(Circulation. 1995;92:1582-1589.)
© 1995 American Heart Association, Inc.
Articles |
Impairment of Endothelium-Dependent Dilation in Rabbit Renal Arteries by Oxidized Lipoprotein(a)
Role of Oxygen-Derived Radicals
From the Department of Medicine, Division of Nephrology, University Hospital of Würzburg and the Department of Medicine, Division of Nephrology, University Hospital of Freiburg (J.B., P.S.),Germany.
Correspondence to Dr J. Galle, Dept of Medicine, Division of Nephrology, University Hospital Würzburg, Joseph-Schneider-Str 2, D-97080 Würzburg, Germany.
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Abstract |
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Background Hyperlipoproteinemia is associated with impairment of nitric oxide (NO)–mediated, endothelium-dependent dilation in renal arteries. In the present study, we assessed and compared the effects of human lipoprotein(a) and LDL on endothelium-dependent and -independent dilation in vitro.
Methods and Results Dilator responses were detected in isolated, saline-perfused, preconstricted arterial segments by a photoelectric device. Acetylcholine-induced, endothelium-dependent dilator responses of rabbit renal arteries were not significantly attenuated after 150 minutes of incubation with native lipoprotein(a) (30 and 100 µg/mL). However, exposure to in vitro oxidized lipoprotein(a) (150 minutes, 30 and 100 µg/mL) suppressed acetylcholine-induced dilator responses in a dose-dependent manner. At similar concentrations, native and oxidized LDL had no effect. Endothelium-independent dilations induced by the NO-donor sodium nitroprusside were also impaired by oxidized lipoprotein(a), whereas forskolin-induced dilator responses were unaffected, indicating that smooth muscle dilator capacity was not impaired. Attenuation of dilator responses by oxidized lipoprotein(a) was potentiated in the presence of superoxide dismutase (SOD). The SOD effect was completely blunted by coincubation with catalase (100 U/mL) or deferoxamine. In the absence of SOD, catalase or deferoxamine had no effect on dilator responses. Using a chemiluminescence assay, we could detect increased O2- production by arteries pretreated with oxidized lipoprotein(a), which suggested that enhanced NO inactivation by O2- could be the underlying mechanism for impairment of endothelium-dependent dilations.
Conclusions These data indicate that oxidized lipoprotein(a) impairs endothelium-dependent dilation and is more potent than oxidized LDL in this effect. The mechanism of the impairment may involve formation of O2- and inactivation of NO.
Key Words: nitric oxide • hypercholesterolemia • hypertension • anions
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Introduction |
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TopAbstractIntroductionMethods Results Discussion References |
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Arterial endothelium contributes to regulation of vascular tone by release of the short-lived endothelium-derived nitric oxide (EDNO).1 2 Pathophysiological conditions associated with attenuation of endothelial function include hypercholesterolemia and atherosclerosis.3 4 In vitro experiments have provided evidence that LDL contributes to impairment of endothelium-dependent dilations. LDL accumulates in the wall of atherosclerotic arteries5 6 and thus is in close vicinity to the endothelium and the vasculature. After entering the prooxidative subendothelial space, LDL is likely to undergo oxidative modification.7 8 Oxidized LDL has been shown to interfere with formation of EDNO9 10 and to directly inactivate it.11 12
Recently, it has been suggested that attenuation of endothelium-dependent dilations in children with familial hypercholesterolemia is linked to plasma levels of lipoprotein(a) [Lp(a)].13 Lp(a) is an atherogenic plasma lipoprotein composed of apolipoprotein B (apoB) and a large glycoprotein termed apolipoprotein(a) [apo(a)]. The distribution of plasma Lp(a) levels is highly skewed toward lower concentrations, with more than two thirds of the population having levels lower than 20 mg/dL. However, several clinical conditions and metabolic states have been shown to modulate Lp(a) plasma levels, including familial hypercholesterolemia14 and kidney diseases such as the nephrotic syndrome and end-stage renal failure.15 Lp(a) deposits, like LDL, have been identified in coronary vessels16 and in glomeruli in various forms of renal disease,17 and they may also undergo oxidative modification. We hypothesized that Lp(a) could influence vascular tone in a fashion similar to that of LDL. To evaluate this hypothesis, we studied the influence of native and oxidized Lp(a) on endothelium-dependent dilation of isolated rabbit arteries and compared its effect with the potency of LDL. Furthermore, since Lp(a) has been shown to induce free radical generation,18 which may play a role in the defective vascular relaxation in atherosclerotic arteries,19 20 21 22 we evaluated whether the effects of Lp(a) on endothelial function are mediated by oxygen-derived radicals.
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Methods |
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TopAbstractIntroductionMethods Results Discussion References |
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Chemicals
Norepinephrine was obtained from Hoechst. Sodium nitroprusside, indomethacin, acetylcholine, forskolin, lucigenin, xanthine, xanthine oxidase, superoxide dismutase (SOD), catalase, EDTA, 4,5-dihydroxy-1,3-benzene disulfonic acid salt, diethyldithiocarbamate (DDC), dithiothreitol, glycerol, SDS, polyacrylamide, NG-nitro-L-arginine, butylated hydroxytoluene (BHT), and substance P were obtained from Sigma Chemical Co. Deferoxamine was from Ciba-Geigy. Forskolin was dissolved in DMSO and, as with all other drugs if not indicated otherwise, further diluted in Tyrode's solution of the following composition (mmol/L): Na+ 144, K2+ 4.0, Ca2+ 1.6, Mg2+ 1.0, Cl- 140, HCO3- 11.9, H2PO4- 0.4, calcium-disodium EDTA 0.025, glucose 11; PO2 120 mm Hg, pH 7.4. Indomethacin was dissolved in ethanol -0.1 mol NaHCO3 (1:3) vol/vol.
Isolation of LDL and Lp(a)
Human LDL was isolated by
sequential
ultracentrifugation and stored at 4°C as
described previously.23 An Lp(a)-enriched regeneration
fluid was obtained from a single patient treated regularly with an LDL
apheresis system based on heparin-induced extracorporeal LDL
precipitation (HELP; Braun Melsungen). The serum Lp(a) concentration of
the patient varied between 180 and 200 mg/dL before HELP treatment and
decreased by 70% during LDL apheresis. The regeneration fluid was
ultracentrifuged first at a density of 1.065 g/mL, then at
1.120 g/mL, and then was subjected to density gradient
ultracentrifugation. Finally, Lp(a) was purified
using Sephadex G-25M gel chromatography and
lysine-Sepharose 4B chromatography and dialyzed
extensively against 150 mmol NaCl, 1 mmol EDTA (pH 7.4). Lp(a) was
analyzed for purity by 0.6% agarose gel electrophoresis as
well as by 4% SDS-PAGE.
Apo(a) Immunoblotting
Apo(a) isoform determination was
performed on serum using a
modification of a previously described sensitive immunoblotting
technique.24 Briefly, serum was reduced with 100 mmol/L
dithiothreitol in 8 mol/L urea and incubated at 37°C for 30 minutes.
The sample was solubilized in a solution containing 75% glycerol,
0.5% bromophenol blue, and 10% SDS and applied to 4% PAGE. Gels were
run on the Phast System (Pharmacia) for approximately 0.5 hours at 60
mA. After transfer of the proteins to
immobilion-polyvenylidene-difluoride transfer
membrane (Millipore), incubation of the membranes with antibodies
was performed using an anti-apo(a) monoclonal antibody (4F3,
1:2000; Cappel Laboratories) as first antibody and the vectastain ABC
anti-mouse test kit (Vector Laboratories) for detection. A standard
of a defined isoform pool (B, S1, S2, S3) was used on the gel (Immuno).
Apo(a) isoforms of the donor were larger than apo B-100 and
corresponded to S1/S2 of the Utermann
classification.25
Oxidation of Lp(a) and LDL
Lp(a) was oxidized in a similar
fashion to LDL by incubation
with CuSO4 (5 µmol) for 24 hours at 23°C as described
previously.23 The degree of oxidation was quantified by
two methods: (1) the increase in relative mobility on agarose gel and
(2) the formation of thiobarbituric acid–reactive substances
(TBARS).26 Homogeneity of lipoproteins was tested by
agarose gel electrophoresis (REP-HDL–plus cholesterol
electrophoresis, Helena Diagnostika). The relative mobility of oxidized
LDL and oxidized Lp(a) as an index for lipoprotein oxidation was 1.3 to
1.6 compared with native LDL and native Lp(a), respectively. Lp(a) was
stored at room temperature in the dark because of its tendency to form
a gel by self-association in the cold. Lipoproteins were prepared
fresh every 2 weeks. During this period, apo(a) isoforms and apo B were
intact and not degraded.
Vessel Preparation and Diameter Determination
Segments of the
aorta and the renal artery (0.8 cm in length)
were obtained from rabbits of either sex (New Zealand White rabbits, 4
to 5 months old, weighing 2.5 to 3.5 kg, n=105). All procedures were
carried out in accordance with the guidelines of the German Ministry of
Agriculture for the use and care of laboratory animals. Intact segments
were cannulated at both ends with steel cannulas and placed in an organ
bath containing oxygenated Tyrode's solution (37°C, pH
7.4) as described previously.23 Perfusion routes for bath
perfusion and intraluminal perfusion were separate. Outer vascular
diameters were recorded by a photoelectric device.27
The transmural pressure was adjusted hydrostatically to 45 mm Hg. In
each experiment, two segments obtained from the same animal were
studied simultaneously, with one segment serving as
time-matched control. Initially, the endothelial
integrity of segments preconstricted with norepinephrine
was tested by intraluminal perfusion with acetylcholine 1 µmol/L.
Only segments with a dilator response of >90% (calculated as the
percent of inhibition of preconstriction) were investigated further.
The segments were then preconstricted by adding
norepinephrine to the organ bath superfusion until a stable
preconstriction of 350 to 550 µm was reached. To study the influence
of oxidized LDL and of Lp(a) on
endothelium-dependent dilations, the segments were
incubated for 150 minutes with native or oxidized LDL (100 µg/mL),
native or oxidized Lp(a) (30 or 100 µg/mL), or their respective
buffers as time-matched control (added to the intraluminal
perfusion) before addition of acetylcholine. Because the amount of
available Lp(a) was limited by the costly preparation procedure, the
intraluminal perfusion rate was reduced from 30 to 2 mL/h during this
incubation period. After the lipoprotein incubation period, before
addition of acetylcholine, the perfusion rate was increased to 30 mL/h.
Control experiments revealed that this reduction had no influence on
vasomotor responses. After washout of the lipoproteins,
endothelium-dependent dilations were elicited by
adding cumulative doses of acetylcholine (1 nmol/L to 3 µmol/L) or of
substance P (1 nmol/L to 0.3 µmol/L) to the intraluminal perfusion.
To investigate the influence of oxidized Lp(a) on
endothelium-independent dilator capacities, sodium
nitroprusside (0.003 to 100 µmol/L) or forskolin (0.01 to 10
µmol/L) was used instead of acetylcholine, and the protocol was
otherwise unchanged. The influence of SOD (10 U/mL),
deferoxamine (1 mmol/L), and catalase (100 U/mL) on
acetylcholine-induced dilations in the presence and absence of 30
µg/mL oxidized Lp(a) was studied by adding the respective substances
to the intraluminal perfusion throughout the experiment. In some
experiments, SOD was added after washout of oxidized Lp(a), just before
stimulation with acetylcholine. All experiments were performed in the
presence of indomethacin (10 µmol/L) added to the
intraluminal perfusate to eliminate the influence of prostanoid
factors. To confirm that acetylcholine-induced dilation was
mediated by EDNO, we studied the inhibitory effect of
NG-nitro-L -arginine on the
dilator responses, as described previously.28
Detection of Chemiluminescence as a Parameter for
O2- Generation
Detection of
chemiluminescence was carried out as described
recently by Ohara et al21 in a scintillation counter with
a single photomultiplier tube (LUMAT LB 9501/16; Berthold). To detect
chemiluminescence in a cell-free O2-
generating system, lucigenin (0.25 mmol) and xanthine (1 to 6 µmol/L)
were dissolved in a final volume of 2 mL Krebs-HEPES buffer of the
following composition (mmol/L): NaCl 118, KCl 4, CaCl2 2.6,
MgCl2 1, KH2PO4 1.2,
NaHCO3 24, glucose 5, pyruvate 2, Na-HEPES 20.0; initially
gassed with 95% O2/5% CO2, pH
7.4. This solution was transferred to scintillation vials and counted
at 1-minute intervals. O2- generation was
induced by addition of xanthine oxidase (0.002 U). To correct for
background, counts obtained before addition of xanthine oxidase were
subtracted from counts obtained after its addition. To determine the
specificity of this assay, we added SOD (10 U/mL) or the
O2- scavenger 4,5-dihydroxy-1,3-benzene
disulfonic acid (10 mmol/L)29 to the xanthine/xanthine
oxidase solution. SOD and 4,5-dihydroxy-1,3-benzene disulfonic acid
blunted the signal obtained by xanthine/xanthine oxidase. To determine
chemiluminescence elicited by arteries treated with native or oxidized
Lp(a) or LDL, segments of the abdominal aorta were prepared (7 mm in
length, treated in an identical manner as the renal arteries) and
incubated for 30 minutes at 37°C in Krebs-HEPES buffer containing 30
µg/mL native or oxidized Lp(a) or LDL or its buffer as control.
Segments of aorta were used because the sensitivity of the system was
too low for signals obtained by renal arteries. Some of the segments
were additionally incubated with 10 mmol DDC, an inhibitor
of the CuZn form of SOD.30 Thereafter, the segments were
transferred to scintillation vials containing 0.25 mmol lucigenin in a
final volume of 2 mL. Counts were obtained at 1-minute intervals at
room temperature. To correct for background, counts obtained from vials
containing all components except the aortic rings were subtracted from
these signals.
Statistical Analyses
Data are presented as mean±SEM.
The n value refers to
the number of animals used. Dilator responses are expressed in percent
of the initial steady state constriction induced by
norepinephrine. Dose-effect curves in the plots in Figs 2 through
6
,
Fig 8, and Fig 9 were compared by use of one-way
ANOVA
for repeated measurements, followed by a point-by-point
comparison using Student's paired t test. For multiple
comparisons of data, the Bonferroni correction was applied. Comparisons
of O2- generation between control and oxidized
Lp(a)–treated arteries were performed by use of Student's paired
t test. A value of P<.05 was considered
significant.
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Results |
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TopAbstractIntroductionMethods Results Discussion References |
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Agarose Gel Electrophoresis and TBARS Levels of Native and Oxidized LDL and Lp(a); Immunoblotting of Lp(a)
Electrophoretic mobility of native and oxidized LDL as well as Lp(a) on agarose gel as a parameter for the extent of Cu2+-induced lipid peroxidation is shown in Fig 1
(left
panel). Relative mobility of the oxidized lipoproteins
was enhanced in a comparable manner, indicating that LDL and Lp(a) were
oxidized to a similar extent. Native Lp(a) was found to migrate in the
pre-ß region. Levels of TBARS, determined in samples containing 0.3
mg lipoprotein per mL, were 0.2±0.01 µmol in native LDL,
0.2±0.01
µmol in native Lp(a), 3.4±0.8 µmol in oxidized LDL, and
4.8±1
µmol in oxidized Lp(a). An immunoblot demonstrating the apo(a)
isoforms of the heterozygous patient is shown in Fig 1 (right
panel).
The two isoforms of the patient migrate in the approximate position of
S1/S2 compared with the standard. In control experiments,
immunoblotting and agarose gel electrophoresis were carried out between
the various steps of isolation. Under all circumstances, apo(a)
isoforms and apo B were intact and not degraded.
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Endothelium-Dependent Dilations
Acetylcholine elicited
endothelium-dependent
dilations in a dose-dependent manner in isolated rabbit renal
arteries preconstricted with norepinephrine.
NG-nitro-L-arginine (1
mmol) inhibited the acetylcholine-induced vasomotor response by
86±4% (6 experiments), identifying the dilation as mediated by EDNO,
as found in previous studies.
Effects of Native and Oxidized LDL and Lp(a) on
Endothelium-Dependent Dilations
Acetylcholine-induced dilations were
not impaired in arteries
following 150 minutes of treatment with 100 µg/mL native or oxidized
LDL (Fig 2
). Native Lp(a) (30 or 100 µg/mL) also had
no significant effect (Fig 2). In contrast, when the arteries
were
incubated with 30 or 100 µg/mL oxidized Lp(a),
endothelium-dependent dilations were attenuated in
a dose-dependent manner (Fig 3). Dilations elicited
by another endothelium-dependent dilator, substance
P, were attenuated by 100 µg/mL oxidized Lp(a) in an identical manner
(Fig 4). In additional experiments (n=3), we
investigated the effect of 100 µg/mL oxidized LDL obtained from the
patient who served as Lp(a) donor to directly compare LDL and Lp(a)
from the same individual. This LDL preparation also had no significant
effect on acetylcholine-induced dilations.
Endothelium-Independent Dilation After Treatment
With Oxidized Lp(a)
In an additional series of experiments, to
determine whether
smooth muscle dilator capacity was altered after treatment of arteries
with oxidized Lp(a), we studied the influence of 30 and 100 µg/mL
oxidized Lp(a) on dilator responses induced by sodium nitroprusside
(0.03 to 100 µmol/L) and of 30 µg/mL oxidized Lp(a) on dilator
responses induced by forskolin (0.01 to 10 µmol/L), a stimulator of
adenylate cyclase in smooth muscle cells. Dilations induced
by the nitric oxide (NO) donor sodium nitroprusside were significantly
inhibited by oxidized Lp(a) (Fig 5). Interestingly, the
nature of this inhibition was different from the effect of oxidized
Lp(a) on acetylcholine-induced dilations, in that high
concentrations of sodium nitroprusside elicited an almost complete
relaxation. Forskolin-induced dilator responses were not impaired
by treatment with oxidized Lp(a), indicating that smooth muscle dilator
functions were fully preserved (Fig 6).
Detection of O2- Generation
Reactive oxygen species may play a crucial role in the
impairment of endothelium-dependent dilations in
atherosclerotic
arteries,19 20 21 22 eg,
through inactivation of
EDNO by O2- or related reaction
products.31 We therefore investigated whether oxidized
Lp(a) stimulated O2- generation in the
isolated arteries. O2- generation detected by
chemiluminescence of lucigenin in a cell-free system and in
arteries treated with oxidized Lp(a) is shown in Fig 7.
In the cell-free system, addition of xanthine oxidase (0.002 U/mL)
to the xanthine- and lucigenin-containing scintillation vials
resulted in a rapid increase of the chemiluminescence signal, the
extent of which depended on the concentration of xanthine.
Chemiluminescence could be completely prevented by SOD (10 U/mL, Fig
7,
top) or the O2- scavenger
4,5-dihydroxy-1,3-benzene disulfonic acid (10 mmol, data not shown).
The chemiluminescence signal obtained from control arteries is shown in
Fig 7, bottom. Treatment of the arteries with 30 µg/mL
oxidized Lp(a)
induced a significant increase in the chemiluminescence signal (Fig
7,
bottom). Preincubation of the arteries with DDC, an
inhibitor of the CuZn form of SOD, further increased the
signal by a factor of 3 (n=4, data not shown). Additional treatment of
the arteries with SOD reduced the signal by 51±9% (n=3,
P<.05). The O2- scavenger
4,5-dihydroxy-1,3-benzene disulfonic acid, which enters the
intracellular space more easily than SOD, almost completely blunted the
chemiluminescence signal (Fig 7, bottom). Treatment of
arterial segments with 30 or 100 µg/mL native Lp(a) or
native or oxidized LDL had no influence on the chemiluminescence signal
(n=4 each). Oxidized Lp(a) in the absence of arteries had no effect on
the background signal.
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Effect of SOD, Catalase, and Deferoxamine on
Endothelium-Dependent Dilations in the Absence and
Presence of Lp(a)
In another series of experiments, we established
that SOD and
catalase, enzymes catabolizing O2- and
H2O2, respectively, and
deferoxamine, which inhibits the conversion of superoxide
anions and hydrogen peroxide to hydroxyl radicals,32 had
no influence on dilator responses in the absence of Lp(a) (n=6
experiments each, data not different from untreated controls). We then
studied the influence of SOD on dilator responses of arteries treated
with 30 µg/mL native or oxidized Lp(a). SOD (10 U/mL) had no
influence on native Lp(a)–treated segments (n=8 experiments, data
not
different from untreated controls). However, in arteries treated with
oxidized Lp(a), the presence of simultaneously given SOD
significantly enhanced the attenuation of
endothelium-dependent dilations (Fig 8). The latter enhancement
of attenuation by SOD was
completely prevented by additional treatment with catalase or
deferoxamine (Fig 8). When SOD was added to oxidized
Lp(a)–treated arteries after washout of the lipoproteins, just before
stimulation with acetylcholine, it improved dilator responses (Fig
8).
In the absence of SOD, catalase had no influence on
dilator responses
in oxidized Lp(a)–treated segments (Fig 9).
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Discussion |
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TopAbstractIntroductionMethods Results Discussion References |
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The present study demonstrates that oxidized but not native Lp(a) attenuates endothelium-dependent dilation in isolated rabbit renal arteries. In this respect, oxidized Lp(a) was far more potent than LDL oxidized under identical conditions. Endothelium-independent dilations elicited by lower concentrations of the NO donor sodium nitroprusside were significantly attenuated by oxidized Lp(a), but endothelium-independent dilator responses induced by forskolin remained unaffected. SOD, when given simultaneously with oxidized Lp(a), enhanced the inhibitory effect. The latter effect could be prevented completely by additional treatment with catalase or deferoxamine, indicating involvement of oxygen-derived radicals. Adding SOD after washout of oxidized Lp(a) improved dilator responses. Increased O2- production of arteries pretreated with oxidized Lp(a) could be detected directly by use of a chemiluminescence assay.
Many recent studies indicate that the vascular endothelium plays an important role in maintenance of physiological blood supply to various organ tissues by releasing EDNO and other dilators, such as prostacyclin. Hypercholesterolemia has frequently been described as a cause of disturbance of endothelial function in human and animal studies,33 34 35 which can result, for example, in impairment of coronary flow reserve.4 In various studies further investigating the mechanism of hypercholesterolemia-induced impairment of endothelial function in vitro, it has been demonstrated that oxidized LDL, which accumulates in atherosclerotic plaques in hypercholesterolemia,6 can interfere with formation10 or activity11 12 of EDNO. However, no study has investigated the effects of native or oxidized Lp(a) on endothelial function. Lp(a) is an independent risk factor for premature atherosclerosis,36 and its blood levels are not strongly correlated with plasma levels of cholesterol or LDL. Thus, it is important to differentiate between the effects of LDL and Lp(a) on endothelial function, especially since there is a wide range for Lp(a) levels within the general population. Although at present there is no direct evidence for the presence of oxidized Lp(a) in vivo, it is tempting to speculate that Lp(a), like LDL, can undergo oxidative modification in the prooxidative subendothelial space. Like LDL, Lp(a) has been localized in atheromatous arteries and in glomeruli16 17 and can easily be oxidized in vitro.37
In hypercholesterolemic vessels, enhanced generation of O2- or related reaction products31 may be responsible for impairment of endothelial function through augmented inactivation of EDNO and/or damage of endothelial cells.19 20 21 22 Furthermore, in a recent study, Lp(a) has been shown to induce formation of oxygen free radicals in human monocytes.18 Therefore, we hypothesized that the mechanism of the effects of oxidized Lp(a) involves formation of reactive oxygen species. Indeed, using a chemiluminescence detection system, we observed a significant increase in O2- production in arteries preincubated with oxidized Lp(a). This O2- production was further enhanced by pretreatment of the arteries with DDC, an inhibitor of SOD, while the O2- scavenger completely blunted the chemiluminescence signal. These data provide direct evidence for O2- generation in oxidized Lp(a)–treated arteries, which might lead to attenuation of endothelium-dependent dilations through inactivation of NO. However, simultaneous treatment of the arteries with SOD and oxidized Lp(a) significantly increased its inhibitory effect on dilator responses. This unexpected effect of SOD could be explained by the deleterious effects of too much SOD activity in relation to H2O2-removing enzymes. Indeed, it has been shown that SOD may favor formation of the most biologically active hydroxyl radical in the absence of catalase.38 39 Hydroxyl radicals might either attack the endothelial cells, destroy NO, or induce further lipid peroxidation.39 The observation that SOD improved dilator responses when added to the arteries after washout of oxidized Lp(a) provides an indirect argument for a hydroxyl radical–mediated propagation of lipid peroxidation during coincubation of oxidized Lp(a) and SOD, which in turn could destroy the endothelial cells. Consistent with the interpretation that hydroxyl radicals contributed to attenuation of endothelium-dependent dilations after coincubation of the arteries with SOD and oxidized Lp(a), additional treatment with catalase completely blunted the effect of SOD, whereas catalase in the absence of SOD had no influence on the inhibitory effect of oxidized Lp(a). Furthermore, additional treatment with the iron chelator deferoxamine, which inhibits the formation of hydroxyl radicals, also prevented attenuation of dilations in arteries treated with oxidized Lp(a) and SOD, providing further evidence for involvement of hydroxyl radicals. Thus, one may speculate that oxidized Lp(a) stimulates the arteries to produce O2- and H2O2. In the absence of sufficient catalase activity, the latter might then serve as the substrate for hydroxyl radical formation.39
Oxidized Lp(a) inhibited acetylcholine-induced dilations, which were characterized as EDNO-mediated by the inhibitory effect of NG-nitro-L -arginine. Endothelium-dependent dilations induced by substance P were attenuated by oxidized Lp(a) in an identical manner, indicating that the inhibitory effect is not selective for muscarinic agonists. Endothelium-independent dilator responses induced by forskolin were not affected by oxidized Lp(a), indicating that vascular smooth muscle function was not attenuated. However, the dilator responses elicited by low to moderate doses of sodium nitroprusside were significantly impaired, whereas high doses induced a complete relaxation of the arteries. A unifying hypothesis explaining both the inhibition of acetylcholine-induced dilations and the equivocal effect on the two different endothelium-independent vasodilators is an enhanced inactivation of NO by oxidized Lp(a) or oxidized Lp(a)–induced formation of oxygen-derived radicals. Whereas acetylcholine releases NO from endothelial cells, sodium nitroprusside relaxes vessels by release of NO as its active compound into the cytosol of smooth muscle cells and into the extracellular space.40 Thus, inactivation of NO in the extracellular space by oxidized Lp(a) or oxygen-derived radicals could explain why sodium nitroprusside–induced dilations were inhibited at lower concentrations, whereas high concentrations of sodium nitroprusside overcame this effect.
Our finding that oxidized Lp(a) inhibited acetylcholine-induced dilation in a dose-dependent manner adds new insights into the pathomechanism of disturbed endothelial function in hyperlipoproteinemia. An important observation of this study is that oxidized Lp(a) was a far more potent inhibitor of acetylcholine-induced, endothelium-dependent dilations than was oxidized LDL. At a concentration of 100 µg/mL, oxidized LDL had no effect on endothelium-dependent dilation in rabbit renal arteries. Although attenuation of endothelial function by oxidized LDL at similar concentrations has been observed by others,10 41 the lack of effect is in agreement with previous studies from this laboratory11 23 and may be due to the relatively mild lipoprotein oxidation conditions. Both lipoproteins were modified according to the same protocol, resulting in comparable TBARS levels and enhancement of electrophoretic mobility as parameters for lipid peroxidation. The reason for the different potency of oxidized Lp(a) and oxidized LDL is unclear. It has been suggested that LDL preparations obtained from different individuals vary in their power to inhibit endothelium-dependent dilations.42 Therefore, in additional experiments, we prepared LDL and Lp(a) from the same patient and oxidized the lipoproteins according to our standardized protocol. However, oxidized Lp(a) remained the more potent agent by far compared with LDL obtained from the same individual.
In conclusion, the present study demonstrates that oxidized Lp(a) impairs endothelium-dependent dilations in rabbit renal arteries and provides evidence that generation of reactive oxygen species is the mechanism of this effect. We hypothesize that impairment of endothelium-dependent dilations by oxidized Lp(a) may contribute significantly to attenuation of endothelial function in humans with high Lp(a) levels.
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Acknowledgments |
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This work was supported by grants from the Deutsche Forschungsgemeinschaft (Ga 431/1-2 and Wa 836/1-1). The skillful technical assistance of Petra Stunz is gratefully acknowledged.
Received February 14, 1995; accepted March 27, 1995.
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