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

Abstract

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 Hin dIII restriction fragment length polymorphism (RFLP) as a marker for genetic variation. Digested genomic DNA, examined by Southern blot analysis and probed with an [α- 32 P]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 (2×10 −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.