© 2000 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Effect of Overexpression of Human Apo A-I in C57BL/6 and C57BL/6 Apo E–Deficient Mice on 2 Lipoprotein-Associated Enzymes, Platelet-Activating Factor Acetylhydrolase and Paraoxonase
Comparison of Adenovirus-Mediated Human Apo A-I Gene Transfer and Human Apo A-I Transgenesis
From the Center for Molecular and Vascular Biology (B.D.G., M.L, M.L, D.C., P.H.), University of Leuven, Leuven, Belgium; and INSERM U321 (D.S., L.L.G., E.N.), Lipoproteins and Atherogenesis, Institut Fédératif Muscle Coeur et Vaisseaux, Hôpital Pitié-Salpêtrière and UFR Médecine Sud (Université Pierre et Marie Curie), Paris, France.
Correspondence to Paul Holvoet, PhD, Center for Molecular and Vascular Biology, Campus Gasthuisberg, Herestraat 49, 3000 Leuven, Belgium. E-mail paul.holvoet{at}med.kuleuven.ac.be
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Abstract |
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Abstract—Various mechanisms may contribute to the antiatherogenic potential of apolipoprotein A-I (apo A-I) and high density lipoproteins (HDLs). Therefore, the effect of adenovirus-mediated human apo A-I gene transfer or human apo A-I transgenesis on platelet-activating factor acetylhydrolase (PAF-AH) and arylesterase/paraoxonase (PON1) was studied in C57BL/6 and C57BL/6 apo E-/- mice. Human apo A-I transgenesis in C57BL/6 mice resulted in a 4.2-fold (P<0.0001) increase of PAF-AH and a 1.7-fold (P=0.0012) increase of PON1 activity. The apo E deficiency was associated with a 1.6-fold (P=0.008) lower PAF-AH and a 2.0-fold (P=0.012) lower PON1 activity. Human apo A-I transgenesis in C57BL/6 apo E-/-mice increased PAF-AH and PON1 activity by 2.1-fold (P=0.01) and 2.5-fold (P=0.029), respectively. After adenovirus-mediated gene transfer of human apo A-I into C57BL/6 apo E-/-mice, a strong correlation between human apo A-I plasma levels and PAF-AH activity was observed at day 6 (r=0.92, P<0.0001). However, PON1 activity failed to increase, probably as a result of cytokine-mediated inhibition of PON 1 expression. In conclusion, this study indicates that overexpression of human apo A-I increases HDL-associated PAF-AH activity. PON1 activity was also increased in human apo A-I transgenic mice, but not after human apo A-I gene transfer, a result that was probably related to cytokine production induced in the liver by the adenoviral vectors. Increased levels of these HDL-associated enzymes may contribute to the anti-inflammatory and antioxidative potential of HDL and thereby to the protection conferred by HDL against atherothrombosis.
Key Words: HDL • gene transfer • platelet-activating factor acetylhydrolase • paraoxonase
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Introduction |
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Plasma levels of apo A-I and HDL cholesterol are negatively correlated with the risk of ischemic cardiovascular disease, the leading cause of death in Western countries.1 2 Studies in human apo A-I–transgenic mice and rabbits have demonstrated that increased HDL cholesterol inhibits the progression of atherosclerosis.3 4 5 6 Various mechanisms may contribute to the antiatherogenic potential of HDL. Reverse cholesterol transport, as originally proposed by Glomset,7 includes the extraction of cholesterol from extrahepatic tissues by HDL and the delivery of cholesterol and cholesterol esters to hepatocytes, which can secrete HDL-derived cholesterol into bile as free cholesterol or as bile acids.
The antiatherogenic potential of HDL may also be related to its anti-inflammatory and antioxidative properties, which are mediated by several mechanisms. First, reactive oxidized lipids may be transferred from LDL to HDL,8 which may inhibit the propagation of an oxidation cascade in LDL. HDL has indeed been shown to be the predominant carrier of cholesterol ester hydroperoxides in humans.9 After transfer to HDL, cholesterol ester hydroperoxides are taken up much more efficiently by hepatocytes than are native cholesterol esters,10 11 providing a link between the antioxidative properties of HDL and reverse cholesterol transport.
Second, several HDL-associated enzymes may protect LDL against oxidative modification. LDL oxidation has been shown to induce fragmentation of the sn-2 residue of phospholipids,12 generating oxidized phospholipids with potent proinflammatory effects. Platelet-activating factor acetylhydrolase (PAF-AH; EC.3.1.1.47), which in humans is associated with both LDL and HDL,13 hydrolyzes not only the ester bond in the sn-2 position of PAF to release acetate but has also been shown to hydrolyze peroxidized fatty acids of phospholipids.12 PAF-AH pretreatment of oxidized phospholipids blocked the mitogenic activity toward smooth muscle cells in culture, which could also be abolished by a PAF receptor antagonist.14 Treatment of mildly oxidized LDL with PAF-AH inhibited the ability of mildly oxidized LDL to induce endothelial cells to bind monocytes and to produce monocyte chemotactic protein-1.15 Lecithin-cholesterol acyltransferase, which circulates mostly in association with HDL, has also been demonstrated to hydrolyze truncated phosphatidylcholines generated during lipoprotein oxidation.16
Paraoxonase (PON1, aryldialkylphosphatase; EC.3.1.8.1) is a glycoprotein of 43-kDa molecular weight, which circulates in an HDL subfraction that also contains apo A-I and clusterin.17 PON1 is a member of a multigene family located on human chromosome 7, which contains 2 additional PON1-like genes, designated PON2 and PON3. PON1 has long been known for its capacity to detoxify the organophosphate-type pesticides and nerve gases by hydrolysis.18 However, the demonstration that formation of total lipid peroxides and thiobarbituric acid–reactive substances, as a result of copper ion–catalyzed LDL oxidation, was reduced by purified PON1 has highlighted a new, physiologically important role for this enzyme.19 PON1 has been shown to have peroxidase activity and to hydrolyze lipid peroxides in oxidized lipoproteins with a preference for cholesteryl linoleate.20 Gene targeting of PON1 has generated unequivocal evidence for the role of PON1 in the protection against LDL oxidation and against the progression of atherosclerosis. HDL isolated from PON1-knockout mice was unable to prevent LDL oxidation, and PON1-knockout mice were more susceptible to diet-induced atherosclerosis than were their wild-type littermates.21
Because oxidized LDLs may promote monocyte adhesion and foam cell generation, induce smooth muscle cell proliferation and migration, and enhance platelet adhesion and aggregation, oxidation of LDL in the arterial wall is thought to be an initiator of atherosclerosis.22 23 Enhancement of the antioxidative and anti-inflammatory potential of HDL induced by elevated levels of apo A-I may therefore be mechanistically linked to the inhibition of atherosclerosis in human apo A-I–transgenic animals3 4 5 6 and the inhibition of injury-induced neointima formation.24 25 26 Therefore, we evaluated the effect of human apo A-I overexpression by transgenesis or adenovirus-mediated gene transfer in C57BL/6 and C57BL/6 apo E-/- mice on PAF-AH activity and on the arylesterase and paraoxonase activity of PON1.
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Methods |
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Generation of AdapoA-I
The recombinant adenovirus AdapoA-I, containing the cytomegalovirus promoter/enhancer, the endogenous 256-bp apo A-I promoter, and the genomic apo A-I sequence, was generated by cotransfecting the rescue plasmid pJM17 and the shuttle plasmid pLpA into 293 cells as described previously.26 To generate the pACCMVpLpA vector containing the genomic apo A-I DNA with inclusion of the 256-bp minimal apo A-I promoter, the pBluescript vector containing the genomic apo A-I DNA was restricted with XbaI and EcoRI. The genomic apo A-I DNA fragment was blunted at 37°C with Klenow DNA polymerase (Boehringer Mannheim). The pACCMVpLpA vector was opened with XbaI, dephosphorylated at 37°C for 1 hour with alkaline phosphatase (Boehringer Mannheim), and blunted by using Klenow DNA polymerase. Ligation was performed with T4 DNA ligase (Boehringer Mannheim). The correct orientation of the genomic apo A-I was determined by restriction with HindIII and was reconfirmed by DNA sequencing. The human tissue-type plasminogen activator virus (Adt-PA) has been described before.27
Animal Experiments
All experimental procedures in animals were performed in
accordance with protocols approved by the Institutional Animal Care and
Research Advisory Committee. All mice used in this study were female
and 3 to 5 months of age. Apo E-/-
mice28 were backcrossed for 10 generations into the
C57BL/6J background and had 99.9% C57BL/6J background. Human apo
A-I–transgenic mice used in this study were originally described by
Rubin et al.29 The human apo A-I–transgenic apo
E-/- mice used here have been described
before.30 Mice were fed normal chow ad libitum. Virus
administration was performed by tail vein injection. Different doses of
recombinant adenovirus were administered in a final volume of 300
µL.
Isolation of Lipoproteins by Gel Filtration
Mice were killed after an overnight fast, and maximal blood
volume was obtained by puncture of the inferior vena cava.
To obtain plasma, anticoagulation was performed with 0.1 volume of 4%
trisodium citrate. Separation of lipoproteins by gel filtration in a
fast pressure liquid chromatography system (Waters
Associates) was performed as described previously.26 For
gel filtration of serum, an isotonic saline buffer (10 mmol/L
Tris-HCl [pH 7.4], 137 mmol/L NaCl, 5 mmol/L KCl, 1
mmol/L CaCl2, and 1 mmol/L
MgCl2) was used.
A pool of fractions 10 to 17 containing VLDL-, IDL-, and LDL-size lipoproteins was used to determine PAF-AH and arylesterase activity of non-HDL–size lipoproteins. Similarly, fractions 18 to 25 and 26 to 33, corresponding to large-size and small-size HDL lipoproteins, respectively, were pooled, and PAF-AH and arylesterase activities were determined on these pools.
For determination of cholesterol levels, cholesterol of fractions obtained after gel filtration was extracted with methanol/chloroform (2:1, vol/vol). Esterified and unesterified cholesterol levels were quantified by high-performance liquid chromatography on a reversed-phase column (Zorbax ODS, Du Pont de Nemours) essentially as described by Vercaemst et al.31
Human Apo A-I ELISA
Human apo A-I levels were determined by sandwich ELISA. In
brief, polystyrene microtiter plates (Costar) were coated with a rabbit
anti-human apo A-I polyclonal antibody. Diluted plasma samples
(1:25 000, 1:50 000, 1:100 000, and 1:200 000) were added to the
wells for 2 hours. After being washed, a 1:15 000 dilution of the
murine monoclonal antibody
A4H4A7
conjugated with peroxidase was placed on the wells for 2 hours.
Peroxidase reaction was performed by adding
H2O2 and
o-phenylenediamine. Finally, absorbance was
measured at 492 nm.
Determination of PAF-AH Activity
Hexadecyl PAF, obtained as a powder from Sigma Chemical Co, was
dissolved at a final concentration of 20 mmol/L in ethanol (80%
vol/vol). This solution was mixed with
1-O-hexadecyl-2-[3H-acetyl]-sn-glycero-3-phosphocholine
(10 Ci/mmol, DuPont–New England Nuclear), dried under a stream of
N2, and redissolved in a solution containing
fatty acid–free bovine serum albumin (0.25% wt/vol in saline)
to obtain a 50 µmol/L [3H-acetyl]PAF
solution. AH activity was measured by the trichloroacetic acid
precipitation procedure as previously described.32 In
brief, the pH of the HEPES-EDTA (2 mmol/L) buffer was adjusted to
7.4, and routine assays were performed for 10 minutes at 37°C in a
total volume of 100 µL. Plasma was diluted 2000-fold and lipoprotein
fractions were diluted 10-fold in HEPES buffer before addition of 10
µL of [3H-acetyl]PAF (50 µmol/L;
specific activity, 
6000 disintegrations per minute per
nanomole).
Determination of Arylesterase and Paraoxonase Activity of
PON1
Arylesterase activity was measured by using phenylacetate as a
substrate.33 Initial rates of hydrolysis were determined
spectrophotometrically at 270 nm in a Power Wave 200 microplate
scanning spectrophotometer (Bio-Tek Instruments). The assays were
performed in a final volume of 250 µL containing 1 mmol/L
phenylacetate and 2 mmol/L CaCl2 in 20
mmol/L Tris-HCl buffer, pH 8.0, in the presence of 0.1 µL of mouse
serum or 10 µL of lipoprotein fraction for 5 minutes. The
extinction coefficient at 270 nm for the reaction was 1307
mol/L-1 · cm-1 for
1 micromole of phenylacetate hydrolyzed per minute.
The rate of hydrolysis of paraoxon was assessed by measuring liberation of p-nitrophenol at 405 nm at 25°C.34 The assays were performed in a final volume of 250 µL containing 5.5 mmol/L paraoxon and 2 mmol/L CaCl2 in 100 mmol/L Tris-HCl buffer, pH 8.0, in the presence of 2 to 4 µL of mouse serum for 4 minutes. The extinction coefficient at 405 nm for the reaction was 17 000 mol/L-1 · cm-1 for 1 nanomole of p-nitrophenol converted per minute.
Evaluation of an Acute-Phase Response and Cytokine
Production After Adenoviral Gene Transfer
High-resolution electrophoresis of serum was carried out by
using Hydragel 15 HR (Sebia Benelux) according to the instructions of
the manufacturer. Albumin,
2-globulins, and complement component
C3 were quantified by densitometric scanning.
Plasma levels of interleukin-1 (IL-1ß) and interleukin-6 (IL-6) were
determined by using the Quantikine M immunoassays (R&D Systems
Europe).
Statistical Analysis
All data are expressed as mean±SEM. Significance of differences
in cholesterol values were assessed by a 2-tailed,
unpaired, alternate Welch t test with the
INSTAT V2.05a statistical program (Graph Pad
Software). Comparison of PAF-AH activity, arylesterase activity, and
paraoxonase activity was performed by the nonparametric
Mann-Whitney U test. The correlation between arylesterase
and paraoxonase activity of PON1 and the correlation between PAF-AH
activity and human apo A-I was calculated by using the
nonparametric Spearman rank correlation in the
INSTAT V2.05a statistical program. A two-sided
P value of <0.05 was considered statistically
significant.
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Results |
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Effect of Human Apo A-I Overexpression on Cholesterol Levels in C57BL/6 and C57BL/6 Apo E-/- Mice
Cholesterol levels of lipoprotein fractions isolated by gel filtration are represented in the Table
. HDL
cholesterol was 1.6-fold (P<0.05) lower in
C57BL/6 apo E-/- mice than in C57BL/6 mice.
Gene transfer with 109 plaque-forming units (pfu)
of AdapoA-I increased HDL cholesterol levels at
6 days after gene transfer by 1.8-fold (P<0.05) and
2.1-fold (P<0.05) in C57BL/6 and C57BL/6 apo
E-/- mice, respectively. HDL
cholesterol levels in human apo A-I C57BL/6 and human apo
A-I C57BL/6 apo E-/- transgenic mice were
3.5-fold (P<0.01) and 3.1-fold (P<0.01) higher,
respectively, than in nontransgenic control mice. HDL
cholesterol levels in human apo A-I C57BL/6 and human apo
A-I C57BL/6 apo E-/- transgenic mice were
1.9-fold (P<0.05) and 1.5-fold (P<0.05) higher,
respectively, than in AdapoA-I–treated C57BL/6 and
AdapoA-I–treated C57BL/6 apo E-/-
mice, respectively (the Table). Less than 5% of HDL
cholesterol and as much as 15% of human apo A-I was
present in small-size HDL particles.
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PAF-AH Activity in C57BL/6 and C57BL/6 Apo E-/-
Mice
PAF-AH activity in C57BL/6 control mice (n=16), in C57BL/6 mice 6
days after gene transfer with 109 pfu of
Adt-PA adenovirus (n=12) or of AdapoA-I
adenovirus (n=11), and in human apo A-I C57BL/6 transgenic mice (n=5)
is shown in Figure IA. PAF-AH activity
was not significantly different in C57BL/6 control mice and C57BL/6
mice treated with Adt-PA control virus. PAF-AH activity
increased 2.0-fold (P<0.0001) after AdapoA-I
transfer and was 4.2-fold (P<0.0001) elevated in human apo
A-I–transgenic mice. PAF-AH activity was 2.1-fold
(P=0.0009) higher in human apo A-I C57BL/6 transgenic mice
than in AdapoA-I–treated mice.
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PAF-AH activity in apo E-/- control mice
(n=9), in apo E-/- mice 6 days after gene
transfer with 109 pfu of Adt-PA
adenovirus (n=11) or AdapoA-I adenovirus (n=12), and in
human apo A-I apo E-/- transgenic mice (n=4) is
shown in Figure
IB. PAF-AH activity was 1.6-fold
(P=0.008) lower in apo E-/- than in
C57BL/6 mice. No significant alteration of PAF-AH activity was observed
after Adt-PA transfer in apo E-/-
mice. PAF-AH activity increased 1.8-fold (P=0.0024) after
AdapoA-I gene transfer and was 2.1-fold (P=0.010)
elevated in human apo A-I apo E-/- transgenic
mice.
To investigate the association between human apo A-I overexpression and
increase in PAF-AH activity in C57BL/6 apo E-/-
mice, 3 different doses of AdapoA-I adenovirus
(5x108, 109, and
2x109 pfu; n=4 for each dose) were administered,
and PAF-AH activity was determined 6 days after gene transfer. The
correlation between human apo A-I plasma levels and PAF-AH activity was
0.92 (P<0.0001; Figure
II).
To investigate the kinetics of increased PAF-AH activity after adenovirus-mediated gene transfer, 2x109 pfu of AdapoA-I was administered to C57Bl/6 apo E-/- mice, and PAF-AH activity was determined at days 3, 6, 14, and 21 after gene transfer. Figures IIIA and IIIB illustrate human apo A-I plasma levels and plasma PAF-AH activity, respectively. PAF-AH activity was increased 2.5-fold at day 3 (P<0.0001), 3.3-fold at day 6 (P<0.0001), 1.7-fold at day 14 (P<0.0001), and 1.4-fold at day 21 (P=0.01).
Lipoprotein Distribution of PAF-AH Activity
Figure IV illustrates the
lipoprotein distribution of PAF-AH activity in C57BL/6 (A) and apo
E-/- (B) mice, respectively. In C57BL/6 control
mice, 83% of PAF-AH activity was recovered in large-size HDL particles
and 13% in small-size HDL particles. This distribution was similar
after Adt-PA gene transfer. PAF-AH activity in large-size
HDL and small-size HDL particles increased 1.8-fold
(P=0.0005) and 3.7-fold (P=0.0005), respectively,
after gene transfer with 109 pfu of
AdapoA-I. PAF-AH activity in large-size HDL and small-size
HDL particles of human apo A-I–transgenic mice was 2.1-fold
(P=0.040) and 18-fold (P=0.0015) higher,
respectively, than in control mice. The activity associated with
small-size HDL particles contributed 25% and 56% of total
lipoprotein-associated activity in AdapoA-I–treated C57BL/6
mice and human apo A-I–transgenic mice, respectively.
In C57BL/6 apoE-/-control mice, 52% of PAF-AH activity was present in large-size HDL and 41% in small-size HDL. PAF-AH activity increased 2.2-fold (P=0.014) in large-size HDL after AdapoA-I gene transfer, whereas no significant change occurred in small-size HDL. PAF-AH activity in large-size HDL and small-size HDL of human apo A-I–transgenic mice was 2.1-fold (P<0.05) and 2.1-fold (P=0.0028) higher, respectively, than in control mice.
Arylesterase and Paraoxonase Activity of PON1 in C57BL/6 and
C57BL/6 Apo E-/- Mice
Arylesterase and paraoxonase activities of PON1 in C57BL/6 control
mice (n=7), in C57BL/6 mice 6 days after gene transfer with
109 pfu of Adt-PA adenovirus (n=4) or
AdapoA-I adenovirus (n=4), and in human apo A-I C57BL/6
transgenic mice (n=9) are shown in Figures
VA and VB, respectively.
Arylesterase activity decreased 1.8-fold (P=0.0061) after
both Adt-PA and AdapoA-I gene transfer and was
1.5-fold (P=0.0003) higher in human apo A-I–transgenic mice
than in C57BL/6 control mice. Paraoxonase activity decreased 2.1-fold
(P=0.0061) and 2.3-fold (P=0.0061) after
Adt-PA and AdapoA-I gene transfer, respectively,
and was 1.7-fold (P=0.0012) higher in human apo
A-I–transgenic mice than in C57BL/6 mice. Arylesterase and paraoxonase
activities of PON1 were highly correlated (r=0.98,
P<0.0001).
Figures VC and VD illustrate the arylesterase and paraoxonase activity, respectively, of PON1 in C57BL/6 apoE-/- control mice (n=4), in C57BL/6 apo E-/- mice 6 days after gene transfer with 109 pfu of Adt-PA virus (n=5) or AdapoA-I virus (n=4), and in human apo A-I C57BL/6 apoE-/- transgenic mice (n=4). Paraoxonase activity was not determined after Adt-PA transfer in apoE-/- mice. Arylesterase and paraoxonase activities were 1.4-fold (P=0.042) and 2.0-fold (P=0.012) lower, respectively, in apo E-/- mice than in C57BL/6 mice. No significant alteration of arylesterase activity was observed after Adt-PA or AdapoA-I gene transfer. Compared with apoE-/- control mice, arylesterase and paraoxonase activity increased 1.8-fold (P=0.029) and 2.5-fold (P=0.029), respectively, in human apo A-I apoE-/- transgenic mice. Arylesterase and paraoxonase activities of PON1 were highly correlated (r=0.90, P=0.0002).
Figure IIIC illustrates the time course of arylesterase
activity after gene transfer with 2x109 pfu of
AdapoA-I in C57BL/6 apo E-/- mice.
Compared with baseline, arylesterase was 1.2-fold lower at day 3
(P<0.05), 1.8-fold lower at day 6 (P<0.0001),
2.2-fold lower at day 14 (P<0.0001), and was not
significantly different at day 21.
Lipoprotein Distribution of Arylesterase Activity
Figure VI illustrates the
lipoprotein distribution of arylesterase activity in C57BL/6 (A) and
apo E-/- mice (B), respectively. Activities
were determined on fractions obtained after gel filtration of pooled
serum samples. In C57BL/6 control mice, 91% of arylesterase activity
was present in large-size HDL particles and 8% in small-size HDL
particles. The 1.8-fold decrease in serum arylesterase activity after
Adt-PA and AdapoA-I gene transfer corresponded
to a 1.5-fold and a 2.2-fold decrease, respectively, of
arylesterase activity associated with large-size HDL particles. The
1.5-fold higher serum arylesterase in human apo A-I–transgenic mice
corresponded to a 1.7-fold increase of activity associated with
large-size HDL particles.
In apo E-/- mice, 88% of arylesterase activity was present in large-size HDL particles and 6% in small-size HDL particles. The 1.8-fold increase in serum arylesterase activity in human apo A-I–transgenic apo E-/- mice corresponded to a 1.5-fold increase of arylesterase activity in large-size HDL particles.
Plasma Cytokines and Acute-Phase Response Proteins After
Gene Transfer
IL-1ß concentration in plasma was below detection (7.5 pg/mL) in
control C57BL/6 mice (n=4) and was 70±44 pg/mL 6 days after gene
transfer with AdapoA-I in C57BL/6 mice. IL-6 was below
detection (15.6 pg/mL) in both control mice and 6 days after gene
transfer with AdapoA-I. High-resolution electrophoresis of
serum proteins in C57BL/6 mice at baseline (n=9) and 6 days after human
apo A-I gene transfer (n=6) showed a 15% (P=0.0004)
decrease of albumin, a 48% (P=0.0008) increase of
2-globulins, and a 13% (P=0.040)
increase of the complement component C3.
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Discussion |
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The main findings of the present study are that (1) PAF-AH, arylesterase, and paraoxonase activities are significantly lower in apo E-/- mice than in wild-type C57BL/6 mice, indicating an impaired anti-inflammatory and antioxidative potential of HDL in C57BL/6 apo E-/- mice; (2) overexpression of human apo A-I by transgenesis or adenovirus-mediated gene transfer increases HDL-associated PAF-AH activity in both C57BL/6 and C57BL/6 apo E-/- mice, and a strong correlation exists between human apo A-I plasma levels and PAF-AH-activity; (3) the arylesterase and paraoxonase activity of PON1 is increased in human apo A-I–transgenic C57BL/6 and apo E-/- mice but not in AdapoA-I–treated mice. This decrease is probably related to the inflammatory response induced by E1-deleted first-generation adenoviral vectors.
PAF-AH was predominantly associated with HDL, and only a minor fraction of the activity was associated with non-HDL. In contrast, approximately two thirds of PAF-AH activity in humans is associated with LDL and one third with HDL.12 Recently, Stafforini et al35 demonstrated that amino acids 205, 115, and 116 are important for the binding of human PAF-AH to LDL and that the carboxyl terminus of apo B-100 plays a key role in the association of PAF-AH with LDL. When residues 115 and 116 of human PAF-AH were introduced into murine PAF-AH, the mutant murine PAF-AH associated with LDL. Therefore, both the amino acid sequence of murine PAF-AH and low levels of apo B-100 in mice may contribute to the predominant association of murine PAF-AH with HDL.
Human apo A-I overexpression in C57BL/6 transgenic mice and in mice treated with the human apo A-I adenovirus was associated with a relative increase in PAF-AH activity in small HDL compared with large HDL. These small, human apo A-I–containing HDL particles isolated by gel filtration may correspond to the very high density lipoprotein-1 subfraction (VHDL-1) isolated by isopycnic density-gradient ultracentrifugation, which has previously been shown to preferentially bind PAF-AH.32 In contrast to C57BL/6 control mice, a significant amount of PAF-AH activity in control apo E-/- mice was associated with small HDL. It is possible that the absence of apo E in apo E-/- mice affects murine PAF-AH distribution in large- and small-size HDL particles. Previously, it has been shown that human PAF-AH associates exclusively with LDL and HDL containing apo E.13 Also, the significantly reduced amount of murine apo A-I in HDL of apo E-/- control mice36 may affect PAF-AH distribution and may cause lower PAF-AH activity associated with HDL in these mice.
In contrast to PAF-AH, PON1 was predominantly associated with
large-size HDL particles, and only a small fraction of activity was in
small-size HDL particles. The association of PON1 with HDL is mediated
through the binding of its retained hydrophobic N-terminal
leader sequence to HDL phospholipids and does not involve a direct
association with apo A-I,37 which may contribute to
the observed differences between the distribution of PAF-AH and PON1.
Arylesterase and paraoxonase activities increased in human apo
A-I–transgenic C57BL/6 and apo E-/- mice but
decreased after human apo A-I gene transfer in C57BL/6 and apo
E-/- mice. This discrepancy may be explained by
the production of inflammatory cytokines in the liver
after gene transfer with the first-generation
E1-deleted adenoviral vectors. Paraoxonase
activity and PON1 mRNA levels in the liver have indeed been
shown to decrease after tumor necrosis factor- and IL-1
administration in Syrian hamsters.38 The presence of
detectable Il-1ß in plasma after gene transfer, the decrease of the
negative acute-phase response protein albumin, and the increase
of 2-globulins and complement component
C3 indicate that cytokine
production was induced in the liver after gene transfer. Thus,
high liver concentrations of cytokines after gene transfer may
have resulted in decreased PON1 expression. It remains to be
investigated whether human apo A-I overexpression induced by gene
transfer with a new-generation, nontoxic adenoviral vector can increase
paraoxonase activity in C57BL/6 and apo E-/-
mice.
Previously, Castellani et al39 reported that the PAF-AH and arylesterase activity is similar in HDL isolated by ultracentrifugation from the plasma of C57BL/6 mice and human apo A-I–transgenic mice, at least when data are normalized for total HDL protein. The PAF-AH and arylesterase activity in HDL isolated by gel filtration in the present study represents the total activity, which was not normalized for HDL protein. Thus, our data in C57BL/6 and human apo A-I–transgenic mice are in accordance with those of Castellani et al.39 The present study also demonstrates that PAF-AH and arylesterase/paraoxonase activity in C57BL/6 apo E-/- mice was significantly lower than in C57BL/6 mice. Decreased paraoxonase activity in apo E-/- mice has been previously described by Hayek et al,40 although the extent of decrease (1.4-fold) was lower than in this study (2.0-fold). This may be related to differences in genetic background or in age of the mice.
The increase in PAF-AH and paraoxonase activity in human apo A-I C57BL/6 apo E-/- transgenic mice to levels equal to or above those of C57BL/6 mice may significantly contribute to the inhibition of progression of atherosclerosis4 5 by restoring an effective anti-inflammatory and antioxidant activity of HDL. Theilmeier et al41 recently demonstrated that human apo A-I transgenesis is associated with reduced oxidative stress in apo E-/- mice, reduced ß-VLDL–triggered endothelial cytosolic Ca2+ signaling through PAF-like bioactivity, and diminished ex vivo leukocyte adhesion. Furthermore, adenoviral gene transfer of PAF-AH reduced in vivo macrophage homing in the absence of increased HDL cholesterol, indicating the potential physiological significance of elevated PAF-AH activity.
In conclusion, this study provides evidence that overexpression of human apo A-I increases HDL-associated PAF-AH activity. In contrast to higher paraoxonase activity in human apo A-I–transgenic mice, paraoxonase activity after human apo A-I gene transfer decreases, probably due to cytokine-mediated inhibition of PON1 expression. Increased levels of these HDL-associated enzymes may improve the anti-inflammatory and antioxidative potential of HDL and may directly contribute to the protection conferred by HDL against atherothrombosis.
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Acknowledgments |
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This work was supported by the Interuniversitaire Attractiepolen Program (P4/34) and by the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen (program G.0110.98). Bart De Geest is a Postdoctoral Fellow of the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen. These studies were partially supported by INSERM and by a research grant from the Actions Intégrée Franco-Belge "Tournesol."
Received May 25, 2000; accepted June 14, 2000.
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Gordon DJ, Rifkind BM. High-density lipoprotein: the clinical implications of recent studies. N Engl J Med. 1989;321:1311–1316.[Medline] [Order article via Infotrieve]
2.
Gordon DJ, Probstfield JL, Garrison RJ, Neaton JD,
Castelli WP, Knoke JD, Jacobs DR, Bangdiwala S, Tyroler HA.
High-density lipoprotein cholesterol and
cardiovascular disease: four prospective American
studies. Circulation. 1989;79:8–15.
3. Rubin EM, Krauss RM, Spangler EA, Verstuyft JG, Clift SM. Inhibition of early atherogenesis in transgenic mice by human apolipoprotein AI. Nature. 1991;353:265–267.[Medline] [Order article via Infotrieve]
4. Paszty C, Maeda N, Verstuyft J, Rubin EM. Apolipoprotein AI transgene corrects apolipoprotein E deficiency-induced atherosclerosis in mice. J Clin Invest. 1994;94:899–903.
5.
Plump AS, Scott CJ, Breslow JL. Human apolipoprotein
A-I gene expression increases high density lipoprotein and suppresses
atherosclerosis in the apolipoprotein E-deficient
mouse. Proc Natl Acad Sci U S A. 1994;91:9607–9611.
6.
Duverger N, Kruth H, Emmanuel F, Caillaud JM,
Viglietta C, Castro G, Tailleux A, Fievet C, Fruchart JC, Houdebine LM,
Denefle P. Inhibition of atherosclerosis development in
cholesterol-fed human apolipoprotein A-I-transgenic
rabbits. Circulation. 1996;94:713–717.
7. Glomset JA. The plasma lecithin:cholesterol acyltransferase reaction. J Lipid Res. 1968;9:155–167.[Abstract]
8. Sattler W, Stocker R. Greater selective uptake by Hep G2 cells of high-density lipoprotein cholesteryl ester hydroperoxides than of unoxidized cholesteryl esters. Biochem J. 1993;294:771–778.
9.
Bowry V, Stanley KK, Stocker R. High density
lipoprotein is the major carrier of lipid hydroperoxides in human blood
plasma from fasting donors. Proc Natl Acad Sci U S A. 1992;89:10316–10320.
10. Fluiter K, Vietsch H, Biessen EAL, Kostner GM, van Berkel TJC, Sattler W. Biochem J. 1996;319:471–476.
11.
Fluiter K, Sattler W, De Beer MC, Connell PM, van der
Westhuyzen DR, van Berkel TJC. Scavenger receptor BI mediates the
selective uptake of oxidized cholesterol esters by rat
liver. J Biol Chem. 1999;274:8893–8899.
12.
Stremler KE, Stafforini DM, Prescott SM, McIntyre TM.
Human plasma platelet-activating factor acetylhydrolase:
oxidatively fragmented phospholipids as substrates. J Biol
Chem. 1991;266:11095–11103.
13.
Stafforini DM, McIntyre TM, Carter ME, Prescott SM.
Human plasma platelet-activating factor acetylhydrolase:
association with lipoprotein particles and role in the degradation of
platelet-activating factor. J Biol Chem. 1987;262:4215–4222.
14. Heery JM, Kozak M, Stafforini DM, Jones DA, Zimmerman GA, McIntyre TM, Prescott SM. Oxidatively modified LDL contains phospholipids with platelet-activating factor-like activity and stimulates the growth of smooth muscle cells. J Clin Invest. 1995;96:2322–2330.
15. Watson AD, Navab M, Hama SY, Sevanian A, Prescott SM, Stafforini DM, McIntyre TM, Du BN, Fogelman AM, Berliner JA. Effect of platelet activating factor-acetylhydrolase on the formation and action of minimally oxidized low density lipoprotein. J Clin Invest. 1995;95:774–782.
16.
Goyal J, Wang K, Liu M, Subbaiah PV. Novel
function of lecithin-cholesterol acyltransferase:
hydrolysis of oxidized polar phospholipids generated during lipoprotein
oxidation. J Biol Chem. 1997;272:16231–16239.
17. Blatter MC, James RW, Messmer S, Barja F, Pometta D. Identification of a distinct human high-density lipoprotein subspecies defined by a lipoprotein-associated protein, K-45: identity of K-45 with paraoxonase. Eur J Biochem. 1993;211:871–879.[Medline] [Order article via Infotrieve]
18. Mackness MI. ‘A’-esterases: enzymes looking for a role? Biochem Pharmacol. 1989;38:385–390.[Medline] [Order article via Infotrieve]
19. Mackness MI, Arrol S, Abbott C, Durrington PN. Protection of low density lipoprotein against oxidative modification by high density lipoprotein associated paraoxonase. Atherosclerosis. 1993;104:129–135.[Medline] [Order article via Infotrieve]
20. Aviram M, Rosenblat M, Bisgaier CL, Newton RS, Primo-Parmo SL, La Du B. Paraoxonase inhibits high density lipoprotein (HDL) oxidation and preserves its functions: a possible peroxidative role for paraoxonase. J Clin Invest. 1998;101:1581–1590.[Medline] [Order article via Infotrieve]
21. Shih DM, Gu L, Xia YR, Navab M, Li WF, Hama S, Castellani LW, Furlong CE, Costa LG, Fogelman AM, Lusis AJ. Mice lacking serum paraoxonase are susceptible to organophosphate toxicity and atherosclerosis. Nature. 1998;394:284–287.[Medline] [Order article via Infotrieve]
22.
Steinberg D. Lewis A. Conner Memorial Lecture:
oxidative modification of LDL and atherogenesis.
Circulation. 1997;95:1062–1071.
23. Holvoet P, Collen D. Oxidized lipoproteins in atherosclerosis and thrombosis. FASEB J. 1994;8:1279–1284.[Abstract]
24.
Ameli S, Hultgardh-Nilsson A, Cercek B, Shah PK,
Forrester JS, Ageland H, Nilsson J. Recombinant apolipoprotein A-I
Milano reduces intimal thickening after balloon injury in
hypercholesterolemic rabbits. Circulation. 1994;90:1935–1941.
25.
Soma MR, Donetti E, Parolini C, Sirtori CR, Fumagalli
R, Franceschini G. Recombinant apolipoprotein A-I Milano dimer inhibits
carotid intimal thickening induced by perivascular manipulation in
rabbits. Circ Res. 1995;76:405–411.
26.
De Geest B, Zhao Z, Collen D, Holvoet P. Effects of
adenovirus-mediated human apo A-I gene transfer on
neointima formation after endothelial
denudation in apo E–deficient mice. Circulation. 1997;96:4349–4356.
27.
Carmeliet P, Stassen JM, Van Vlaenderen I, Meidell RS,
Collen D, Gerard RD. Adenovirus-mediated transfer of tissue-type
plasminogen activator augments
thrombolysis in tissue-type plasminogen
activator-deficient and plasminogen
activator inhibitor-1-overexpressing mice.
Blood. 1997;90:1527–1534.
28.
Piedrahita JA, Zhang SH, Hagaman JR, Oliver PM, Maeda
N. Generation of mice carrying a mutant apolipoprotein E gene
inactivated by gene targeting in embryonic stem cells.
Proc Natl Acad Sci U S A. 1992;89:4471–4475.
29.
Rubin EM, Ishida BY, Clift SM, Krauss RM. Expression of
human apolipoprotein A-I in transgenic mice results in reduced plasma
levels of murine apolipoprotein A-I and the appearance of two new high
density lipoprotein size subclasses. Proc Natl Acad Sci
U S A. 1991;88:434–438.
30. Holvoet P, Danloy S, Deridder E, Lox M, Bernar H, Dhoest A, Collen D. Substitution of the carboxyl-terminal domain of apo AI with apo AII sequences restores the potential of HDL to reduce the progression of atherosclerosis in apo E knockout mice. J Clin Invest. 1998;102:379–385.[Medline] [Order article via Infotrieve]
31. Vercaemst R, Union A, Rosseneu M, De Craene I, De Backer G, Kornitzer M. Quantitation of plasma free cholesterol and cholesteryl esters by high performance liquid chromatography: study of a normal population. Atherosclerosis. 1989;78:245–250.[Medline] [Order article via Infotrieve]
32.
Tselepis AD, Dentan C, Karabina SA, Chapman MJ, Ninio
E. PAF-degrading acetylhydrolase is preferentially associated with
dense LDL and VHDL-1 in human plasma: catalytic characteristics and
relation to the monocyte-derived enzyme. Arterioscler Thromb Vasc
Biol. 1995;15:1764–1773.
33. Gan KN, Smolen A, Eckerson HW, La Du BN. Purification of human serum paraoxonase/arylesterase: evidence for one esterase catalyzing both activities. Drug Metab Dispos. 1991;19:100–106.[Abstract]
34. Mackness MI, Harty D, Bhatnagar D, Winocour PH, Arrol S, Ishola M, Durrington PN. Serum paraoxonase activity in familial hypercholesterolaemia and insulin-dependent diabetes mellitus. Atherosclerosis. 1991;86:193–199.[Medline] [Order article via Infotrieve]
35.
Stafforini DM, Tjoelker LW, McCormick SP, Vaitkus D,
McIntyre TM, Gray PW, Young SG, Prescott SM. Molecular basis of the
interaction between plasma platelet-activating factor
acetylhydrolase and low density lipoprotein. J Biol
Chem. 1999;274:7018–7024.
36. Zhang SH, Reddick RL, Burkey B, Maeda N. Diet-induced atherosclerosis in mice heterozygous and homozygous for apolipoprotein E gene disruption. J Clin Invest. 1994;94:937–945.
37.
Sorenson RC, Bisgaier CL, Aviram M, Hsu C, Billecke S,
La Du BN. Human serum paraoxonase/arylesterase’s retained hydrophobic
N-terminal leader sequence associates with HDLs by binding
phospholipids: apolipoprotein A-I stabilizes activity.
Arterioscler Thromb Vasc Biol. 1999;19:2214–2225.
38. Feingold KR, Memon RA, Moser AH, Grunfeld C. Paraoxonase activity in the serum and hepatic mRNA levels decrease during the acute phase response. Atherosclerosis. 1998;139:307–315.[Medline] [Order article via Infotrieve]
39. Castellani LW, Navab M, Lenten BJV, Hedrick CC, Hama SY, Goto AM, Fogelman AM, Lusis AJ. Overexpression of apolipoprotein AII in transgenic mice converts high density lipoproteins to proinflammatory particles. J Clin Invest. 1997;100:464–474.[Medline] [Order article via Infotrieve]
40.
Hayek T, Fuhrman B, Vaya J, Rosenblat M, Belinky P,
Coleman R, Elis A, Aviram M. Reduced progression of
atherosclerosis in apolipoprotein E–deficient mice
following consumption of red wine, or its polyphenols quercetin or
catechin, is associated with reduced susceptibility of LDL to oxidation
and aggregation. Arterioscler Thromb Vasc Biol. 1997;17:2744–2752.
41.
Theilmeier G, De Geest B, Van Veldhoven P,
Stengel D, Michiels C, Lox M, Landeloos M, Chapman MJ, Ninio E, Collen
D, Himpens B, Holvoet P. HDL associated PAF-AH reduces
endothelial adhesiveness in apo
E-/- mice. FASEB J. 2000;14:2032–2039.
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