Genotype-specific transcriptional regulation of PAI-1 expression by hypertriglyceridemic VLDL and Lp(a) in cultured human endothelial cells.
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 [alpha-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 micrograms/mL) or Lp(a) (0 to 50 micrograms/mL) at 37 degrees 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 approximately 2- to 3-fold by HTG-VLDL and approximately 1.6 to 2-fold by Lp(a); mRNA levels were increased approximately 3- to 4.5-fold by HTG-VLDL and approximately 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 x 10(-6) mol/mL) or puromycin (1 microgram/mL). In addition, nuclear transcription run-on assays typically demonstrated that HTG-VLDL increased PAI-1 gene transcription rates by approximately 5- to 6-fold and approximately 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 approximately 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.
Li
X N
XN
Division of Cardiovascular Disease, University of Alabama at Birmingham 35294-2170, USA.
Grenett
H E
HE
Benza
R L
RL
Demissie
S
S
Brown
S L
SL
Tabengwa
E M
EM
Gianturco
S H
SH
Bradley
W A
WA
Fless
G M
GM
Booyse
F M
FM
D001161:Arteriosclerosis / Q000453:epidemiology / Q000235:genetics; D002478:Cells, Cultured; D003327:Coronary Disease / Q000453:epidemiology / Q000235:genetics; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D005342:Fibrinolysis; D014644:Genetic Variation; D005838:Genotype; D006801:Humans; D015228:Hypertriglyceridemia / Q000097:blood*; D017270:Lipoprotein(a) / Q000097:blood / Q000494:pharmacology*; D008079:Lipoproteins, VLDL / Q000097:blood / Q000494:pharmacology*; D017395:Plasminogen Activator Inhibitor 1 / Q000096:biosynthesis / Q000235:genetics*; D012150:Polymorphism, Restriction Fragment Length; D012333:RNA, Messenger / Q000096:biosynthesis; D012307:Risk Factors; D019851:Thrombophilia / Q000453:epidemiology / Q000235:genetics; D014158:Transcription, Genetic; D014471:Umbilical Veins; D014568:Urokinase-Type Plasminogen Activator / Q000032:analysis
D017270:Lipoprotein(a); D008079:Lipoproteins, VLDL; D017395:Plasminogen Activator Inhibitor 1; D012333:RNA, Messenger; D014568:Urokinase-Type Plasminogen Activator