The effect of number of days in culture and plasminogen activator inhibitor-1 (PAI-1) 4G/5G genotype on PAI-1 antigen release by cultured human umbilical vein endothelial cells.
An insertion/deletion (4G/5G) polymorphism in what has been shown to be an enhancer/repressor binding site in the promoter region of the PAI-1 gene has been related to plasma PAI-1 activity. Transfection studies demonstrated increased interleukin-1 stimulated PAI-1 synthesis in cells containing the 4G sequence. To study this response in endothelial cells, first passage HUVEC from 26 umbilical cords were stimulated with interleukin-1 and tumor necrosis factor-alpha. PAI-1 antigen was measured in 24-hour conditioned medium and allele-specific PCR utilized to determine genotype at the 4G/5G locus. Analysis of covariance was used to determine whether the effect of a variable time in culture was masking a difference between genotypes. A trend towards higher PAI-1 levels with increasing time in culture was observed. The geometric mean (95% confidence interval) of the basal rate of PAI-1 release was, 4G/4G 9.7 (7.0, 13.5) ng/24 hours (n=11), 4G/5G 9.5 (6.5, 13.9) ng/24 hours (n=9), and 5G/5G 10.9 (7.8, 15.1) ng/24 hours (n=6). In cells of the same cultures, the interleukin-1 stimulated levels were 25.9 (23.1, 29.1), 27.2 (23.6, 31.3), and 23.1 (19.5, 27.3) ng/24 hours, respectively, corresponding to ratios of stimulated to basal levels of 2.68, 2.87, and 2.12. After adjustment for time in culture the basal PAI-1 release was 4G/4G 10.7, 4G/5G 9.1, and 5G/5G 9.7 ng/24 hours. For interleukin-1 stimulated release the adjusted levels were 26.3, 27.0, and 22.7 ng/24 hours, respectively. Adjusted levels in 4G/4G genotype cells were non-significantly greater than those in cells of 5G/5G genotype by a factor of 1.16 (0.95, 4.08). This study did not demonstrate a significant difference in basal or cytokine stimulated PAI-1 release from cells of different PAI-1 promoter (4G/5G) genotypes but does not exclude increased interleukin-1 stimulated PAI-1 release in the 4G/4G compared with the 5G/5G genotype.
McCormack
L J
LJ
Unit of Molecular Vascular Medicine, Division of Medicine, University of Leeds, United Kingdom. medljm@medphysics.leeds.ac.uk
Semple
J I
JI
Stickland
M H
MH
Barrett
J H
JH
Grant
P J
PJ
D000704:Analysis of Variance; D000941:Antigens / Q000378:metabolism; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D005838:Genotype; D006801:Humans; D007231:Infant, Newborn; D007375:Interleukin-1 / Q000494:pharmacology; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics* / Q000276:immunology / Q000502:physiology*; D015842:Serine Proteinase Inhibitors / Q000235:genetics* / Q000276:immunology / Q000502:physiology*; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014471:Umbilical Veins
D000941:Antigens; D007375:Interleukin-1; D017395:Plasminogen Activator Inhibitor 1; D015842:Serine Proteinase Inhibitors; D014409:Tumor Necrosis Factor-alpha