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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:1688.)
© 2000 American Heart Association, Inc.

Thrombosis

Impact of Antioxidants and HDL on Glycated LDL–Induced Generation of Fibrinolytic Regulators From Vascular Endothelial Cells

Song Ren; Garry X. Shen

From the Departments of Internal Medicine and Physiology, University of Manitoba, Winnipeg, Canada.

Correspondence to Garry X. Shen, MD, PhD, Departments of Internal Medicine and Physiology, University of Manitoba, BS432 730 William Ave, Winnipeg, Manitoba R3E 0W3, Canada. E-mail gshen{at}ms.umanitoba.ca

	Abstract

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Abstract—Hyperglycemia and dyslipoproteinemia are biochemical markers of diabetes mellitus (DM). Elevated levels of plasminogen activator inhibitor-1 (PAI-1) with and without reduction of tissue plasminogen activator (tPA) in plasma have been frequently found in patients with DM. Our previous studies indicated that glycation enhances low density lipoprotein (LDL)-induced production of PAI-1 and further decreases tPA generation in vascular endothelial cells (ECs). The present study demonstrated that treatment with antioxidants, butylated hydroxytoluene or vitamin E, blocked native LDL– and glycated LDL–induced changes in PAI-1 and tPA generation in ECs. Native or glycated high density lipoprotein (HDL) did not significantly alter tPA generation in ECs. Glycated but not native HDL (100 µg/mL) moderately increased PAI-1 release from ECs. Cotreatment with native or glycated HDL inhibited LDL-induced or glycated LDL–induced changes in PAI-1 and tPA generation in ECs. The abundance of conjugated dienes was increased in glycated or EC-modified LDL. Treatment with butylated hydroxytoluene, vitamin E, or HDL reduced the abundance of conjugated dienes in glycated or EC-modified LDL. The effects of antioxidants and HDL on LDL-induced or its glycated LDL–induced changes in the generation of PAI-1 and tPA were also found in cultured human coronary artery ECs. The findings of the present study suggest that antioxidants and HDL may attenuate native LDL– or glycated LDL–induced changes in the generation of fibrinolytic regulators from vascular ECs, which possibly results from their inhibition on the lipid peroxidation of LDL particles. Treatment with antioxidants or hypolipidemic agents potentially improves fibrinolytic activity and reduces thrombotic tendencies in patients with DM.

Key Words: LDL • glycation • antioxidants • HDL • plasminogen activator inhibitor-1

	Introduction

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Hyperglycemia and dyslipoproteinemia, 2 major biochemical markers in diabetes mellitus (DM), have been implicated in the development of cardiovascular complications in patients with DM. Intravascular thrombosis plays a critical role in the development of ischemic events with and without the presence of atherosclerotic lesions. The formation of thrombus in the vasculature mainly depends on the balance between coagulation and fibrinolysis. The release of fibrinolytic activity from the vascular wall responding to temporary occlusion was impaired in patients with DM.^{1 2} The production of plasminogen activator inhibitor-1 (PAI-1) and tissue plasminogen activator (tPA) in vascular endothelial cells (ECs) is modulated by thrombin,³ glucose,⁴ proinsulin,⁵ growth factors,⁶ and several types of plasma lipoproteins.^{7 8 9 10 11 12 13} Elevated levels of glycated LDL were detected in the plasma of patients with DM.^{14 15} Previous studies by our group demonstrated that glycation enhanced LDL-induced overproduction of PAI-1 and further reduced the generation of tPA from cultured vascular ECs.¹³

Glycation increases the oxidative stress of lipoproteins.¹⁶ Treatment with antioxidants has been found to attenuate diet-induced atherosclerosis in experimental animal models in some studies.^{17 18} Results from epidemiological studies have demonstrated a strong negative correlation between the levels of HDL and the incidence of atherosclerotic cardiovascular diseases.¹⁹ The presence of HDL protects LDL from oxidative modification.²⁰ The influence of HDL or antioxidants on the generation of fibrinolytic regulators from ECs induced by LDL or its modified forms has not been documented.

We hypothesize that treatment with antioxidants and HDL may affect the glycated LDL–induced generation of fibrinolytic regulators from vascular ECs. The present study examined the effects of antioxidants and HDL on native LDL– and glycated LDL–induced PAI-1 and tPA generation from human umbilical vein ECs (HUVECs) and human coronary artery ECs (HCAECs). The impact of antioxidants and HDL on lipid peroxidation in LDL was also investigated.

	Methods

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Isolation of Lipoproteins
LDL (density 1.019 to 1.063) and HDL (density 1.063 to 1.210) were isolated from fresh human plasma of healthy donors by sequential flotation ultracentrifugation.²¹ Lp(a) was removed from LDL and HDL fractions by lysine-Sepharose-CL 4B affinity chromatography as previously described.²² LDL and HDL were dialyzed at 4°C against 150 mmol/L NaCl and 0.01% EDTA (pH 7.4) and stored in sealed tubes overlaid with nitrogen at 4°C in the dark to prevent auto-oxidation. The levels of endotoxin in lipoproteins were monitored by a Limulus amebocyte lysate test with the use of commercially available kits (E-TOXATE, Sigma Chemical Co). The low limit for the detection of endotoxin is 0.05 ng/mL. No detectable amount of endotoxin was found in the lipoprotein preparations used in the present study.

Modification of Lipoproteins
LDL and HDL preparations were diluted to 2 mg of protein per milliliter with 0.1 mol/L phosphate buffer (pH 7.4) containing 0.01% EDTA and 0.01% sodium azide and then incubated with 50 mmol/L glucose and equimolar amounts of sodium cyanoborohydride for 2 weeks at 37°C in the dark with nitrogen.¹³ Native LDL and HDL were prepared identically except for the exposure to glucose. At the end of glycation, lipoproteins were dialyzed to remove free glucose. The extent of glycation in lipoproteins was evaluated by measuring the abundance of glucitollysine by use of high-performance liquid chromatography as previously described.¹³ The ratio of glucitollysine/lysine in glycated LDL modified by the condition described above was 1.07±0.09, and ratios in native LDL, native HDL, and glycated HDL were 0.16±0.04, 0.17±0.05, and 0.25±0.07 (mean±SD, n=4), respectively.

Cell Culture and Experimental Stimulation
HUVECs were obtained by collagenase digestion as previously described,²³ and the types of cells were verified by morphology and the presence of factor VIII antigen. Cells were grown to confluence in medium 199 supplemented with 10% heat-inactivated FBS, 30 µg/mL EC growth supplements (Sigma), 100 µg/mL heparin, 0.1 mmol/L nonessential amino acids, 200 U/mL penicillin, and 200 µg/mL streptomycin in a humidified incubator with 95% air/5% CO₂ at 37°C. Confluent cells were treated with indicated reagents supplemented in heparin-free medium 199. Seed HCAECs were obtained from Clonetics. Cells were cultured in endothelial growth medium MV (Clonetics) and used within passage 8. Cytotoxicity of lipoproteins was determined by incubating cells with 5x10⁶ dpm per well of [³H]leucine (54 Ci/mmol/L, ICN Radiochemical) in leucine-free medium for 2 hours after treatment with lipoproteins. No detectable reduction in the incorporation of radioactive leucine was found in ECs treated with the tested concentrations of native or glycated lipoproteins.

Measurement of PAI-1 and tPA Antigens
Conditioned media of HUVECs were collected at the end of the incubations. Cells were harvested in PBS (pH 7.4) containing 0.1% SDS and 0.5% Triton X-100. Total PAI-1 and tPA antigens (free and complex forms) in the media were measured by using IMUBIND PAI-1 or tPA ELISA kits (American Diagnostica Inc) and expressed in micrograms of antigen per milligram of total cellular proteins.¹³

Northern Blotting Analysis
Northern blotting analysis of PAI-1 mRNA in total RNA from HUVECs was conducted as previously described.¹² Plasmids containing cDNA fragment–encoded human PAI-1 or ß-actin were labeled with [³²P]dCTP (>111 TBq/mmol/L, New England Nuclear). The abundance of mRNA on blots was visualized by autoradiography. The levels of specific PAI-1 mRNA were quantified by density scanning with adjustment of ß-actin mRNA in the same lanes of stripped blots.

Analysis of Conjugated Dienes
Lipids of lipoproteins were extracted by using chloroform/methanol (2:1). The absorbance of lipid extracts resuspended in ethanol was measured over 220 to 330 nm against an ethanol blank by using a UV spectrophotometer and expressed in arbitrary units.^{13 24} The extent of lipid peroxidation was estimated from absorbance minima at 233 nm for the quantification of conjugated dienes as previously described.^{24 25}

Statistical Analysis
Values are presented as mean±SD generated from experiments that used quadruplicated cultures or lipoprotein preparations. Probability between 2 groups was estimated by the Student t test. Comparisons among multiple groups were achieved by 1-way ANOVA, followed by the Duncan test. The level of significance was defined as P<0.05.

	Results

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Effect of Antioxidants on Native LDL– and Glycated LDL–Induced PAI-1 and tPA Generation
Our previous study demonstrated that treatment with native and glycated LDL at 100 µg protein/mL for 48 hours induced significant changes in the generation of PAI-1 and tPA from HUVECs.¹³ Butylated hydroxytoluene (BHT) is a structural analogue of probucol, a potent antioxidant and cholesterol-lowering drug, that is free of hypolipidemic effect.²⁶ In the present study, HUVECs were treated with 1 to 100 µmol/L BHT in the presence and absence of 100 µg/mL glycated LDL for 48 hours (Figure 1). BHT alone up to 100 µmol/L did not significantly affect PAI-1 or tPA generation from ECs. Treatment with 10 µmol/L BHT significantly reduced the release of PAI-1 induced by glycated LDL and increased tPA generation compared with treatment with glycated LDL alone (P<0.01 or P<0.001). The inhibitory effects of BHT on glycated LDL–induced changes in the generation of PAI-1 and tPA reached plateaus >40 µmol/L (Figure 1). Incubation with 100 µg/mL native LDL for 48 hours significantly increased PAI-1 production (2.57±0.10 µg/mg) and decreased tPA generation (3.05±0.21 µg/mg) in HUVECs compared with control cultures (PAI-1 1.72±0.13 µg/mg, tPA 3.76±0.32 µg/mg; mean±SD, n=4; P<0.01). Treatment with 80 µmol/L BHT almost completely inhibited the increase in PAI-1 release and the decrease in tPA generation induced by native LDL (PAI-1 1.76±0.05 µg/mg, tPA 3.71±0.17 µg/mg; mean±SD, n=4; P<0.01).

View larger version (16K): [in this window] [in a new window]   Figure 1. Dose response of BHT on glycated LDL (gly-LDL)–induced generation of PAI-1 and tPA in HUVECs. Cells were incubated with 1 to 100 µmol/L BHT in the presence or absence of 100 µg/mL of gly-LDL for 48 hours. The levels of PAI-1 and tPA antigens in the medium after culture were determined by ELISA. Values were expressed as microgram per milligram protein (mean±SD, n=4). Comparable results were obtained from 2 independent experiments. *P<0.05, **P<0.01, and ***P<0.001 vs cultures without addition of BHT; +P<0.05, ++P<0.01, and +++P<0.001 vs control cultures treated with equal concentrations of BHT without exposure to gly-LDL.

The effects of antioxidants on the generation of fibrinolytic regulators induced by native or glycated LDL in HUVECs were further investigated by using vitamin E (-tocopherol), a natural antioxidant abundant in lipoprotein particles. Cells were treated with up to 100 µmol/L -tocopherol succinate (Sigma) in the presence and absence of 100 µg/mL native or glycated LDL for 48 hours. Treatment with 10 µmol/L vitamin E significantly reduced native LDL– or glycated LDL–induced PAI-1 release and increased tPA generation compared with treatment with LDL or its glycated form alone (P<0.05 or P<0.01, Figure 2).

View larger version (17K): [in this window] [in a new window]   Figure 2. Effect of vitamin E on native LDL– and gly-LDL–induced PAI-1 and tPA generation from HUVECs. Cells were treated with1 to 100 µmol/L -tocopherol succinate (vitamin E) in the presence or absence of 100 µg/mL native LDL or gly-LDL for 48 hours. The levels of PAI-1 and tPA antigens in the medium after culture were expressed as micrograms per milligram (mean±SD, n=4). Comparable results were found in 2 independent experiments. *P<0.05 and **P<0.01 vs control (equal concentrations of vitamin E alone); +P<0.05 and ++P<0.01 vs cultures without addition of vitamin E; and xP<0.05 vs cultures treated LDL (native) plus equimolar amounts of vitamin E.

The effects of BHT and vitamin E on glycated LDL–induced PAI-1 production in HUVECs was verified by detecting the steady-state levels of PAI-1 mRNA with the use of Northern blot analysis. Incubation with 100 µg/mL glycated LDL increased the PAI-1/ß-actin mRNA ratio by 3- to 3.5-fold. Treatment with 80 µmol/L BHT or 20 µmol/L vitamin E completely inhibited the glycated LDL–induced increase in PAI-1 mRNA in ECs. The levels of ß-actin mRNA in ECs treated with glycated LDL and/or antioxidants were not noticeably altered compared with control levels (Figure 3). The effect of antioxidants on tPA mRNA was not examined because earlier studies indicated that glycated LDL reduced de novo synthesis but not mRNA levels of tPA in HUVECs.¹³

View larger version (41K): [in this window] [in a new window]   Figure 3. Effects of antioxidants on gly-LDL–induced changes in PAI-1 mRNA in HUVECs. Cells were treated with 100 µg/mL gly-LDL for 48 hours with or without the addition of 80 µmol/L BHT or 20 µmol/L vitamin E (Vit.E). Total RNA (20 µg per lane) extracted from ECs was analyzed on 1% agarose-formaldehyde gel electrophoresis. Membrane blots were hybridized with [32P]dCTP-labeled human PAI-1 cDNA probe (top). The levels of ß-actin mRNA on stripped blots were analyzed as internal control (bottom). Control indicates no addition. Comparable results were obtained from 2 independent experiments.

Influence of Antioxidants on Lipid Peroxidation in Native and Glycated LDL
Lipid peroxidation of polyunsaturated fatty acids at the sn-2 position of phospholipids or cholesteryl ester of lipoproteins causes the formation of conjugated dienes and peroxy radicals.²⁰ Previous observations by our group demonstrated that glycation increased the formation of conjugated dienes in LDL.¹³ The pattern of conjugated dienes in glycated LDL is distinct from that in Cu²⁺-oxidized LDL. The most abundant form of conjugated dienes in glycated LDL is detected at 233 nm instead of at 242 nm in Cu²⁺-oxidized LDL.¹³ Incubation with HUVECs for 48 hours significantly increased the absorbance minima at 233 nm in native or glycated LDL (P<0.001). Treatment of native and glycated LDL with 80 µmol/L BHT or 20 µmol/L vitamin E during the incubation with ECs significantly reduced the abundance of conjugated dienes at 233 nm compared with the lipoproteins treated with ECs without exposure to the antioxidants (P<0.001, Table 1).

View this table: [in this window] [in a new window]   Table 1. Impact of Antioxidants and HDL on Abundance of Conjugated Dienes in Native and Glycated LDL

Impact of HDL on Native LDL– and Glycated LDL–Induced Generation of Fibrinolytic Regulators and Lipid Peroxidation in LDL
Native HDL alone up to 140 µg/mL did not significantly affect the generation of PAI-1 or tPA from HUVECs. Glycated HDL at levels of 100 or 140 µg/mL (22% and 27%, respectively), not 35 or 70 µg/mL, moderately but significantly increased PAI-1 release from ECs (P<0.05). The levels of tPA released from ECs were not significantly changed by up to 140 µg/mL of glycated HDL (data not shown). Vascular endothelium was exposed to HDL with the presence of LDL in vivo. The ratio of HDL/LDL proteins in plasma is 1.4:1 in normal conditions. Cotreatment with 35 to 140 µg/mL native or glycated HDL significantly inhibited the elevation of PAI-1 release and the reduction in tPA generation induced by 100 µg/mL native or glycated LDL after 48 hours of incubation (Figure 4). No detectable amount of conjugated dienes was found in native HDL. Increases in the formation of conjugated dienes were found in glycated HDL compared with native HDL after 48 hours of incubation with HUVECs. The abundance of conjugated dienes in glycated HDL was significantly lower than in LDL modified by the identical procedure (P<0.001). Cotreatment of native or glycated HDL (140 µg/mL) with native or glycated LDL (100 µg/mL) significantly reduced the abundance of conjugated dienes in LDL reisolated from the media after 48 hours of incubation with ECs (P<0.001, Table 1).

View larger version (24K): [in this window] [in a new window]   Figure 4. Effects of native HDL and gly-HDL on the generation of PAI-1 and tPA induced by LDL or its glycated form in HUVECs. Cells were treated with 35 to 200 µg/mL of native HDL or gly-HDL in the presence of 100 µg/mL of native LDL or gly-LDL for 48 hours. The levels of PAI-1 and tPA antigens in the medium after culture were expressed as percentage of control cultures without addition (mean±SD, n=4). *P<0.05, **P<0.01, and ***P<0.001 vs cultures without addition of native HDL or gly-HDL. A and B, PAI-1. C and D, tPA. A and C, HDL (native) or gly-HDL+LDL (native). B and D, HDL or gly-HDL+gly-LDL. Comparable results were obtained from 2 independent experiments.

Impact of Antioxidants and HDL on Native LDL– or Glycated LDL–Induced Generation of PAI-1 and tPA From HCAECs
Treatment with 80 µmol/L BHT or 20 µmol/L vitamin E significantly reduced PAI-1 release and increased tPA generation from HCAECs induced by 100 µg/mL native or glycated LDL (P<0.05 or P<0.01). HDL did not significantly affect PAI-1 or tPA generation in HCAECs. Cotreatment with 140 µg/mL native or glycated HDL normalized the PAI-1 and tPA generation induced by 100 µg/mL native or glycated LDL in HCAECs (P<0.01 or P<0.001, Table 2).

View this table: [in this window] [in a new window]   Table 2. Effects of Antioxidants or HDL on Native LDL– or Glycated LDL–Induced Generation of PAI-1 and tPA in HCAECs

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Hyperglycemia has been considered to be a key contributing factor in the development of diabetic vascular complications. Dyslipoproteinemia, characterized by increased levels of LDL/VLDL and decreased levels of HDL, is commonly found in patients with poorly controlled DM. The levels of glycated lipoproteins were increased in humans and diabetic animal models.^{14 15 27} Nonenzymatic glycation slows the clearance of LDL from blood circulation.²⁸ Glycation increases the chemotactic activity of LDL for monocytes, which promotes the deposition of monocytes on the endothelial surface.²⁹ Previous studies by our group indicated that glycated LDL augmented PAI-1 production and reduced tPA generation in vascular ECs.¹³ Imbalance between EC-derived PAI-1 and tPA potentially reduces intravascular fibrinolytic activity and enhances thrombotic tendency. The present study for the first time demonstrated that the disorders of the generation of fibrinolytic regulators in vascular ECs induced by LDL or its glycated form may be prevented by pharmacological or physiological interventions.

Previous studies have indicated that antioxidants, including probucol, BHT, and vitamin E, retarded diet-induced atherosclerosis in several types of animal models,^{17 18 26} although the antiatherogenic effect of antioxidants in humans remains controversial. Very high doses of vitamin E (10 g/kg in the diet) potentiated atherosclerotic lesions compared with lower doses of vitamin E (0.04 g/kg) in rabbits.³⁰ The results of the animal studies have suggested that the antiatherogenic effects of vitamin E and BHT are mainly due to their antioxidative activity.^{26 30} Previous studies have indicated that incubation with ECs causes oxidative modification of LDL.³¹ Glycation increases the oxidative susceptibility of LDL. The present study demonstrated that the presence of BHT or vitamin E during incubation with ECs effectively inhibited the native LDL– and glycated LDL–induced changes in the generation of PAI-1 and tPA in ECs. Our findings suggest that cell-mediated oxidative modification plays an important role in native LDL– and glycated LDL–induced changes in the generation of fibrinolytic regulators in ECs. Treatment with BHT or vitamin E reduced the abundance of conjugated dienes in EC-modified native and glycated LDL; this reduction possibly contributes to the preventive effect of antioxidants on the changes in EC-derived fibrinolytic activity induced by LDL or its glycated form. Involvement of other types of oxidative modification in native and glycated LDL particles has not been excluded by the results of the present study. PAI-1 and tPA may be produced by multiple types of cells in the blood or vascular wall. Further investigation in a suitable in vivo model may provide additional information about the impact of antioxidants on fibrinolytic activity in the blood circulation.

Several lines of evidence suggest that HDL protects the oxidative modification of LDL. Injection of HDL₃ into hypercholesterolemic rabbits reduced the levels of conjugated dienes in plasma.³² The levels of HDL cholesterol significantly correlated with the levels of conjugated dienes in the plasma of healthy subjects and patients with coronary artery disease.³³ The results of the present study demonstrated that HDL attenuates the formation of conjugated dienes in LDL after incubation with ECs. Our observations indicated that native HDL did not affect EC-derived PAI-1 or tPA. The changes in the generation of fibrinolytic regulators in ECs induced by glycated HDL were significantly less than those induced by equal protein amounts of LDL glycated with the identical procedure. This is possibly due to the relatively lower oxidative susceptibility of HDL compared with LDL.³⁴ It should be pointed out that the abundance of conjugated dienes in glycated HDL was comparable to that in EC-modified LDL (Table 1), whereas the effects of glycated HDL on PAI-1 and tPA generation were significantly weaker than the effects of EC-modified or "native" LDL. This implies that other unidentified factors besides the formation of conjugated dienes in lipoproteins may also contribute to this process.

The present study is the first to demonstrate that HDL may improve vascular EC–derived fibrinolytic activity attenuated by LDL or its glycated form. Cotreatment with native or glycated HDL dose-dependently reduced PAI-1 production and augmented the generation of tPA induced by native or glycated LDL. The findings imply that increased levels of HDL in normal or diabetic status may improve EC-derived fibrinolytic activity deteriorated by LDL or its modified forms. The beneficial effect of HDL on EC-derived fibrinolytic activity is possibly due to the prevention of lipid peroxidation, which is suggested by the reduced abundance of conjugated dienes in EC-modified LDL with the presence of HDL. The inhibitory effect of HDL on lipid peroxidation may result from certain enzymes associated with HDL particles, including paraoxonase.³⁵

The present study compared the impact of antioxidants and HDL on native LDL– and glycated LDL–induced generation of fibrinolytic regulators from venous and arterial ECs. HCAECs release similar amounts of tPA but considerably more PAI-1 compared with HUVECs at basal conditions. Treatment with native or glycated LDL resulted in relatively weak responses regarding the changes of the generation of PAI-1 and tPA in HCAECs compared with HUVECs in the tested conditions. However, the production of PAI-1 and tPA in cultured ECs may also be affected by other variations, such as the medium and the passages of cultured cells.

In conclusion, EC-mediated oxidative modification may play a critical role in native LDL– and glycated LDL–induced alterations in PAI-1 and tPA generation in vascular ECs. HDL in the native or glycated form may neutralize LDL-induced or glycated LDL–induced changes in PAI-1 and tPA generation in vascular ECs. Treatment with antioxidants or correction of dyslipoproteinemia by physiological (eg, exercises) or pharmacological interventions may potentially improve vascular cell–derived fibrinolytic activity and thus reduce the thrombotic tendency in patients with DM.

	Acknowledgments

 
The authors thank Jianying Zhang (University of Boston) for the contribution of relevant studies, Dr Paul E. DiCorleto (Cleveland Clinic Research Institute) for providing the cDNA probes, and the Canadian Diabetes Association for operating grants. This study is dedicated to the memory of the late Archibald Mitchell.

Received January 1, 2000; accepted February 17, 2000.

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