Departments of Internal Medicine and Physiology, University of Manitoba, Winnipeg, Manitoba, Canada R3E OW3
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ABSTRACT |
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Elevated levels of low-density lipoproteins (LDL) and
lipoprotein(a) [Lp(a)] have been considered strong risk
factors for atherosclerotic cardiovascular disease. Increased
production of plasminogen activator inhibitor-1 (PAI-1) has been
implicated in the development of thrombosis and atherosclerosis.
Previous studies by our group and others demonstrated that oxidation
enhances LDL- and Lp(a)-induced production of PAI-1 in human umbilical vein endothelial cells (HUVEC). The present study examined the involvement of protein kinase C (PKC) and its isoform in vascular endothelial cells (EC) induced by native or oxidized LDL and Lp(a). Treatment with Lp(a) or LDL transiently increased PKC activity at 15 min and 5.5 h after the start of lipoprotein treatment in EC.
Copper-oxidized LDL and Lp(a) induced greater PKC activation in EC
compared with comparable forms of those lipoproteins. Additions of 1 µM calphostin C, a PKC-specific inhibitor, at the beginning or 5 h,
but not
9 h, after the initiation of lipoprotein treatment, blocked
native and oxidized LDL- or Lp(a)-induced increases in PKC activity and
PAI-1 production. Treatment of LDL, Lp(a), or their oxidized forms was
induced in translocation of PKC-
1 from cytosol to membrane in HUVEC.
Treatments with 60 nM 379196, a PKC-
-specific inhibitor, effectively
prevented PAI-1 production induced by LDL, Lp(a), or their oxidized
forms in HUVEC and human coronary artery EC. The results suggest that
activation of PKC-
may mediate the production of PAI-1 in cultured
arterial and venous EC induced by LDL, Lp(a), or their oxidized forms.
low-density lipoproteins; lipoprotein(a); oxidation; plasminogen activator inhibitor-1
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INTRODUCTION |
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ELEVATION OF PLASMA LOW-DENSITY lipoprotein (LDL) has been considered a strong risk factor for the development of coronary artery disease (40). Lipoprotein(a) [Lp(a)] is a structural homolog of LDL with a unique apolipoprotein (Apo), Apo(a). Results from epidemiological studies suggest that increased plasma Lp(a) is an independent lipoprotein risk factor for premature cardiovascular disease (2, 4). It has been widely accepted that oxidative modification increases atherogenicity of lipoproteins (33). Multiple lines of evidence suggest that oxidation also increases thrombogenicity of lipoproteins. For example, Cu2+ oxidation enhanced the effect of LDL on the expression of tissue factor in macrophages (39). Oxidized Lp(a) was stronger than its native form in terms of the inhibition on the binding of plasminogen to U937 monocytes (23). Those findings suggest that oxidative modification of LDL may superimpose disorders in the coagulation and fibrinolysis induced by these atherogenic lipoproteins.
Intravascular thrombosis is a consequence of an imbalance between coagulation and fibrinolysis. Vascular endothelial cells (EC) synthesize tissue plasminogen activator, urokinase plasminogen activator, and their major physiological inhibitor, plasminogen activator inhibitor-1 (PAI-1). The expression of PAI-1 was increased in thrombotic vascular wall (1). Elevated PAI-1 activity in plasma has been considered a risk factor for coronary artery disease (7). The production of PAI-1 from vascular EC was stimulated by thrombin (20), heparin, EC growth factor (17), activated protein C (38), interleukin-1, endotoxin (8), and plasma lipoproteins (5, 14, 18, 19, 26, 27, 33, 37). The release of PAI-1 from cultured vascular EC was increased by LDL in native or oxidized form (5, 18, 19, 37). Lp(a) is a stronger agonist than LDL on the stimulation of PAI-1 production in EC (9, 26). Previous studies by our group demonstrated that oxidation enhanced the effects of Lp(a) and LDL on PAI-1 production in vascular EC (26). Protein kinase C (PKC) mediates PAI-1 production in vascular EC induced by thrombin (11). The signal transduction mechanism for PAI-1 overproduction induced by lipoproteins or their oxidized forms remains unknown. The present study examined the involvement of PKC and its isoform in PAI-1 production induced by LDL, Lp(a), or their oxidized forms in cultured human venous and arterial EC.
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METHODS |
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Isolation and modification of lipoproteins. Plasma fractions <1.21 in density were separated from fresh human plasma by ultracentrifugation in the presence of 1 mM EDTA. Lipoprotein fractions were applied to a lysine-Sepharose 4B affinity chromatography column (32). Lp(a) bound to the affinity column was eluted with 20 mM 6-amino hexanoic acid in 0.1 M phosphate buffer (pH 7.4) containing 1 mM benzamidine and 0.01% EDTA. Concentrations of Lp(a) were estimated by a Macro Lp(a) enzyme immunoassay kit (Terumo, Elkon, MD). Apo(a) isoforms were determined by means of SDS-PAGE in combination with Western blotting analysis, as previously described (29). Lp(a)-free LDL (density 1.019-1.063) was isolated from lipoprotein fractions eluted from the affinity column by sequential floating ultracentrifugation. For oxidative modification, lipoproteins were thoroughly dialyzed against EDTA-free buffer and then were treated with 5 µM CuSO4 for 24 h at 22°C. The reaction was terminated by the addition of EDTA to the final concentration of 0.6 mM (22). The extent of lipid peroxidation was verified by measurement of the levels of thiobarbituric acid reactive substance. Tetramethoxypropane was applied as a standard, as previously described (25). Lipoproteins were stored in sealed tubes filled with nitrogen and kept in the dark at 4°C to prevent oxidation during storage. Endotoxin in lipoprotein preparations was undetectable with a Limulus Amebocyte Lysate assay kit (Sigma, St. Louis, MO) with a low detection limit of 0.05 ng endotoxin/ml. Protein concentrations of the lipoproteins were estimated by a modified Lowry method (10). Molar concentrations of LDL and Lp(a) were determined from protein contents of lipoproteins and molecular weights of their apolipoproteins.
Cell culture and stimulation.
Human umbilical vein EC (HUVEC) were isolated by collagenase digestion
and verified by nonoverlapping cobblestone-like morphology and the
presence of factor VIII antigen. HUVEC were grown in M-199 medium
supplemented with 10% fetal bovine serum, 30 µg/ml EC growth supplements (Sigma), 100 µg/ml heparin, 0.1 mM nonessential amino acids, and 4 mM L-glutamine in a humidified incubator under
95% air-5% CO2 at 37°C. Human coronary arterial EC
(HCAEC) were originally received from Clonetics (San Diego, CA). The
cells were cultured in endothelial growth medium-MV (Clonetics) and
used within passage 8. Cells were seeded and stimulated in
60-mm dishes for experiments detecting PKC activity and in 12-well
plates for measuring PAI-1 release. Equivalent density of HUVEC or
HCAEC was seeded in cell culture dishes. The effects of LDL and Lp(a)
on PAI-1 production were not significantly affected by treatments with
EC growth supplements or hirudin (a specific thrombin inhibitor). No
detectable amount of endotoxin was found in media before or after
incubations with lipoproteins. Confluent cells were treated with
lipoproteins at indicated doses in medium without heparin under 5%
CO2 at 37°C (26). Calphostin C (Sigma), a
photoactivatable PKC inhibitor (15), was used in cultured cells under
light exposure. PKC--specific inhibitor, 379196, was kindly
provided by Dr. Kirk Ways and James Gillig (Eli Lilly,
Indianapolis, IN).
ELISA for PAI-1 antigen. Postcultural media were collected at the end of incubations for PAI-1 measurement. Cells were harvested with 0.1 N NaOH for protein determination. The levels of total PAI-1 antigen (free + complexed forms) in the medium were determined with the use of Imubind PAI-1 ELISA kits with monoclonal antibody against human PAI-1 (American Diagnostica, Greenwich, CT) according to the manufacturer's instructions. Absorbency at 490 nm was read on a microplate reader. PAI-1 antigen was presented in micrograms per milligram of total cellular proteins or percentage of controls without addition (26).
PKC activity assay.
At the end of incubation, cells were harvested by means of Triton X-100
and 0.1% SDS. The cell lysates were incubated with 20 mM
Tris · HCl (pH 7.5) supplemented with 10 mM
MgCl2, 0.5 mM EDTA, 0.5 mM EGTA, 2 mM sodium pyrophosphate,
0.1 mM phenylmethylsulfonyl fluoride, 25 µg/ml leupeptin, 100 µM
CaCl2, 320 µg/ml phosphatidylserine, 30 µg/ml
dioctanoylglycerol, 100 µM [-32P]ATP
(1,000 cpm/pmol), and 5 µM neurogrinin (CalBiochem, La Jolla, CA), a
PKC-specific peptide substrate (31), at 25°C for 10 min (6).
Aliquots of the reaction mixture were spotted on membrane filters. The
filters were thoroughly washed and then were subjected to scintillation
counting. The activity of PKC was presented in picomoles per minute per
milligram of cellular proteins.
Detection of translocation of PKC isoform by Western blotting
analysis.
After experimental treatment, HUVEC were harvested by a rubber
policeman and homogenized in a glass homogenizer. Cytosolic and
membrane fractions of the cells were separated by TLX-100 ultracentrifugation (Beckman Canada, Ontario, Canada) at 100,000 g at 4°C for 1 h. Proteins in cytosol and membrane pellets
were analyzed on 10% SDS-PAGE and then blotted to nitrocellulose
membrane. PKC isoforms in the subcellular fractions were detected by
polyclonal antibodies against human PKC-1 or -
2 (Santa Cruz, CA)
in combination with appropriate second antibodies conjugated with
alkaline phosphatase.
Statistical analysis. Values were expressed as means ± SD from quadruplicate wells. Student's t-test was used to compare probability between two groups. For multiple groups, ANOVA was performed by one-way ANOVA followed by Duncan's test. A P value of <0.05 was considered to be statistically significant.
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RESULTS |
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Effects of LDL, Lp(a), and their oxidized forms on generation of
PAI-1 from EC.
Previous studies in this laboratory demonstrated that the increases in
PAI-1 release from HUVEC reached plateau after 48 h of incubation with
10 nM native or oxidized Lp(a). Native LDL at the 10 nM level did not
significantly increase PAI-1 generation from EC (26). Initial
experiments in the present study examined the effects of 100 nM native
and oxidized LDL for 48 h on PAI-1 generation from HUVEC compared with
10 nM LDL or Lp(a) in native or oxidized form (Fig.
1). The levels of PAI-1 antigen in the EC
medium were significantly elevated after treatment with 100 nM native
LDL (P < 0.01) but not with 10 nM native LDL. Native Lp(a) at
the level of 10 nM stimulated PAI-1 release (P < 0.01), as
previously described (23). Cu2+ oxidation enhanced PAI-1
generation in EC induced by LDL or Lp(a) under all tested conditions
(P < 0.05 or 0.01).
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Changes of PKC activity in lipoprotein-treated EC.
The levels of PKC activity in cell lysates of HUVEC were monitored
during the incubation with LDL, Lp(a), or their oxidized forms. Cells
were treated with 10 nM Lp(a) or 100 nM LDL in native or oxidized form
for a time period of from 5 min to 24 h. The levels of PKC activity in
cell lysates were transiently increased at ~15 min and 5.5 h after
the addition of Lp(a), LDL, or their oxidized forms and quickly
returned to the basal level. The elevations of PKC activity at 15 min
and at 5.5 h in cells treated with oxidized Lp(a) or LDL were
significantly greater than those in cells treated with native Lp(a) or
LDL under equivalent conditions (Fig.
2).
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Characterization of activation of PKC associated with PAI-1
production induced by lipoproteins.
Treatments of EC with 1 µM calphostin C, a PKC inhibitor (15), begun
at the start (0 h) of lipoprotein stimulation, blocked the
overproduction of PAI-1 induced by LDL (100 nM), Lp(a) (10 nM), or
their oxidized forms in HUVEC (Fig. 3).
Addition of calphostin C at 5 h (Fig. 3), which was only 30 min before
the second PKC peak but hours after the first one (Fig. 1), prevented
the generation of PAI-1 induced by native and oxidized LDL or Lp(a).
Interestingly, the addition of calphostin C at 9 h or at 16 h after the
initiation of lipoprotein treatment did not significantly alter LDL-,
Lp(a)-, or their oxidized form-induced PAI-1 generation from EC (Fig. 3).
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Translocation of PKC-1 in EC induced by lipoprotein
treatment.
PKC-
1, but not -
2, was detected in subcellular fractions of
HUVEC. At basal condition, PKC-
1 was predominantly found in cytosol
but was undetectable in membrane fractions. After treatment with 100 nM
LDL, 10 nM Lp(a), or their oxidized forms for 5.5 h, substantial
amounts of PKC-
1 were detected in membrane fractions of EC (Fig.
4, A and B).
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Involvement of PKC- in lipoprotein-induced PKC
activation and PAI-1 overproduction.
379196 is one of the PKC-
-specific inhibitors recently developed at
Eli Lilly (J. Gillig and co-workers, unpublished observations). Treatment with 60 nM 379196 blocked the elevation of PKC activity in
HUVEC at 5.5 h after the addition of 100 nM LDL, 10 nM Lp(a), or their
oxidized forms (Fig. 5A). An
identical dose of the PKC-
inhibitor effectively prevented
generation of PAI-1 induced by LDL, Lp(a), or their oxidized forms from
EC (Fig. 5B). Treatment with 379196 alone did not significantly
affect PKC activity or PAI-1 production in HUVEC (Fig. 5).
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Involvement of PKC- in PAI-1 production induced by
LDL, Lp(a), and their oxidized forms in arterial EC.
Because atherosclerosis occurs predominantly in middle- to large-size
arteries, the effects of the lipoproteins on PAI-1 production and the
involvement of PKC-
were verified in EC isolated from human coronary
artery. The basal level of PAI-1 antigen in the medium of HCAEC after
48 h of incubation was 2 to 3 times greater than that of HUVEC.
Treatments with native and oxidized LDL (100 nM) or Lp(a) (10 nM) for
48 h significantly increased PAI-1 release from HCAEC (P < 0.01 or 0.001). Oxidation significantly enhanced the effects of LDL and
Lp(a) on PAI-1 generation from HCAEC (P < 0.05). Increases in
PAI-1 generation induced by LDL, Lp(a), or their oxidized forms from
HCAEC (+23 to +67%) were less extensive than those from HUVEC (+52 to
+113%). PKC-
-specific inhibitor, 379196 (60 nM), effectively
prevented PAI-1 overproduction induced by LDL, Lp(a), and their
oxidized forms in HCAEC (Fig. 6).
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DISCUSSION |
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The results of the present study demonstrate for the first time that PKC mediates the regulation of PAI-1 generation induced by atherogenic lipoproteins in vascular EC. Previous studies indicated that PAI-1 production induced by oxidized LDL in EC may be mimicked by the addition of lysophosphatidylcholine (lyso-PC), a peroxidation product of phosphatidylcholine (7). The content of lyso-PC was increased in oxidized LDL compared with native LDL (22). PKC activation is involved in lyso-PC-induced production of monocyte chemoattractant protein-1 in HUVEC (34). The present study indicates that physiological levels of LDL and Lp(a) elevate PKC activity and stimulate PAI-1 production, and the effects of both types of lipoproteins are enhanced by Cu2+ oxidation. The involvement of PKC in this process was verified by the effectiveness of PKC-specific inhibitors on PKC activation and PAI-1 generation induced by the atherogenic lipoproteins. Lipid peroxidation products, including lyso-PC, potentially contribute to PKC activation and PAI-1 production induced by oxidized lipoproteins in EC. LDL and Lp(a) share extensive structural homology with the major difference in the presence of Apo(a) in Lp(a) but not in LDL. Lp(a) particles were more susceptible to oxidation than LDL (24). LDL particles may be oxidatively modified by EC (34). EC-mediated oxidative modification may contribute to native LDL- and Lp(a)- induced PKC activation and PAI-1 production in EC. This hypothesis is supported by a separate study by our group, which demonstrated that antioxidants, BHT or vitamin E, inhibited native LDL-induced generation of PAI-1 from HUVEC (30).
The results of the present study indicate that LDL, Lp(a), and their
oxidized forms induce two PKC peaks in EC within 24 h of stimulation.
The first peak of PKC activity was detected at 15 min after the start
of lipoprotein stimulation. The early PKC activation does not likely
mediate native or oxidized lipoprotein-induced PAI-1 production in EC,
on the basis of the fact that the addition of calphostin C at 5 h after
the first PKC peak still effectively prevented lipoprotein-induced
PAI-1 production. The second PKC peak occurred ~5.5 h after
initiation of the stimulation. The addition of the PKC-specific
inhibitor at 30 min before the second PKC peak effectively prevented
PAI-1 production induced by LDL, Lp(a), or their oxidized forms in EC.
The additions of PKC inhibitor at 9 h after initiation of lipoprotein
stimulation failed to inhibit lipoprotein-induced PAI-1 overproduction.
Those findings imply that a delayed elevation of PKC activity occurring
between 5 and 9 h after the initiation of lipoprotein stimulation may mediate PAI-1 production induced by LDL, Lp(a), or their oxidized forms
in vascular EC.
Recent studies reported by Lopez et al. (21) provided indirect evidence
that PKC- may mediate phorbol myristic acetate (PMA, a PKC
agonist)-induced PAI-1 production in HL-60 promyolocytes by the fact
that PMA failed to increase PAI-1 production in a PKC-
-deficient
cell line, HL-525. The results of the present study demonstrated for
the first time that lipoproteins induced translocation of PKC-
1 in
vascular EC. PKC-
2 is undetectable in subcellular fractions of HUVEC
at basal or stimulated conditions; however, the possibility of the
involvement of PKC-
2 in lipoprotein-induced PAI-1 production has not
been completely excluded by the data of this study.
The PKC- inhibitor 379196, as well as LY-333531 (3), is a
dimethylamine analog and specifically inhibits the activity of PKC-
1
and -
2. The ED50 of 379196 for PKC-
1 (50 nM) and
-
2 (30 nM) was 12- to 23-fold lower than that for PKC-
(0.6 µM), PKC-
(0.6 µM), and PKC-
(0.7 µM) assessed with histone
H1. Therefore, cellular response inhibited by <200 nM 379196 may be
considered PKC-
-specific activity (J. Gillig and co-workers,
unpublished observations). The results of the present study demonstrate
that 60 nM 379196 prevented PKC activation and PAI-1 overproduction in
EC induced by LDL, Lp(a), or their oxidized forms. This suggests that
PAI-1 overproduction induced by native and oxidized LDL and Lp(a) in EC
may be mediated via a PKC-
-specific pathway. Previous studies
indicated that LY-333531 prevented the development of vascular
complications in diabetic animals (13). Hyperglycemia promotes the
glycation of plasma proteins, including lipoproteins. Glycation
augments the susceptibility of lipoproteins to oxidation (16). Our
studies indicated that glycation enhanced LDL-induced PAI-1 production
in vascular EC (42). PKC-
inhibitors may prevent vascular
complications in diabetic animals partially by inhibiting the
generation of PAI-1 induced by glycooxidized lipoproteins.
The results of the present study demonstrate that LDL, Lp(a), and their
oxidized forms increase PAI-1 generation not only in HUVEC but also in
HCAEC. The production of PAI-1 induced by the lipoproteins in both
HUVEC and HCAEC was effectively inhibited by a PKC--specific
inhibitor. Those findings suggest that PKC-
mediates PAI-1
overproduction induced by native and oxidized LDL or Lp(a) in both
venous and arterial EC. In an unstimulated condition, HCAEC produced
greater amounts of PAI-1 than HUVEC did. The relative increases in
PAI-1 generation induced by LDL, Lp(a), or their oxidized forms from
HCAEC were generally less than those from HUVEC; however, differences
in the types of media and passage numbers between HCAEC and HUVEC
potentially affect the PAI-1 generation at basal level or their
responses to the lipoproteins. Besides, fibrinolytic activity is known
to be affected by plasminogen activators and other components of the
fibrinolytic system, including the receptors and binding proteins of
plasminogen activators (28), which are beyond the scope of the present study.
Impaired fibrinolytic activity characterized by elevated levels of
PAI-1 with or without a reduction in tissue plasminogen activator has
been found frequently in patients with coronary artery disease (7, 12).
Increases in PAI-1 protein and mRNA were detected in atherosclerotic
and thrombotic lesions in arteries and veins (1, 41). Elevation of
PAI-1 generation from vascular EC may attenuate fibrinolytic activity
and promote the development of thrombosis with and without the presence
of atherosclerotic lesions. The results of the present study provide
original evidence that PKC- may be involved in atherogenic
lipoprotein-induced overproduction of PAI-1 in coronary arterial EC.
PKC-
-specific inhibitors may also be considered a type of candidate
for the prevention of ischemic events in patients with coronary artery disease.
In summary, the present study indicates that a delayed activation of
PKC- mediates PAI-1 overproduction induced by LDL, Lp(a), and their
oxidized forms in vascular EC. Treatment with PKC-
inhibitor
effectively prevented the generation of PAI-1 induced by the
atherogenic lipoproteins in cultured venous and arterial EC. The
findings of this study provide a rationale to investigate the effect of
PKC-
inhibitors on the prevention of thrombotic events in vivo.
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ACKNOWLEDGEMENTS |
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The authors acknowledge with thanks Dr. Kirk Ways and James Gillig (Eli Lilly) for the supply of important reagents, and George King (Joselin Diabetes Center, Boston, MA) for helpful advice.
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FOOTNOTES |
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This study was supported by operating grants from the Medical Research Council of Canada, Canadian Diabetes Association (in memory of the late Archibald Mitchell), Manitoba Health Research Council, and the University of Manitoba (to G. X. Shen).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: G. X. Shen, Departments of Internal Medicine and Physiology, University of Manitoba, BS4 730 William Ave, Winnipeg, Manitoba, Canada R3E 0W3 (E-mail: gshen{at}ms.umanitoba.ca).
Received 7 July 1999; accepted in final form 12 November 1999.
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