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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:3215-3223.)
© 1997 American Heart Association, Inc.

Articles

Genotype-Specific Transcriptional Regulation of PAI-1 Expression by Hypertriglyceridemic VLDL and Lp(a) in Cultured Human Endothelial Cells

Xin-Nong Li; Hernan E. Grenett; Raymond L. Benza; Sossiena Demissie; Stephen L. Brown; Edlue M. Tabengwa; Sandra H. Gianturco; William A. Bradley; Gunther M. Fless; ; Francois M. Booyse

From the Divisions of Cardiovascular Disease X.-N.L., H.E.G., R.L.B., S.D., S.L.B., E.M.T., F.M.B.) and of Gerontology and Geriatric Medicine (S.H.G., W.A.B.), Department of Medicine, University of Alabama at Birmingham, and the Lipoprotein Study Unit, Department of Medicine, University of Chicago, Chicago, Ill (G.M.F.).

Correspondence to Francois M. Booyse, PhD, 809 Bevill Biomedical Research Building, University of Alabama at Birmingham, 845 19th St S, Birmingham, AL 35294-2170.

Abstract The hypothesized relationships between plasminogen activator inhibitor (PAI-1) genotypes, PAI-1 levels, and their potential regulation by hypertriglyceridemic (HTG) very low density lipoprotein (VLDL) and lipoprotein(a) [Lp(a)] was examined in a PAI-1 genotyped human umbilical vein endothelial cell (HUVEC) culture model system. Individual human umbilical veins were used to obtain cultured ECs and were genotyped for PAI-1 by using the HindIII restriction fragment length polymorphism (RFLP) as a marker for genetic variation. Digested genomic DNA, examined by Southern blot analysis and probed with an [-³²P]dCTP–labeled 2.2-kb PAI-1 cDNA, yielded three RFLPs designated 1/1 (22-kb band only), 1/2 (22- plus 18-kb bands), and 2/2 (18-kb band only). Individual PAI-1 genotyped HUVEC cultures were incubated in the absence or presence of HTG-VLDL (0 to 50 µg/mL) or Lp(a) (0 to 50 µg/mL) at 37°C for various times (4 to 24 hours), followed by analyses of PAI-1 antigen (by ELISA) and mRNA (by ribonuclease protection assay) levels, EC surface–localized plasmin generation assays, and nuclear run-on transcription assays. Secreted PAI-1 antigen levels were increased 2- to 3-fold by HTG-VLDL and 1.6 to 2-fold by Lp(a); mRNA levels were increased 3- to 4.5-fold by HTG-VLDL and 2.5- to 3.2-fold by Lp(a) compared with medium-incubated controls, primarily in the 2/2 PAI-1 genotype HUVEC cultures. Increases in PAI-1 mRNA induced by HTG-VLDL or Lp(a) could be abolished by coincubation with actinomycin D (2x10^-6 mol/mL) or puromycin (1 µg/mL). In addition, nuclear transcription run-on assays typically demonstrated that HTG-VLDL increased PAI-1 gene transcription rates by 5- to 6-fold and 4- to 5-fold, respectively, primarily in the 2/2 PAI-1 genotype HUVEC cultures compared with 1/1 PAI-1 genotype HUVEC cultures or medium-incubated controls. The positive control interleukin-1 increased both 2/2 and 1/1 PAI-1 mRNA levels by 5- to 6-fold. Increased PAI-1 antigen and mRNA expression were associated with a concomitant 50% to 60% decrease in plasmin generation. These combined results demonstrate the genotype-specific regulation of PAI-1 expression by HTG-VLDL and Lp(a) and further indicate that these risk factor–associated components regulate PAI-1 gene expression at the transcriptional level in cultured HUVECs. Results from these studies further suggest that individuals with this responsive 2/2 PAI-1 genotype may reflect the additional inherent potential for later HTG-VLDL- or Lp(a)-induced fibrinolytic dysfunction, resulting in the early initiation of thrombosis, atherogenesis, and coronary artery disease.

Key Words: fibrinolysis • PAI-1 gene regulation • hypertriglyceridemic VLDL • lipoprotein(a) • endothelial cells

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