© 1998 American Heart Association, Inc.
Original Contributions |
Influence of Glycation on LDL-Induced Generation of Fibrinolytic Regulators in Vascular Endothelial Cells
From the Departments of Internal Medicine and Physiology, University of Manitoba, Winnipeg, Manitoba, Canada.
Correspondence to Dr Garry X. Shen, Departments of Internal Medicine and Physiology, University of Manitoba, BS439 730 William Ave, Winnipeg, Manitoba, Canada R3E 3J7. E-mail gshen{at}ms.umanitoba.ca
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Abstract—Hyperglycemia and dyslipidemia are two biochemical markers of diabetes mellitus. Increased incidence of cardiovascular disease and impaired fibrinolytic activity have been found in diabetic subjects. Previous studies have demonstrated that low density lipoproteins (LDLs) stimulate the production of plasminogen activator inhibitor-1 (PAI-1) and reduce the generation of tissue plasminogen activator (tPA) in vascular endothelial cells (ECs). The present study investigated the effect of glycated LDL on the production of PAI-1 and tPA in cultured human umbilical vein ECs (HUVECs). Glycation increased the abundance of glucitollysine and conjugated dienes in LDL and amplified the overproduction of PAI-1 and the reduction in tPA generation from HUVECs induced by LDL. The steady-state levels of PAI-1 mRNA in glycated LDL–treated ECs were significantly higher than those in native LDL–treated cells. Actinomycin D blocked the increase in PAI-1 generation induced by glycated LDL. Glycated LDL did not significantly reduce the levels of tPA mRNA but attenuated de novo synthesis of tPA in ECs. Treatment with 25 mmol/L aminoguanidine, an antioxidant and inhibitor of the formation of advanced glycation end products, during glycation normalized glycated LDL–induced generation of PAI-1 and tPA in ECs. The results of the present study indicate that glycation enhances the production of PAI-1 and attenuates tPA synthesis in ECs induced by LDL, which may contribute to the increased incidence of cardiovascular complications in diabetes. Formation of advanced glycation end products or peroxidation may be involved in glycated LDL–induced alterations in the generation of fibrinolytic regulators from ECs.
Key Words: glycated LDL • plasminogen activator inhibitor-1 • tissue plasminogen activator • endothelial cells • aminoguanidine
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Diabetes mellitus is the most common metabolic disorder in worldwide populations. The risk for developing cardiovascular complications is increased by 2- to 6-fold in diabetic subjects.1 Disorders in lipid metabolism, coagulation, and fibrinolysis have been implicated in the development of diabetic vascular complications.2 It is generally accepted that hemostasis is determined by a balance between coagulation and fibrinolysis. Imbalance between the two antagonistic systems may result in thrombosis or hemorrhage. Plasmin, the active product of the fibrinolytic system, functions by dissolving fibrin clots and maintains the fluidity of blood.3 The generation of plasmin is regulated by plasminogen activators and their inhibitors. tPA is the main activator for plasminogen in the blood circulation.4 The activity of tPA is modulated by its major physiological inhibitor, PAI-1.5 The levels of PAI-1 mRNA are significantly increased in atherosclerotic vessels and are correlated with the severity of atherosclerotic lesions.6 An increase in PAI-1 production or a decrease in tPA generation from vascular ECs may reduce fibrinolytic activity in the blood. Attenuated fibrinolytic activity or reduced fibrinolytic response to ischemic stimulus has been found in diabetic subjects.2 7
Vascular ECs synthesize both PAI-1 and tPA. The generation of PAI-1 or tPA from ECs is regulated by a variety of biological agonists, including plasma lipoproteins. The release of PAI-1 from ECs has been increased by treatments with VLDLs isolated from hypertriglyceridemic individuals.8 LDLs and their oxidized forms, modified by acetylation, UV radiation, or CuSO4, stimulate the production of PAI-1 in ECs.9 10 11 Lp(a), an LDL-like lipoprotein, stimulates PAI-1 production in ECs, and oxidization amplifies the effect of Lp(a).12 13 Treatment with native LDL reduces the generation of tPA in ECs.14 Glycated LDLs are susceptible to oxidization in vitro.15 Increased levels of glycated LDLs have been detected in subjects with poorly controlled diabetes.16 17 The influence of glycated lipoproteins on the production of fibrinolytic regulators in vascular ECs has not been documented. The present study examined the effect of glycation on LDL-induced generation of PAI-1 and tPA from cultured human vascular ECs.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Isolation of Lipoproteins
LDL fractions (d=1.024 to 1.063 g/mL) were isolated from fresh human plasma of healthy donors by sequential floating ultracentrifugation. Lp(a) was removed from LDL by lysine–Sepharose CL 4B affinity chromatography as previously described.18 Lp(a)-free LDLs were dialyzed at 4°C against 150 mmol/L NaCl and 0.01% EDTA (pH 7.4) and stored in sealed tubes overlaid with N2 at 4°C in the dark. The levels of endotoxin in lipoproteins were monitored by the Limulus amebocyte lysate test using commercially available kits (E-TOXATE, Sigma Chemical Co). No detectable amount of endotoxin was found in lipoprotein preparations used in this study.
Modification of Lipoproteins
LDL 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% NaN3 and then incubated with 5 to 200
mmol/L glucose and equimolar amounts of NaBH3CN
for 1 to 3 weeks at 37°C in the dark under N2.
Native LDLs were processed identically except without the addition of
glucose. At the end of glycation, lipoproteins were dialyzed to remove
free glucose. Radiolabeled [14 C]glucose (10
µCi/mL) was mixed with unlabeled glucose during glycation to monitor
the incorporation of glucose. Oxidized LDLs were modified by 5
µmol/L CuSO4 at 22°C for 24
hours.13
Analysis of Amino Acid Profile of Lipoproteins
Glycated and native LDLs were hydrolyzed in 2N HCl for 24
hours at 100°C, washed with coupling buffer
(acetonitrile/ethanol/triethylamine/water, 10:5:2:3, vol/vol/vol/vol),
and dried by evaporation. The samples were resuspended in 100 µL of
the coupling buffer containing 0.45 mol/L phenylisothiocyanate,
incubated at room temperature for 5 minutes, and then dried. Residues
were resuspended in 400 µL of 70 mmol/L sodium acetate (pH 6.5)
and analyzed on an Ultrasphere ODS C18
column (4.6 mmx25 cm) using a gradient solvent system (0% to
30% acetonitrile, 100% to 70% of 70 mmol/L sodium acetate) on a
high-performance liquid chromatography (Gold
System, Beckman Instruments Inc) and monitored at 245
nm.19
Preparation of Glucitollysine
-N-Z-L-Lysine (Sigma) was incubated
with 27 mmol/L glucose for 17 days in 0.2 mol/L sodium phosphate
buffer (pH 8.0) as previously described.20 The
mixture was passed through a Dowex 50 WX8 column followed by a Sephadex
G-15 column. The protecting group was removed by catalytic transfer
hydrogenation.
Cell Culture and Experimental Incubation
HUVECs were obtained by collagenase digestion
as previously described.21 Cell type was verified
by morphology and the presence of factor VIII antigen. Cells were grown
in medium 199 supplemented with 10% heat-inactivated FBS,
30 µg/mL of EC growth stimulator, 100 µg/mL of heparin, 0.1
mmol/L nonessential amino acids, 200 U/mL of penicillin, and 200
µg/mL of streptomycin in a humidified incubator under 95% air–5%
CO2 at 37°C. Confluent cells were incubated in
heparin-free medium 199 with or without LDL.
Measurement of PAI-1 and tPA Antigen
Conditioned media of HUVECs were collected at the end of
incubation. Cells were harvested in PBS (pH 7.4) containing 0.1% SDS
and 0.5% Triton X-100. Total amounts of PAI-1 and tPA antigen (free
and complex forms) in the media were estimated by using IMUBIND PAI-1
or tPA ELISA kits (American Diagnostica Inc). The levels of
PAI-1 and tPA antigen were read on a microtest plate spectrophotometer
at 490 nm and were expressed in micrograms of antigen per milligram of
cellular proteins.13
Northern Blotting Analysis
Total cellular RNA was extracted from cells at the end of
incubation by the guanidine isothiocyanate–CsCl
method.22 RNA was denatured, subjected to
electrophoresis on a 1% agarose-formaldehyde gel, and then transferred
to Zeta-Probe GT blotting membranes (Bio-Rad). Plasmid containing the
cDNA fragment–encoded human tPA, PAI-1,23 or
ß-actin was labeled with [32P]dCTP (>111
TBq/mmol/L, New England Nuclear) by using random-primer labeling kits.
Blots were prehybridized in 0.25 mol/L
Na2HPO4 (pH 7.2) and 7%
SDS for 10 minutes at 42°C and then hybridized with denatured probe
for 16 hours at 42°C.24 After hybridization,
blots were washed and subjected to autoradiography. The
levels of PAI-1 and tPA mRNA were quantified from
autoradiogram by density scanning and then adjusted
with ß-actin mRNA on rehybridized blots.
Metabolic Labeling and Immunoprecipitation
Confluent cells in 60-mm dishes were treated with 100
µCi/mL of Tran35 S label (38 TBq/mmol/L, 85%
methionine, and 15% cystine, ICN) in methionine- and cystine-free
supplemented with 2 mmol/L glutamine and 10% serum with or
without lipoproteins as previously described.24
Cells and media were harvested at the end of incubation and diluted
with a buffer containing 0.5 ml/L NaCl, 1 mmol/L EDTA, 50
mmol/L Tris, 0.1% NP-40, 2.5% gelatin, and 0.5% BSA. Diluted media
and cell lysates were first incubated with 30 µg/mL of rabbit IgG at
25°C for 1 hour and then with 40 µL of a 50% slurry of protein
A–Sepharose for 30 minutes. Sepharose beads in the mixtures were
removed by centrifugation. Resultant supernatant was
incubated at 25°C for 2 hours with goat anti-human tPA IgG or rabbit
anti-human PAI-1 IgG (American Diagnostica Inc). Immune
complexes of tPA or PAI-1 were recovered by using protein A–Sepharose.
The beads were washed sequentially with, as described above, the buffer
dilution supplemented with 0.5% sodium deoxycholate followed by
10 mmol/L Tris (pH 7.5) containing 0.1% NP-40. Proteins were
recovered from the beads with 125 mmol/L Tris buffer (pH 6.8)
containing 20% glycerol and 4.6% SDS and then analyzed by
12% SDS-PAGE. De novo synthesized tPA or PAI-1 was detected on dried
gels by autoradiography and quantified by density
scanning.13
Estimation of Cytotoxicity of Lipoproteins
Cytotoxicity of lipoproteins was examined by incubating cells
with 5x106 disintegrations per minute per well
of [3H]leucine [54
Ci/(mmol/L), ICN Radiochemicals] in leucine-free medium for 2 hours
after treatment with lipoproteins. Cellular proteins were precipitated
with 5% trichloroacetic acid. Radioactivity incorporated into cellular
precipitates was analyzed by scintillation counting. No
detectable reduction in the incorporation of radioactive leucine was
found in HUVECs treated with the indicated amounts of native or
glycated lipoproteins (data not shown).
Analysis of CDs
Lipids in modified or native lipoproteins were extracted in
chloroform/methanol, 2:1, vol/vol. The organic phase of lipid extracts
was dried under N2 at room temperature. The lipid
residue was resuspended in absolute ethanol. Absorbance of lipid
extracts was measured from 220 to 330 nm against an ethanol blank by
using a UV spectrophotometer and was expressed in arbitrary
units.25 The extent of peroxidation was estimated
from absorbance minima at 242 and 233 nm for the quantitative
analysis of CDs as previously
described.25 26
Measurement of TBARS
Lipid peroxidation in lipoproteins and the postculture media of
ECs was determined by measuring the amount of TBARS and expressed as
nanomoles of malondialdehyde per milligram of protein in LDL as
previously described.27 28
Measurement of Protein Concentrations in Lipoproteins and
Cells
Protein contents in lipoproteins were measured by using a
modified Lowry method.29 For analyzing total
proteins in cultured cells, cells were lysed in PBS containing 0.5%
Triton X-100 and 0.1% SDS.
Statistical Analysis
Data are presented as mean±SD. Probability between
paired data was estimated with Student's t test.
Comparisons among multiple groups were achieved by one-way ANOVA
followed by Duncan's test. The level of significance was defined as
P<0.05.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Biochemical Characterization of Glycated LDL
The abundance of lysine in glycated LDL (Figure 1B
) was greatly reduced compared with
native LDL (Figure 1A). An extra amino acid peak (Figure 1B) was
detected in glycated LDL but not in native LDL. This product
comigrated with synthesized glucitollysine (Figure 1C), a glycation
product of lysine. The glucitollysine-to-lysine ratio was elevated
3-fold in LDL glycated by 25 mmol/L glucose compared with
nonglycated LDL (Table 1). Radioactivity
of [14C]glucose in
glucitollysine was 5 to 6 times higher than in background, lysine, or
phenylalanine. The glucitollysine-to-lysine ratio is only one of
several potential markers for the pathogenic alterations in glycated
LDL. Glycated LDL migrated apparently faster than did native LDL toward
the positively charged anode on 1% agarose electrophoresis gel, as
expected (data not shown). The levels of TBARS in glycated LDL were
very low or undetectable (0.10±0.09 nmol malondialdehyde per milligram
protein, mean±SD, n=4), which was slightly higher than but not
significantly different from that in native LDL (0.06±0.06 nmol/mg).
The levels of TBARS in oxidized LDL were 46.16±3.85 nmol/mg. In the
lipid extracts of native LDL, no CDs were detected. Oxidized LDL
contained CDs at 233 and 242 nm (Figure 2). The absorbance minimum of CDs at 233
nm in glycated LDL was significantly higher than that in oxidized LDL,
but no CDs were found at 242 nm in glycated LDL (Table 2, Figure 2).
|
|
|
|
Effect of Glycated LDL on PAI-1 and tPA Secretion
The influence of the extent of glycation of LDL on the generation
of PAI-1 and tPA was examined in HUVECs. Cells were treated with 100
µg/mL of glycated LDL modified with 25 to 200 mmol/L glucose for
1 week. Glycated LDL (100 µg/mL) modified by 
50 mmol/L glucose
significantly increased the levels of PAI-1 antigen in the media of
HUVECs after 48 hours of treatment (6.43±0.39, 6.58±0.27, or
6.76±0.14 µg/mg by 50, 100, or 200 mmol/L, respectively, of
glucose-modified LDL) compared with native LDL (5.37±0.37 µg/mg,
n=4, P<0.01, Figure 3, top).
To evaluate the influence of the length of glycation on glycated
LDL–induced PAI-1 generation, ECs were treated with 100 µg/mL of
glycated LDL modified by a lower concentration of glucose for a longer
time (25 mmol/L glucose for 3 weeks) for 48 hours compared with
glycated LDL modified by 25 mmol/L glucose for 1 week or 50
mmol/L glucose for 2 weeks. The generation of PAI-1 in ECs treated with
glycated LDL modified by 25 mmol/L glucose for 3 weeks or by
50 mmol/L glucose for 2 weeks was significantly higher than in ECs
treated with matching native LDL (P<0.01). Native LDL
proceeded in parallel with glycated LDL for 3 weeks without exposure to
glucose-stimulated PAI-1 production compared with control
cultures (P<0.05), which was possibly due to peroxidation.
However, native LDLs allowed to proceed for 1 or 2 weeks under the same
condition did not significantly affect PAI-1 production (Table 3).
|
|
The generation of tPA from HUVECs was significantly reduced by a
24-hour treatment with 100 µg/mL of glycated LDL modified by 25
mmol/L glucose for 1 week (0.75±0.03 µg/mg) compared with native LDL
(0.84±0.06 µg/mg, n=4, P<0.05, Figure 3
, bottom). The
decrease in tPA generation was more evident in ECs treated with LDL
modified by 50 mmol/L glucose for 1 week (0.48±0.06,
0.51±0.05, or 0.56±0.06 µg/mg by 50, 100, or 200 mmol/L,
respectively, of glucose-modified LDL) compared with native LDL
(P<0.01).
After 24 hours of incubation with 100 µg/mL of native LDL, the
levels of PAI-1 (4.52±0.26 µg/mg after 24 hours, 5.31±0.31 µg/mg
after 48 hours) in the media were significantly higher than in
time-matched controls (3.71±0.17 µg/mg after 24 hours, 4.09±0.26
µg/mg after 48 hours, n=4, P<0.01). Treatment with equal
amounts of glycated LDL (modified by 50 mmol/L glucose for 2
weeks) induced significantly greater increases of PAI-1 released from
HUVECs (5.35±0.37 µg/mg after 24 hours, 6.28±0.19 µg/mg after 48
hours) compared with cells treated with native LDL for matching periods
(Figure 4, top). Dose-response curves for
the effect of glycated LDL on PAI-1 generation were observed in HUVECs
treated with 10 to 100 µg/mL of glycated or native LDL. The levels of
PAI-1 antigen in the medium of HUVECs treated with 50 µg/mL of
glycated LDL (4.66±0.25 µg/mg for 50 µg/mL, 5.41±0.35 µg/mg for
100 µg/mL) were significantly higher than those in cells treated with
equal amounts of native LDL (3.37±0.22 µg/mL for 50 µg/mL,
4.28±0.16 µg/mg for 100 µg/mL, n=4, P<0.01, Figure 4, bottom).
|
To answer the question whether the transcription of PAI-1 was
stimulated during this process, HUVECs were treated with 0.2 µg/mL of
actinomycin D in addition to 100 µg/mg of native or glycated
LDL.13 Actinomycin D treatment inhibited native
and glycated LDL–induced PAI-1 generation from HUVECs to levels
(native LDL, 5.34±0.51 µg/mg versus native LDL+actinomycin D,
4.28±0.32 µg/mg, n=4, P<0.01; glycated LDL, 6.74±0.42
µg/mg versus glycated LDL+actinomycin D, 4.43±0.40 µg/mg,
P<0.01; actinomycin D alone, 4.50±0.23 µg/mg) close to
those of controls (4.10±0.10 µg/mg, Figure 5).
|
The levels of tPA antigen in the media of HUVECs were elevated with the
elongation of incubation time. The secretion of tPA from ECs was
significantly reduced by treatment with 100 µg/mL of native LDL for
16 hours (0.91±0.05, 1.17±0.12, or 2.07±0.13 µg/mg for 16, 24,
or 48 hours, respectively) compared with time-matched controls
(1.12±0.07, 1.62±0.10, or 2.47±0.07 µg/mg for 16, 24, or 48 hours,
P<0.05). Glycated LDL (modified by 50 mmol/L glucose
for 2 weeks) further attenuated tPA release from ECs (0.69±0.07,
0.76±0.07, or 1.64±0.12 µg/mg for 16, 24, or 48 hours) compared
with native LDL incubated for matching periods (P<0.05,
Figure 6, top). Treatment with 25
µg/mL of native or glycated LDL for 24 hours significantly reduced
tPA secretion from HUVECs compared with no-addition controls
(1.15±0.09 µg/mg, P<0.01). The amounts of tPA secreted
from ECs treated with 25 to 100 µg/mL of glycated LDL (0.75±0.05,
0.58±0.02, or 0.57±0.05 µg/mg by 25, 50, or 100 µg/mL) were
significantly less than those from cells treated with corresponding
amounts of native LDL (0.90±0.04, 0.71±0.04, or 0.67±0.04 µg/mg by
25, 50, or 100 µg/mL, P<0.05, Figure 6, bottom).
|
Effect of Glycated LDL on PAI-1 and tPA mRNA
PAI-1 mRNA in HUVECs was present as two distinguishable
species, 
3.4 and 2.4 kb, as previously
described.13 Treatment with 100 µg/mL of native
LDL for 48 hours increased the level of 2.4-kb PAI-1 mRNA by 2-fold
(P<0.001) but slightly reduced the level of 3.4-kb PAI-1
mRNA. Treatment with an equal amount of glycated LDL evidently
augmented the level of 2.4-kb PAI-1 mRNA compared with controls and
native LDL–treated cells (Figure 7, top). The levels of 2.4-kb PAI-1 mRNA in glycated LDL–treated ECs were
>3.25±0.21-fold higher than the mean of controls and were
significantly greater than that in native LDL–treated cells
(2.13±0.16-fold, n=3, P<0.001, Figure 7, bottom). This
finding is consistent with the inhibitory effect of
actinomycin D on glycated LDL–induced PAI-1 generation (Figure 5).
However, the level of tPA mRNA in HUVECs treated with 100 µg/mL of
glycated or native LDL did not significantly differ from controls
(Figure 8).
|
|
Effect of Glycated LDL on De Novo Synthesis of tPA and
PAI-1
To determine whether the synthesis of fibrinolytic regulators was
altered by glycated LDL treatment, HUVECs were
metabolically labeled with
[35S]methionine/cystine in the presence of
native or glycated LDL. De novo synthesized PAI-1 was mainly detected
in the cell-associated pool of HUVECs. Radioactivity of PAI-1 was weak
in the medium and not visualized by using
autoradiography. Native LDL (100 µg/mL for 48 hours)
moderately increased the level of de novo synthesized PAI-1 in the
cell-associated compartment (3.47±0.9-fold) compared with controls
(1.00±0.07-fold of the mean, n=4, P<0.05). Treatment with
glycated LDL significantly increased the level of de novo synthesized
PAI-1 (13.95±4.2-fold) compared with native LDL (n=4,
P<0.05, Figure 9). De novo
synthesized tPA was mainly found in the media of HUVECs. Treatment with
100 µg/mL of native LDL reduced tPA synthesis after 24 hours of
incubation. Glycated LDL at the same concentration further attenuated
tPA synthesis in HUVECs compared with native LDL (P<0.01,
Figure 10).
|
|
Effects of Oxidized and Aminoguanidine-Treated Glycated LDL on the
Generation of PAI-1 and tPA
In the media of HUVECs treated with 100 µg/mL of oxidized
LDL for 48 hours, the levels of PAI-1 were 23% higher than in native
LDL–treated cells and 51% higher than controls (P<0.001)
but did not significantly differ from those of cells treated with
glycated LDL. The generation of tPA in oxidized LDL–treated ECs was
35% lower than that from native LDL–treated cells
(P<0.001) and 17% less than in glycated LDL–treated ECs
(P<0.05, Figure 11).
|
Aminoguanidine is an antioxidant and an inhibitor for the
formation of AGEs.30 31 Addition of 25
mmol/L aminoguanidine during LDL glycation effectively inhibited the
glycation of lysine (Figure 1C). The inhibitory effect of
aminoguanidine on the glycation of lysine was evident in LDLs treated
with 25 mmol/L aminoguanidine (data not shown). Glycated
LDL–induced changes in the secretion of PAI-1 and tPA antigen (PAI-1,
148.5±5.3%; tPA, 52.8±5.2% of control) were effectively reduced by
treatment with 25 mmol/L aminoguanidine during glycation (PAI-1,
122.7±7.9%; tPA, 72.0±5.3% of control, P<0.01) to
levels that were not significantly different from those of native
LDL–treated cells (PAI-1, 122.5±3.6%; tPA, 66.4±3.5% of control,
Figure 11). Additionally, aminoguanidine treatment normalized the de
novo synthesis of PAI-1 and tPA in ECs induced by glycated LDL (Figures 9 and 10).
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Attenuation of fibrinolytic activity has been frequently found in diabetic patients and has been implicated in the development of cardiovascular complications in diabetes.32 33 Alternations in the generation of PAI-1 and tPA from vascular ECs may directly affect fibrinolytic activity in the blood circulation. The results of the present studies demonstrate that glycation enhances the production of PAI-1 and reduces the generation of tPA induced by LDL in cultured vascular ECs. Increased generation of PAI-1 accompanied by a decreased release of tPA from vascular ECs may result in hypofibrinolysis and lead to intravascular thrombogenesis. The findings of the present study may help investigators understand the mechanism for attenuated fibrinolytic activity and increased thrombotic vascular complications in diabetes. It should be pointed out that the present study only examined the effects of LDL and its glycated form from healthy individuals. The composition of LDL in diabetes may differ from that from nondiabetic subjects with respect to factors other than glycation products. Investigation of the effects of LDL from various types of diabetic individuals may provide additional information on the modulation of EC-derived fibrinolytic regulators in diabetes.
The results of the present study indicate that the generation of PAI-1 and tPA in ECs is significantly altered by glycated LDL that has been modified by 25 to 50 mmol/L glucose for extended periods. This range of blood glucose occurs in uncontrolled diabetes. Previous studies reported that the levels of glucose may be elevated to a range of 200 to 2000 mg/dL (11 to 111 mmol/L) in patients with diabetic ketoacidosis.34 The extent of glycation of plasma proteins is correlated with the length of glycation time as well as glucose concentrations.35 This concept is supported by the results of the present study that LDL glycated by 25 mmol/L glucose for 3 weeks significantly increased PAI-1 generation compared with LDL glycated by the same concentration of glucose for a shorter period (1 week). We speculate that the generation of PAI-1 may be increased by LDL modified by even lower concentrations of glucose for longer periods in vivo.
Levin et al14 described that the generation
of tPA from HUVECs was reduced by treatment with native LDL. The effect
of native LDL on the secretion of tPA from HUVECs observed in the
present study supports their observation. The level of tPA mRNA was
inhibited by LDL in HUVECs in medium with the addition of 100 µg/mL
of heparin in the previous study.14 It has been
shown that 50 µg/mL of heparin effectively inhibits the levels of
tPA mRNA in baboon aortic smooth muscle cells.36
In the present study, heparin was supplemented only in the growth
medium but not in the stimulation medium to avoid interference with the
regulation of tPA generation. Pilot studies in our laboratory have
found that treatment with 200 µg/mL of native or glycated LDL in
heparin-free medium evidently impairs
[3H]leucine incorporation into HUVECs (data not
shown). Glycated LDL did not reduce the mRNA level of tPA mRNA but did
inhibit the synthesis of tPA in ECs. The difference between the effects
of LDL on tPA mRNA in the present and the previous
study14 is likely due to the variation in
stimulation conditions.
The present study demonstrates that glycated LDL stimulates the steady-state levels of PAI-1 mRNA in ECs that are associated with increases in de novo synthesis and secretion of PAI-1. Actinomycin D, a known inhibitor of transcription, blocked glycated and native LDL–induced generation of PAI-1. The combination of these findings strongly suggests that both glycated and native LDL regulate PAI-1 production in ECs at the transcriptional level. In contrast, the steady-state level of tPA mRNA in ECs was not affected by glycated or native LDL. De novo synthesis of tPA was reduced in native and glycated LDL–treated ECs. Therefore, the reduction in tPA generation from ECs induced by native and glycated LDL may result from the decrease in tPA synthesis or secretion.
Previous studies have demonstrated that oxidized LDL stimulates
the generation of PAI-1 in ECs.10 11 13 The
results of the present study indicate that oxidized LDL also
reduces tPA generation in HUVECs. Glycated LDL may be more susceptible
to oxidation than is native LDL.9 Although the
TBARS assay did not reveal a significant increase in peroxidation in
glycated LDL, CDs were detected at 233 nm in lipid extracts of glycated
LDL in our studies. The pattern of CDs in glycated LDL apparently
differs from that in oxidized LDL (Figure 2). The findings suggest the
presence of excess lipid peroxidation products in glycated LDL, and
glycoxidation may be a major contributing factor for glycated
LDL–induced alterations in PAI-1 and tPA generation in ECs.
Extended glycation induces the generation of AGEs in blood components and tissues. AGEs promote cross-linking between glycation products and arterial wall proteins, which may be involved in atherogenesis.37 AGE complexes also enable the generation of reactive oxygen intermediates.38 Receptors for AGEs have been identified in several types of cells, including ECs.39 Aminoguanidine inhibits the formation of AGEs by forming a complex with early glycation products.31 Treatment with aminoguanidine has prevented the formation of AGEs in the aortic wall of diabetic rats.37 The results of the present study for the first time demonstrate that treatment with aminoguanidine during glycation normalized glycated LDL–induced overproduction of PAI-1 and the reduction of tPA synthesis in HUVECs. Aminoguanidine is also an inhibitor of NO synthesis40 and diamine oxidase.41 Previous studies indicated that treatment with aminoguanidine prevented the oxidation of LDL.42 Both AGEs and oxidation may contribute to glycated LDL–induced generation of fibrinolytic regulators in vascular ECs, and the levels of AGEs are higher in both apoB and the lipid moiety of LDLs isolated from diabetic patients than from nondiabetic subjects.43 Determination of interactions between AGEs, LDL-specific structure, and the EC surface may help investigators define the regulatory mechanism for glycated LDL–induced alterations in the generation of fibrinolytic regulators from ECs.
The present study was performed in ECs isolated from umbilical veins. Previous studies have demonstrated that HUVECs are functionally closer to arterial ECs than to ECs isolated from other veins.44 This is possibly due to the unique circumstance of the umbilical vein, which carries oxygenated and nutrient-bearing blood instead of regular venous blood from the placenta to fetus. It is reasonable, therefore, to predict that HUVECs may respond to glycated LDL in a way similar to arterial ECs than do other venous ECs.
In summary, glycation enhances the overproduction of PAI-1 and further reduces the generation of tPA induced by LDL in vascular ECs. The effect of glycated LDL on the generation of PAI-1 and tPA was effectively prevented by treatment with aminoguanidine. Our findings suggest that increased levels of glycated LDL in the blood circulation may attenuate fibrinolytic activity. Management of hyperglycemia and hyperbetalipoproteinemia or the reduction of glycation of lipoproteins through pharmacological intervention potentially prevents the development of thrombotic vascular complications in diabetic subjects.
|
Selected Abbreviations and Acronyms |
|---|
|
|
Acknowledgments |
|---|
This study was supported by operating grants from the Canadian Diabetes Association in the memory of the late Archibald Mitchell and from the Dr Paul H.T. Thorlakson Foundation to G.X.S. We would like to thank Dr H.R. Knull (University of North Dakota, Grand Forks) for help with analysis of the amino acid profile and Dr P.E. DiCorleto (The Cleveland Clinic Foundation, Cleveland, Ohio) for providing the cDNA probes.
Received May 13, 1997; accepted February 5, 1998.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Ruderman NB, Williamson JR, Brownlee M. Glucose and diabetic vascular disease. FASEB J.. 1992;6:2905–2914.[Abstract]
2. Garcia-Frade LJ, de la Calle H, Torrado MC, Lara JI, Cuellar L, Garcia-Avello A. Hypofibrinolysis associated with vasculopathy in non insulin dependent diabetes mellitus. Thromb Res.. 1990;59:51–59.[Medline] [Order article via Infotrieve]
3. Henkin JH, Marcotte P, Yang H. The plasminogen-plasmin system. Prog Cardiovasc Dis.. 1991;34:179–192.[Medline] [Order article via Infotrieve]
4. Saksela O, Rifkin DB. Cell-associated plasminogen activation: regulation and physiological functions. Annu Rev Cell Biol.. 1988;4:93–126.
5. Loskutoff DJ, Aken BEV, Seiffert D. Abnormalities in the fibrinolytic system of the vascular wall associated with atherosclerosis. Ann N Y Acad Sci.. 1994;748:177–184.[Medline] [Order article via Infotrieve]
6.
Schneiderman J, Sawdey MS, Keeton MR, Bordin GM,
Bernstein EF, Dilley RB, Loskutoff DJ. Increased type 1
plasminogen activator inhibitor
gene expression in atherosclerotic human arteries. Proc Natl Acad
Sci U S A.. 1992;89:6998–7002.
7. Carmassi F, Morale M, Paccent R, DeNegre F, Monzani F, Navalesi R, Mariani G. Coagulation and fibrinolytic system impairment in insulin dependent diabetic mellitus. Thromb Res.. 1992;67:643–654.[Medline] [Order article via Infotrieve]
8.
Stiko-Rahm A, Wiman B, Hamsten A, Nilsson J. Secretion
of plasminogen activator
inhibitor-1 from cultured human umbilical vein
endothelial cells is induced by very low density
lipoprotein. Arteriosclerosis.. 1990;10:1067–1073.
9.
Tremoli E, Camera M, Maderna P, Sironi L, Prati L,
Colli S, Piovella F, Bernini F, Corsini A, Mussoni L. Increased
synthesis of plasminogen activator
inhibitor-1 by cultured human endothelial
cells exposed to native and modified LDLs: an LDL receptor–independent
phenomenon. Arterioscler Thromb.. 1993;13:338–346.
10.
Latron Y, Chautan M, Anfosso F, Alessi MC, Nalbone G,
Lafont H, Juhan-Vague I. Stimulating effect of oxidized low density
lipoprotein on plasminogen activator
inhibitor-1 synthesis by endothelial cells.
Arterioscler Thromb.. 1991;11:1821–1829.
11.
Kugiyama K, Sakamoto T, Misumi I, Sugiyama S, Ohgushi
M, Ogawa H, Horiguchi M, Yasue H. Transferable lipids in oxidized
low-density lipoprotein stimulate plasminogen
activator inhibitor-1 and inhibit tissue type
plasminogen activator release from
endothelial cells. Circ Res.. 1993;73:335–343.
12. Etingin OR, Hajjar DP, Hajjar KA, Harpel PC, Nachman RL. Lipoprotein(a) regulates plasminogen activator inhibitor-1 expression in endothelial cells. J Biol Chem.. 1991;226:2459–2465.
13. Ren S, Man RYK, Angel A, Shen GX. Oxidative modification enhances lipoprotein(a)-induced overproduction of plasminogen activator inhibitor-1 in cultured vascular endothelial cells. Atherosclerosis.. 1997;128:1–10.[Medline] [Order article via Infotrieve]
14.
Levin EG, Miles LA, Fless GM, Scanu AM, Baynham P,
Curtiss LK, Plow EF. Lipoproteins inhibit the secretion of tissue
plasminogen activator from human
endothelial cells. Arterioscler Thromb.. 1994;14:438–442.
15. Kobayashi K, Watanabe J, Umeda F, Nawata H. Glycation accelerates the oxidation of low density lipoprotein by copper ions. Endocrinol J.. 1995;42:461–465.
16.
Bunn HF, Gabbay KH, Gallop PM. The glycosylation of
haemoglobin: relevance to diabetes mellitus. Science.. 1978;200:21–27.
17. Lyons TJ, Baynes JW, Patrick JS, Colwell JA, Lopes-Virella MF. Glycosylation of low density lipoprotein in patients with type I (insulin-dependent) diabetes: correlation with other parameters of glycaemic control. Diabetologia.. 1986;29:685–689.[Medline] [Order article via Infotrieve]
18.
Snyder ML, Polacek D, Scanu AM, Fless GM. Comparative
binding and degradation of lipoprotein(a) and low density lipoprotein
by human monocyte-derived macrophages. J Biol
Chem.. 1992;267:339–346.
19. Brown MR, Keith TJ, Knull HR. Nonenzymatic incorporation of glucose and galactose into brain cytoskeletal proteins in vitro. Neurochem Int.. 1992;21:177–183.[Medline] [Order article via Infotrieve]
20. Treb B, Hughes GJ, Winterhalter KH. Synthesis and quantitation of glucitollysine, a glycosylated amino acid elevated in proteins from diabetics. Anal Biochem.. 1982;119:330–334.[Medline] [Order article via Infotrieve]
21. Shen X-Y, Figard PH, Kaduce TL, Spector AA. Conversion of 15-hydroxyeicosatetraenoic acid to 11-hydroxyhexadecatrienoic acid by endothelial cells. Biochemistry.. 1988;27:996–1004.[Medline] [Order article via Infotrieve]
22. Chirgwin JM, Pryzbyla AE, MacDonald RJ, Rutter WJ. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry.. 1979;18:5294–5299.[Medline] [Order article via Infotrieve]
23. Wun TC, Kretzmer KK. cDNA cloning and expression in E. coli of a plasminogen activator inhibitor (PAI) related to a PAI produced by HepG2 hepatoma cell. FEBS Lett.. 1987;210:11–16.[Medline] [Order article via Infotrieve]
24. Cockell KA, Ren S, Sun J, Angel A, Shen GX. Effect of thrombin on release of plasminogen activator inhibitor-1 from cultured primate arterial smooth muscle cells. Thromb Res.. 1995;77:119–131.[Medline] [Order article via Infotrieve]
25. Corongiu FP, Lai M, Milla A. Carbon tetrachloride, bromotrichloromethane and ethanol acute intoxication. Biochem J.. 1983;212:625–631.[Medline] [Order article via Infotrieve]
26. Sevanian A, Hodis HN, Hwang J, McLeod LL, Peterson H. Characterization of endothelial cell injury by cholesterol products in oxidized LDL. J Lipid Res.. 1995;36:1971–1986.[Abstract]
27. Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissue by thiobarbituric acid reaction. Anal Biochem.. 1979;95:351–358.[Medline] [Order article via Infotrieve]
28. Liu S-Y, Lu X, Choy S, Dembinski TC, Hatch GM, Mymin D, Shen X, Angel A, Choy PC, Man RYK. Alteration of lysophosphatidylcholine content in low density lipoprotein after oxidative modification: relationship to endothelium dependent relaxation. Cardiovasc Res.. 1994;28:1476–1481.[Medline] [Order article via Infotrieve]
29.
George R, Barbar DL, Schneider WJ.
Characterization of the chicken oocyte receptor for low and very low
density lipoproteins. J Biol Chem. 1987;262:16838–16847.
30.
Picard S, Parthasarathy S, Fruebis J, Witztum JL.
Aminoguanidine inhibits oxidative modification of low density
lipoprotein and the subsequent increase in uptake by macrophage
scavenger receptors. Proc Natl Acad Sci U S A.. 1992;89:6876–6880.
31. Edelstein D, Brownlee M. Mechanistic studies of advanced glycosylation end product inhibition by aminoguanidine. Diabetes.. 1992;41:26–29.[Abstract]
32. Juhan-Vague I, Roul C, Alessi MC, Ardissone JP, Heim M, Vague P. Increased plasminogen activator inhibitor activity in non insulin dependent diabetic patients: relationship with plasma insulin. Thromb Haemost.. 1989;61:370–373.[Medline] [Order article via Infotrieve]
33. Schneider DJ, Nordt TK, Sobel BE. Attenuated fibrinolysis and accelerated atherogenesis in type II diabetic patients. Diabetes. 1993;42:1–7.[Abstract]
34. Fulop M, Tannenbaum H, Dreyer N. Ketotic hyperosmolar coma. Lancet.. 1973;2:635–639.[Medline] [Order article via Infotrieve]
35. Kim HJ, Kurup IV. Nonenzymatic glycosylation of human plasma low density lipoprotein: evidence for in vitro and in vivo glucosylation. Metabolism.. 1982;31:348–353.[Medline] [Order article via Infotrieve]
36.
Au YTP, Kenagy RD, Clowes AW. Heparin selectively
inhibits the transcription of tissue-type plasminogen
activator in primate arterial smooth muscle
cells during mitogenesis. J Biol Chem.. 1992;267:3438–3444.
37.
Brownlee M, Vlassara H, Kooney T, Ulrich P, Cerami A.
Aminoguanidine prevents diabetes-induced arterial wall
protein crosslinking. Science.. 1986;232:1629–1632.
38.
Schmidt AM, Hori O, Brett J, Yan SD, Wautier J-L, Stern
D. Cellular receptors for advanced glycation end products:
implications for induction of oxidant stress and cellular dysfunction
in the pathogenesis of vascular lesions. Arterioscler
Thromb.. 1994;14:1521–1528.
39. Brett J, Schmidt AM, Yan SD, Zou YS, Weidman E, Pinsky D, Nowygrod R, Neeper M, Przysiecki C, Shaw A, Migheli A, Stern D. Survey of the distribution of a newly characterized receptor for advanced glycation end products in tissues. Am J Pathol.. 1993;143:1699–1712.[Abstract]
40. Corbett JA, Tilton RG, Chang K, Hasan KS, Ido Y, Wang JL, Sweetland MA, Lancaster JR Jr, Williamson JR, McDaniel ML. Aminoguanidine, a novel inhibitor of nitric oxide formation, prevents diabetic vascular dysfunction. Diabetes.. 1992;41:552–556.[Abstract]
41. Barlier AM, Benamouzig R, Tome D. Putrescine transport in differentiated Caco-2 cells. Gastroenterol Clin Biol.. 1996;20:178–184.[Medline] [Order article via Infotrieve]
42.
Philis-Tsimikas A, Parthasarathy S, Picard S, Palinski
W, Witztum JL. Aminoguanidine has both pro-oxidant and antioxidant
activity toward LDL. Arterioscler Thromb Vasc Biol.. 1995;15:367–376.
43.
Bucala R, Makita Z, Koschinsky T, Cerami A, Vlassara H.
Lipid advanced glycosylation: pathway for lipid oxidation in vivo.
Proc Natl Acad Sci U S A.. 1993;90:6434–6438.
44. Vargas FF, Caviedes PF, Grant DS. Electrophysiological characteristics of cultured human umbilical vein endothelial cells. Microvasc Res.. 1994;47:153–165.[Medline] [Order article via Infotrieve]
This article has been cited by other articles:
 |
|
 |
|
  G. P. Van Guilder, G. L. Hoetzer, J. J. Greiner, B. L. Stauffer, and C. A. DeSouza Acute and chronic effects of vitamin C on endothelial fibrinolytic function in overweight and obese adult humans J. Physiol., July 15, 2008; 586(14): 3525 - 3535. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Zhao and G. X. Shen Involvement of Heat Shock Factor-1 in Glycated LDL-Induced Upregulation of Plasminogen Activator Inhibitor-1 in Vascular Endothelial Cells Diabetes, May 1, 2007; 56(5): 1436 - 1444. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. K. Ray, C. P. Cannon, D. A. Morrow, A. J. Kirtane, J. Buros, N. Rifai, C. H. McCabe, C. M. Gibson, and E. Braunwald Synergistic relationship between hyperglycaemia and inflammation with respect to clinical outcomes in non-ST-elevation acute coronary syndromes: analyses from OPUS-TIMI 16 and TACTICS-TIMI 18 Eur. Heart J., April 2, 2007; (2007) ehm010v1. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. K. Ray, C. P. Cannon, R. Cairns, D. A. Morrow, N. Rifai, A. J. Kirtane, C. H. McCabe, A. M. Skene, C. M. Gibson, P. M. Ridker, et al. Relationship Between Uncontrolled Risk Factors and C-Reactive Protein Levels in Patients Receiving Standard or Intensive Statin Therapy for Acute Coronary Syndromes in the PROVE IT-TIMI 22 Trial J. Am. Coll. Cardiol., October 18, 2005; 46(8): 1417 - 1424. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Veiraiah Hyperglycemia, Lipoprotein Glycation, and Vascular Disease Angiology, July 1, 2005; 56(4): 431 - 438. [Abstract] [PDF]
|
|
|
|
 |
|
 B. E. Sobel, D. J. Taatjes, and D. J. Schneider Intramural Plasminogen Activator Inhibitor Type-1 and Coronary Atherosclerosis Arterioscler Thromb Vasc Biol, November 1, 2003; 23(11): 1979 - 1989. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Velarde, A. J. Jenkins, J. Christopher, T. J. Lyons, and A. A. Jaffa Activation of MAPK by modified low-density lipoproteins in vascular smooth muscle cells J Appl Physiol, September 1, 2001; 91(3): 1412 - 1420. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Zimmermann, U. Panzenbock, A. Wintersperger, S. Levak-Frank, W. Graier, O. Glatter, G. Fritz, G. M. Kostner, and R. Zechner Lipoprotein Lipase Mediates the Uptake of Glycated LDL in Fibroblasts, Endothelial Cells, and Macrophages Diabetes, July 1, 2001; 50(7): 1643 - 1653. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Ren and G. X. Shen Impact of Antioxidants and HDL on Glycated LDL-Induced Generation of Fibrinolytic Regulators From Vascular Endothelial Cells Arterioscler Thromb Vasc Biol, June 1, 2000; 20(6): 1688 - 1693. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Ren, S. Shatadal, and G. X. Shen Protein kinase C-beta mediates lipoprotein-induced generation of PAI-1 from vascular endothelial cells Am J Physiol Endocrinol Metab, April 1, 2000; 278(4): E656 - E662. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. M Wileman, N. A Booth, N. Moore, B. Redmill, J. V Forrester, and R. M Knott Regulation of plasminogen activation by TGF-beta in cultured human retinal endothelial cells Br J Ophthalmol, April 1, 2000; 84(4): 417 - 422. [Abstract] [Full Text]
|
|
|
|
|
|
L. H. Kuller, P. Velentgas, J. Barzilay, N. J. Beauchamp, D. H. O’Leary, and P. J. Savage Diabetes Mellitus : Subclinical Cardiovascular Disease and Risk of Incident Cardiovascular Disease and All-Cause Mortality Arterioscler Thromb Vasc Biol, March 1, 2000; 20(3): 823 - 829. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||