Lipoprotein-Associated Phosphatidylethanol Increases the
Plasma Concentration of Vascular Endothelial
Growth Factor
Marja K. Liisanantti, Minna L. Hannuksela, Maria E. Rämet, Markku J. Savolainen
Downloaded from http://atvb.ahajournals.org/ by guest on July 31, 2017
Objective—To study whether qualitative changes in high-density lipoprotein (HDL) phospholipids mediate part of the
beneficial effects of alcohol on atherosclerosis, we investigated whether phosphatidylethanol (PEth) in HDL particles
affects the secretion of vascular endothelial growth factor (VEGF) from endothelial cells.
Methods and Results—PEth increased the secretion of VEGF into the culture medium of EA.hy 926 endothelial cells. The
mitogen-activated protein kinase (MAPK) phosphorylation increased by 3.3-fold and protein kinase C (PKC) by
2.2-fold by PEth-containing HDL. Moreover, we showed that intravenous injection of PEth incorporated into HDL
particles increased plasma concentration of VEGF by 2.4-fold in rats in vivo. Similar effect was observed when the rats
were injected with HDL particles isolated from alcohol drinkers.
Conclusions—HDL particles containing PEth affect endothelial cells by MAPK and PKC signaling. This may mediate the
effects of ethanol on the arterial wall by increasing VEGF secretion from endothelial vascular cells. That may explain,
at least in part, the beneficial effect of moderate alcohol consumption on atherosclerosis. (Arterioscler Thromb Vasc
Biol. 2004;24:1037-1042.)
Key Words: alcohol 䡲 endothelial cells 䡲 vascular endothelial growth factor 䡲 lipoproteins 䡲 phospholipids
A
s with plasma high-density lipoprotein (HDL) cholesterol concentration, HDL phospholipid concentration
also correlates inversely with the severity of coronary artery
disease.1 In addition, the ability of reconstituted HDL particles to inhibit endothelial cell adhesion molecule expression
depends on their phospholipid composition, which also emphasizes the importance of phospholipids for the putative
antiatherogenic functions of HDL particles.2
Previous epidemiological studies have shown that even
light or moderate alcohol consumption has protective effects
against coronary heart disease.3 A large part of the protective
effects could be mediated by an increase in HDL cholesterol,
but the exact mechanisms are not known.4 – 8 HDL may play
a crucial role in reverse cholesterol transport. Some data
suggest, however, that alcohol does not enhance reverse
cholesterol transport despite its increasing effect on the
plasma HDL cholesterol concentration.9 –11
HDL may also have other functions unrelated to its role in
reverse cholesterol transport, for example, HDL particles
seem to be able to regulate the expression or abundance of
various growth factors, endothelial nitric oxide synthase,
cytokines, and adhesion molecules.2,12–17 Alcohol drinkers
have elevated serum levels of adhesion molecules, but the
mechanism of increase is not known.18
The fact that even light alcohol consumption of 1 drink
twice per week protects against atherosclerosis compared
with total abstinence is difficult to explain, because such a
low level of drinking does not affect the HDL cholesterol
concentration.8,19 A plausible explanation for the effect of
moderate drinking may be provided by our new hypothesis
suggesting that the sustained action of occasional drinking is
caused by the involvement of lipoproteins as carriers of
phosphatidylethanol (PEth).
PEth, structurally an ethanol adduct of phosphatidic acid (a
lipid second messenger), is formed in the presence of ethanol
and affects cellular metabolism and function.20 –24 Ethanol
itself has been shown to induce in vitro angiogenesis in the
cultured endothelial cells by the activation of protein kinase C
(PKC) and mitogen-activated protein kinase (MAPK).25
The present research demonstrates that PEth markedly
increases the secretion of vascular endothelial growth factor
(VEGF) in cultured endothelial cells and increases its plasma
concentration in experimental animals in vivo. This effect of
lipoprotein-associated PEth is mediated mainly by MAPK.
Methods
Please see online Methods section at http://atvb.ahajournals.org for
further details.
Subjects
Male alcoholic subjects (n⫽17) referred by general practitioners for
withdrawal therapy to the Alcoholism Treatment Unit of Oulu and
Received January 21, 2004; revision accepted March 19, 2004.
From the Department of Internal Medicine, University of Oulu, Oulu, Finland.
Correspondence to Prof Markku J. Savolainen, Department of Internal Medicine, University of Oulu, P.O. Box 5000, FIN-90014 Oulu, Finland. E-mail
[email protected]
© 2004 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org
1037
DOI: 10.1161/01.ATV.0000128409.62292.d6
1038
Arterioscler Thromb Vasc Biol.
June 2004
healthy nonalcoholic men (n⫽17) recruited from among the local
paper mill employees were recruited for blood tests. Venous blood
samples were taken from subjects after an overnight fast. The clinical
characteristics of the study subjects are shown in Table I (available
online at http://atvb.ahajournals.org).
Animals
Anesthetized male Sprague-Dawley rats were injected intravenously
with native and PEth-modified HDL particles. Retro-orbital blood
(0.3 mL) was collected into EDTA-containing tubes for
VEGF determinations.
Isolation and Labeling of Lipoproteins
Lipoproteins were isolated from plasma by sequential ultracentrifugation on the basis of their density, as described.26 –28 PEth was
labeled,29 extracted,30 and separated by thin-layer chromatography
(TLC). Iodination of the PEth–HDL was performed using IODO
Beads Iodination Reagent (Perbio Science).
Chemical Analyses
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Plasma and lipoprotein compositions were determined by kits of
Roche Diagnostics GmbH (cholesterol, triglycerides), Wako Chemicals GmbH (phospholipids), and Bio-Rad Laboratories (proteins).
Cell Culture Experiments
Human endothelial EA.hy 926 cells were grown under standard
conditions and incubated for different time points in the serum-free
medium containing ethanol, phosphatidylcholine (PC), PEth, HDL,
PEth–HDL, or alcoholic HDL.
Determination of PEth–HDL Cell Association and
Selective PEth Uptake
EA.hy 926 cells were incubated for 5 hours with [125I]PEth–HDL
and [14C]-labeled PEth-HDL, and the cell association of PEth-HDL
and selective PEth uptake was determined, as described previously,
detailed by Acton and Rigotti.31
Immunoassays
The VEGF concentration was measured from the human and rat
plasma and cell culture medium by kits of R&D Systems.
Western Blot Analysis of PKC and p44/42 MAPK
Please see online Methods section at http://atvb.ahajournals.org for
further details.
Statistical Analysis
The results are expressed as means and standard deviation (SD). The
statistical significance of the differences between the means was
assessed by independent samples 2-tailed Student t test or 1-way
analysis of variance (ANOVA, Tukey test), as appropriate. P⫽0.05
was considered significant.
Results
Characteristics of the Study Subjects
Age, body mass index (BMI), and daily alcohol intake data
for the subjects are shown in Table I. Age and BMI did not
differ between the groups, nor did VLDL cholesterol, plasma
total triglyceride, or serum bilirubin concentration. The alcoholics had a 19% lower total cholesterol concentration
(P⬍0.002) and a 39% lower LDL cholesterol concentration
(P⬍0.0001) than the controls. The HDL cholesterol concentration was 28% higher in the group of alcoholics than in
controls (P⫽0.062).
Figure 1. Plasma concentration of VEGF is high in alcoholic
subjects and is increased after injection of alcoholic HDL or
PEth-modified HDL into rats. a, The mean plasma VEGF values
(n⫽17) are marked with horizontal bars. f indicates alcoholic
subjects; E, controls. b, The rats were injected with PBS (▫ indicates PBS, n⫽9), control HDL (Œ indicates control HDL, n⫽9),
alcoholic HDL (〫 indicates alcoholic HDL, n⫽8), or PEth–HDL
(F indicates PEth-HDL, n⫽9).
VEGF Concentration in the Plasma of the
Study Subjects
To examine the hypothesis that alcohol drinking is capable of
affecting the VEGF secretion or concentration in circulation,
we collected plasma samples from controls and alcoholics.
The plasma VEGF concentration was 39% higher in the
alcoholics (74⫾37 pg/mL, n⫽17) than in the controls (Figure
1a) (53⫾23 pg/mL, n⫽17, P⫽0.056), although there was
pronounced overlapping.
PEth Increases the Plasma VEGF Concentration
in Rats
To prove that PEth present in lipoprotein particles increases
the secretion of VEGF in vivo, the effects of PEth-modified
HDL on plasma VEGF concentration were determined in
vivo in rats. One group of rats (PBS) was injected intravenously with PBS buffer, whereas another group (control
HDL) received injections of human HDL isolated from
control subjects. The third group of rats (alcoholic HDL) was
injected with HDL isolated from the plasma samples of
alcoholics, and the fourth group (PEth–HDL) received intravenous injections of control HDL containing PEth added in
vitro. Alcoholic HDL increased the plasma VEGF concentration 2-fold (from 41⫾5 to 79⫾8 pg/mL, P⬍0.003) compared
with the control rats (PBS) 2 hours after the injection (Figure
1b). PEth–HDL increased the VEGF concentration by 144%
(from 41⫾5 to 100⫾19 pg/mL) compared with the control
rats (PBS) 3 hours after the injection (P⬍0.0001).
Transfer of PEth From HDL Into
Endothelial Cells
To find out if PEth is transferred from HDL particles into the
endothelial cells, we used radiolabeled PEth–HDL particles.
125
I-PEth–HDL was used to follow the fate of the protein
component and 14C-PEth–HDL was used to determine the fate
of the phospholipid component. To determine PEth–HDLO
Liisanantti et al
Phosphatidylethanol Increases VEGF Secretion
1039
PEth–HDL Heparin-Released Binding, PEth–HDL Holoparticle
Uptake, and Selective HDL–PEth Uptake by EA.hy 926 Cells
PEth–HDL Protein-Bound
or Taken-Up ng/mg
Cell Protein
% of Total
PEth–HDL
PEth–HDL binding
49 (14)
0.20 (0.06)
PEth–HDL particle uptake
54 (4)
0.22 (0.02)
156 (15)*
0.62 (0.04)*
Selective PEth uptake†
The data represent the means (SD) from 2 experiments performed in
triplicate wells. The statistical significance of the differences between the
means was assessed by Student t test.
*P⬍0.004 compared with PEth–HDL binding or PEth–HDL holoparticle
uptake.
†The selective uptake of PEth from the HDL particles was calculated as the
amount of HDL protein.
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cell association (binding and internalization) and selective
PEth uptake, we compared the fates of the phospholipid and
protein components (Table). Heparin was used to separate the
binding of HDL to a cell surface from the amount of
internalized HDL. Lipoprotein binding was performed at
37°C and the cell-surface HDL release was determined at
4°C. Selective PEth uptake from HDL particle to the endothelial cells was more efficient (0.62% of the radioactivity)
than the uptake of intact PEth–HDL particles (0.22% of the
radioactivity) (P⬍0.004).
Secretion of VEGF From Endothelial Cells
VEGF is an angiogenic growth factor and potentially also an
endogenous vascular protective factor.32 Previous studies
have shown that PEth formation in mononuclear cells is
accompanied by responses in cell function.22 We hypothesized that PEth, either formed inside cells or associated with
lipoproteins taken-up by cells, enhances VEGF secretion
from cells. To test this hypothesis, we treated EA.hy 926
endothelial cells with different HDL particles and PEth
liposomes. The VEGF concentration in the cell culture
medium was 14% higher in the presence of alcoholic HDL
(2.4⫾0.4 pg/mL) than in the presence of control HDL
(2.1⫾0.2 pg/mL) (Figure 2a, P⬍0.05). The VEGF concentration in the cell culture medium was not affected by the
presence of ethanol or HDL alone at the concentration of 0.2
mg/mL compared with nontreated cells (Figure 2b). By
contrast, the VEGF concentration in the medium increased
significantly (from 2.4⫾1.3 to 3.6⫾0.4 pg/mL, P⬍0.04)
when both HDL and ethanol were added together into the
culture medium (Figure 2b). The same effect was produced
by PEth–HDL particles, which increased the VEGF concentration by 2.4-fold as compared with the control cells (from
2.4⫾1.3 to 5.7⫾1.2 pg/mL, P⬍0.002) (Figure 2b). The
addition of ethanol into the medium did not further enhance
the effect of PEth-HDL (Figure 2b). To prove that the
increasing effect of VEGF secretion is caused by PEth per se,
another set of experiments with phospholipid liposomes was
performed (Figure 2c). The VEGF concentration in the cell
culture medium was markedly increased in the presence of
PEth (from 2.1⫾0.9 to 20.7⫾1.5 pg/mL, P⬍0.0002) (Figure
2c), whereas PC increased the VEGF concentration only from
2.1⫾0.9 to 5.2⫾3.0 pg/mL (P⬍0.05).
Figure 2. VEGF secretion increased from EA.hy 926 cells by
alcoholic HDL and PEth. a, Alcoholic HDL (0.2 mg/mL)
increased the VEGF secretion more than control HDL (0.2
mg/mL) (n⫽12). b, PEth–HDL increased the VEGF concentration
in the absence (white columns) and presence (black columns) of
100 mmol/L ethanol and HDL only in the presence of ethanol
(n⫽6). c, PC (0.5 mg/mL) increased slightly and PEth (0.5
mg/mL) increased markedly the VEGF concentration (n⫽4).
*P⬍0.05, **P⬍0.002, ***P⬍0.0002.
PEth Stimulates MAPK Kinase Activity in
EA.hy 926 Cells
Ethanol induces in vitro angiogenesis by stimulating the
MAPK activity in EA.hy 926 cells.25 Therefore, the effect of
PEth–HDL on the MAPK phosphorylation was investigated.
As shown in Figure 3, incubation of serum-starved EA.hy
926 cells in medium containing PEth–HDL for 10 minutes
resulted in a 3.6-fold (P⬍0.006) increase in the MAPK
(Erk1/Erk2) phosphorylation compared with control cells
treated with PBS. HDL increased the Erk1/Erk2 phosphorylation by 3.3-fold (P⬍0.03) compared with control cells
treated with PBS. The increase in the Erk1/Erk2 phosphorylation remained significantly elevated above control (PBS)
levels when the cells were incubated with HDL for up to 15
minutes. PEth–HDL had a more extended effect on the
Erk1/Erk2 phosphorylation than HDL alone. The phosphor-
Figure 3. PEth stimulated MAPK phosphorylation in EA.hy 926
cells. a, Representative blots of phosphorylated and total
p44/42 MAPK. ␤-actin was used as a control. b, Quantitative
analysis of the MAPK phosphorylation results. Phospho-MAPK
in relation to ␤-actin (black columns, n⫽6) and total MAPK in
relation to ␤-actin (white columns, n⫽6) are shown. *P⬍0.05,
**P⬍0.03, ***P⬍0.006.
1040
Arterioscler Thromb Vasc Biol.
June 2004
tion by 5-fold compared with cells treated with PBS (Figure
4b) (P⬍0.004). To ascertain whether the PEth-induced
VEGF secretion is mediated through PKC and MAPK, the
effect of inhibiting PKC and MAPK on this phenomenon was
investigated. The maximal effect of the inhibitors was seen at
the 15-minute time point (Figure 4b). The increase in VEGF
secretion induced by PEth–HDL was inhibited by 40% in the
presence of the PKC inhibitor, Gö6983, and by 25% in the
presence of the MAPK inhibitor, PD098059.
Discussion
Figure 4. PEth-induced VEGF secretion is inhibited by 1 ␮mol/L
Gö6983 and 10 ␮mol/L PD098059. a, Representative blots of
MAPK and PKC phosphorylation. ␤-actin was used as a control.
b, Quantitative analysis of the effects of MAPK and PKC inhibitors on the PEth–HDL induced VEGF secretion (n⫽3). *P⬍0.05,
**P⬍0.004.
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ylation induced by PEth–HDL remained elevated for up to 24
hours compared with control cells. PD098059 inhibited
PEth–HDL-mediated stimulation of Erk1/Erk2 phosphorylation by 84% (Figure 4a) (P⬍0.004).
PEth Stimulates PKC Activity in EA.hy 926 Cells
Because ethanol stimulates PKC activity in endothelial
cells,25 the effect of PEth–HDL on the PKC phosphorylation
was investigated. As shown in Figure 5, incubation of
serum-starved EA.hy 926 cells in medium containing PEth–
HDL for 10 minutes resulted in a 2.2-fold (P⬍0.05) increase
in the PKC phosphorylation compared with control cells
treated with PBS. HDL increased the phosphorylation by
2-fold (P⬍0.05) compared with control cells treated with
PBS. PEth–HDL mediated stimulation of the PKC phosphorylation was slightly reduced by a PKC inhibitor Gö6983, but
the result was not statistically significant (Figure 4a).
PEth-Induced VEGF Secretion Is Inhibited by
PKC and MAPK Inhibitors
Incubation of serum-starved EA.hy 926 cells for 15 minutes
in a medium containing PEth–HDL increased VEGF secre-
Figure 5. PEth stimulated PKC phosphorylation in EA.hy 926
cells. a, Representative blots of phosphorylated and total PKC
protein. ␤-actin was used as a control. b, Quantitative analysis
of the PKC phosphorylation results. Phospho-PKC in relation to
␤-actin (black columns, n⫽6) and total PKC in relation to ␤-actin
(white columns, n⫽6) are shown. *P⬍0.05.
PEth is formed in mammalian organs in the presence of
ethanol and it is also present in the blood of subjects who
have been drinking alcohol.20,33–35 PEth is formed especially
in the lungs and the intestine, but also in vascular endothelial
cells.36 –38 The present study suggests that PEth affects vascular endothelial cells and may mediate at least part of the
cardioprotective effects of ethanol. As a proof of principle,
the present study demonstrates that lipoprotein-associated
PEth increases the production of VEGF in endothelial cell
cultures. VEGF was chosen as an end point marker because
its function is considered beneficial in the later phases of
atherosclerosis because of its ability to inhibit neointima
formation.32 In addition, moderate levels of ethanol induce
the expression and secretion of VEGF in cultured vascular
smooth muscle cells and chick embryo chorioallantoic membranes25,39 and ethanol, at least at high concentrations, induces the in vitro angiogenesis through PKC and MAPK
signaling pathways in endothelial cells.25 The present cell
culture and animal experiments suggest that the increased
plasma VEGF concentrations observed in alcohol drinkers
may be caused by ethanol-induced qualitative alterations in
the HDL particles of alcohol drinkers, and that this effect may
be mediated by PEth through the MAPK and PKC signaling
pathways.
The stimulation of the MAPK activity, as measured as an
increase in the phosphorylation of Thr202/Tyr204 residues of
Erk1/Erk2, was prolonged by PEth–HDL compared with the
stimulation caused by native HDL. In addition, the partial
inhibition of PEth-induced VEGF secretion by specific
MAPK inhibitor PD098059 indicates that the MAPK pathway could be one of the signaling routes between PEth and
VEGF secretion in endothelial cells. This is in accordance to
a recent report in hepatocytes.40 The PEth-induced stimulation of the phosphorylation of PKC and the partial inhibition
by Gö6983 of the PEth-induced VEGF secretion from the
endothelial cells indicate that the PKC pathway could also
contribute to the PEth-induced VEGF secretion in endothelial
cells. Moreover, this study gives a plausible explanation for
the ethanol-induced increase in plasma VEGF level by
demonstrating in an animal model that the addition of an
ethanol-induced abnormal lipid, PEth, into HDL particles is
also capable of elevating plasma VEGF levels. However, we
cannot exclude the contribution of other compounds present
in HDL isolated from plasma of alcohol drinkers, despite the
apparent similarity in the VEGF response in vivo.
The present study provides evidence that PEth–HDL may
interfere with the vascular endothelium in a different manner
than native HDL. This qualitative difference leads into
Liisanantti et al
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increased concentration of VEGF in plasma in vivo and also
in endothelial cells, which line the blood vessel and are in
close contact with smooth muscle cells and macrophages. All
of these cells may sense changes in the environment when the
composition and concentration of lipoproteins are altered, eg,
by ethanol.
The mechanism of the cardioprotective effect of moderate
alcohol drinking has been elusive. For most hours of the day,
ethanol is not present in the body of a social drinker.
Therefore, the direct effects of ethanol on vascular endothelial cells might be only temporary if they are confined to the
period of ethanol abundance, even though PEth may be
metabolized more slowly than PC. The relatively long halflife of lipoproteins may prolong the presence of PEth in the
body from a few hours to several days even in an occasional
drinker.8,19 This kind of “memory molecule” might help to
explain the beneficial effects of light or moderate drinking on
cardiovascular diseases. The effect of PEth may last even
longer, because erythrocytes may act as a reservoir of
PEth,41,42 and because this abnormal phospholipid is transported between lipoproteins,23 lipoproteins may transport
PEth from erythrocytes to vascular wall cells.41 More sensitive methods are needed for the measurement of PEth content
in lipoprotein fractions, however. This work is currently
underway in our laboratory.
In conclusion, ethanol not only alters the quantity of
lipoproteins or their components but also may cause incorporation of abnormal derivatives of lipids and proteins into
lipoproteins, and may therefore have marked and sustained
effects on all vascular cells.43 Further studies are needed to
elucidate more detailed mechanisms of how PEth regulates
the VEGF function and secretion in endothelial and in other
vascular cells.
Acknowledgments
We acknowledge the technicians of the laboratory in the Department
of Internal Medicine at the University of Oulu for their skillful
technical assistance. We are also very grateful to the patients and
staff of the Kiviharju Alcoholism Treatment Unit and Stora Enso Oyj
Fine Paper, Oulu Mill for their cooperation. This work was financially supported by the Academy of Finland, Sigrid Juselius Foundation, Inkeri and Mauri Vänskä Foundation, and the Finnish
Foundation for Alcohol Studies.
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Downloaded from http://atvb.ahajournals.org/ by guest on July 31, 2017
Lipoprotein-Associated Phosphatidylethanol Increases the Plasma Concentration of
Vascular Endothelial Growth Factor
Marja K. Liisanantti, Minna L. Hannuksela, Maria E. Rämet and Markku J. Savolainen
Arterioscler Thromb Vasc Biol. 2004;24:1037-1042; originally published online April 15, 2004;
doi: 10.1161/01.ATV.0000128409.62292.d6
Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272
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Liisanantti et al. Phosphatidylethanol increases VEGF secretion
1
Data supplement
Methods
Subjects
A series of 17 male alcoholics referred by general practitioners for withdrawal therapy to the
Alcoholism Treatment Unit of Oulu were recruited for blood tests. This unit treats ambulatory
patients who seek assistance for terminating their dependence on alcohol and have no signs or
symptoms of other diseases. Healthy non-alcoholic men (n = 17) recruited from among the local
paper mill employees served as control subjects. Venous blood samples were taken from both the
alcoholics and the controls after an overnight fast. The intake of alcohol was measured by an
extensive interview concerning the amount of beer, wine, and strong alcoholic beverages
consumed during the previous two weeks. The mean alcohol intake with a range was calculated
and expressed in grams of pure alcohol per day. The study was approved by the Ethical Committee
of the University of Oulu. The clinical characteristics of the study subjects are shown in Table I.
Animals
Nine-week old male Sprague-Dawley rats (330 ± 27 grams in body weight; mean ± SD, n = 35)
were housed in a controlled environment with free access to food and water. The experiments were
approved by the Animal Care and Use Committee, University of Oulu. The rats were anesthetized
with fentanyl-fluanisone and midazolam before injecting them intravenously with HDL particles
isolated by ultracentrifugation and dialyzed in phosphate-buffered saline (PBS), pH 7.4. HDL
particles were isolated from the plasma of control subjects and alcoholics as described previously.1
These HDL fractions (0.70 µmol cholesterol per 100 g body weight) or control HDL supplemented
with phosphatidylethanol (PEth) were injected into the tail vein. Retro-orbital blood (0.3 ml) was
collected into EDTA-containing tubes at the indicated times.
Liisanantti et al. Phosphatidylethanol increases VEGF secretion
2
Isolation of lipoproteins
Blood samples were obtained after an overnight fast from control subjects and alcoholics, and
plasma was separated by centrifugation. Lipoproteins were isolated from plasma by sequential
ultracentrifugation on the basis of their density as described earlier.1-3 1,2-Dioleoyl-sn-glycero-3phosphoethanol (sodium salt) (Avanti Polar Lipids) and 1,2-dioleoyl-sn-glycero-3-phosphocholine
(Avanti Polar Lipids) were incorporated into human HDL particles (5 % of total lipoprotein
phospholipids). Phospholipid-modified HDL particles were first centrifugated at a density of 1.006
g/ml in order to remove the unincorporated phospholipids from the HDL fraction, and then at a
density of 1.21 g/ml. All isolated lipoproteins were dialyzed against PBS, pH 7.4 before use.
Lipoprotein labeling procedure
PEth was labeled by the phospholipase D-catalyzed transphosphatidylation reaction using 1,2-di[114C]oleoyl-sn-glycero-3-phosphocholine (100 mCi/mmol, Amersham Biosciences) as a labeled
substrate.4 Phospholipids were extracted as described by Meacci et al5 and separated by thin-layer
chromatography (TLC) in ethylacetate:iso-octane:acetic acid (81:45:18). The [14C]labeled PEthcontaining spot was transferred to a clean tube, extracted as previously, dissolved in ethanol, and
incorporated to HDL as described earlier. Iodination of the PEth-HDL (5 mg protein) was
performed using IODO Beads Iodination Reagent (Perbio Science) and 1 mCi of Na125I (17.5
Ci/mg, NEN Life Science Products Inc.). The labeling reaction was carried out in PBS, pH 7.4
buffer according to the manufacturer’s instructions. Unincorporated
125
I2 and excess Na125I were
removed from the iodinated HDL by a PD10 desalting column (Amersham Biosciences). Labeled
PEth-HDL particles were stored at 4 oC and used within one week.
Liisanantti et al. Phosphatidylethanol increases VEGF secretion
3
Chemical analyses
The concentrations of cholesterol, triglycerides, and phospholipids in the plasma and the
lipoprotein fractions were determined by enzymatic colorimetric methods using a Kone Specific
Selective Chemistry Analyser (Kone Oy) and kits of Roche Diagnostics GmbH (cholesterol,
triglycerides) and Wako Chemicals GmbH (phospholipids). The protein concentrations were
determined by Lowry method6 using a kit (Bio-Rad Laboratories).
Cell culture experiments
Human endothelial EA.hy 926 cells (kindly provided by Dr. Cora-Jean S. Edgell, University of
North Carolina, NC, USA) were cultured in DMEM high glucose supplemented with 10 % fetal
bovine serum, 2 mM L-glutamine, 50 U/ml penicillin, 50 mg/ml streptomycin, and HAT
supplement. The cells were incubated at 37 °C in a humidified atmosphere of 5 % CO2 and 95 %
air. For the VEGF determinations, the cells were seeded at a concentration of 1-2 x 106 cells/ml on
plates (Ø 10 cm), grown for three days, washed twice with PBS, and incubated for 24 hours in the
serum-free medium containing 20-400 mM ethanol, 200 µg/ml phosphatidylcholine (PC), 200
µg/ml PEth, 200 µg/ml HDL, 200 µg/ml PEth-HDL or 200 µg/ml alcoholic HDL. For VEGF
determinations, samples of cell culture medium were collected at indicated time points,
concentrated by an Ultrafree-4 centrifugal filter unit (Millipore Corporation, Bedford,
Massachusetts, USA), and stored at –70 °C.
Determination of PEth-HDL cell association and selective PEth uptake
To determine cell association (heparin-non-releasable binding and internalization) of HDL and
selective HDL-PEth uptake, EA.hy 926 cells were grown to confluence in 6-well plates, and
incubated with 25 µg of PEth-HDL protein labeled with
125
I in the protein moiety and with
[14C]PEth in the lipid moiety in separate incubations as described earlier for 5 hours.7 The amount
Liisanantti et al. Phosphatidylethanol increases VEGF secretion
4
of specific PEth-HDL binding, holoparticle uptake and selective PEth uptake were determined by
the difference between the total radioactivity and the amount of nonspecific radioactivity in the
presence of excess unlabeled lipoprotein as described detailed by Acton and Rigotti.8
Immunoassays
The VEGF concentration was measured from the human plasma and cell culture medium (4-fold
concentrated) by a Quantikine human VEGF kit (R&D Systems Inc.). The VEGF concentration in
rat plasma was measured by a Quantikine M VEGF kit (R&D Systems).
Western Blot Analysis of PKC and p44/42 MAPK
Serum-starved EA.hy 926 cells were incubated with 0.2 mg/ml HDL, 0.2 mg/ml 0.5 % PEth-HDL,
1 µM Gö6983 (a PKC inhibitor, Calbiochem-Novabiochem), or 10 µM PD098059 (a Erk1/Erk2
MAPK inhibitor, Sigma) for the indicated amounts of time. After treatment, cells were lysed with
lysis buffer (62.5 mM Tris-HCl, pH 6.8, 2 % w/v SDS, 10 % glycerol, 50 mM DTT, 0.01 %
bromphenol blue). Aliquotes of cell lysates (20 µl) were loaded onto 10 % polyacrylamide gels for
electrophoresis. After electrophoresis proteins were transferred to PVDF membranes (Millipore),
and incubated with the following antibodies: Phospho-PKC (pan) (Cell Signaling Technology),
PKC (Ab-1) (Oncogene Research Products), Phospho-p44/42 Map Kinase (Thr202/Tyr204) (Cell
Signaling Technology), MAP Kinase-2 (Erk-2) (Clone 1B3B9) (Sigma), and β-actin (Clone AC15) (Sigma). Horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences)
and ECL Western blotting detection reagents (Amersham Biosciences) were used to for detection,
and the signals were analyzed by Kodak 1D Imaging System (Eastman Kodak Company).
Liisanantti et al. Phosphatidylethanol increases VEGF secretion
5
Statistical analysis
The results are expressed as means and standard deviation (SD). The alcohol intake and plasma
triglyceride values were not normally distributed and were therefore log-transformed before the
statistical analysis. The statistical significance of the differences between the means was assessed
by independent samples’ 2-tailed Student’s t-test or one-way analysis of variance (ANOVA,
Tukey’s test), as appropriate. P values were considered significant at 0.05.
Liisanantti et al. Phosphatidylethanol increases VEGF secretion
6
References
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2. Hannuksela M, Marcel YL, Kesäniemi YA, Savolainen MJ. Reduction in the concentration
and activity of plasma cholesteryl ester transfer protein by alcohol. J Lipid Res 1992;33:737744.
3. Liinamaa MJ, Kesäniemi YA, Savolainen MJ. Lipoprotein composition influences cholesteryl
ester transfer in alcohol abusers. Ann Med 1998;30:316-322.
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1967;242:477-484.
5. Meacci E, Vasta V, Farnararo M, Bruni P. Bradykinin increases ceramide and sphingosine
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6. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol
reagent. J Biol Chem 1951;193:265-275.
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Zechner R, Frank S. Endothelial cell-derived lipase mediates uptake and binding of highdensity lipoprotein (HDL) particles and the selective uptake of HDL- associated cholesterol
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Liisanantti et al. Phosphatidylethanol increases VEGF secretion
7
Table I Characteristics of the study subjects
Alcoholics
Controls
(n=17)
(n=17)
Age (years)
45.7 (10.5)
48.6 (5.6)
BMI (kg/m2)
25.4 (4.5)
25.7 (3.0)
Alcohol intake (g/day)
136 (50-302)
20 (0-63) ‡
Total cholesterol (mmol/l)
4.64 (0.98)
5.71 (0.84) ‡
VLDL cholesterol (mmol/l)
0.40 (0.34)
0.36 (0.22)
LDL cholesterol (mmol/l)
2.10 (0.91)
3.42 (0.81) ‡
HDL cholesterol (mmol/l)
1.94 (0.79)
1.51 (0.47) *
Plasma triglycerides (mmol/l)
1.79 (1.19)
1.28 (0.52)
γ-glutamyltransferase (mmol/l)
232 (345)
38 (18) †
Aspartate aminotransferase (U/l)
78 (62)
32 (13) †
Alanine aminotransferase (U/l)
57 (39)
32 (12) †
Alkaline phosphatase (U/l)
224 (95)
151 (41) †
Bilirubin (µmol/l)
18 (9)
14 (6)
The data are presented as means (SD). The ethanol consumption is given as mean with a range. The
statistical significance of the differences between the means was assessed by Student’s t-test. * P =
0.061, † P < 0.04, ‡ P < 0.009.