© 1999 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Membrane Active Lipids in Remnant Lipoproteins Cause Impairment of Endothelium-Dependent Vasorelaxation
From the Departments of Cardiovascular Medicine (H.D., K.K., M.O., S.S., T.M., Y.O., H.O., N.O., H.Y.) and Ophthalmology (A.H.), Kumamoto University School of Medicine, Kumamoto; and the Research Center for Advanced Science and Technology (Y.Y.), University of Tokyo, Japan.
Correspondence to Kiyotaka Kugiyama, MD, Division of Cardiovascular Medicine, Kumamoto University School of Medicine, 1-1-1 Honjo, Kumamoto City, Kumamoto 860-8556, Japan. E-mail kiyo{at}gpo.kumamoto-u.ac.jp
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Abstract |
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Abstract—We have recently found that remnant lipoproteins (RLPs) and their lipid fractions impair endothelium-dependent vasorelaxation (EDR). This study was aimed at clarifying mechanisms responsible for RLP-induced endothelial dysfunction in isolated rabbit aortas. RLPs were isolated from plasma in hyperlipidemic subjects by use of the immunoaffinity gel mixture of anti-ApoA1 and anti-ApoB100 monoclonal antibodies and ultracentrifugation. Organ chamber experiments showed that EDR impairment was restored by addition of reduced glutathione (GSH) or N-acetylcysteine, antioxidants, into the incubation buffer containing isolated rabbit aortas and RLPs (0.75 mg of triglyceride/mL). Furthermore, the incubation of isolated human red blood cells (RBCs) with RLP and its lipids converted the normal shape of RBCs to echinocytes, but coincubation with antioxidants suppressed the RLP-induced RBC transformation, suggesting that they exerted oxidative damage on RBC surface membranes. Studies with HPLC and the postcolumn chemiluminescence method showed that RLPs contain a substantial amount of phosphatidylcholine hydroperoxides. Peroxidized phosphatidylcholine also impaired EDR and had echinocytogenic action, both of which were suppressed by N-acetylcysteine. RLPs isolated from the plasma of patients under treatment with
-tocopherol, an
antioxidant, had a lower level of phosphatidylcholine hydroperoxides
(15% of the amount in nontreated patients), which was associated with
a lack of the inhibitory action on EDR and with lesser
effect on RBC transformation. Oxidative damage caused by lipid
components in RLPs, especially peroxidized phospholipids, deteriorates
cell surface membrane and may be at least partly responsible for
RLP-induced impairment of EDR.
Key Words: endothelium • lipoproteins • antioxidants • endothelial-derived factors • hyperlipoproteinemia
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Introduction |
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We recently found that lipids in remnant lipoproteins (RLPs), remmant-like lipoprotein particles in the strict sense of the word, from postprandial plasma caused impairment of endothelium-dependent vasorelaxation (EDR) in an apoprotein receptor–independent manner.1 In a clinical study, we further showed that levels of RLP were an independent predictor of coronary endothelial vasomotor dysfunction, an early event leading to the formation and development of atherosclerosis.2 Thus, the endothelial dysfunction caused by RLPs may play a role in the pathogenesis of atherosclerotic coronary artery disease in postprandial hypertriglyceridemic patients. However, the precise mechanisms underlying RLP-induced endothelial dysfunction remain to be clarified.
It was previously difficult to isolate RLPs because they have heterogeneous properties.3 However, Nakajma et al have recently developed a simple and reliable method to isolate RLPs in the unbound fraction by an immunoaffinity gel mixture of human anti-ApoA1 and anti-ApoB100 monoclonal antibodies.1 2 3 4 5 The unique anti-ApoB100 antibody has been shown to recognize ApoB100 in LDLs and most VLDLs but not in ApoE-enriched VLDLs.1 2 3 4 5 The current method with anti-ApoB100 antibody is capable of isolating remnants of VLDLs and chylomicron, neither of which binds to the gel. Our previous reports confirmed that lipoproteins isolated from plasma taken 5 hours after meals by using this immunoaffinity gel consisted mainly of VLDL remnants.1 2
Oxidative stress is a common feature of various coronary risk factors for atherosclerosis.6 Further, several clinical studies showed that antioxidant therapy improved endothelial dysfunction in patients with hyperlipidemia and in normocholesterolemic subjects after a single fatty meal.7 8 In this study, we thus tested the hypothesis that peroxidized lipids in RLPs may have a causative role in EDR impairment by RLPs. The present study shows that antioxidants, added to the incubation mixture containing isolated rabbit aortas and RLPs, suppressed EDR impairment. RLPs were found to contain peroxidized phospholipids that were effective components for the inhibitory action of RLPs, and they caused perturbation of cell surface membranes that was also attenuated by antioxidants.
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Methods |
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Lipoprotein Preparations
EDTA plasma (1 mg/mL EDTA) was obtained at 5 hours after the test meal from 14 hypertriglyceridemic subjects who had >5 mg of cholesterol/dL (or 20 mg of triglyceride/dL,
75th percentile of the distribution of
the RLP levels in the population) of RLP levels in a plasma sample
after overnight fasting. The energy content of the test meal was
490 kcal/m2 of body surface area (86.8% from
fat, 8.7% from carbohydrate, 4.5% from protein, and the ratio of
polyunsaturated fatty acids to saturated fatty acids was 0.51). They
had no other serious diseases and did not take
cardiovascular medications, antioxidants, and estrogen
for >7 days before the overnight fast. The mean level of RLPs
in the postprandial plasma was 84.2±15 mg of
triglyceride/dL (21.5±5 mg of cholesterol/dL)
in these subjects. Plasma from each subject was applied to the immunoaffinity-mixed gel containing anti-ApoA1 and anti ApoB100 monoclonal antibodies (Japan Immunoresearch Laboratories).1 2 3 4 5 The epitope for ApoB100 monoclonal antibody has been localized to an amphipathic helical region of ApoB100 encompassing residues 2291 to 2318, ie, at about ApoB51.5 This antibody can recognize ApoB100 in LDL and most VLDLs but not in ApoE-enriched VLDLs.1 2 3 4 5 The unbound fraction containing ApoE-enriched lipoproteins and albumin was eluted with PBS (mmol/L: 138 NaCl, 2.7 KCl, 8.1 Na2HPO4, 1.1 KH2PO4, pH 7.4). The bound fraction was then eluted with 3 mol/L NaSCN containing 1 mg/mL BSA (fatty acid-free BSA). The unbound fraction was then ultracentrifuged (d<1.006) to isolate RLPs from the unbound fraction containing albumin. Bound VLDL was also isolated from the bound fraction by ultracentrifugation (d<1.006). The fraction in the d<1.006 g/mL was also obtained from the EDTA plasma by ultracentrifugation, and then the fraction in the d<1.006 g/mL was subjected to the immunoaffinity gel column to isolate RLPs. These 2 protocols for isolation of RLPs were aimed at testing the effect of the presence of albumin during RLP elution on the vasoactivity of RLP. The prepared lipoproteins were extensively dialyzed for 24 hours at 4°C against PBS (mmol/L: 137 NaCl, 2.7 KCl, 1.4 NaH2PO4, 4.3 Na2HPO4, pH 7.4) containing EDTA (50 µmol/L) and then sterilized by filtration (filter pore size, 0.22 µm; Millipore). According to analyses with SDS–polyacrylamide gel electrophoretograms, elution profiles with HPLC, agarose gel electrophoretograms, electron photomicrographs, and composition of lipids and apoproteins, the lipoproteins isolated from the unbound fraction after ultracentrifugation consisted mainly of VLDL RLPs, as shown in our previous reports.1 2 3 4 5 The affinity of this antibody to ApoB100 in oxidized lipoproteins remains to be determined. However, it is less likely that oxidized LDLs may be contaminated in the remnants fraction, because the unbound fraction was further ultracentrifuged (d<1.006) to isolate remnants from the unbound fraction. Lipids were extracted from RLPs and bound VLDL with chloroform/methanol (2:1, vol/vol).1 9
Total cholesterol and triglycerides were
measured enzymatically by commercially available kits (Nos. 439-17501
and 274-69802, Wako Chemicals).10 11 Final concentrations
of the prepared lipoproteins were 25 to 35 mg of
triglyceride in lipoproteins per milliliter. The
-tocopherol level of lipoproteins was measured by
HPLC.12
Twelve subjects who had fasting RLP levels of >20 mg of
triglyceride/dL were treated orally with
-tocopherol (300 mg/d, n=6) or placebo (n=6) for 4
weeks. The 2 treatment groups of subjects were matched with the respect
to age, sex, and other coronary risk factors. They were advised
to adhere to their usual diet and exercise activity throughout the
4-week treatment. Before and after the 4-week treatment, EDTA
plasma was obtained at 5 hours after the test meal. Postprandial levels
of RLP were comparable between the -tocopherol and
placebo treatment groups both before the treatment (85.4±7 and 80.3±8
mg of triglyceride/dL, respectively) and after the 4-week
treatment (85.4±12 and 84.2±8 mg of triglyceride/dL,
respectively).
Peroxidation of Synthetic Phosphatidylcholine
Synthetic phosphatidylcholine (PC) mixture in 1 mL of ethanol (5
mg PC oleate, 10 mg PC linoleate, and 10 mg PC
arachidonate) was peroxidized in the presence of free
radical initiator 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN) under
aerobic conditions at 37°C for 5 hours.13 Thiobarbituric
acid–reactive substances (TBARS), a marker of lipid peroxidation, in
the incubation mixture were increased after the incubation (1.5±1
pmol/mg PC before AMVN versus 326.4±26 pmol/mg PC after AMVN,
P<0.01, n=6).
1-Palmitoyl-2-(5-oxovaleroyl)-sn-glycerophosphocholine
(5-CHO-PC), 1 of the peroxidized PCs that includes a 5-carbon aldehyde
at the sn-2 position, was synthesized by ozonizing
1-palmitoyl-2-(5-arachidonoyl)-sn-glycerophosphocholine and
purified by HPLC as described previously.14
Organ Chamber Experiments
Male New Zealand White rabbits weighing from 2.5 to 3.0 kg were
killed under pentobarbital anesthesia (30 mg/kg IV) and the
thoracic aortas were isolated from the rabbits. The aortas were
dissected free from adherent connective tissues and cut into ring
segments 3 mm wide, and then aortic strips of 3-mm width were
prepared from each ring segment. During the preparation, any contacts
with the luminal surface were avoided to preserve
endothelial cell integrity. The strips were suspended
by stainless steel clips in the organ chambers, which were filled with
Krebs–Henseleit solution (mmol/L: 118.0 NaCl, 4.0 KCl, 1.5
CaCl2, 1.2 MgSO4, 1.2
NaH2PO4, 25.0
NaHCO3, and 5.0 glucose). The solution was
aerated with 15% O2/5%
CO2/80% N2
(PO2100 mm Hg) and maintained at pH 7.4
and 37°C. The resting tension of rabbit thoracic aortas was adjusted
to 3g, and changes of isometric tension were monitored by
using a force transducer (Minebea and Nihon Kohden) and recorded by
a polygraph machine (Nihon Kohden) as described in our previous
reports.1 9 After 1-hour equilibration, aortic strips
were incubated for 2 hours with 1 preparation of lipoproteins and
lipids in the organ chambers. In other sets of experiments, the strips
were incubated for 1 hour with RLP (0.75 mg of
triglyceride/mL) or a preparation of PC (15 µmol/L)
in the presence or absence of N-acetylcysteine (NAC; 1
mmol/L) or reduced glutathione (GSH; 1 mmol/L). Control studies
were performed to expose the aortic strips in the organ chamber to the
same volume of PBS (as a vehicle) and were otherwise performed
in the same manner as the studies using lipoproteins or lipids. After
incubation, the strips were repeatedly washed, then they were
contracted with phenylephrine (1 µmol/L) and relaxed
with increasing concentrations of various vasodilators in the absence
of the preparations of lipids and lipoproteins and the antioxidants.
All of the bioassay experiments were performed in the arteries with the
intact endothelium. When the effect of 1 lipoprotein
preparation was compared with that of other lipoprotein preparations,
the same batch of lipoprotein preparations isolated from the same
subject were used. Vasorelaxation was expressed as a percent change of
the reduction from precontraction elicited by
phenylephrine. For relaxations, the effective
concentrations of agonists causing 20% (EC20)
and 50% (EC50) relaxation of the contraction by
phenylephrine were calculated from each
concentration–response curve.
Measurement of Susceptibility of RLP to Oxidative
Modification
The susceptibility of RLP to oxidative modification was
determined by measuring Cu2+-induced formation of
conjugated dienes. The conjugated dienes formation in RLP (0.1 mg of
triglyceride/mL) was monitored by spectrophotometric
change in absorbance at 234 nm, as described
previously.15
Measurement of Phosphatidylcholine Hydroperoxides and
Lysophosphatidylcholine in RLP
Lipids in RLP were extracted with methanol and hexane in
sequence as described previously.13 16 The hexane layer
was removed and evaporated under vacuum and then redissolved in HPLC
eluent. Measurement of phosphatidylcholine hydroperoxides (PC-OOH) was
performed by using the HPLC and isoluminol chemiluminescence detection
system as described in our previous reports.13 16
Concentration of lysophosphatidylcholine (lysoPC) in RLP was
measured by using thin layer chromatography as
described in our previous report.9
Transformation of Red Blood Cell (RBC) Shape
The erythrocyte surface membranes are known to be susceptible to
peroxidation and serve as a good model for the oxidative damage of
biological membrane.17 To test whether RLPs may have
membrane active molecules, RLPs were incubated with RBCs devoid of
lipoprotein receptors. RBCs freshly obtained from healthy volunteers
were washed 3 times with PBS and resuspended at a hematocrit of 5% in
the buffer (mmol/L: 150 NaCl, 20 Tris-Cl, 1
CaCl2, 10 glucose, pH 7.4).9 RBC
suspension was incubated with one of the preparations of lipids
and lipoproteins, bound VLDL, lipid extracts of RLP, and preparations
of PC in the presence or absence of NAC (1 mmol/L) or GSH (1
mmol/L) for 1 hour at 37°C. After incubation, the cell suspensions
were fixed with 2.5% glutaraldehyde. RBC morphology
was examined by phase-contrast microscopy and scanning electron
microscopy. Differential counts on the shape of transformed RBCs were
performed by using the morphological criteria for echinocytes as
described previously by Brecher and Bessis.18 For each
preparation, counts were made on 100 consecutive cells in each of 5
microscopic fields.
Materials
AMVN was obtained from Wako Chemicals. GSH (G4251), NAC (A7250),
and all other chemical reagents were from Sigma Chemicals.
Statistic Analysis
All data were expressed as mean±SEM values unless
otherwise indicated. The mean values in >3 groups were compared by
using 1-way ANOVA, then the difference between the 2 mean values was
analyzed with Fisher's protected least significant difference
test. Two-way ANOVA for the repeated measures, followed by the
Bonferroni multiple comparison test, was used for comparison of the
curves for vasorelaxation. The difference between 2 mean values in the
curves was analyzed by unpaired Student's t test. A
value of P<0.05 was considered statistically
significant.
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Results |
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Effects of Lipoproteins and Lipids Preparations on Relaxation of Rabbit Aortas
Incubation of the aortic strips with RLP inhibited relaxation of the aortas in response to acetylcholine (ACh) and A23187, whereas bound VLDL isolated from the same subject had no effect, as shown in Table 1
. Incubation with the lipids (0.75 mg of
triglyceride/mL) extracted from RLP but not those from
bound VLDL (up to 1.5 mg of triglyceride/mL) also had an
inhibitory action on vasorelaxation (maximum relaxation
[%]: control, 72.2±2; bound VLDL lipid, 71.5±2; RLP lipid,
41.1±5; P<0.001, RLP lipid versus control, n=6 to
8). Inhibition of vasorelaxation was induced at concentrations
of RLP observed in the postprandial plasma of patients with
coronary artery disease (50 to 200 mg of
triglyceride in RLP/dL). On the other hand,
vasorelaxation in response to sodium nitroprusside, a direct
smooth-muscle dilator, was fully preserved after incubation with RLP
(up to 1.5 mg of triglyceride/mL), as shown in Table 1. Furthermore, Figure 1 shows
that combined incubation with GSH (1 mmol/L) or NAC (1
mmol/L) attenuated the RLP-induced impairment of vasorelaxation in
response to ACh.
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RLP isolated from patients under treatment with
-tocopherol lost its inhibitory action on
vasorelaxation in response to ACh, whereas RLP from patients treated
with placebo still had the inhibitory action, as shown in
Figure 2. The -tocopherol
level in RLP was increased after treatment with
-tocopherol compared with that before treatment
(289.7±36 µg -tocopherol/mg of
triglyceride in RLP before
-tocopherol versus 1053.0±92 µg
-tocopherol/mg of triglyceride in RLP
after -tocopherol, P<0.001, n=6), whereas it
was not changed after placebo treatment compared with that before
treatment (307.8±30 µg -tocopherol/mg of
triglyceride in RLP before placebo versus 325.8±48
µg -tocopherol/mg of triglyceride
in RLP after placebo, P=NS, n=6). Lag time of the
oxidation of RLP isolated from patients under treatment with
-tocopherol was longer than that of RLP before treatment
(267.2±23 minutes before -tocopherol versus 456.1±111
minutes after -tocopherol, P<0.05, n=6). In
the present study, RLP was eluted with or without the
albumin fraction during the isolation procedure.
However, the inhibitory effect of RLP eluted with the
albumin fraction on vasorelaxation in response to ACh was
comparable with that of RLP without the albumin fraction
(maximum relaxations were 38.4±4% with albumin fraction
versus 35.8±3% without albumin fraction, P=NS,
n=6), suggesting that the presence of albumin during isolation
of RLP and bound VLDL did not affect the inhibitory action
of RLP on vasorelaxation. In the present study, we were very
careful to prevent intraexperimental autoxidation of RLP during
preparation of the lipoproteins. It is unlikely that intraexperimental
artifacts including autoxidation of RLP would cause EDR impairment
because another lipoprotein, bound VLDL, which was prepared in the same
manner as RLP, had no effect on EDR, and TBARS, an indicator of lipid
peroxidation, was not detected in the incubation mixture after
incubation with the aortas.
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Measurement of Peroxidized PC and its Effects on
Vasorelaxation
RLPs contained a substantial amount of PC-OOH, detected by HPLC
and isoluminol chemiluminescence detection system, as shown in Figure 3. LysoPC, an active component of
oxidized LDL,7 was not detectable in RLP (<5 nmol/mg of
protein). The level of PC-OOH contained in RLPs was
significantly decreased in RLPs isolated from subjects under treatment
with -tocopherol compared with that level before
treatment (PC-OOH in RLP, 292.5±66 pmol/mg of triglyceride
in RLP before -tocopherol versus 49.6±6 pmol/mg of
triglyceride in RLP after -tocopherol,
P<0.01, n=5). TBARS were not detectable in the isolated
RLPs.
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Incubation of the artery with peroxidized PC but not untreated PC
significantly impaired vasorelaxation in response to ACh, as shown in
Table 2. Incubation of the artery with
5-CHO-PC (10 µmol/L) also impaired vasorelaxation in response to
ACh (maximum relaxations were 77.1±2% for control versus 53.9±3%
for 5-CHO-PC, P<0.001, n=6). Furthermore, the combined
incubation with NAC (1 mmol/L) significantly suppressed the
impairment of vasorelaxation induced by peroxidized PC, as shown in
Table 2. On the other hand, vasorelaxation to sodium
nitroprusside was fully preserved after incubation with peroxidized PC,
as shown in Table 2.
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Transformation of RBC Shape
RLPs (0.5 mg of triglyceride/mL) and their lipid
extracts (0.5 mg of triglyceride/mL) transformed RBC shape
to echinocytes, whereas bound VLDL (0.5 mg of
triglyceride/mL) had less effect on the shape of RBCs, as
shown in Figures 4 and 5. Coincubation with GSH (1 mmol/L)
or NAC (1 mmol/L) suppressed the RLP-induced RBC transformation
(Figure 5). Furthermore, RLPs from patients under treatment with
-tocopherol had lower effect on RBC
transformation, as shown in Figures 4 and 5. Incubation of RBCs
with peroxidized PC but not untreated PC significantly transformed the
shape of RBCs (Figure 5). Coincubation with NAC (1 mmol/L)
also suppressed the RBC transformation induced by peroxidized PC
(Figure 5). Incubation of RBCs with 5-CHO-PC (10 µmol/L)
also transformed RBC shape to echinocytes (transformation of RBC shape
was 4.3±1% for control versus 93.6±2% for 5-CHO-PC,
P<0.001, n=6). Transformed RBCs on stages I to III
according to Brecher and Bessis18
were considered echinocytes.
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P<0.001 versus RLP; #P<0.001
versus peroxidized PC. |
Discussion |
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The present study showed that RLPs and their extracted lipids impaired EDR of isolated rabbit aortas at the same concentrations of RLP as in postprandial plasma in patients with coronary artery disease (50 to 200 mg of triglyceride in RLP/dL), whereas the bound VLDL fraction, which is the remaining VLDL after removing RLP, had no effect on EDR. These results are compatible with those in our previous report showing that lipid components of RLP from postprandial plasma inhibited EDR in an apoprotein receptor–independent manner.1 This study further showed that coincubation with NAC and GSH, antioxidants that were added to incubation mixtures containing isolated rabbit aortas and RLP, almost completely suppressed EDR impairment, suggesting that oxygen radicals may play a significant role in the impairment of EDR by RLP.
The study with HPLC and the isoluminol chemiluminescence
detection system showed that RLPs contained a substantial amount of
PC-OOH. The peroxidized phospholipids are known to be a reactive oxygen
species capable of initiating and propagating a chain of free radical
reactions, especially in polyunsaturated fatty acids, in lipoproteins
and cell surface membranes, leading to deterioration of the cell
surface membrane and intracellular thiol depletion both of which result
in cellular dysfunction.17 19 20 In fact, the present
study showed that the incubation of RBCs with RLP or peroxidized PC
including 5-CHO-PC produced a quantifiable transformation of the
erythrocytes from their discoidal to echinocytic configuration.
Further, the present study showed that peroxidized PC including
5-CHO-PC also caused EDR impairment. For all lipoproteins and lipid
preparations tested, there was an apparent concordance between
echinocytogenic action and EDR inhibition. Thus, oxidative damage,
which induced perturbation of RBC surface membranes, could occur in
surface membranes and in other constituents such as sulfur-containing
enzymes of the endothelium, and may lead to the
endothelial dysfunction. This is supported by the
present results that the echinocytogenic action and EDR impairment
by RLPs and peroxidized PC were both inhibited by GSH and NAC,
antioxidants that improve the intracellular redox state and terminate
the chain reaction of peroxidation by scavenging chain propagation
radicals and reducing lipid hydroperoxides.17 20 21 22 23
Furthermore, the present study showed that RLPs isolated from
plasma of patients under treatment with a usual pharmacological dose of
-tocopherol, an antioxidant, had a lower level
of phospholipid hydroperoxides and resistance of the RLPs to
Cu2+-induced oxidation. These effects of
-tocopherol were associated with a lack of
inhibitory action on EDR and of echinocytogenic action.
Taken together, these results suggest that oxidative damage mediated by
the peroxidized phospholipids, primarily contained in RLPs, may be at
least partly responsible for the impairment of EDR and RBC
transformation by RLP. Nonspecific cytotoxicity of the lipoproteins is
unlikely to cause dysfunction at the present dose of remnants
because our previous experiments1 showed that incubation
of the cultured endothelial cells with RLPs caused
neither LDH release into the medium nor cell death, and cell lining was
preserved after incubation of the aortas with RLPs by electron
micrograph. One possibility is that RLPs may perturb surface
receptor–mediated signal transduction as proposed for oxidized
LDLs,9 which also caused EDR impairment and
echinocytogenic effect.
This study cannot exclude the possibility that other oxidized lipid products possibly contained in remnants, such as degradation products of sphingomyelin, short chains of free fatty acids, peroxidized and fragmented polyunsaturated fatty acid–containing triglycerides, and oxysterols, could also play a role in the remnants-induced endothelial dysfunction. In fact, we previously reported that sphingosine, a sphingomyelin-hydrolyzed product, induced impairment of endothelium-dependent relaxation of the isolated rabbit aortas.24 During peroxidation of PC, phospholipid hydroperoxides are produced as a first step, then they are instantly decomposed to phospholipids with a short chain of aldehydes in the presence of transition metals.25 In the present study, we quantified the amount of only phospholipid hydroperoxides, precursors of phospholipids including aldehyde residues, in remnants. We did not determine the species of fatty acids and the degree of their degradation in the peroxidized phospholipids responsible for the endothelial dysfunction. 5-CHO-PC, a peroxidized PC, is reported to have various biological effects and it is contained in oxidized LDL26 27 and in atherosclerotic arterial walls.28 5-CHO-PC was found to impair endothelium-dependent relaxation of the isolated rabbit aortas, as shown in the present study. Thus, 5-CHO-PC may be 1 candidate of peroxidized phospholipids responsible for endothelial dysfunction. TBARS are known to be more hydrophilic and transferable compared with peroxidized phospholipids.29 Thus, TBARS produced in remnants in vivo can be transferred to plasma, albumin, other lipoproteins, or phospholipid layers of cell surface membranes in the circulation or in the arterial walls and they are dialyzed during the isolation procedure. These are reasons why TBARS were not detectable in the isolated RLPs. It remains to be evaluated whether peroxidized phospholipids taken up intracellularly through the phospholipid receptors may also cause the injurious effects of remnants on endothelial function.
Lipoproteins are believed to be oxidized in the subendothelial space that is sequestered from plasma antioxidants, but they are not oxidized in the circulation.25 There is a possibility that some of the circulating RLPs may enter into the subendothelial space and reemerge from the intima into the circulation and that lipid hydroperoxides may be produced in RLPs in the subendothelial space in the meantime. In patients with high RLP levels, the prolonged retention of RLPs in the circulation, as a result of the delayed hepatic uptake and/or the increased hepatic secretion of VLDL, may augment susceptibility of RLPs to oxidative modification in the arterial intima. Oxidative damage against endothelial cells, as observed in the present organ chamber experiment, could be induced by RLPs entering into the subendothelial space, leading to endothelial dysfunction in postprandial hyperlipidemic patients. In the present analysis, RLPs had very little lysoPC compared with oxidized LDLs.9 Thus, the extent of the oxidation of RLPs seems to be minimal. However, the inhibitory effect on EDR was comparable between RLPs and oxidized LDLs.9 30 31 This is the case in the minimally oxidized LDLs. Minimally oxidized LDLs have multiple atherogenic effects on vascular cells, and some peroxidized phospholipids also contribute to these effects.32
In conclusion, oxidative damage caused by lipid components in RLPs, especially peroxidized phospholipids, deteriorates cell surface membranes and may be at least partly responsible for RLP-induced impairment of EDR.
Received July 7, 1998; accepted January 14, 1999.
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