immunocytochemical staining for UAE-1 was also positive. Moreover, oncoproteins, such as Ki-67 and p53, were expressed in these cells. Cell cycle analysis by flow cytometry revealed that the percentages of G1, S, and G2/M stages in cycling transfected-GEN culture in RPMI 1640 medium supplemented with 10% fetal calf serum were 34%, 52.9%, and 13.1%, respectively. The conditioned medium from confluent transfected-GEN stimulated [3H]thymidine incorporation into glomerular mesangial cells. This cell line may provide a useful tool for examining modulators of mesangial cell growth. Thus this cell line is the first immortalized bovine GEN that retain the morphologic, phenotypic, and functional characteristics of bovine GEN.
Nitta
K
K
Department of Medicine, Tokyo Women's Medical College, Japan.
Horiba
N
N
Uchida
K
K
Tsutsui
T
T
Horita
S
S
Murai
K
K
Kawashima
A
A
Yumura
W
W
Nihei
H
H
D000818:Animals; D000956:Antigens, Viral; D002417:Cattle; D002453:Cell Cycle; D002460:Cell Line; D004727:Endothelium / Q000166:cytology / Q000201:enzymology; D007678:Kidney Glomerulus / Q000166:cytology* / Q000201:enzymology; D007703:Peptidyl-Dipeptidase A / Q000378:metabolism; D013539:Simian virus 40 / Q000276:immunology; D014162:Transfection
D000956:Antigens, Viral; D007703:Peptidyl-Dipeptidase A