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(Circulation. 1995;92:767-772.)
© 1995 American Heart Association, Inc.

Articles

Attenuation of the Synthesis of Plasminogen Activator Inhibitor Type 1 by Niacin

A Potential Link Between Lipid Lowering and Fibrinolysis

Steven L. Brown, MD, PhD; Burton E. Sobel, MD; Satoshi Fujii, MD, PhD

From the Cardiovascular Division, Washington University School of Medicine, St Louis, Mo, and the Cardiovascular Division, the University of Vermont College of Medicine, Burlington.

Correspondence to Satoshi Fujii, MD, PhD, Cardiovascular Division, C-350 Given Bldg, University of Vermont, College of Medicine, Burlington, VT 05405.

	Abstract

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Background Plasminogen activator inhibitor type 1 (PAI-1), the primary physiological inhibitor of endogenous plasminogen activators, has been implicated as a potentiating factor in atherogenesis as well as in coronary thrombosis. We and others have observed attenuation of PAI-1 expression by gemfibrozil both in vivo and in vitro.

Methods and Results To determine whether other lipid-lowering agents with different mechanisms of action exert similar effects, we exposed Hep G2 cells, a highly differentiated human hepatoma cell line, to selected concentrations of niacin. Accumulation of PAI-1 protein, assayed with an ELISA, decreased in conditioned media by 72% in 48 hours in a specific, concentration-dependent fashion. Metabolic labeling experiments demonstrated a decrease in the rate of PAI-1 synthesis. Northern blot analysis demonstrated a preceding, parallel, and specific decrease in the concentration of PAI-1 mRNA. Niacin attenuated the increased PAI-1 synthesis induced by mediators released from thrombi as well. Thus, with 4.25 ng/mL transforming growth factor-ß₁, PAI-1 accumulation increased 4.5-fold in conditioned media in 48 hours. However, niacin attenuated the increase by 65%. Again, both the rate of PAI-1 synthesis and PAI-1 mRNA were reduced. The increased plasma PAI-1 activity and PAI-1 mRNA in liver induced by dexamethasone (0.8 mg IP) in vivo in rats were attenuated by 3 weeks of pretreatment with niacin.

Conclusions These results suggest that niacin, by decreasing PAI-1 expression, may potentiate fibrinolysis, thereby decreasing the stimulation of atherogenesis by clot-associated mitogens associated with microthrombi. Furthermore, the results imply that a pathogenetic link may exist between intracellular lipid metabolism and regulation of expression of fibrinolytic system components.

Key Words: atherosclerosis • fibrinolysis • niacin • plasminogen activators • lipids

	Introduction

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Plasminogen activator inhibitor type 1 (PAI-1) is a primary inhibitor of tissue-type (TPA) and urokinase plasminogen activators. Increased plasma concentrations of PAI-1 have been associated with premature coronary artery disease,^{1 2 3 4} acute myocardial infarction,^{5 6 7} and restenosis after percutaneous transluminal coronary angioplasty.⁸ Gemfibrozil, a lipid-lowering fibrate, decreases early cardiac events and cardiac mortality^{9 10 11 12} in hypertriglyceridemic patients and decreases plasma PAI-1 in subjects with elevated triglycerides.¹³ We found¹⁴ that it decreases PAI-1 elaboration and mRNA expression and attenuates increased PAI-1 synthesis mediated by platelet-associated growth factors in cells in culture and in vivo in laboratory animals. Thus, the salutary clinical effects of gemfibrozil may be related, at least in part, to induction of an altered shift toward thrombolysis in the balance between thrombosis and thrombolysis.^{13 14}

Niacin lowered plasma cholesterol¹⁵ and decreased total mortality in secondary prevention trials,¹⁶ perhaps because it inhibits lipolysis, hepatic triglyceride and cholesterol synthesis, and apolipoprotein B synthesis.^{17 18} After patients are treated with niacin,^{19 20} the rate of lysis of their whole blood clots in vitro is accelerated. Thus, niacin may influence the fibrinolytic system directly or secondarily via its hypolipidemic effects.

Although diverse cell types can synthesize PAI-1, hepatocytes are a primary source of plasma PAI-1.²¹ A highly differentiated human hepatoma cell line, Hep G2, simulates liver cells in vivo in terms of changes in PAI-1 expression in response to agonists and antagonists.^{14 22} Accordingly, the present study was performed to determine whether PAI-1 synthesis in Hep G2 cells in vitro and in rat liver in vivo is attenuated directly by niacin.

	Methods

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Cell Culture
Studies were performed in Hep G2 cells obtained from American Type Culture Collection (ATCC) or from Dr Alan Schwartz (Washington University). Results obtained with cells from both sources were identical. The cells were grown to confluence in minimal essential medium supplemented with Earle's salts and 2 mmol/L L-glutamine (Life Technologies), 10% NuSerum (Collaborative Biomedical), 30 U/mL penicillin, and 30 µg/mL streptomycin (Washington University Medical School Tissue Culture Support Center [WUMSTCSS]). Confluent monolayers were grown in Dulbecco's modified Eagle's medium with Ham's nutrient mixture F12 with HEPES (DME/F12) (WUMSTCSS) in the absence of serum for 4 to 24 hours, an interval sufficient for the decline of PAI-1 synthesis to basal levels. After serum starvation, cells were washed with PBS (WUMSTCSS) and exposed to DME/F12 with selected concentrations of niacin (Sigma Chemical Co) prepared at 30 g/L in distilled water with pH adjusted to 7.4 with NaOH in a sterile, Millipore-filtered solution stored at 4°C. Transforming growth factor-ß₁ (TGF-ß) (Boehringer Mannheim), found in platelet -granules and shown to mediate increased PAI-1 expression secondary to platelet activation,^{23 24 25} was added directly to cell media with siliconized pipettes to stimulate the cells in some experiments in a final concentration of 4.25 ng/mL.

Procedures in Animals
Protocols used conformed to the "Position of the American Heart Association on Research Animal Use" (November 11, 1984) and were approved by the Animal Studies Committee at Washington University. Adult Sprague-Dawley rats (250 to 300 g, Charles River) were fed a niacin supplement (0%, 1%, or 5% wt/wt) in the standard laboratory diet (Purina) for 3 weeks. This dose was equivalent to a human dose on the basis of body surface area.²⁶ PAI-1 activity in plasma and PAI-1 mRNA expression in parenchymal liver cells were induced by dexamethasone as previously described.²⁷ In brief, tissue culture grade dexamethasone (Sigma) was diluted in sterile PBS and filtered (0.22 µm), and 1 mL (0.8 mg/mL) was administered. Sterile PBS alone was used in the control animals. Both PBS and dexamethasone solution had <0.01 ng/mL endotoxin as determined by a limulus amebocyte lysate gel clot assay (Associates of Cape Cod). Rats were injected intraperitoneally. After 4 hours, rats were anesthetized with ketamine (30 mg/kg IM) and xylazine (5 mg/kg IM), and blood for PAI-1 plasma levels was obtained from the cardiac puncture immediately after the abdominal cavity was opened. Blood was collected into sodium citrate–containing tubes, centrifuged at 7500g for 15 minutes, and stored at -70°C until use. Liver was obtained immediately after blood collection, washed briefly in saline, snap-frozen in liquid nitrogen, and stored in airtight containers at -70°C until use. Plasma PAI-1 activity was determined spectrophotometrically as previously described²³ and expressed as arbitrary units, with 1 arbitrary unit defined as the amount of PAI-1 that could inhibit 1 IU of TPA within 10 minutes.

Quantification of PAI-1 Protein
After serum starvation for 4 to 24 hours, confluent monolayers of Hep G2 cells in 9.6-cm² wells were washed with PBS and fed with fresh DME/F12 with or without niacin and TGF-ß. Preliminary time-course study revealed that niacin (20 µmol/L) attenuated basal PAI-1 secretion by 45±5%, 59±11%, and 74±20% at 18, 24, and 48 hours (n=3 for each condition), and the following experiments were performed after 48 hours of exposure to niacin. Conditioned media were harvested and supplemented with Tween 80 (final concentration, 0.01%). Cellular debris was removed by centrifugation at 13 600g for 1 minute at 4°C. Samples were stored at -70°C until assay. The concentration of PAI-1 antigen was determined by ELISA (TintElize PAI-1, Biopool) with a monoclonal goat antibody, MA7D4B7, that detects latent, active, and TPA-complexed PAI-1 with equal sensitivity. For each experiment, at least five wells were used for each condition, with averages of at least three experiments reported for each condition (means as a percentage of control means). To determine whether changes in PAI-1 antigen accumulation were specific, total protein was assayed in conditioned medium (Bio-Rad Protein Assay).

Quantification of PAI-1 mRNA
After serum starvation from 4 to 24 hours, confluent monolayers in 25-cm² flasks were washed with PBS and fed with DME/F12 with or without niacin. In some experiments, TGF-ß was added either immediately or after 1 hour. Preincubation of cells with niacin for 1 hour before addition of TGF-ß did not accentuate its effects. After 6 to 48 hours, total cellular RNA was isolated with RNAzol B (Tel-Test), extracted with chloroform (Fisher), precipitated with isopropanol (Fisher), washed with 70% ethanol (Quantum), and size-fractionated on 1.5% formaldehyde agarose gels. Rat liver tissue samples were pulverized under liquid nitrogen with a frozen stainless steel crucible. The final pulverized powder was transferred immediately to RNAzol B, and total RNA was isolated. RNA was transferred to nylon membranes (Biodyne B, Pall Filter) by Northern blotting. Northern blots were prehybridized at 42°C for 4 to 18 hours and hybridized at 42°C for 12 to 24 hours. The 0.9-kb EcoRI–to–Sal I fragment of the human PAI-1 cDNA (kindly provided by K. Kretzmer of Monsanto Corp), the 0.9-kb Pvu II fragment of the rat PAI-1 cDNA,²⁸ and the 0.6-kb Xba I–to–HindIII fragment of the glyceraldehyde-3-phosphate (GAP) cDNA (ATCC 57091) were purified from agarose gels with Elutip-d minicolumns (Schleicher and Schuell), random primed (Labeling Kit, Boehringer Mannheim) with -³²P-dCTP (Amersham), and separated from unincorporated nucleotides with NucTrap push columns (Stratagene). The probes were coincubated with membranes, human or rat PAI-1 at 4x10⁵ cpm/mL, and GAP at 2x10⁵ cpm/mL. Membranes were washed three times at room temperature in 0.3 mol/L sodium chloride/0.3 mol/L sodium citrate followed by one wash at 60°C in 0.3 mol/L sodium chloride/0.3 mol/L sodium citrate and 1% sodium dodecyl sulfate (SDS) for 6 minutes. Blots were subjected to autoradiography at -70°C, and bands were quantified by laser densitometry (Ultrascan XL; Pharmacia LKB) and by radioisotopic scanning (AMBIS, Inc). Differences in RNA integrity, loading, and transfer were controlled according to the ratio of PAI-1 mRNA to GAP mRNA. Confounding effects of interventions on GAP expression were excluded by correspondence between GAP hybridization signals and the intensity of ethidium bromide–stained membrane ribosomal RNA bands.

Metabolic Labeling and Immunoprecipitation of Newly Synthesized PAI-1
After serum starvation for 12 to 18 hours, cells were washed with PBS and fed with DME/F12 either with or without niacin. In some experiments, cells were exposed to TGF-ß (4.25 ng/mL). After 6 hours, ³⁵S-methionine (Tran ³⁵S-label, ICN) was added (10 µCi/mL). After 20 minutes, the supernatant fraction was removed, and the cells were lysed with ice-cold buffer [10 mmol/L Tris, pH 8.4, 20 mmol/L sodium chloride, 0.5% Triton X-100, 1 mmol/L EDTA, 5 µg/mL leupeptin, 5 µg/mL antipain, 5 µg/mL 4-(2-aminoethyl)benzene-sulfonyl fluoride hydrochloride]. The lysates were scraped, transferred to Eppendorf tubes, vortexed, and centrifuged at 13 600g for 5 minutes at 4°C. The supernatant fractions were stored at -20°C.

PAI-1 was immunoprecipitated with a mouse monoclonal anti-human PAI-1 antibody that binds to active, latent, and complexed PAI-1 (3780, American Diagnostica) (20 µg/mL) with gentle agitation at 4°C for 12 to 18 hours in the presence of 0.1% BSA to reduce nonspecific binding. Agarose-linked goat anti-mouse IgG was added, and gentle agitation was continued for 2 hours at room temperature. Agarose beads were pelleted by centrifugation at 13 600g; washed twice with PBS containing 0.1% SDS, 0.5% Nonidet P-40, and 0.1% deoxycholate and once with PBS alone; suspended in reducing buffer; and heated to 95°C for 3 minutes. Supernatant fractions were subjected to SDS-PAGE with 4% polyacrylamide stacking and 10% polyacrylamide separating gels (Protogel, National Diagnostics). After electrophoresis, the gels were stained with rapid Coomassie blue (Diversified Biotech), dried (Drygel Jr SE540; Hoefer), and subjected to autoradiography at -70°C. The individual bands of PAI-1 and vitronectin–PAI-1 complex were identified as previously described.²² The intensities of individual bands were quantified by laser densitometry. Equal loading of samples was ensured by Coomassie blue staining of goat IgG derived from agarose beads.

Statistical Analysis
Results are expressed as mean±SEM. Differences between groups were tested for statistical significance by subjecting data to ANOVA. The significance of differences between groups was defined as P<.05.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Effects of Niacin on PAI-1 in Conditioned Medium
Hep G2 cells were exposed to niacin at selected concentrations from 4 to 20 µmol/L or to medium alone (control) (n=3 for each condition). After 48 hours, conditioned medium was assayed for PAI-1 and total protein concentrations. Niacin did not alter the microscopic morphology of the cells or the concentration of total protein in the medium at any concentration. However, it decreased PAI-1 expression significantly in a concentration-dependent fashion (Fig 1A), with r=.954 on a semilog plot (Fig 1B).

View larger version (49K): [in this window] [in a new window]   Figure 1. Graphs showing relation between amount of plasminogen activator inhibitor (PAI)-1 (hatched bars) or total protein (open bars) in the conditioned medium of Hep G2 cells and concentration of niacin (A and B). After serum starvation, confluent monolayers were exposed to 0, 4, 6, 10, or 20 µmol/L niacin for 48 hours. Conditioned medium was harvested and assayed for PAI-1 (n=3 for each condition) and, in separate experiments, for total protein (n=3 for each condition). Cells were exposed also to 4.25 ng/mL transforming growth factor (TGF)-ß (C and D). Total protein and PAI-1 protein are shown as percentages of values of control cells in medium without niacin. Niacin did not alter the concentration of total protein but decreased PAI-1 significantly with (C) or without (A) TGF-ß (*P<.05). B and D demonstrate a linear relation (semilog plot) typical of a conventional dose-response relation.

To determine whether niacin attenuates augmented PAI-1 expression induced by mediators implicated in vivo, Hep G2 cells were exposed to TGF-ß (4.25 ng/mL) in the presence or absence of 4 to 20 µmol/L niacin, and conditioned medium was assayed for PAI-1 48 hours later. In the absence of niacin, PAI-1 increased 3.4-fold with TGF-ß, from 212 to 721 ng/mL. Niacin attenuated the increase by up to 65±5% (Fig 1C). Again, the attenuation was concentration dependent (r=.865 on a semilog plot, Fig 1D).

Effects of Niacin on PAI-1 Synthesis
The changes in PAI-1 accumulation in conditioned medium could be attributable to changes in rates of PAI-1 synthesis, secretion, degradation, or a combination. To determine whether niacin altered PAI-1 synthesis, Hep G2 cells were serum starved from 12 to 18 hours and exposed to fresh medium with or without 20 µmol/L niacin (n=6 for each condition). After 6 hours, ³⁵S-methionine was added. Twenty minutes later, the cells were lysed, and PAI-1 was immunoprecipitated and assayed by SDS-PAGE. Niacin reduced newly synthesized PAI-1 by 39±8% (Fig 2A).

View larger version (86K): [in this window] [in a new window]   Figure 2. A representative autoradiogram (SDS-PAGE) of immunoprecipitated 35S-methionine-labeled, newly synthesized plasminogen activator inhibitor (PAI)-1. After serum starvation, confluent monolayers of Hep G2 cells were exposed to medium with or without (A) 20 µmol niacin and with or without (B) 4.25 ng/mL transforming growth factor (TGF)-ß. After 6 hours, the cells were exposed to 35S-methionine for 20 minutes. The band at approximately 50 kD represents PAI-1, the band at 75 kD vitronectin, and the bands at 125 and 180 kD PAI-1/vitronectin complexes.22 In the left lane of each gel are 14C-methylated molecular weight markers (Amersham). As illustrated in these representative autoradiograms, niacin decreased the rate of PAI-1 synthesis (by 39±8%, n=6) and significantly attenuated the increase in PAI-1 synthesis induced by TGF-ß (by 44±12%, n=6).

To determine whether niacin attenuated the increased PAI-1 synthesis in Hep G2 cells exposed to TGF-ß, confluent monolayers were serum starved and exposed to fresh medium with or without 20 µmol/L niacin. TGF-ß was then added (4.25 ng/mL). After 6 hours, ³⁵S-methionine was added for 20 minutes, and the cells were lysed. PAI-1 was immunoprecipitated and assayed by SDS-PAGE. Niacin reduced the augmentation in newly synthesized PAI-1 by 44±12% (n=6) (Fig 2B).

Effects of Niacin on Steady-State Concentrations of PAI-1 mRNA
The decreased PAI-1 synthesis could be attributable to changes in steady-state levels of mRNA, changes in translation, or both. To determine whether niacin altered levels of steady-state PAI-1 mRNA, Hep G2 cells were exposed to increasing concentrations of niacin after serum starvation. After 6 hours, total RNA was isolated and assayed by Northern blotting. The yield was unaffected by niacin. Concentrations of niacin of 6 µmol/L decreased steady-state mRNA significantly (Fig 3A and 3B). With 20 µmol/L niacin, PAI-1 mRNA decreased by 49±9% after 6 hours of exposure. Niacin (20 µmol/L) decreased PAI-1 mRNA levels by 25±9% and 24±8% after 24 and 48 hours of exposure (n=3 for each condition).

View larger version (81K): [in this window] [in a new window]   Figure 3. Representative autoradiograms and graphs showing the concentrations of plasminogen activator inhibitor (PAI)-1 mRNA in Hep G2 cells exposed to 0, 4, 6, 10, or 20 µmol/L niacin for 6 hours with (C and D) or without (A and B) 4.25 ng/mL transforming growth factor (TGF)-ß. In A and C, bands at 3.2 and 2.2 kb represent PAI-1 mRNA. The 1.3-kb band represents glyceraldehyde-3-phosphate (GAP) mRNA. The 28S rRNA on the same ethidium bromide–stained membrane is shown as a further control. B and D show results from three experiments. Hybridized bands were quantified by radioisotopic scanning. PAI-1 mRNA (the sum of the 3.2- and 2.2-kb forms) was determined relative to GAP mRNA, and the signals were compared with those from medium of cells not exposed to niacin (*P<.05).

To determine whether changes in steady-state mRNA levels occurred with niacin in the presence of TGF-ß, Hep G2 cells were serum starved and then exposed to TGF-ß in the presence of selected concentrations of niacin. At 6 hours, TGF-ß (4.25 ng/mL) increased mRNA expression by 4.4±1-fold (n=3), but the increase was attenuated by niacin by 22±6% (Fig 3C and 3D). At 24 and 48 hours, TGF-ß increased mRNA expression by 1.3±0.2-fold and 1.2±0.1-fold (n=3), and the increase was attenuated by niacin by 26±12% and 30±7%, respectively (n=3).

Effects of Niacin on Plasma PAI-1 Activity and Liver PAI-1 mRNA In Vivo
PAI-1 activity was determined in plasma from animals treated with dexamethasone. Four hours after treatment with dexamethasone (0.8 mg), plasma PAI-1 activity was approximately 2.7-fold greater than that measured in saline-treated animals (P<.05, Fig 4A). These values were similar to those reported previously.²⁷ Pretreatment with 1% and 5% niacin for 3 weeks attenuated the response to dexamethasone by 43% and 69%, respectively (P<.05 compared with the nonniacin group). To determine whether the dexamethasone-induced increase in plasma PAI-1 activity and its attenuation by niacin is accompanied by alterations in PAI-1 gene expression in liver, total liver RNA was isolated and evaluated for PAI-1 mRNA expression. PAI-1 mRNA was undetectable by Northern blot analysis of total liver RNA from saline-treated rats, consistent with previous observations.²⁷ With dexamethasone treatment, there was an induction of PAI-1 mRNA in liver (n=8, Fig 4B). Pretreatment with 1% and 5% niacin for 3 weeks attenuated the increase induced by dexamethasone by 52% and 64%, respectively (P<.05 compared with the nonniacin group, Fig 4C).

View larger version (50K): [in this window] [in a new window]   Figure 4. Bar graph showing relation between the plasminogen activator inhibitor (PAI)-1 activity in plasma and the amount of niacin (A). Rats were fed niacin (0%, 1%, and 5%) for 3 weeks. PAI-1 activity in plasma was induced by dexamethasone (DEX, 0.8 mg IP). After 4 hours, rats were anesthetized, and blood was drawn for PAI-1 activity assay. There was a 2.7-fold increase in PAI-1 activity in dexamethasone-treated rats (n=8) compared with saline-treated rats (n=3, *P<.05). The increase in PAI-1 activity induced by dexamethasone was attenuated in niacin-fed rats (1%, n=8; 5%, n=9) compared with non–niacin-treated, dexamethasone-stimulated rats (**P<.05). B, Representative autoradiogram showing concentration of PAI-1 mRNA in liver tissue exposed to 0.8 mg dexamethasone. In rats, only 3.1-kb PAI-1 mRNA is present. The 1.3-kb band represents GAP mRNA. The 18S rRNA on the same ethidium bromide–stained membrane is shown as a further control. Hybridized bands were quantified by radioisotopic scanning. PAI-1 mRNA was determined relative to GAP mRNA (C). The signals were decreased in niacin-fed rats (1%, n=8; 5%, n=9) compared with those from the non–niacin-treated group (n=8, *P<.05).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Our results demonstrate that niacin decreases PAI-1 elaboration by Hep G2 cells in vitro as a result of decreased PAI-1 synthesis. Both decreased PAI-1 elaboration and decreased PAI-1 synthesis appear to reflect at least partly decreased gene expression, manifested by decreased steady-state mRNA levels. Since the reductions in PAI-1 mRNA levels throughout a 48-hour exposure were small relative to the magnitude of the effects observed in PAI-1 secretion detected by ELISA, and more marked effects of niacin on accumulation of immunoreactive PAI-1 in conditioned medium were observed, additional mechanisms must be operating at the posttranscriptional level. The response of Hep G2 cells to TGF-ß, a potent stimulus for PAI-1 gene expression, was attenuated by niacin as well, with consequently decreased PAI-1 synthesis and elaboration, as demonstrated by immunoprecipitation of newly synthesized PAI-1 and quantification of PAI-1 protein by ELISA.

The reported peak plasma level of niacin in patients given a single 1-g oral dose is 1 µmol/L.^{17 29} Niacin is taken up rapidly by red blood cells. Thus, 50% of an injected dose is removed from plasma within 1.5 minutes,³⁰ and the ratio of whole blood to plasma niacin at steady state is 110 to 1.³¹ As with other compounds given orally, transient concentrations in portal blood markedly exceed those in peripheral blood.

In mice given niacin intravenously, the highest concentrations 20 and 60 minutes later occurred in liver at a time when blood levels were virtually nil.³² Thus, the concentrations of niacin used in our study in vitro are not inconsistent with concentrations likely to occur in the liver after pharmacological dosing.

Animal studies in vivo demonstrated a marked effect on circulating PAI-1 levels and PAI-1 mRNA in liver at pharmacologically meaningful concentrations of niacin,²⁶ confirming that alterations observed in vitro in an established transformed liver cell line are relevant to what happens in vivo upon niacin administration in PAI-1 levels in circulation and PAI-1 mRNA levels in liver. Basal PAI-1 levels in normal rat plasma and basal PAI-1 mRNA expression in normal rat liver were low,²⁷ and accurate assessment of the effects of pharmacological interventions on PAI-1 expression was difficult. Therefore, the effects of niacin on PAI-1 levels in plasma and PAI-1 mRNA expression in dexamethasone-treated animals were assessed. Further studies in patients will be helpful in assessing the effect of this agent on plasma PAI-1 levels.

Elevated concentrations of PAI-1 have been implicated in atherogenesis and in coronary thrombosis. If the balance between thrombosis and thrombolysis is shifted toward thrombosis, elevated levels of PAI-1 may increase exposure of vessel walls to intermittent platelet activation, with release of platelet- and clot-associated mitogens.²² Thus, direct diminution of expression of PAI-1 by niacin may play a role, in addition to its hypolipidemic effects, in the decreased mortality observed in the Coronary Drug Project.¹⁶

Because gemfibrozil^{13 14} and niacin have now been shown to decrease PAI-1 expression directly and because both decrease hepatic triglyceride and cholesterol biosynthesis,¹⁶ coordinate regulation of lipoprotein and PAI-1 gene expression by intracellular alterations in lipid metabolism may be involved, despite the otherwise dissimilar pharmacological effects of these agents on lipid metabolism. In a study of young survivors of myocardial infarction, plasma PAI-1 increased in hypertriglyceridemic patients (Fredrickson types IIb and IV) but not in patients with isolated, increased LDL (Fredrickson type IIa).⁹ In hypercholesterolemic patients, elevated PAI-1 levels were reduced by an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase.³³ HMG-CoA reductase and PAI-1 mRNAs are found in the same type of cells in rabbit liver.³⁴ Again, these results support the view that coordinate regulation of lipoprotein and PAI-1 gene expression may be involved.

In the arterial wall, both thrombosis and lipid deposition appear to contribute to atherosclerosis and its complications. Elucidation of links between the two and potential modulation of both by pharmacological alterations of intracellular lipid metabolism are attractive targets for improved prevention and treatment of vascular disease. Our results suggest that the links between lipoprotein and PAI-1 gene expression may be particularly critical for development of atherosclerosis and its complications.

	Acknowledgments

 
This work was supported in part by NIH grant HL-17646, SCOR in Coronary and Vascular Diseases, and NIH training grant 5-T32-HL-07081-19 (Dr Brown). The authors thank Dr Thomas Gelehrter (University of Michigan) for kindly supplying rat PAI-1 cDNA; Drs Hirofumi Sawa and Craig Lundgren for assistance in preparing cDNA probes; Denise Nachowiak, Jeffrey Labuda, John Botz, Pamela Lundins, and Amy Guala for technical assistance; and Barbara Donnelly, Kelly Hall, and Kathryn Quackenbush for secretarial support.

Received November 16, 1994; revision received February 14, 1995; accepted February 20, 1995.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Aznar J, Estellés A, Tormo G, Sapena P, Tormo V, Blanch S, España F. Plasminogen activator inhibitor activity and other fibrinolytic variables in patients with coronary artery disease. Br Heart J. 1988;59:535-541. [Abstract/Free Full Text]
   
2. Olofsson BO, Dahlén G, Nilsson TK. Evidence for increased levels of plasminogen activator inhibitor and tissue plasminogen activator in plasma of patients with angiographically verified coronary artery disease. Eur Heart J. 1989;10:77-82. [Abstract/Free Full Text]
   
3. Bick RL, Baker WF. Tissue plasminogen activator inhibitor type 1 (PAI-1) abnormalities in patients with precocious and premature coronary artery disease. Blood. 1992;80:507a. Abstract.
   
4. Graf S, Beckmann R, Christ G, Geppert A, Kaindl F, Binder BR, Huber K. Possible causes of a defective fibrinolytic system in young patients with coronary artery disease. J Am Coll Cardiol. 1993;21:211A. Abstract.
   
5. Verheugt FWA, ten Cate JW, Sturk A, Imandt L, Verhorst PMJ, van Eenige MJ, Verwey W, Ross JP. Tissue plasminogen activator activity and inhibition in acute myocardial infarction and angiographically normal coronary arteries. Am J Cardiol. 1987;59:1075-1079. [Medline] [Order article via Infotrieve]
   
6. Hamsten A, Wiman B, de Faire U, Blombäck M. Increased plasma levels of a rapid inhibitor of tissue plasminogen activator in young survivors of myocardial infarction. N Engl J Med. 1985;313:1557-1563. [Abstract]
   
7. Hamsten A, de Faire U, Walldius G, Dahlén G, Szasmosi A, Landou C, Blombäck M, Wiman B. Plasminogen activator inhibitor in plasma: risk factor for recurrent myocardial infarction. Lancet. 1987;2:3-9. [Medline] [Order article via Infotrieve]
   
8. Huber K, Jörg M, Probst P, Schuster E, Lang I, Kaindl F, Binder BR. A decrease in plasminogen activator-1 activity after successful percutaneous transluminal coronary angioplasty is associated with a significantly reduced risk for coronary restenosis. Thromb Haemost. 1993;67:209-213.
   
9. Heikki Frick M, Elo O, Haapa K, Heinonen OP, Heinsalmi P, Helo P, Huttunen JK, Kaitaniemi P, Koskinen P, Manninen V, Mäenpää H, Mälkönen M, Mänttäri M, Norola S, Pasternack A, Pikkarainen J, Romo M, Sjöblom T, Nikkilä E. Helsinki Heart Study: primary-prevention trial with gemfibrozil in middle-aged men with dyslipidemia. N Engl J Med. 1987;317:1237-1245. [Abstract]
   
10. Manninen V, Elo O, Heikki Frick M, Haapa K, Heinonen OP, Heinsalmi P, Helo P, Huttunen JK, Kaitaniemi P, Koskinen P, Mäenpää H, Mälkönen M, Mänttäri M, Norola S, Pasternack A, Pikkarainen J, Romo M, Sjöblom T, Nikkilä EA. Lipid alterations and decline in the incidence of coronary heart disease in the Helsinki Heart Study. JAMA. 1988;260:641-651. [Abstract]
    
11. Manninen V, Huttunen JK, Heinonen OP, Tenkanen L, Heikki Frick M. Relation between baseline lipid and lipoprotein values and the incidence of coronary heart disease in the Helsinki Heart Study. Am J Cardiol. 1989;63:42H-47H. [Medline] [Order article via Infotrieve]
    
12. Frick MH, Manninen V, Huttunen JK, Heinonen OP, Tenkanen L, Mänttäri M. HDL-cholesterol as a risk factor in coronary heart disease: an update of the Helsinki Heart Study. Drugs. 1990;40(suppl 1):7-12.
    
13. Andersen P, Smith P, Seljeflot I, Brataker S, Arnesen H. Effects of gemfibrozil on lipids and haemostasis after myocardial infarction. Thromb Haemost. 1990;63:174-177. [Medline] [Order article via Infotrieve]
    
14. Fujii S, Sobel BE. Direct effects of gemfibrozil on the fibrinolytic system. Circulation. 1992;85:1888-1893. [Abstract/Free Full Text]
    
15. Altschul R, Hoffer A, Stephen JD. Influence of nicotinic acid on serum cholesterol in man. Arch Biochem Biophys. 1955;54:558-559.
    
16. Canner PL, Berge KG, Wenger NK, Stamler J, Friedman L, Prineas RJ, Friedewald W, for the Coronary Drug Project Research Group. Fifteen year mortality in coronary drug project patients: long-term benefit with niacin. J Am Coll Cardiol. 1986;8:1245-1255. [Abstract]
    
17. DiPalma JR, Thayer WS. Use of niacin as a drug. Annu Rev Nutr. 1991;11:169-187. [Medline] [Order article via Infotrieve]
    
18. Shepherd J, Packard CJ. Pharmacological modification of lipoprotein metabolism. Biochem Soc Trans. 1986;15:199-201.
    
19. Weiner M, Redisch W, Steele JM. Occurrence of fibrinolytic activity following administration of nicotinic acid. Proc Soc Exp Biol Med. 1958;98:755-757.
    
20. Weiner M, DeCrinis K, Redisch W, Steele JM. Influence of some vasoactive drugs on fibrinolytic activity. Circulation. 1959;19:845-848. [Abstract/Free Full Text]
    
21. Sawdey MS, Loskutoff DJ. Regulation of murine type 1 plasminogen activator inhibitor gene expression in vivo. J Clin Invest. 1991;88:1346-1353.
    
22. Nordt TK, Schneider DJ, Sobel BE. Augmentation of the synthesis of plasminogen activator inhibitor type-1 by precursors of insulin: a potential risk factor for vascular disease. Circulation. 1994;89:321-330. [Abstract/Free Full Text]
    
23. Fujii S, Sobel BE. Induction of plasminogen activator inhibitor by products released from platelets. Circulation. 1990;82:1485-1493. [Abstract/Free Full Text]
    
24. Fujii S, Hopkins WE, Sobel BE. Mechanisms contributing to increased synthesis of plasminogen activator inhibitor type 1 in endothelial cells by constituents of platelets and their implications for thrombolysis. Circulation. 1991;83:645-651. [Abstract/Free Full Text]
    
25. Hopkins WE, Westerhausen DR, Fujii S, Billadello JJ, Sobel BE. Mediators of induction of augmented expression of plasminogen activator inhibitor type-1 in Hep G2 cells by platelets. Thromb Haemost. 1991;66:239-245. [Medline] [Order article via Infotrieve]
    
26. Holland RE, Rahman K, Morris AI, Coleman R, Billington D. Effects of niacin on biliary lipid output in the rat. Biochem Pharmacol. 1993;45:43-49. [Medline] [Order article via Infotrieve]
    
27. Konkle BA, Schuster SJ, Kelly MD, Harjes K, Hassett DE, Bohrer M, Tavassoli M. Plasminogen activator inhibitor-1 messenger RNA expression is induced in rat hepatocytes in vivo by dexamethasone. Blood. 1992;79:2636-2642. [Abstract/Free Full Text]
    
28. Bruzdzinski CJ, Riordan-Johnson M, Nordby EC, Suter SM, Gelehrter TD. Isolation and characterization of the rat plasminogen activator inhibitor-1 gene. J Biol Chem. 1990;265:2078-2085. [Abstract/Free Full Text]
    
29. Weiner M, Van Eys J. Nicotinic Acid: Nutrient-Cofactor-Drug. New York, NY: Marcel Dekker; 1983:227-232.
    
30. Lee KW, Abelson DM, Kwon YO. Nicotinic acid-6-¹⁴C metabolism in man. Am J Clin Nutr. 1968;21:223-225. [Medline] [Order article via Infotrieve]
    
31. Frank O, Baker H, Sobotka H. Blood- and serum-levels of water-soluble vitamins in man and animals. Nature. 1963;197:490-491. [Medline] [Order article via Infotrieve]
    
32. Carlson LA, Hanngren A. Initial distribution in mice of ³H-labeled nicotinic acid studied with autoradiography. Life Sci. 1964;3:867-871.
    
33. Wada H, Mori Y, Kaneko T, Wakita Y, Nakase T, Minamikawa K, Ohiwa M, Tamaki S, Tanigawa M, Kageyama S, Deguchi K, Nakano T, Shirakawa S, Suzuki K. Elevated plasma levels of vascular endothelial cell markers in patients with hypercholesterolemia. Am J Hematol. 1993;44:112-116. [Medline] [Order article via Infotrieve]
    
34. Rea TJ, DeMattos RB, Pape ME. Hepatic expression of genes regulating lipid metabolism in rabbits. J Lipid Res. 1993;34:1901-1910.[Abstract]
    

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