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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.

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These results demonstrate that each of these phenolics up-regulates both t-PA and u-PA gene transcription, which results in the sustained increased expression of surface-localized fibrinolytic activity in cultured HUVECs. Wine phenolics increase fibrinolytic activity, independent of ethanol, and it is likely that the overall cardioprotective benefits associated with moderate red wine consumption are attributable to the combined, additive, or perhaps synergistic effects of alcohol and other wine components.

SummaryArterial endothelial cells were obtained from bovine aortae by mild treatment with collagenase and medium perfusion. These cells were cultured in RPMI-1640 medium containing 15 mM Hepes buffer and 35% fetal calf serum at pH 7.35. Essentially ah (90–95%) the effluent cells were viable and 80% of these cells attached to the substratum within 1 hour. Small patches of attached cells coalesced to form confluent monolayers in 3–5 days. Confluent monolayers of endothelial cells consisted of a homogeneous population of tightly packed, polygonal cells. Selected cultures were serially subcultured (trypsin-EDTA) for 12–14 months (30–35 passages) without any apparent change in morphology or loss of growth characteristics. Primary and three-month old (15 passages) cultures had population doubling times of 32–34 hours and 29–31 hours, respectively. These cells (primary and subcultures) did not require a minimum cell number to become established in culture. Bovine endothelial cells (primary, first, fifth and thirteenth passages) were characterized ultrastructurally by the presence of Weibel-Palade bodies, pinocytotic vesicles and microfilaments and immunologically by the presence of thrombosthenin-like contractile proteins and Factor VIII antigen. The intercellular junctions of post-confluent cultures stained specifically with silver nitrate. From these data, we concluded that identifiable endothelial cells could be obtained from bovine aortae and cultured and maintained for prolonged periods of time.

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