Quercetin induced tissue-type plasminogen activator expression is mediated through Sp1 and p38 mitogen-activated protein kinase in human endothelial cells.

BACKGROUND: Wine polyphenol quercetin upregulates tissue-type plasminogen activator (t-PA) transcription in cultured human umbilical cord vein endothelial cells (HUVECs). However, the regulatory elements and signaling pathways involved in this regulation are unknown. OBJECTIVES: We aimed to localize quercetin-responsive t-PA promoter elements, identify the proteins that bind these elements, and decipher signaling pathways involved in the regulation of t-PA. METHODS: To localize quercetin-responsive elements, HUVECs were transiently transfected with various t-PA promoter-reporter constructs. Element functionality was evaluated by mutational analysis. Nuclear protein-t-PA element interactions were evaluated by electrophoretic mobility shift assays (EMSAs) and chromatin immunoprecipitation (ChIP) analysis. Mitogen-activated protein kinase (MAPK) inhibitors were used to determine the signaling pathways involved in t-PA regulation. MAPK inhibition effects were evaluated by real-time PCR, immunoblotting analysis, and transfections. Coimmunoprecipitation was used to evaluate MAPK and transcription factor interaction. RESULTS: Deletion of the t-PA promoter region - 288 to - 250 resulted in loss of quercetin responsiveness. This region contains putative Sp1-binding elements, which we termed Sp1a and Sp1b. Sp1b mutation abolished the quercetin-inducible response, whereas Sp1a mutation had no effect. EMSA and ChIP analysis demonstrated quercetin-enhanced Sp1 binding to Sp1b. Inhibition of p38 MAPK abrogated basal and quercetin-induced t-PA expression and promoter activity, as well as quercetin-induced Sp1 binding to Sp1b. Quercetin enhanced p38 MAPK and Sp1 physical association, which was similarly diminished by p38 MAPK inhibition. CONCLUSIONS: We showed, for the first time, the presence of a functional Sp1-binding element in the t-PA promoter controlling quercetin induction via the p38 MAPK pathway. Understanding these mechanisms may provide new insights into polyphenol cardioprotective effects.

PCR-RFLP genotyping assay for the Bcl I polymorphism of the beta-fibrinogen gene.

Polymorphisms of the beta-fibrinogen gene have been shown to affect plasma fibrinogen levels and risk of coronary artery disease (CAD). We were interested in developing an automated, PCR-based genotyping assay for the purpose of exploring relationships between CAD and CAD-associated aortic stiffness and the Bcl I allele of the beta-fibrinogen gene. We have developed a rapid PCR-RFLP assay for the Bcl I polymorphism of the beta-fibrinogen gene. We carried out direct PCR of genomic DNA to facilitate sequencing of the flanking region of the beta-fibrinogen gene. Using this new sequence information, primers were designed which border the site of the Bcl I polymorphism. One of the primers was labeled with a fluorophore to facilitate detection of the fragments. DNA fragment analysis was carried out using an automated capillary electrophoresis instrument (ABI310). We have developed an improved PCR-RFLP high-sample-throughput assay for the semiautomated detection of the Bcl I polymorphism of the beta-fibrinogen gene. This assay will support screening of large sample sizes required for population studies.

Identification of a 251-bp fragment of the PAI-1 gene promoter that mediates the ethanol-induced suppression of PAI-1 expression.

BACKGROUND: Moderate alcohol consumption reduces the risk for coronary heart disease. This cardioprotection may be due to ethanol enhancement of fibrinolysis. Fibrinolysis involves the interaction of plasminogen activators (PAs) and the plasminogen activator inhibitor type-1 (PAI-1). Factor(s) that decrease endothelial cell (EC) PAI-1 expression increase fibrinolysis and may decrease the risk for cardiovascular disease. METHODS: Five promoter deletion fragments were generated from a 1.1-kb PAI-1 promoter fragment and ligated to a luciferase reporter gene. Cultured human umbilical vein endothelial cells (HUVECs) were transiently transfected with these PAI-1 deletion constructs. A 251-base pair (bp) fragment of the PAI-1 promoter, positions -800 to -549, was cloned upstream of a heterologous promoter/enhancer. ECs luciferase activity was measured in the absence/presence of 20 mM ethanol. Electrophoresis mobility shift assays were performed with nuclear extracts from untreated and ethanol-treated ECs using this 251-bp fragment. RESULTS: Deletion analysis showed a region between position -800 and -549 mediated ethanol repression of luciferase activity. This 251-bp promoter fragment also repressed the activity of a heterologous promoter/enhancer in the presence of ethanol. Using the labeled 251-bp fragment, nuclear extracts from ethanol-treated ECs contained two inducible bands and one enhanced band. Non-ethanol treated nuclear extracts also contained a band that was not observed in ethanol-treated samples. Competition using 100-fold molar excess of unlabeled probe abolished these four bands. CONCLUSIONS: Repression of PAI-I gene transcription in cultured HUVECs exposed to ethanol may involve the interaction of several transcription factors with binding sites localized between positions -800 and -549 of the PAI-1 gene promoter.

Ethanol-induced increased surface-localized fibrinolytic activity in cultured human endothelial cells: kinetic analysis.

BACKGROUND: Moderate alcohol consumption is associated with reduced risk for coronary heart disease and this cardioprotection may be due, in part, to increased fibrinolysis. We have previously demonstrated that low concentrations of ethanol (0.1%, v/v) induce the short-term (<1 hr) and sustained, long-term (24 hr) increase in surface-localized fibrinolytic activity; it up-regulates t-PA, u-PA, and the candidate plasminogen receptor (PmgR), annexin II, and gene transcription in cultured human umbilical vein endothelial cells (HUVECs). These studies describe the short- and long-term effects of low concentrations of ethanol on the kinetics of cell-bound 125I-labeled Glu-plasminogen (Glu-Pmg) activation by receptor (R)-bound t-PA, resulting in increased fibrinolytic activity in cultured HUVECs. METHODS: Live cultured HUVECs were incubated with varying concentrations of Glu-Pmg (0.25-2 /M) and ethanol (0.025-0.1%, v/v) (in the presence of Aprotinin and alpha2-antiplasmin) and the direct activation of cell-bound 125I-labeled Glu-Pmg quantitated by measurement of 125I-labeled Mr 20 kDa plasmin light-chain, after reduction/SDS-PAGE. The effects of ethanol on '25I-labeled Glu-Pmg and t-PA ligand binding were determined by Scatchard analysis (Bmax, sites/cell). RESULTS: Cell-bound t-PA (endogenous/exogenous) activation of cultured HUVEC-bound 125I-labeled Glu-Pmg (short- and long-term) obeyed Michaelis-Menten kinetics, both in the absence/presence of low ethanol, as shown by Lineweaver-Burke plot analysis. In the short-term, ethanol (at 0.1%) increased the Vmax (2.5-fold), kcat (2-fold) and the apparent kcat/Km (4-fold), commensurate with a significant decrease in the apparent Km (6-fold) and increase in '25I-labeled Glu-Pmg ligand binding, Bmax (2-fold). In the long-term, ethanol increased the Vmax (2- to 3-fold), kcat (2.5-fold), apparent kcat/Km (5-fold), and Bmax (2-fold) for 125I-labeled Glu-Pmg ligand binding, without significantly affecting the apparent K . CONCLUSIONS: Low concentrations of ethanol induce the short- versus long-term increase in surface-localized fibrinolytic activity in cultured HUVECs via different mechanisms. Short-term effects may be mediated by ethanol-induced membrane conformational changes that simultaneously facilitate increased surface-localized HUVEC PmgR availability and fibrinolytic protein/receptor interactions, resulting in the increased affinity of t-PA for Glu-Pmg and the accelerated activation of Glu-Pmg (increased Bmax, decreased apparent Km). The long-term effects may be attributed primarily to the ethanol-induced increased availability of both newly synthesized t-PA and PmgR and, hence, the accelerated activation of Glu-Pmg (increased Bmax)

Polyphyenolics increase t-PA and u-PA gene transcription in cultured human endothelial cells.

BACKGROUND: Moderate red wine consumption has been associated with a reduced risk for coronary heart disease, and this cardioprotection may be mediated, in part, by promoting fibrinolysis. This protection may be attributed to the combined or perhaps synergistic effects of alcohol and other red wine components (i.e., polyphenolics). These studies were carried out to determine whether individual phenolics (i.e., catechin, epicatechin, quercetin, and resveratrol) affect fibrinolytic protein (tissue-type plasminogen activator [t-PA] and urokinase-type PA [u-PA]) expression and surface-localized fibrinolytic activity in cultured human umbilical vein endothelial cells (HUVECs). METHODS: Cultured HUVECs were preincubated (1 hr, 37 degrees C) in the absence or presence of varying concentrations of catechin, epicatechin, quercetin, and resveratrol (0.001-10 microM) and then were washed and incubated for various times in the absence of phenolics. Secreted t-PA/u-PA antigen (24 hr, enzyme-linked immunoadsorbent assay) and mRNA [0-16 hr, reverse transcription-polymerase chain reaction(RT-PCR)] levels and fibrinolytic activity (direct activation of HUVEC-bound 125I-labeled glutamylplasminogen, quantitation of 125I-labeled Mr 20 kDa plasmin light-chain) were measured. Transient transfections of cultured HUVECs were carried out with the pt-PA222/luc and pu-PA236/luc promoter constructs, by using lipofectamine. RESULTS: Each of the phenolics similarly increased t-PA and u-PA antigen (2- to 3-fold) and mRNA (3- to 4-fold) levels, concomitant with an increase (2- to 3-fold) in sustained (24 hr), surface-localized fibrinolytic activity. Transcription inhibitor actinomycin D abolished the induction of t-PA and u-PA mRNA expression by these phenolics. Transfections with the pt-PA222/luc and pu-PA236/luc promoter constructs showed 2- to 3-fold and 2- to 4-fold increases in luciferase activity for t-PA and u-PA, respectively. CONCLUSIONS: 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.

Ethanol-induced up-regulation of the urokinase receptor in cultured human endothelial cells.

BACKGROUND: Moderate alcohol consumption has been correlated to reduced coronary artery disease (CAD) risk and mortality. This alcohol effect may be mediated in part by an increased endothelial cell (EC) fibrinolysis. ECs synthesize fibrinolytic proteins, tissue plasminogen activator (t-PA), urokinase type plasminogen activator (u-PA), and plasminogen activator inhibitor type-1(PAI-1). In addition, they synthesize and regulate receptors for fibrinolytic proteins, namely (t-PA and plasminogen receptor) Annexin II and u-PA receptor (u-PAR). These receptors play an important role in the regulated expression of receptor-bound plasminogen activator conversion of receptor-bound plasminogen to receptor-bound plasmin on the EC surface (surface-localized fibrinolytic activity). Therefore, systemic factors, such as ethanol, that affect the level, or activity or interaction of one or more of these components, resulting in the increased expression of surface-localized EC fibrinolytic activity, will be expected to reduce the risk for thrombosis, CAD, and myocardial infarction (MI). We have previously shown that low ethanol up-regulates t-PA and u-PA gene transcription, while it down-regulates PAI-1, hence resulting in increased (sustained, 24 hr) surface-localized EC fibrinolytic activity. The current studies were carried out to determine whether low ethanol increased u-PAR expression in cultured human umbilical cord vein ECs (HUVECs). METHODS: Cultured HUVECs were preincubated (1 hr) in the absence/presence of ethanol (0.025-0.2%, v/v); u-PAR mRNA (RT-PCR), antigen (western blot), and activity (125I-u-PA ligand binding/Scatchard analysis) levels were then measured after 0-24 hr. To determine whether the ethanol-induced changes in the u-PAR expression were transcriptional, transient transfection studies were carried out using a u-PAR/ luciferase promoter construct (pu-PAR120/luc [1.2-kb u-PAR promoter fragment ligated to a promoterless luciferase vector]). RESULTS: uPAR mRNA levels increased 2- to 3-fold and antigen levels (western blot) increased 2- to 4-fold while u-PA binding activity increased 36% (1.25 vs. 1.7 x 10(5) sites/cell, Bmax) without significantly affecting the Kd (1-2 nM). Transient transfection of cultured HUVECs with a pu-PAR120/luc construct resulted in a 2- to 3-fold increase in promoter activity in ethanol-induced cultures, compared with controls. CONCLUSION: These combined results demonstrate that low ethanol (< or =0.1%, v/v) induces the up-regulation of u-PAR gene transcription, resulting in increased u-PAR ligand binding activity. These results also further identify/define the contribution and role of another fibrinolytic protein in the overall ethanol-induced increase in surface-localized EC fibrinolysis that may underlie and contribute, in part, to the cardioprotection attributed to moderate alcohol consumption.

Ethanol-induced up-regulation of candidate plasminogen receptor annexin II in cultured human endothelial cells.

INTRODUCTION: Epidemiological studies indicate that moderate alcohol consumption reduces the risk for coronary heart disease and that this cardioprotective benefit may be mediated, in part, by increased fibrinolysis. Endothelial cells (ECs) synthesize plasminogen activators, tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA), receptors for plasminogen activators, and a receptor for plasminogen, annexin II (Ann-II). These receptors localize and facilitate receptor-bound plasminogen activator-mediated conversion of receptor-bound plasminogen to receptor-bound plasmin on the EC surface, which results in the regulated expression of surface-localized EC fibrinolytic activity. Ethanol is a systemic factor that affects these components, which increases EC fibrinolysis and hence reduces the risk for thrombosis, coronary heart disease, and myocardial infarction (MI). METHODS: This study was carried out to determine whether low ethanol (0.1% v/v) increased plasminogen receptor, Ann-II antigen (western blot), messenger ribonucleic acid (mRNA) (reverse transcription polymerase chain reaction; RT-PCR) expression, activity (ligand binding/Scatchard analysis), and hence fibrinolysis (plasmin generation) in cultured human ECs. RESULTS: Plasminogen receptor activity increased approximately 2-fold (2.5 vs. 5.6 x 10(6) sites/cell), as evidenced by increased 125I-labeled Glu-plasminogen ligand binding/Scatchard analysis. In addition, western blot analyses indicated an increase in Ann-II antigen, and mRNA levels increased approximately 2-fold (RT-PCR). This increase in Ann-II expression was concomitant with approximately 2- to 3-fold sustained increase (approximately 24 hr) in surface-localized EC fibrinolytic activity. Nuclear transcription run-on assays showed an approximately 5- to 6-fold increase in new 32P-labeled Ann-II mRNA levels, compared with controls (no ethanol). CONCLUSIONS: These results demonstrated that low ethanol increased Ann-II antigen/mRNA levels and up-regulated Ann-II gene expression at the transcriptional level. The results further identify and define the contribution and role of the plasminogen receptor, Ann-II, in the ethanol-induced mechanism of increased EC fibrinolysis that may underlie and contribute, in part, to the cardioprotective benefit associated with moderate alcohol consumption.

Hypertriglyceridemic VLDL downregulates tissue plasminogen activator gene transcription through cis-repressive region(s) in the tissue plasminogen activator promoter in cultured human endothelial cells.

The relationship between tissue plasminogen activator (tPA) levels and the potential regulation by hypertriglyceridemic very low density lipoprotein (HTG-VLDL) was examined in a human umbilical vein endothelial cell (HUVEC) culture model system. HUVEC cultures were incubated in the absence/presence of HTG-VLDL or normal (NTG)-VLDL (0 to 50 microg/mL) at 37 degrees C for various times (0 to 24 hours), followed by analyses of tPA antigen (ELISA), mRNA (reverse transcription-polymerase chain reaction), endothelial cell surface-localized plasmin generation assays, and nuclear transcription run-on assays. Secreted tPA antigen levels decreased approximately 53% (3.3+/-0.14 versus 6.97+/-0.42 microg/mL) and mRNA levels decreased approximately 70% in HTG-VLDL-treated HUVECs compared with NTG-VLDL-treated and culture medium control cells. Decreased tPA antigen and mRNA expression was associated with a concomitant approximately 98% decrease in tPA-mediated plasmin generation in HTG-VLDL-treated HUVEC cultures. Nuclear transcription run-on assays demonstrated that HTG-VLDL decreased tPA gene transcription approximately 73% (tPA mRNA/GAPDH mRNA) in cultured HUVECs. To identify and localize the repressive element(s) in the tPA promoter responsive to HTG-VLDL, a tPA promoter/luciferase construct (ptPA222/luc) was generated. HUVECs transiently transfected with this construct were incubated in the absence/presence of HTG-VLDL or NTG-VLDL (20 microg/mL). HTG-VLDL decreased promoter activity approximately 52% to 57% in the ptPA222/luc-transfected cells compared with NTG-VLDL-treated or buffer control cells. These results indicate that the 2.2-kb fragment of the promoter and 5' flanking region of the tPA gene contains the repressive sequences that direct the transcriptional downregulation of the tPA promoter. Data from these studies suggest that the repression of tPA gene expression by HTG-VLDL may contribute to the impaired fibrinolysis often associated with hypertriglyceridemia.

Identification of the Hind III polymorphic site in the PAI-1 gene: analysis of the PAI-1 Hind III polymorphism by PCR.

The identification of the Hind III polymorphic site in the 3' end of the plasminogen activator inhibitor 1 (PAI-1) gene and a simple method to identify the Hind III polymorphism rapidly in the PAI-1 gene using PCR is described. The Hind III restriction site was identified by restriction site mapping and sequence analysis from a cosmid DNA clone. Genomic DNA was isolated from individual human umbilical cords and a 754-bp fragment of the human PAI-1 gene was amplified by PCR. Aliquots of the PCR products were digested with Hind III and analyzed by agarose gel electrophoresis. The presence of two fragments, 754 and 567 bp, was identified, and they were designated as 1/1 (750-bp band), 1/2 (754- and 567-bp bands), and 2/2 (567-bp band). The PCR method is considerably less time consuming than the conventional DNA genotyping using Southern blot analysis. To ensure that this new method identified the same PAI-1 genotypes as previously identified by Hind III restriction fragment length polymorphism (RFLP), samples were simultaneously genotyped by PCR and Southern blot analysis. Both methods identified the same Hind III genotypes in all the samples, confirming the reliability of this new PCR method for the rapid identification of the Hind III polymorphism in the human PAI-1 gene.

Ethanol downregulates transcription of the PAI-1 gene in cultured human endothelial cells.

Human endothelial cells are a major site of synthesis for plasminogen activator inhibitor type-1. Elevated plasminogen activator inhibitor type-1 levels in young survivors of myocardial infarction [1] suggest that plasminogen activator inhibitor type-1 may have an important pathologic role in the development of coronary artery disease. Epidemiological studies indicate that moderate alcohol consumption (1-2 drinks/day) reduces the risk for cardiovascular mortality. This cardioprotective benefit has been attributed in part to an increase in fibrinolysis, which decreases fibrin-based thrombosis. The studies described herein were performed to determine whether moderate levels of ethanol affect plasminogen activator inhibitor type-1 gene expression. Cultured human endothelial cells were exposed to 0.1% v/v ethanol for 1 hour. Following incubation in the absence of ethanol plasminogen activator inhibitor type-1, mRNA levels were decreased in a time- and dose-dependent manner, reaching a maximum decrease of 3- to 4-fold at 2 to 4 hours following ethanol challenge. This decline in mRNA occurs at the transcription level; therefore, nuclear transcription run-on assays were performed. A 2.5- to 5-fold decrease in the rate of plasminogen activator inhibitor type-1 gene transcription was measured at 2 and 4 hours following ethanol challenge. Next, a 3.4- and a 1.1-kb fragment from the plasminogen activator inhibitor type-1 promoter region were linked to a luciferase reporter gene, and these constructs were transfected into human endothelial cells. Treatment of these transiently transfected human endothelial cells with ethanol showed a 2- to 3.5-fold decrease in promoter activity, respectively. These results indicate that low doses of ethanol downregulate transcription of the plasminogen activator inhibitor type-1 gene in cultured human endothelial cells. However, the mechanism(s) for this transcriptional decrease is currently unknown.

Automated, PCR-RFLP genotyping of the urokinase gene.

The urokinase-type plasminogen activator (u-PA) has been suggested to play a role in the early initiation and progression of atherosclerosis and coronary artery disease (CAD) (Grenett et aL, 1998). Recently, a common genetic polymorphism in the untranslated region of the u-PA gene was shown to be associated with syptomatic CAD. To study the possible role of this common genetic polymorphism in the u-PA gene, we have developed an automated, PCR-based assay. Automation of the PCR-RFLP genotyping of the BamHI polymorphism of the urokinase gene will support the screening of the large sample sizes required to do the population-based studies necessary to uncover disease susceptibility associations.

Expression of plasminogen activator inhibitor type I in genotyped human endothelial cell cultures: genotype-specific regulation by insulin.

Patients with non-insulin-dependent diabetes mellitus frequently have been associated with elevation in plasma levels of PAI-1. Part of the variations in individual plasma PAI-1 levels have been attributed to variations in the PAI-1 gene. In order to determine whether insulin regulates PAI-1 expression in a genotype-specific manner, individual human umbilical vein ECs (HUVECs) were genotyped using a Hind III RFLP and incubated in the absence/presence of insulin. Treatment of 1/1 PAI-1 genotype HUVECs with insulin increased secretion of PAI-1 antigen approximately 1.7 to 2.2-fold and mRNA levels were increased approximately 1.8 to 2.8-fold. Treatment of HUVECs with actinomycin D or puromycin completely abolished the induction of PAI-1 by insulin. The nuclear run-on assays indicated approximately 3-4 fold increase in PAI-1 transcription rates. These in vitro studies with the 1/1 PAI-1 genotyped cultured HUVECs, suggests that hyperinsulinemia may be expected to increase EC PAI-1 synthesis in those patients with the responsive 1/1 genotype.

Endothelial cell fibrinolysis: transcriptional regulation of fibrinolytic protein gene expression (t-PA, u-PA, and PAI-1) by low alcohol.

Epidemiological studies have associated moderate alcohol consumption with a reduced risk for coronary artery disease (CAD) and myocardial infarction (MI). This cardioprotection may be attributed to alcohol-induced changes in a variety of cellular functions, including increased fibrinolysis. Fibrinolysis is important in regulating normal hemostasis. Endothelial cells (ECs) synthesize fibrinolytic proteins, t-PA, u-PA, and PAs inhibitor, PAI-1. Systemic factors, i.e., alcohol, that affect one or more of these components, resulting in increased EC fibrinolysis, will reduce the risk for thrombosis, CAD, and MI and afford cardioprotection. These studies will identify/define the effects of low ethanol (< 0.1%, v/v) on the expression of PAs, PAI-1, and surface-localized fibrinolytic activity in cultured ECs. Low ethanol exerted a short-term time- and dose-dependent increase (approximately 5- to 8-fold) in activity at approximately 20 min and 0.05% ethanol, which was sustained for approximately 1 hr. On the other hand, a single brief exposure to low ethanol (< 0.1%, < 120 min), followed by 4-24 hr incubation in the absence of ethanol, showed a time- and dose-dependent increase (approximately 2- to 3-fold) in PAs antigen/mRNA and a concomitant approximately 2- to 3-fold sustained increase (approximately 24 hr) in fibrinolytic activity. Further nuclear transcription run-on assays and transient transfection experiments, using pPAs/luc and pPAI-1/luc promoter constructs, demonstrated that low ethanol transcriptionally upregulates t-PA and u-PA gene expression and downregulates PAI-1 gene expression. These combined studies have described a feasible molecular mechanism by which low ethanol can induce and sustain increased surface-localized EC fibrinolysis that may underlie and contribute, in part, to the cardioprotective benefit associated with moderate alcohol consumption.

Gene polymorphisms for plasminogen activator inhibitor-1/tissue plasminogen activator and development of allograft coronary artery disease.

BACKGROUND: Impaired fibrinolytic activity has been linked to the presence and severity of allograft vasculopathy (Tx CAD). This impairment may be associated with the presence of certain fibrinolytic protein gene polymorphisms. METHODS AND RESULTS: To investigate the relation between donor-specific fibrinolytic protein genotypes and Tx CAD, we identified donor plasminogen activator inhibitor-1 (PAI-1) HindIII and tissue plasminogen activator (TPA) EcoRI restriction fragment length polymorphisms-based genotypes by Southern blot analysis in 48 recipients of cardiac allografts and correlated these genotypes with the development of CAD. No association was found between donor TPA genotypes and the presence of Tx CAD. Among the 48 patients, 17% were homozygous for the 1/1 PAI-1 genotype, 51% for the 2/2 PAI-1 genotype, and 32% for the 1/2 PAI-1 genotype. The actuarial freedom from any CAD for the recipients with each respective donor PAI-1 genotype at 12 and 24 months was 100% and 100% for the 1/1 PAI-1 genotype, 92% and 92% for the 1/2 PAI-1 genotype, and 75% and 45% for the 2/2 PAI-1 genotype (P=0.03). Recipients with a diseased 2/2 PAI-1 genotyped allograft had longer ischemic times (P=0.02) than those recipients with a Tx CAD-free allograft. CONCLUSIONS: These data suggest that recipients with a 2/2 PAI-1 genotype are at a significant risk of developing Tx CAD. This genotype may serve as a useful screening tool for predicting the future development of Tx CAD.

Genotype-specific transcriptional regulation of PAI-1 gene by insulin, hypertriglyceridemic VLDL, and Lp(a) in transfected, cultured human endothelial cells.

Plasminogen activator inhibitor-1 (PAI-1) has been shown to be an independent risk factor for coronary artery disease. Variations in plasma PAI-1 levels have been attributed to variations in the PAI-1 gene, and associations between PAI-1 levels and PAI-1 genotypes suggest that PAI-1 expression may be regulated in a genotype-specific manner by insulin, hypertriglyceridemic (HTG) very low density lipoprotein (VLDL), or lipoprotein(a) [Lp(a)]. Polymerase chain reaction-amplified 1106-bp fragments of the promoter of the 1/1 and 2/2 PAI-1 genotypes were sequenced and showed 5 regions of small nucleotide differences in the 1/1 versus 2/2 PAI-1 promoters that consistently occurred with high frequency. These fragments were ligated into the luciferase reporter gene, and 1/1 and 2/2 PAI-1 genotype human umbilical vein endothelial cell (HUVEC) cultures were transiently transfected with their respective p1PAI110/luc and p2PAI110/luc constructs and vice versa. Insulin induced an approximately 12- to 16-fold increase in luciferase activity in both the 1/1 and 2/2 PAI-1 genotype HUVEC cultures transfected with the p1PAI110/luc construct. HTG-VLDL and Lp(a) induced luciferase activity by approximately 14- to 16- and approximately 8- to 11-fold, respectively, in both the 1/1 and 2/2 PAI-1 genotype HUVEC cultures transfected with the p2PAI110/luc construct. The positive control interleukin-1 showed an approximately 7- to 12-fold response in the 1/1 and 2/2 PAI-1 genotype HUVEC cultures transfected with either of the constructs. These cross-over results demonstrate that regulation of either the 1/1 or 2/2 PAI-1 genotype by its respective inducer is due to the promoter itself and not to some factor(s) expressed differently in the 1/1 or 2/2 PAI-1 genotype HUVEC cultures.

Ethanol transcriptionally upregulates t-PA and u-PA gene expression in cultured human endothelial cells.

Epidemiological studies have suggested that moderate alcohol consumption reduces the risk of cardiovascular mortality. This cardioprotective benefit may be mediated, in part, by promoting fibrinolysis through changes in fibrinolytic components and/or activity, resulting in the decreased risk for thrombosis, coronary artery disease, and eventual myocardial infarction. Endothelial cells (ECs) play a pivotal role in maintaining normal hemostasis by regulating fibrinolysis through the synthesis of plasminogen activators (PAs), tissue-type plasminogen activator (t-PA), and urokinase-type plasminogen activator (u-PA). The studies described herein were conducted to determine whether a single brief preincubation (1 hr, 37 degrees C) of cultured human umbilical vein ECs (HUVECs) with low ethanol (0.1%, v/v), will upregulate t-PA and/or u-PA gene expression at the transcriptional level, using a combination of nuclear transcription run-on assays and transient transfections of cultured HUVECs with the pPA/luc promoter constructs. Nuclear run-on assays showed approximately 2- to 3-fold and approximately 6- to 7-fold increase in the transcription of new t-PA and u-PA mRNAs, respectively. In addition, transient transfections of cultured HUVECs with the pt-PA363/luc and pu-PA236/luc promoter constructs, using lipofectamine, demonstrated approximately 4- to 6-fold and approximately 6- to 9-fold increase in luciferase activity for t-PA and u-PA, respectively. These combined results demonstrate that low ethanol transcriptionally upregulates both t-PA and u-PA gene expression in cultured HUVECs and provides a molecular basis for the ethanol-induced increase in EC-mediated fibrinolytic activity that may underlie and contribute, in part, to the cardioprotective benefit associated with moderate alcohol consumption.

Alcohol-induced upregulation of plasminogen activators and fibrinolytic activity in cultured human endothelial cells.

Clinical studies suggest that moderate alcohol consumption may decrease the risk for coronary artery disease and myocardial infarction. This effect may be attributed, in part, to the alcohol-mediated increase in endothelial cell (EC)-mediated fibrinolytic activity mediated by the increase in synthesis and/or activity of tissue-type plasminogen activators (t-PAs) and/or urokinase-type PA (u-PAs). To determine whether low alcohol levels (0.01 to 0.1%, v/v) induced the expression of these proteins, cultured human saphenous vein ECs (HSVECs) were preincubated in the absence/presence of ethanol for 5 to 120 min at 37 degrees C, washed, refed, and further incubated for 8 and 24 hr without alcohol. PA mRNA (reverse transcriptase-polymerase chain reaction) and secreted antigen (ELISA) levels were analyzed after incubation for 8 and 24 hr and the net expression of (sustained) endogenous PA-mediated surface-localized HSVEC fibrinolytic activity (plasmin generation) quantitated by activation of 125I-Glu-plasminogen after incubation for 24 hr. A brief 5 to 30 min preincubation (induction) of both t-PA and u-PA antigen increased approximately 3-fold (t-PA control, 14.2 +/- 1.7, plus alcohol, 25.4 +/- 5 ng/ml; u-PA control, 15 +/- 0.8, plus alcohol, 46.4 +/- 1.3 ng/ml) and mRNA levels approximately 2-fold, as compared with controls. Increased PA expression was associated with a significant concomitant approximately 2-fold increase in surface-localized fibrinolytic activity (control, 96 +/- 2.8, plus alcohol, 255 +/- 42 fmol/ well). These combined results indicate that a brief exposure (<30 min) to low levels of alcohol can induce synthesis of EC-produced t-PA and u-PA resulting in an increased expression of HSVEC surface-localized fibrinolytic activity and may account, in part, for the apparent cardioprotective benefit associated with moderate alcohol consumption.

Ethanol increases surface-localized fibrinolytic activity in cultured endothelial cells.

Epidemiological studies demonstrated a positive association between moderate alcohol consumption and reduced cardiovascular mortality that may be mediated, in part, through increased fibrinolysis. These studies were conducted to determine whether low concentrations of alcohol (0.025 to 0.1%, v/v) directly affected the surface-localized versus secreted/solution phase fibrinolytic activity in live cultured endothelial cell (EC) types. Confluent live cultured ECs [human umbilical vein ECs (HUVECs), human saphenous vein ECs (HSVECs), and porcine aortic ECs (PAECs)] were preincubated (0 to 20 min, 4 degrees C) in the absence or presence of varying concentrations of alcohol (0 to 0.1%, v/v), in the presence of saturating levels of 125I-labeled Glu-plasminogem (2 microM) and 125I-Plasmin M(r) 20-kDA light-chain formation quantitated by phosphorimaging autoradiography analysis. Endogenous plasminogen activator (PA)-mediated fibrinolytic activity was time- and dose-dependent; reached a maximum approximately 5- to 10-fold increase at 0.05% alcohol in HUVECs, HSVECs, and PAECs; was completely inhibited by anti-t-PA IgG in HUVECs; and partially inhibited by both anti-t-PA (approximately 40%) and anti-u-PA IgG (approximately 60%) in HSVECs. Complete inhibition of alcohol-induced (0.05%) fibrinolytic activity in cultured HUVECs by 2 mM tranexamic acid (an antagonist of plasminogen binding) indicated that the increased fibrinolytic activity was receptor-bound and localized to the EC surface, rather than present in or secreted into the medium (solution phase). Finally, the alcohol-induced increased fibrinolytic activity in cultured HUVECs returned to essentially normal control levels in approximately 1 hr. These studies have demonstrated a direct effect of low alcohol on EC fibrinolytic activity that may contribute, in part, to the decreased risk for thrombosis, coronary artery disease, and myocardial infarction associated with moderate alcohol consumption.

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.